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    Normalizing glycosphingolipids restores function in CD4+ T cells from lupus patients. McDonald Georgia,Deepak Shantal,Miguel Laura,Hall Cleo J,Isenberg David A,Magee Anthony I,Butters Terry,Jury Elizabeth C The Journal of clinical investigation Patients with the autoimmune rheumatic disease systemic lupus erythematosus (SLE) have multiple defects in lymphocyte signaling and function that contribute to disease pathogenesis. Such defects could be attributed to alterations in metabolic processes, including abnormal control of lipid biosynthesis pathways. Here, we reveal that CD4+ T cells from SLE patients displayed an altered profile of lipid raft-associated glycosphingolipids (GSLs) compared with that of healthy controls. In particular, lactosylceramide, globotriaosylceramide (Gb3), and monosialotetrahexosylganglioside (GM1) levels were markedly increased. Elevated GSLs in SLE patients were associated with increased expression of liver X receptor β (LXRβ), a nuclear receptor that controls cellular lipid metabolism and trafficking and influences acquired immune responses. Stimulation of CD4+ T cells isolated from healthy donors with synthetic and endogenous LXR agonists promoted GSL expression, which was blocked by an LXR antagonist. Increased GSL expression in CD4+ T cells was associated with intracellular accumulation and accelerated trafficking of GSL, reminiscent of cells from patients with glycolipid storage diseases. Inhibition of GSL biosynthesis in vitro with a clinically approved inhibitor (N-butyldeoxynojirimycin) normalized GSL metabolism, corrected CD4+ T cell signaling and functional defects, and decreased anti-dsDNA antibody production by autologous B cells in SLE patients. Our data demonstrate that lipid metabolism defects contribute to SLE pathogenesis and suggest that targeting GSL biosynthesis restores T cell function in SLE. 10.1172/JCI69571
    Enhanced functional response of CD133+ circulating progenitor cells in patients early after acute myocardial infarction. Vöö Stefan,Eggermann Juliane,Dunaeva Marina,Ramakers-van Oosterhoud Carolien,Waltenberger Johannes European heart journal AIMS:Circulating progenitor cells (PC) may contribute to myocardial recovery following infarction. Growth factors including VEGF are produced during ischaemia and stimulate PC release and activation. In this study, we focused on the functional chemotactic response of PC to VEGF in subjects early after myocardial ischaemia. METHODS AND RESULTS:Number and phenotype of PC were characterized using flow-cytometry. CD133(+)PC were isolated from peripheral blood using positive MACS isolation. The chemotactic response towards members of the VEGF family (VEGF-A, PlGF-1, and VEGF-E) was analysed in three groups: (i) early period following acute myocardial infarction (days 2-4) treated with primary PCI (AMI) (n = 35), (ii) stable coronary artery disease (CAD) (n = 35), and (iii) controls (CTR) (n = 20). CD133(+)PC number was 2-fold higher in AMI when compared with CAD and CTR (P = 0.0001), whereas CAD was not different from CTR. The chemotactic response of CD133(+)PC to VEGF-A, PlGF-1, and VEGF-E was significantly enhanced (2-fold) in AMI when compared with CAD (P = 0.0001). While the increase of the VEGFR-1-mediated/PlGF-triggered response was rapid (2 days following infarction), the VEGFR-2-mediated/VEGF-E-triggered response was maximally increased on day 4 post-AMI, thus correlating with the kinetics of maximal inflammatory activation reflected by increased CRP levels (P = 0.019). CONCLUSION:The enhanced chemotactic response of CD133(+)PC following myocardial infarction represents a novel principle potentially involved in cardiovascular repair early after myocardial infarction. Acute inflammatory processes are closely associated with this increased cellular function. 10.1093/eurheartj/ehm542
    Global impairment of CD4+CD25+FOXP3+ regulatory T cells in idiopathic pulmonary fibrosis. Kotsianidis Ioannis,Nakou Evangelia,Bouchliou Irene,Tzouvelekis Argyrios,Spanoudakis Emmanouil,Steiropoulos Paschalis,Sotiriou Ioannis,Aidinis Vassilis,Margaritis Dimitrios,Tsatalas Costas,Bouros Demosthenes American journal of respiratory and critical care medicine RATIONALE:The implication of T cells in the pathogenesis of idiopathic pulmonary fibrosis (IPF) is controversial. CD4(+)CD25(+)FOXP3(+) regulatory T cells (Tregs) are pivotal in maintaining immune homeostasis, but their role in IPF pathophysiology has not yet been studied. OBJECTIVES:To explore Treg dynamics and function in IPF. METHODS:Treg levels and dynamics were analyzed by flow cytometry in the peripheral blood (PB) and bronchoalveolar lavage (BAL) of 21 patients with IPF, 35 patients with lung diseases other than IPF (patients without IPF), 20 patients with collagen vascular diseases with pulmonary parenchymal involvement (CVD-IP), and 28 healthy volunteers. The suppression of autologous CD4(+)CD25(-) cell-proliferative responses and cytokine release by magnetic bead-isolated Tregs was evaluated by proliferation assays and cytometric bead array. Correlations of Treg function and levels with lung function parameters were also performed. MEASUREMENTS AND MAIN RESULTS:In patients with IPF, both BAL and PB Tregs were reduced compared with those of healthy volunteers and patients without IPF, although not always significantly. Treg levels were not affected by the administration of low-dose prednisone in four nonresponding patients. The suppressor potential of BAL and PB Tregs was compromised in patients with IPF and patients with CVD-IP, compared with healthy volunteers and patients without IPF. Similarly, the Treg-induced suppression of helper T-cell type 1 and 2 cytokine secretion was impaired in the BAL of patients with IPF and patients with CVD-IP. Moreover, the defective function of BAL Tregs correlated highly with parameters of disease severity. CONCLUSIONS:This study provides the first evidence of global Treg impairment in IPF that strongly correlates with disease severity, suggesting a role for Tregs in the fibrotic process. 10.1164/rccm.200812-1936OC
    ClampFISH detects individual nucleic acid molecules using click chemistry-based amplification. Rouhanifard Sara H,Mellis Ian A,Dunagin Margaret,Bayatpour Sareh,Jiang Connie L,Dardani Ian,Symmons Orsolya,Emert Benjamin,Torre Eduardo,Cote Allison,Sullivan Alessandra,Stamatoyannopoulos John A,Raj Arjun Nature biotechnology Methods for detecting single nucleic acids in cell and tissues, such as fluorescence in situ hybridization (FISH), are limited by relatively low signal intensity and nonspecific probe binding. Here we present click-amplifying FISH (clampFISH), a method for fluorescence detection of nucleic acids that achieves high specificity and high-gain (>400-fold) signal amplification. ClampFISH probes form a 'C' configuration upon hybridization to the sequence of interest in a double helical manner. The ends of the probes are ligated together using bio-orthogonal click chemistry, effectively locking the probes around the target. Iterative rounds of hybridization and click amplify the fluorescence intensity. We show that clampFISH enables the detection of RNA species with low-magnification microscopy and in RNA-based flow cytometry. Additionally, we show that the modular design of clampFISH probes allows multiplexing of RNA and DNA detection, that the locking mechanism prevents probe detachment in expansion microscopy, and that clampFISH can be applied in tissue samples. 10.1038/nbt.4286
    Statins do not influence clinical response and B cell depletion after rituximab treatment in rheumatoid arthritis. Das Sudipto,Fernandez Matilla Meritxell,Dass Shouvik,Buch Maya H,Rawstron Andrew C,Vital Edward M,Emery Paul Annals of the rheumatic diseases 10.1136/annrheumdis-2012-202454
    Asymmetric segregation of polarized antigen on B cell division shapes presentation capacity. Thaunat Olivier,Granja Aitor G,Barral Patricia,Filby Andrew,Montaner Beatriz,Collinson Lucy,Martinez-Martin Nuria,Harwood Naomi E,Bruckbauer Andreas,Batista Facundo D Science (New York, N.Y.) During the activation of humoral immune responses, B cells acquire antigen for subsequent presentation to cognate T cells. Here we show that after mouse B cells accumulate antigen, it is maintained in a polarized distribution for extended periods in vivo. Using high-throughput imaging flow cytometry, we observed that this polarization is preserved during B cell division, promoting asymmetric antigen segregation among progeny. Antigen inheritance correlates with the ability of progeny to activate T cells: Daughter cells receiving larger antigen stores exhibit a prolonged capacity to present antigen, which renders them more effective in competing for T cell help. The generation of progeny with differential capacities for antigen presentation may have implications for somatic hypermutation and class switching during affinity maturation and as B cells commit to effector cell fates. 10.1126/science.1214100
    FACS purification and transcriptome analysis of drosophila neural stem cells reveals a role for Klumpfuss in self-renewal. Berger Christian,Harzer Heike,Burkard Thomas R,Steinmann Jonas,van der Horst Suzanne,Laurenson Anne-Sophie,Novatchkova Maria,Reichert Heinrich,Knoblich Juergen A Cell reports Drosophila neuroblasts (NBs) have emerged as a model for stem cell biology that is ideal for genetic analysis but is limited by the lack of cell-type-specific gene expression data. Here, we describe a method for isolating large numbers of pure NBs and differentiating neurons that retain both cell-cycle and lineage characteristics. We determine transcriptional profiles by mRNA sequencing and identify 28 predicted NB-specific transcription factors that can be arranged in a network containing hubs for Notch signaling, growth control, and chromatin regulation. Overexpression and RNA interference for these factors identify Klumpfuss as a regulator of self-renewal. We show that loss of Klumpfuss function causes premature differentiation and that overexpression results in the formation of transplantable brain tumors. Our data represent a valuable resource for investigating Drosophila developmental neurobiology, and the described method can be applied to other invertebrate stem cell lineages as well. 10.1016/j.celrep.2012.07.008
    The central nervous system-restricted transcription factor Olig2 opposes p53 responses to genotoxic damage in neural progenitors and malignant glioma. Mehta Shwetal,Huillard Emmanuelle,Kesari Santosh,Maire Cecile L,Golebiowski Diane,Harrington Emily P,Alberta John A,Kane Michael F,Theisen Matthew,Ligon Keith L,Rowitch David H,Stiles Charles D Cancer cell High-grade gliomas are notoriously insensitive to radiation and genotoxic drugs. Paradoxically, the p53 gene is structurally intact in the majority of these tumors. Resistance to genotoxic modalities in p53-positive gliomas is generally attributed to attenuation of p53 functions by mutations of other components within the p53 signaling axis, such as p14(Arf), MDM2, and ATM, but this explanation is not entirely satisfactory. We show here that the central nervous system (CNS)-restricted transcription factor Olig2 affects a key posttranslational modification of p53 in both normal and malignant neural progenitors and thereby antagonizes the interaction of p53 with promoter elements of multiple target genes. In the absence of Olig2 function, even attenuated levels of p53 are adequate for biological responses to genotoxic damage. 10.1016/j.ccr.2011.01.035
    A PML–PPAR-δ pathway for fatty acid oxidation regulates hematopoietic stem cell maintenance. Ito Keisuke,Carracedo Arkaitz,Weiss Dror,Arai Fumio,Ala Ugo,Avigan David E,Schafer Zachary T,Evans Ronald M,Suda Toshio,Lee Chih-Hao,Pandolfi Pier Paolo Nature medicine Stem-cell function is an exquisitely regulated process. Thus far, the contribution of metabolic cues to stem-cell function has not been well understood. Here we identify a previously unknown promyelocytic leukemia (PML)–peroxisome proliferator-activated receptor δ (PPAR-δ)–fatty-acid oxidation (FAO) pathway for the maintenance of hematopoietic stem cells (HSCs). We have found that loss of PPAR-δ or inhibition of mitochondrial FAO induces loss of HSC maintenance, whereas treatment with PPAR-δ agonists improved HSC maintenance. PML exerts its essential role in HSC maintenance through regulation of PPAR signaling and FAO. Mechanistically, the PML–PPAR-δ–FAO pathway controls the asymmetric division of HSCs. Deletion of Ppard or Pml as well as inhibition of FAO results in the symmetric commitment of HSC daughter cells, whereas PPAR-δ activation increased asymmetric cell division. Thus, our findings identify a metabolic switch for the control of HSC cell fate with potential therapeutic implications. 10.1038/nm.2882
    Retention of the NLRP3 Inflammasome-Primed Neutrophils in the Bone Marrow Is Essential for Myocardial Infarction-Induced Granulopoiesis. Circulation BACKGROUND:Acute myocardial infarction (MI) results in overzealous production and infiltration of neutrophils to the ischemic heart. This is mediated in part by granulopoiesis induced by the S100A8/A9-NLRP3-IL-1β signaling axis in injury-exposed neutrophils. Despite the transcriptional upregulation of the NLRP3 (Nod Like Receptor Family Pyrin Domain-Containing 3) inflammasome and associated signaling components in neutrophils, the serum levels of IL-1β (interleukin-1β), the effector molecule in granulopoiesis, were not affected by MI, suggesting that IL-1β is not released systemically. We hypothesize that IL-1β is released locally within the bone marrow (BM) by inflammasome-primed and reverse-migrating neutrophils. METHODS:Using a combination of time-dependent parabiosis and flow cytometry techniques, we first characterized the migration patterns of different blood cell types across the parabiotic barrier. We next induced MI in parabiotic mice by permanent ligation of the left anterior descending artery and examined the ability of injury-exposed neutrophils to permeate the parabiotic barrier and induce granulopoiesis in noninfarcted parabionts. Last, using multiple neutrophil adoptive and BM transplant studies, we studied the molecular mechanisms that govern reverse migration and retention of the primed neutrophils, IL-1β secretion, and granulopoiesis. Cardiac function was assessed by echocardiography. RESULTS:MI promoted greater accumulation of the inflammasome-primed neutrophils in the BM. Introducing a time-dependent parabiotic barrier to the free movement of neutrophils inhibited their ability to stimulate granulopoiesis in the noninfarcted parabionts. Previous priming of the NLRP3 inflammasome is not a prerequisite, but the presence of a functional CXCR4 (C-X-C-motif chemokine receptor 4) on the primed-neutrophils and elevated serum S100A8/A9 levels are necessary for homing and retention of the reverse-migrating neutrophils. In the BM, the primed-neutrophils secrete IL-1β through formation of gasdermin D pores and promote granulopoiesis. Pharmacological and genetic strategies aimed at the inhibition of neutrophil homing or release of IL-1β in the BM markedly suppressed MI-induced granulopoiesis and improved cardiac function. CONCLUSIONS:Our data reveal a new paradigm of how circulatory cells establish a direct communication between organs by delivering signaling molecules (eg, IL-1β) directly at the sites of action rather through systemic release. We suggest that this pathway may exist to limit the off-target effects of systemic IL-1β release. 10.1161/CIRCULATIONAHA.121.056019
    Tumor-associated Macrophage-derived Interleukin-23 Interlinks Kidney Cancer Glutamine Addiction with Immune Evasion. Fu Qiang,Xu Le,Wang Yiwei,Jiang Qi,Liu Zheng,Zhang Junyu,Zhou Quan,Zeng Han,Tong Shanyou,Wang Tao,Qi Yangyang,Hu Baoying,Fu Hangcheng,Xie Huyang,Zhou Lin,Chang Yuan,Zhu Yu,Dai Bo,Zhang Weijuan,Xu Jiejie European urology BACKGROUND:Glutamine addiction is a hallmark of clear cell renal cell carcinoma (ccRCC); yet whether glutamine metabolism impacts local immune surveillance is unclear. This knowledge may yield novel immunotherapeutic opportunities. OBJECTIVE:To seek a potential therapeutic target in glutamine-addicted ccRCC. DESIGN, SETTING, AND PARTICIPANTS:Tumors from ccRCC patients from a Shanghai cohort and ccRCC tumor data from The Cancer Genome Atlas (TCGA) cohort were analyzed. In vivo and in vitro studies were conducted with fresh human ccRCC tumors and murine tumor cells. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS:Immune cell numbers and functions were analyzed by flow cytometry. Glutamine and cytokine concentrations were determined. Survival was compared between different subpopulations of patients using Kaplan-Meier and Cox regression analyses. RESULTS AND LIMITATIONS:We found that in ccRCC, high interleukin (IL)-23 expression was significantly associated with poor survival in both TCGA (overall survival [OS] hazard ratio [HR]=2.04, cancer-specific survival [CSS] HR=2.95; all p<0.001) and Shanghai (OS HR=2.07, CSS HR=3.92; all p<0.001) cohorts. IL-23 blockade prolongs the survival of tumor-bearing mice, promotes T-cell cytotoxicity in in vitro cultures of human ccRCC tumors, and augments the therapeutic benefits of anti-PD-1 antibodies. Mechanistically, glutamine consumption by ccRCC tumor cells results in the local deprivation of extracellular glutamine, which induces IL-23 secretion by tumor-infiltrating macrophages via the activation of hypoxia-inducible factor 1α (HIF1α). IL-23 activates regulatory T-cell proliferation and promotes IL-10 and transforming growth factor β expression, thereby suppressing tumor cell killing by cytotoxic lymphocytes. The positive correlations between glutamine metabolism, IL-23 levels, and Treg responses are confirmed in both TCGA cohort and tumors from Shanghai ccRCC patients. Study limitations include the unclear impacts of glutamine deprivation and IL-23 on other immune cells. CONCLUSIONS:Macrophage-secreted IL-23 enhanced Treg functions in glutamine-addicted tumors; thus, IL-23 is a promising target for immunotherapy in ccRCC. PATIENT SUMMARY:In this study, we analyzed the immune components in glutamine-addicted clear cell renal cell carcinoma (ccRCC) tumors from two patient cohorts and conducted both in vitro and in vivo studies. We found that ccRCC tumor cell-intrinsic glutamine metabolism orchestrates immune evasion via interleukin (IL)-23, and IL-23-high patients had significantly poorer survival than IL-23-low patients. IL-23 should thus be considered a therapeutic target in ccRCC, either alone or in combination with immune checkpoint inhibitors. 10.1016/j.eururo.2018.09.030
    Reduction of Squalene Epoxidase by Cholesterol Accumulation Accelerates Colorectal Cancer Progression and Metastasis. Jun Soo Young,Brown Andrew J,Chua Ngee Kiat,Yoon Ji-Yong,Lee Jeong-Ju,Yang Jin Ok,Jang InSu,Jeon Su-Jin,Choi Tae-Ik,Kim Cheol-Hee,Kim Nam-Soon Gastroenterology BACKGROUND & AIMS:Squalene epoxidase (SQLE), a rate-limiting enzyme in cholesterol biosynthesis, is suggested as a proto-oncogene. Paradoxically, SQLE is degraded by excess cholesterol, and low SQLE is associated with aggressive colorectal cancer (CRC). Therefore, we studied the functional consequences of SQLE reduction in CRC progression. METHODS:Gene and protein expression data and clinical features of CRCs were obtained from public databases and 293 human tissues, analyzed by immunohistochemistry. In vitro studies showed underlying mechanisms of CRC progression mediated by SQLE reduction. Mice were fed a 2% high-cholesterol or a control diet before and after cecum implantation of SQLE genetic knockdown/control CRC cells. Metastatic dissemination and circulating cancer stem cells were demonstrated by in vivo tracking and flow cytometry analysis, respectively. RESULTS:In vitro studies showed that SQLE reduction helped cancer cells overcome constraints by inducing the epithelial-mesenchymal transition required to generate cancer stem cells. Surprisingly, SQLE interacted with GSK3β and p53. Active GSK3β contributes to the stability of SQLE, thereby increasing cell cholesterol content, whereas SQLE depletion disrupted the GSK3β/p53 complex, resulting in a metastatic phenotype. This was confirmed in a spontaneous CRC metastasis mice model, where SQLE reduction, by a high-cholesterol regimen or genetic knockdown, strikingly promoted CRC aggressiveness through the production of migratory cancer stem cells. CONCLUSIONS:We showed that SQLE reduction caused by cholesterol accumulation aggravates CRC progression via the activation of the β-catenin oncogenic pathway and deactivation of the p53 tumor suppressor pathway. Our findings provide new insights into the link between cholesterol and CRC, identifying SQLE as a key regulator in CRC aggressiveness and a prognostic biomarker. 10.1053/j.gastro.2020.09.009
    Bat3 promotes T cell responses and autoimmunity by repressing Tim-3–mediated cell death and exhaustion. Rangachari Manu,Zhu Chen,Sakuishi Kaori,Xiao Sheng,Karman Jozsef,Chen Andrew,Angin Mathieu,Wakeham Andrew,Greenfield Edward A,Sobel Raymond A,Okada Hitoshi,McKinnon Peter J,Mak Tak W,Addo Marylyn M,Anderson Ana C,Kuchroo Vijay K Nature medicine T cell immunoglobulin and mucin domain–containing 3 (Tim-3) is an inhibitory receptor that is expressed on exhausted T cells during infection with HIV-1 and hepatitis C virus. By contrast, Tim-3 expression and function are defective in multiple human autoimmune diseases. However, the molecular mechanisms modulating Tim-3 function are not well understood. Here we show that human leukocyte antigen B (HLA-B)-associated transcript 3 (Bat3) binds to, and represses the function of, Tim-3. Bat3 protects T helper type 1 (TH1) cells from galectin-9–mediated cell death and promotes both proliferation and proinflammatory cytokine production. Bat3-deficient T cells have elevated expression of exhaustion-associated molecules such as Tim-3, Lag3, Prdm1 and Pbx3, and Bat3 knockdown in myelin-antigen–specific CD4+ T cells markedly inhibits the development of experimental autoimmune encephalomyelitis while promoting the expansion of a dysfunctional Tim-3hi, interferon-γ (IFN-γ)loCD4+ cell population. Furthermore, expression of Bat3 is reduced in exhausted Tim-3+ T cells from mouse tumors and HIV-1–infected individuals. These data indicate that Bat3 acts as an inhibitor of Tim-3–dependent exhaustion and cell death. Bat3 may thus represent a viable therapeutic target in autoimmune disorders, chronic infections and cancers. 10.1038/nm.2871
    Neutrophils mediate insulin resistance in mice fed a high-fat diet through secreted elastase. Talukdar Saswata,Oh Da Young,Bandyopadhyay Gautam,Li Dongmei,Xu Jianfeng,McNelis Joanne,Lu Min,Li Pingping,Yan Qingyun,Zhu Yimin,Ofrecio Jachelle,Lin Michael,Brenner Martin B,Olefsky Jerrold M Nature medicine Chronic low-grade adipose tissue and liver inflammation is a major cause of systemic insulin resistance and is a key component of the low degree of insulin sensitivity that exists in obesity and type 2 diabetes. Immune cells, such as macrophages, T cells, B cells, mast cells and eosinophils, have all been implicated as having a role in this process. Neutrophils are typically the first immune cells to respond to inflammation and can exacerbate the chronic inflammatory state by helping to recruit macrophages and by interacting with antigen-presenting cells. Neutrophils secrete several proteases, one of which is neutrophil elastase, which can promote inflammatory responses in several disease models. Here we show that treatment of hepatocytes with neutrophil elastase causes cellular insulin resistance and that deletion of neutrophil elastase in high-fat-diet–induced obese (DIO) mice leads to less tissue inflammation that is associated with lower adipose tissue neutrophil and macrophage content. These changes are accompanied by improved glucose tolerance and increased insulin sensitivity. Taken together, we show that neutrophils can be added to the extensive repertoire of immune cells that participate in inflammation-induced metabolic disease. 10.1038/nm.2885
    Phase I active immunotherapy with combination of two chimeric, human epidermal growth factor receptor 2, B-cell epitopes fused to a promiscuous T-cell epitope in patients with metastatic and/or recurrent solid tumors. Kaumaya Pravin T P,Foy Kevin Chu,Garrett Joan,Rawale Sharad V,Vicari Daniele,Thurmond Jennifer M,Lamb Tammy,Mani Aruna,Kane Yahaira,Balint Catherine R,Chalupa Donald,Otterson Gregory A,Shapiro Charles L,Fowler Jeffrey M,Grever Michael R,Bekaii-Saab Tanios S,Carson William E Journal of clinical oncology : official journal of the American Society of Clinical Oncology PURPOSE To evaluate the maximum-tolerated dose (MTD), safety profile, and immunogenicity of two chimeric, B-cell epitopes derived from the human epidermal growth factor receptor (HER2) extracellular domain in a combination vaccine with a promiscuous T-cell epitope (ie, MVF) and nor-muramyl-dipeptide as adjuvant emulsified in SEPPIC ISA 720. PATIENTS AND METHODS Eligible patients with metastatic and/or recurrent solid tumors received three inoculations on days 1, 22, and 43 at doses of total peptide that ranged from 0.5 to 3.0 mg. Immunogenicity was evaluated by enzyme-linked immunosorbent assay, flow cytometry, and HER2 signaling assays. Results Twenty-four patients received three inoculations at the intended dose levels, which elicited antibodies able to recognize native HER2 receptor and inhibited both the proliferation of HER2-expressing cell lines and phosphorylation of the HER2 protein. The MTD was determined to be the highest dose level of 3.0 mg of the combination vaccine. There was a significant increase from dose level 1 (0.5 mg) to dose level 4 (3.0 mg) in HER2-specific antibodies. Four patients (one each with adrenal, colon, ovarian, and squamous cell carcinoma of unknown primary) were judged to have stable disease; two patients (one each with endometrial and ovarian cancer) had partial responses; and 11 patients had progressive disease. Patients with stable disease received 6-month boosts, and one patient received a 20-month boost. CONCLUSION The combination vaccines were safe and effective in eliciting antibody responses in a subset of patients (62.5%) and were associated with no serious adverse events, autoimmune disease, or cardiotoxicity. There was preliminary evidence of clinical activity in several patients. 10.1200/JCO.2009.22.3883
    Microanatomic Distribution of Myeloid Heme Oxygenase-1 Protects against Free Radical-Mediated Immunopathology in Human Tuberculosis. Chinta Krishna C,Rahman Md Aejazur,Saini Vikram,Glasgow Joel N,Reddy Vineel P,Lever Jeremie M,Nhamoyebonde Shepherd,Leslie Alasdair,Wells Ryan M,Traylor Amie,Madansein Rajhmun,Siegal Gene P,Antony Veena B,Deshane Jessy,Wells Gordon,Nargan Kievershen,George James F,Ramdial Pratistadevi K,Agarwal Anupam,Steyn Adrie J C Cell reports Heme oxygenase-1 (HO-1) is a cytoprotective enzyme that controls inflammatory responses and redox homeostasis; however, its role during pulmonary tuberculosis (TB) remains unclear. Using freshly resected human TB lung tissue, we examined the role of HO-1 within the cellular and pathological spectrum of TB. Flow cytometry and histopathological analysis of human TB lung tissues showed that HO-1 is expressed primarily in myeloid cells and that HO-1 levels in these cells were directly proportional to cytoprotection. HO-1 mitigates TB pathophysiology by diminishing myeloid cell-mediated oxidative damage caused by reactive oxygen and/or nitrogen intermediates, which control granulocytic karyorrhexis to generate a zonal HO-1 response. Using whole-body or myeloid-specific HO-1-deficient mice, we demonstrate that HO-1 is required to control myeloid cell infiltration and inflammation to protect against TB progression. Overall, this study reveals that zonation of HO-1 in myeloid cells modulates free-radical-mediated stress, which regulates human TB immunopathology. 10.1016/j.celrep.2018.10.073
    Transcriptional code and disease map for adult retinal cell types. Siegert Sandra,Cabuy Erik,Scherf Brigitte Gross,Kohler Hubertus,Panda Satchidananda,Le Yun-Zheng,Fehling Hans Jörg,Gaidatzis Dimos,Stadler Michael B,Roska Botond Nature neuroscience Brain circuits are assembled from a large variety of morphologically and functionally diverse cell types. It is not known how the intermingled cell types of an individual adult brain region differ in their expressed genomes. Here we describe an atlas of cell type transcriptomes in one brain region, the mouse retina. We found that each adult cell type expressed a specific set of genes, including a unique set of transcription factors, forming a 'barcode' for cell identity. Cell type transcriptomes carried enough information to categorize cells into morphological classes and types. Several genes that were specifically expressed in particular retinal circuit elements, such as inhibitory neuron types, are associated with eye diseases. The resource described here allows gene expression to be compared across adult retinal cell types, experimenting with specific transcription factors to differentiate stem or somatic cells to retinal cell types, and predicting cellular targets of newly discovered disease-associated genes. 10.1038/nn.3032
    Large-scale mapping of sequence-function relations in small regulatory RNAs reveals plasticity and modularity. Peterman Neil,Lavi-Itzkovitz Anat,Levine Erel Nucleic acids research Two decades into the genomics era the question of mapping sequence to function has evolved from identifying functional elements to characterizing their quantitative properties including, in particular, their specificity and efficiency. Here, we use a large-scale approach to establish a quantitative map between the sequence of a bacterial regulatory RNA and its efficiency in modulating the expression of its targets. Our approach generalizes the sort-seq method, introduced recently to analyze promoter sequences, in order to accurately quantify the efficiency of a large library of sequence variants. We focus on two small RNAs (sRNAs) in E. coli, DsrA and RyhB, and their regulation of both repressed and activated targets. In addition to precisely identifying functional elements in the sRNAs, our data establish quantitative relationships between structural and energetic features of the sRNAs and their regulatory activity, and characterize a large set of direct and indirect interactions between nucleotides. A core of these interactions supports a model where specificity can be enhanced by a rigid molecular structure. Both sRNAs exhibit a modular design with limited cross-interactions, dividing the requirements for structural stability and target binding among modules. 10.1093/nar/gku863
    Identification of SARS-CoV-2-specific immune alterations in acutely ill patients. Rébillard Rose-Marie,Charabati Marc,Grasmuck Camille,Filali-Mouhim Abdelali,Tastet Olivier,Brassard Nathalie,Daigneault Audrey,Bourbonnière Lyne,Anand Sai Priya,Balthazard Renaud,Beaudoin-Bussières Guillaume,Gasser Romain,Benlarbi Mehdi,Moratalla Ana Carmena,Solorio Yves Carpentier,Boutin Marianne,Farzam-Kia Negar,Descôteaux-Dinelle Jade,Fournier Antoine Philippe,Gowing Elizabeth,Laumaea Annemarie,Jamann Hélène,Lahav Boaz,Goyette Guillaume,Lemaître Florent,Mamane Victoria Hannah,Prévost Jérémie,Richard Jonathan,Thai Karine,Cailhier Jean-François,Chomont Nicolas,Finzi Andrés,Chassé Michaël,Durand Madeleine,Arbour Nathalie,Kaufmann Daniel E,Prat Alexandre,Larochelle Catherine The Journal of clinical investigation Dysregulated immune profiles have been described in symptomatic patients infected with SARS-CoV-2. Whether the reported immune alterations are specific to SARS-CoV-2 infection or also triggered by other acute illnesses remains unclear. We performed flow cytometry analysis on fresh peripheral blood from a consecutive cohort of (a) patients hospitalized with acute SARS-CoV-2 infection, (b) patients of comparable age and sex hospitalized for another acute disease (SARS-CoV-2 negative), and (c) healthy controls. Using both data-driven and hypothesis-driven analyses, we found several dysregulations in immune cell subsets (e.g., decreased proportion of T cells) that were similarly associated with acute SARS-CoV-2 infection and non-COVID-19-related acute illnesses. In contrast, we identified specific differences in myeloid and lymphocyte subsets that were associated with SARS-CoV-2 status (e.g., elevated proportion of ICAM-1+ mature/activated neutrophils, ALCAM+ monocytes, and CD38+CD8+ T cells). A subset of SARS-CoV-2-specific immune alterations correlated with disease severity, disease outcome at 30 days, and mortality. Our data provide an understanding of the immune dysregulation specifically associated with SARS-CoV-2 infection among acute care hospitalized patients. Our study lays the foundation for the development of specific biomarkers to stratify SARS-CoV-2-positive patients at risk of unfavorable outcomes and to uncover candidate molecules to investigate from a therapeutic perspective. 10.1172/JCI145853
    Impaired plasmacytoid dendritic cell maturation and differential chemotaxis in chronic hepatitis C virus: associations with antiviral treatment outcomes. Mengshol J A,Golden-Mason L,Castelblanco N,Im K A,Dillon S M,Wilson C C,Rosen H R, Gut BACKGROUND:Dendritic cell (DC) defects may contribute to chronicity in hepatitis C virus (HCV) infection and determine response to PEG-interferon and ribavirin therapy via poor T cell stimulation. Studies to date have produced inconsistent results regarding DC maturation and function: no large study has examined DCs before and after therapy. AIMS:We examined if DC defects in maturation and chemotaxis are present by comparing therapeutic responders to non-responders. METHODS:We analysed peripheral DCs of 64 HCV genotype 1-infected patients from the Virahep-C study 2 weeks before and 24 weeks after therapy. We used flow cytometry to enumerate plasmacytoid DC (pDC) and myeloid DCs (mDC) and quantify expression of chemokine receptors and maturation markers. Chemotaxis was measured with an in vitro assay. RESULTS:Pre-treatment frequencies of pDCs and mDCs were significantly lower in HCV patients than controls and successful therapy normalised pDCs. Levels of CXCR3 and CXCR4 on pDCs were higher at baseline compared to normal controls and decreased with therapy. Pre-therapy levels of co-stimulatory marker CD40 and the maturation marker CD83 were higher in pDCs of patients chronically infected with HCV compared to normal patients, and levels of both markers dropped significantly with therapy in the SVR+ group only. Other maturation markers (CD86 and CCR7) were not elevated suggesting a partially activated phenotype. Baseline chemotaxis of pDCs to CXCL12 and CXCL10 predicted failure of antiviral response and correlated with the histological activity index inflammation score. CONCLUSIONS:Plasmacytoid DC defects exist in chronic HCV and successful antiviral therapy normalises many phenotypic and functional abnormalities. 10.1136/gut.2008.168948
    Genetic predisposition directs breast cancer phenotype by dictating progenitor cell fate. Proia Theresa A,Keller Patricia J,Gupta Piyush B,Klebba Ina,Jones Ainsley D,Sedic Maja,Gilmore Hannah,Tung Nadine,Naber Stephen P,Schnitt Stuart,Lander Eric S,Kuperwasser Charlotte Cell stem cell Women with inherited mutations in the BRCA1 gene have increased risk of developing breast cancer but also exhibit a predisposition for the development of aggressive basal-like breast tumors. We report here that breast epithelial cells derived from patients harboring deleterious mutations in BRCA1 (BRCA1(mut /+) give rise to tumors with increased basal differentiation relative to cells from BRCA1+/+ patients. Molecular analysis of disease-free breast tissues from BRCA1(mut /+) patients revealed defects in progenitor cell lineage commitment even before cancer incidence. Moreover, we discovered that the transcriptional repressor Slug is an important functional suppressor of human breast progenitor cell lineage commitment and differentiation and that it is aberrantly expressed in BRCA1(mut /+) tissues. Slug expression is necessary for increased basal-like phenotypes prior to and after neoplastic transformation. These findings demonstrate that the genetic background of patient populations, in addition to affecting incidence rates, significantly impacts progenitor cell fate commitment and, therefore, tumor phenotype. 10.1016/j.stem.2010.12.007
    Hair follicle stem cells provide a functional niche for melanocyte stem cells. Tanimura Shintaro,Tadokoro Yuko,Inomata Ken,Binh Nguyen Thanh,Nishie Wataru,Yamazaki Satoshi,Nakauchi Hiromitsu,Tanaka Yoshio,McMillan James R,Sawamura Daisuke,Yancey Kim,Shimizu Hiroshi,Nishimura Emi K Cell stem cell In most stem cell systems, the organization of the stem cell niche and the anchoring matrix required for stem cell maintenance are largely unknown. We report here that collagen XVII (COL17A1/BP180/BPAG2), a hemidesmosomal transmembrane collagen, is highly expressed in hair follicle stem cells (HFSCs) and is required for the maintenance not only of HFSCs but also of melanocyte stem cells (MSCs), which do not express Col17a1 but directly adhere to HFSCs. Mice lacking Col17a1 show premature hair graying and hair loss. Analysis of Col17a1-null mice revealed that COL17A1 is critical for the self-renewal of HFSCs through maintaining their quiescence and immaturity, potentially explaining the mechanism underlying hair loss in human COL17A1 deficiency. Moreover, forced expression of COL17A1 in basal keratinocytes, including HFSCs, in Col17a1-null mice rescues MSCs from premature differentiation and restores TGF-β signaling, demonstrating that HFSCs function as a critical regulatory component of the MSC niche. 10.1016/j.stem.2010.11.029
    Stage-specific optimization of activin/nodal and BMP signaling promotes cardiac differentiation of mouse and human pluripotent stem cell lines. Kattman Steven J,Witty Alec D,Gagliardi Mark,Dubois Nicole C,Niapour Maryam,Hotta Akitsu,Ellis James,Keller Gordon Cell stem cell Efficient differentiation of embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) to a variety of lineages requires step-wise approaches replicating the key commitment stages found during embryonic development. Here we show that expression of PdgfR-α segregates mouse ESC-derived Flk-1 mesoderm into Flk-1(+)PdgfR-α(+) cardiac and Flk-1(+)PdgfR-α(-) hematopoietic subpopulations. By monitoring Flk-1 and PdgfR-α expression, we found that specification of cardiac mesoderm and cardiomyocytes is determined by remarkably small changes in levels of Activin/Nodal and BMP signaling. Translation to human ESCs and iPSCs revealed that the emergence of cardiac mesoderm could also be monitored by coexpression of KDR and PDGFR-α and that this process was similarly dependent on optimal levels of Activin/Nodal and BMP signaling. Importantly, we found that individual mouse and human pluripotent stem cell lines require optimization of these signaling pathways for efficient cardiac differentiation, illustrating a principle that may well apply in other contexts. 10.1016/j.stem.2010.12.008
    Regulation of CXCL16 expression and secretion by myeloid cells is not altered in rheumatoid arthritis. van Lieshout A W T,van der Voort R,Toonen L W J,van Helden S F G,Figdor C G,van Riel P L C M,Radstake T R D J,Adema G J Annals of the rheumatic diseases OBJECTIVE:Chemokine (C-X-C motif) ligand 16 (CXCL16) is secreted by macrophages and dendritic cells (DCs) to attract memory type T cells. CXCL16 expression is increased in arthritic joints of patients with rheumatoid arthritis (RA) and a role for CXCL16 has been suggested in the pathogenesis of RA. To date, little is known about the regulation of CXCL16 on monocytes/macrophages and DCs. The aim of this study was to elucidate how CXCL16 expression is regulated in healthy donors and patients with RA. METHODS:CD14+cells were isolated from the peripheral blood or synovial fluid of patients with RA and healthy controls, differentiated into different types of dendritic cells or macrophages and stimulated with various cytokines or lipopolysaccharide (LPS). Cell surface proteins, including surface CXCL16, were measured by flow cytometry and soluble CXCL16 was measured by ELISA. RESULTS:Distinct types of dendritic cells constitutively express and secrete CXCL16, which is not affected by maturation. Monocytes rapidly upregulate membrane-bound CXCL16 expression and release soluble CXCL16 upon culture. CXCL16 expression by monocytes is transiently inhibited by the Toll-like receptor (TLR)4 ligand LPS. Th2 type cytokines inhibit soluble CXCL16, whereas T helper (Th)1 cell stimulus enhances its release. In RA monocytes/macrophages, neither CXCL16 expression, nor CXCL16 regulation is different from healthy controls. CONCLUSIONS:Culture of monocytes is the main trigger for CXCL16 surface expression in vitro, which is not altered in RA. Together our data suggest that the increased CXCL16 expression in patients with RA is likely to be caused by increased influx of monocytes rather than intrinsic differences in CXCL16 regulation. 10.1136/ard.2007.086611
    Residual inflammation after rituximab treatment is associated with sustained synovial plasma cell infiltration and enhanced B cell repopulation. Teng Y K O,Levarht E W N,Toes Rene E M,Huizinga Tom W J,van Laar Jacob M Annals of the rheumatic diseases OBJECTIVE:To investigate the clinical effects of rituximab treatment in relation to immunological effects of rituximab on tissue-derived B lineage cells and repopulation of circulating B cells. METHODS:A total of 24 patients with rheumatoid arthritis (RA) were treated with 2x1000 mg rituximab and assessed clinically at 4, 12, 18 and 24 weeks using a 44-joint Disease Activity Score (DAS(44)). Synovial biopsies were analysed with immunohistochemistry at baseline and 12 weeks after treatment. Peripheral blood mononuclear cells were analysed by high sensitivity flow cytometry at all timepoints. RESULTS:In this study, a cohort of patients was dichotomised according to those who achieved a low disease activity score (DAS(44)<2.4: LoA group) and those with persistent disease activity (DAS(44)>2.4: HiA group) at any time after rituximab treatment. At baseline, the low activity (LoA) group had significantly lower DAS(44) scores (median 3.33, range 2.84 to 4.23) than the high activity (HiA) group (median 3.73, range 3.03 to 5.23; p = 0.022) and significantly less histological inflammation in synovium (median 6.7, range 1 to 15 vs 16.6, range 4 to 22; p = 0.036). DAS(44) scores before and after rituximab treatment were associated with synovial infiltration of CD79a+ CD20- B cells, morphologically resembling plasma cells. Following treatment with rituximab, the LoA group had significantly reduced repopulation of circulating pre-switched IgD+ B cells (median 0.044%, range 0.002 to 0.66 vs 0.45%, range 0.07 to 9.47; p = 0.006) and post-switched CD27+ B cells (median 0.17%, range 0.04 to 0.39 vs 0.67, range 0.08 to 2.05; p = 0.005) compared to the HiA group. CONCLUSION:The present study demonstrated that a low disease activity state following rituximab was associated with reduced infiltration of CD79a+ CD20- plasma cells in synovium and reduced B cell repopulation. 10.1136/ard.2008.092791
    ΔNp63α is an oncogene that targets chromatin remodeler Lsh to drive skin stem cell proliferation and tumorigenesis. Keyes William M,Pecoraro Matteo,Aranda Victoria,Vernersson-Lindahl Emma,Li Wangzhi,Vogel Hannes,Guo Xuecui,Garcia Elvin L,Michurina Tatyana V,Enikolopov Grigori,Muthuswamy Senthil K,Mills Alea A Cell stem cell The p53 homolog p63 is essential for development, yet its role in cancer is not clear. We discovered that p63 deficiency evokes the tumor-suppressive mechanism of cellular senescence, causing a striking absence of stratified epithelia such as the skin. Here we identify the predominant p63 isoform, ΔNp63α, as a protein that bypasses oncogene-induced senescence to drive tumorigenesis in vivo. Interestingly, bypass of senescence promotes stem-like proliferation and maintains survival of the keratin 15-positive stem cell population. Furthermore, we identify the chromatin-remodeling protein Lsh as a new target of ΔNp63α that is an essential mediator of senescence bypass. These findings indicate that ΔNp63α is an oncogene that cooperates with Ras to promote tumor-initiating stem-like proliferation and suggest that Lsh-mediated chromatin-remodeling events are critical to this process. 10.1016/j.stem.2010.12.009
    Targeting the crosstalk between cytokine-induced killer cells and myeloid-derived suppressor cells in hepatocellular carcinoma. Yu Su Jong,Ma Chi,Heinrich Bernd,Brown Zachary J,Sandhu Milan,Zhang Qianfei,Fu Qiong,Agdashian David,Rosato Umberto,Korangy Firouzeh,Greten Tim F Journal of hepatology BACKGROUND & AIMS:Cytokine-induced killer (CIK) cell-based immunotherapy is effective as an adjuvant therapy in early stage hepatocellular carcinoma (HCC) but lacks efficacy in advanced HCC. We aimed to investigate immune suppressor mechanisms in HCC, focusing on the role of myeloid-derived suppressor cells (MDSCs) in response to CIK therapy. METHODS:MDSCs were quantified by flow cytometry and quantitative real-time PCR. Cytokines were detected by cytokine array. A lactate dehydrogenase cytotoxicity assay was performed in the presence or absence of MDSCs to study CIK function against HCC cells in vitro. An FDA-approved PDE5 inhibitor, tadalafil, was used to target MDSCs in vitro and in vivo. Two different murine HCC cell lines were tested in subcutaneous and orthotopic tumor models in C57BL/6 and BALB/c mice. The antitumor effects of human CIKs and MDSCs were also tested in vitro. RESULTS:Adoptive cell transfer of CIKs into tumor-bearing mice induced inflammatory mediators (e.g., CX3CL1, IL-13) in the tumor microenvironment and an increase of tumor-infiltrating MDSCs, leading to impaired antitumor activity in 2 different HCC models. MDSCs efficiently suppressed the cytotoxic activity of CIKs in vitro. In contrast, treatment with a PDE5 inhibitor reversed the MDSC suppressor function via ARG1 and iNOS blockade and systemic treatment with a PDE5 inhibitor prevented MDSC accumulation in the tumor microenvironment upon CIK cell therapy and increased its antitumor efficacy. Similar results were observed when human CIKs were tested in vitro in the presence of CD14HLA-DR MDSCs. Treatment of MDSCs with a PDE5 inhibitor suppressed MDSC suppressor function and enhanced CIK activity against human HCC cell lines in vitro. CONCLUSION:Our results suggest that targeting MDSCs is an efficient strategy to enhance the antitumor efficacy of CIKs for the treatment of patients with HCC. LAY SUMMARY:Cytokine-induced killer cells are a mixture of immune cells given to eliminate cancer cells. However, not all patients respond to this treatment. Herein, we show in 2 different liver cancer models that myeloid-derived suppressor cells are increased in response to cytokine-induced killer cell therapy. Targeting these myeloid-derived suppressor cells may provide an additional therapeutic benefit alongside cytokine-induced killer cell therapy. 10.1016/j.jhep.2018.10.040
    Host alloreactive memory T cells influence tolerance to kidney allografts in nonhuman primates. Nadazdin Ognjenka,Boskovic Svjetlan,Murakami Toru,Tocco Georges,Smith Rex-Neal,Colvin Robert B,Sachs David H,Allan James,Madsen Joren C,Kawai Tatsuo,Cosimi A Benedict,Benichou Gilles Science translational medicine Transplant tolerance, defined as indefinite allograft survival without immunosuppression, has been regularly achieved in laboratory mice but not in nonhuman primates or humans. In contrast to laboratory mice, primates regularly have high frequencies of alloreactive memory T cells (TMEMs) before transplantation. These TMEMs are poorly sensitive to conventional immunosuppression and costimulation blockade, and the presence of donor-reactive TMEMs in primates may account for their resistance to transplant tolerance protocols that have proven consistently effective in mice. We measured the frequencies of anti-donor TMEMs before and after transplantation in a series of rejecting and tolerant monkeys that underwent nonmyeloablative conditioning, short-term immunosuppression, and combined allogeneic kidney/cell transplantation. Transplants were acutely rejected in all the monkeys with high numbers of donor-specific TMEMs before transplantation. In contrast, long-term survival was observed in the recipients harboring lower frequencies of anti-donor TMEMs before transplantation. Similar amounts of TMEM homeostatic expansion were recorded in all transplanted monkeys upon hematopoietic reconstitution; however, only the tolerant monkeys had no expansion or activation of donor-reactive TMEMs after transplantation. These results indicate that the presence of high frequencies of host donor-reactive TMEMs before transplantation impairs tolerance induction to kidney allografts in this nonhuman primate model. Indeed, recipients harboring a low anamnestic reactivity to their donor before transplantation were successfully rendered tolerant via infusion of donor cells and short-term immunosuppression. This suggests that selection of allogeneic donors with low memory responses in recipients may be essential to successful transplant tolerance induction in patients. 10.1126/scitranslmed.3002093
    Misfolded GPI-anchored proteins are escorted through the secretory pathway by ER-derived factors. Zavodszky Eszter,Hegde Ramanujan S eLife We have used misfolded prion protein (PrP*) as a model to investigate how mammalian cells recognize and degrade misfolded GPI-anchored proteins. While most misfolded membrane proteins are degraded by proteasomes, misfolded GPI-anchored proteins are primarily degraded in lysosomes. Quantitative flow cytometry analysis showed that at least 85% of PrP* molecules transiently access the plasma membrane to lysosomes. Unexpectedly, time-resolved quantitative proteomics revealed a remarkably invariant PrP* interactome during its trafficking from the endoplasmic reticulum (ER) to lysosomes. Hence, PrP* arrives at the plasma membrane in complex with ER-derived chaperones and cargo receptors. These interaction partners were critical for rapid endocytosis because a GPI-anchored protein induced to misfold at the cell surface was not recognized effectively for degradation. Thus, resident ER factors have post-ER itineraries that not only shield misfolded GPI-anchored proteins during their trafficking, but also provide a quality control cue at the cell surface for endocytic routing to lysosomes. 10.7554/eLife.46740
    Actions of a picomolar short-acting S1P₁ agonist in S1P₁-eGFP knock-in mice. Cahalan Stuart M,Gonzalez-Cabrera Pedro J,Sarkisyan Gor,Nguyen Nhan,Schaeffer Marie-Therese,Huang Liming,Yeager Adam,Clemons Bryan,Scott Fiona,Rosen Hugh Nature chemical biology Sphingosine 1-phosphate receptor 1 (S1P(1)) is critical for lymphocyte recirculation and is a clinical target for treatment of multiple sclerosis. By generating a short-duration S1P(1) agonist and mice in which fluorescently tagged S1P(1) replaces wild-type receptor, we elucidate physiological and agonist-perturbed changes in expression of S1P(1) at a subcellular level in vivo. We demonstrate differential downregulation of S1P(1) on lymphocytes and endothelia after agonist treatment. 10.1038/nchembio.547
    Systems serology for evaluation of HIV vaccine trials. Ackerman Margaret E,Barouch Dan H,Alter Galit Immunological reviews The scale and scope of the global epidemic, coupled to challenges with traditional vaccine development approaches, point toward a need for novel methodologies for HIV vaccine research. While the development of vaccines able to induce broadly neutralizing antibodies remains the ultimate goal, to date, vaccines continue to fail to induce these rare humoral immune responses. Conversely, growing evidence across vaccine platforms in both non-human primates and humans points to a role for polyclonal vaccine-induced antibody responses in protection from infection. These candidate vaccines, despite employing disparate viral vectors and immunization strategies, consistently identify a role for functional or non-traditional antibody activities as correlates of immunity. However, the precise mechanism(s) of action of these "binding" antibodies, their specific characteristics, and their ability to be selectively induced and/or potentiated to result in complete protection merits parallel investigation to neutralizing antibody-based vaccine design approaches. Ultimately, while neutralizing and functional antibody-based vaccine strategies need not be mutually exclusive, defining the specific characteristics of "protective" functional antibodies may provide a target immune profile to potentially induce more robust immunity against HIV. Specifically, one approach to guide the development of functional antibody-based vaccine strategies, termed "systems serology", offers an unbiased and comprehensive approach to systematically survey humoral immune responses, capturing the array of functions and humoral response characteristics that may be induced following vaccination with high resolution. Coupled to machine learning tools, large datasets that explore the "antibody-ome" offer a means to step back from anticipated correlates and mechanisms of protection and toward a more fundamental understanding of coordinated aspects of humoral immune responses, to more globally differentiate among vaccine candidates, and most critically, to identify the features of humoral immunity that distinguish protective from non-protective responses. Overall, the systematic serological approach described here aimed at broadly capturing the enormous biodiversity in antibody profiles that may emerge following vaccination, complements the existing cutting edge tools in the cellular immunology space that survey vaccine-induced polyfunctional cellular activity by flow cytometry, transcriptional profiling, epigenetic, and metabolomic analysis to offer a means to develop both a more nuanced and a more complete understanding of correlates of protection to support the design of functional vaccine strategies. 10.1111/imr.12503
    Identification and specificity of broadly neutralizing antibodies against HIV. McCoy Laura E,Burton Dennis R Immunological reviews Beginning in 2009, studies of the humoral responses of HIV-positive individuals have led to the identification of scores, if not hundreds, of antibodies that are both broadly reactive and potently neutralizing. This development has provided renewed impetus toward an HIV vaccine and led directly to the development of novel immunogens. Advances in identification of donors with the most potent and broad anti-HIV serum neutralizing responses were crucial in this effort. Equally, development of methods for the rapid generation of human antibodies from these donors was pivotal. Primarily these methods comprise single B-cell culture coupled to high-throughput neutralization screening and flow cytometry-based sorting of single B cells using HIV envelope protein baits. In this review, the advantages and disadvantages of these methodologies are discussed in the context of the specificities targeted by individual antibodies and the need for further improvements to evaluate HIV vaccine candidates. 10.1111/imr.12484
    Short-term memory in gene induction reveals the regulatory principle behind stochastic IL-4 expression. Mariani Luca,Schulz Edda G,Lexberg Maria H,Helmstetter Caroline,Radbruch Andreas,Löhning Max,Höfer Thomas Molecular systems biology Although cell-to-cell variability has been recognized as an unavoidable consequence of stochasticity in gene expression, it may also serve a functional role for tuning physiological responses within a cell population. In the immune system, remarkably large variability in the expression of cytokine genes has been observed in homogeneous populations of lymphocytes, but the underlying molecular mechanisms are incompletely understood. Here, we study the interleukin-4 gene (il4) in T-helper lymphocytes, combining mathematical modeling with the experimental quantification of expression variability and critical parameters. We show that a stochastic rate-limiting step upstream of transcription initiation, but acting at the level of an individual allele, controls il4 expression. Only a fraction of cells reaches an active, transcription-competent state in the transient time window determined by antigen stimulation. We support this finding by experimental evidence of a previously unknown short-term memory that was predicted by the model to arise from the long lifetime of the active state. Our analysis shows how a stochastic mechanism acting at the chromatin level can be integrated with transcriptional regulation to quantitatively control cell-to-cell variability. 10.1038/msb.2010.13
    Evidence that tissue resident human enthesis γδT-cells can produce IL-17A independently of IL-23R transcript expression. Cuthbert Richard James,Watad Abdulla,Fragkakis Evangelos M,Dunsmuir Robert,Loughenbury Peter,Khan Almas,Millner Peter A,Davison Adam,Marzo-Ortega Helena,Newton Darren,Bridgewood Charlie,McGonagle Dennis G Annals of the rheumatic diseases OBJECTIVES:Murine models of interleukin (IL)-23-driven spondyloarthritis (SpA) have demonstrated entheseal accumulation of γδT-cells which were responsible for the majority of local IL-17A production. However, IL-23 blockers are ineffective in axial inflammation in man. This study investigated γδT-cell subsets in the normal human enthesis to explore the biology of the IL-23/17 axis. METHODS:Human spinous processes entheseal soft tissue (EST) and peri-entheseal bone (PEB) were harvested during elective orthopaedic procedures. Entheseal γδT-cells were evaluated using immunohistochemistry and isolated and characterised using flow cytometry. RNA was isolated from γδT-cell subsets and analysed by qPCR. Entheseal γδT-cells were stimulated with phorbol 12-myristate 13-acetate (PMA) and ionomycin, anti-CD3/28 or IL-23 and IL-17A production was measured by high-sensitivity ELISA and qPCR. RESULTS:Entheseal γδT-cells were confirmed immunohistochemically with Vδ1 and Vδ2 subsets that are cytometrically defined. Transcript profiles of both cell populations suggested tissue residency and immunomodulatory status. Entheseal Vδ2 cells expressed high relative abundance of IL-23/17-associated transcripts including IL-23R, RORC and CCR6, whereas the Vδ1 subset almost completely lacked detectable IL-23R transcript. Following PMA stimulation IL-17A was detectable in both Vδ1 and Vδ2 subsets, and following CD3/CD28 stimulation both subsets showed IL-17A and IL-17F transcripts with neither transcript being detectable in the Vδ1 subset following IL-23 stimulation. CONCLUSION:Spinal entheseal Vδ1 and Vδ2 subsets are tissue resident cells with inducible IL-17A production with evidence that the Vδ1 subset does so independently of IL-23R expression. 10.1136/annrheumdis-2019-215210
    AirLab: a cloud-based platform to manage and share antibody-based single-cell research. Catena Raúl,Özcan Alaz,Jacobs Andrea,Chevrier Stephane,Bodenmiller Bernd Genome biology Single-cell analysis technologies are essential tools in research and clinical diagnostics. These methods include flow cytometry, mass cytometry, and other microfluidics-based technologies. Most laboratories that employ these methods maintain large repositories of antibodies. These ever-growing collections of antibodies, their multiple conjugates, and the large amounts of data generated in assays using specific antibodies and conditions makes a dedicated software solution necessary. We have developed AirLab, a cloud-based tool with web and mobile interfaces, for the organization of these data. AirLab streamlines the processes of antibody purchase, organization, and storage, antibody panel creation, results logging, and antibody validation data sharing and distribution. Furthermore, AirLab enables inventory of other laboratory stocks, such as primers or clinical samples, through user-controlled customization. Thus, AirLab is a mobile-powered and flexible tool that harnesses the capabilities of mobile tools and cloud-based technology to facilitate inventory and sharing of antibody and sample collections and associated validation data. 10.1186/s13059-016-1006-0
    Platelets generated from human embryonic stem cells are functional in vitro and in the microcirculation of living mice. Lu Shi-Jiang,Li Feng,Yin Hong,Feng Qiang,Kimbrel Erin A,Hahm Eunsil,Thon Jonathan N,Wang Wei,Italiano Joseph E,Cho Jaehyung,Lanza Robert Cell research Platelets play an essential role in hemostasis and atherothrombosis. Owing to their short storage time, there is constant demand for this life-saving blood component. In this study, we report that it is feasible to generate functional megakaryocytes and platelets from human embryonic stem cells (hESCs) on a large scale. Differential-interference contrast and electron microscopy analyses showed that ultrastructural and morphological features of hESC-derived platelets were indistinguishable from those of normal blood platelets. In functional assays, hESC-derived platelets responded to thrombin stimulation, formed microaggregates, and facilitated clot formation/retraction in vitro. Live cell microscopy demonstrated that hESC-platelets formed lamellipodia and filopodia in response to thrombin activation, and tethered to each other as observed in normal blood. Using real-time intravital imaging with high-speed video microscopy, we have also shown that hESC-derived platelets contribute to developing thrombi at sites of laser-induced vascular injury in mice, providing the first evidence for in vivo functionality of hESC-derived platelets. These results represent an important step toward generating an unlimited supply of platelets for transfusion. Since platelets contain no genetic material, they are ideal candidates for early clinical translation involving human pluripotent stem cells. 10.1038/cr.2011.8
    Interrogation of the Microenvironmental Landscape in Brain Tumors Reveals Disease-Specific Alterations of Immune Cells. Klemm Florian,Maas Roeltje R,Bowman Robert L,Kornete Mara,Soukup Klara,Nassiri Sina,Brouland Jean-Philippe,Iacobuzio-Donahue Christine A,Brennan Cameron,Tabar Viviane,Gutin Philip H,Daniel Roy T,Hegi Monika E,Joyce Johanna A Cell Brain malignancies encompass a range of primary and metastatic cancers, including low-grade and high-grade gliomas and brain metastases (BrMs) originating from diverse extracranial tumors. Our understanding of the brain tumor microenvironment (TME) remains limited, and it is unknown whether it is sculpted differentially by primary versus metastatic disease. We therefore comprehensively analyzed the brain TME landscape via flow cytometry, RNA sequencing, protein arrays, culture assays, and spatial tissue characterization. This revealed disease-specific enrichment of immune cells with pronounced differences in proportional abundance of tissue-resident microglia, infiltrating monocyte-derived macrophages, neutrophils, and T cells. These integrated analyses also uncovered multifaceted immune cell activation within brain malignancies entailing converging transcriptional trajectories while maintaining disease- and cell-type-specific programs. Given the interest in developing TME-targeted therapies for brain malignancies, this comprehensive resource of the immune landscape offers insights into possible strategies to overcome tumor-supporting TME properties and instead harness the TME to fight cancer. 10.1016/j.cell.2020.05.007
    Transplantation tolerance: memories that haunt us. Ford Mandy L,Larsen Christian P Science translational medicine Transplant tolerance, which allows grafts--allogeneic cells, tissues, or organs--to be accepted without host immunosuppression, can be achieved in mice but not in primates. In this issue of Science Translational Medicine, Nadazdin et al. report that a high pretransplant frequency of graft-reactive memory T cells may inhibit the induction of transplant tolerance in nonhuman primates and lead to transplant rejection. Knowing the frequency of allograft-specific memory T cells in potential transplant recipients could aid clinical decision-making by guiding selection of the antigenic profile of the donor organ or by influencing the type of tolerance-induction protocol pursued. 10.1126/scitranslmed.3002504
    Dynamics of IFN-β Responses during Respiratory Viral Infection. Insights for Therapeutic Strategies. Watson Alastair,Spalluto C Mirella,McCrae Christopher,Cellura Doriana,Burke Hannah,Cunoosamy Danen,Freeman Anna,Hicks Alex,Hühn Michael,Ostridge Kristoffer,Staples Karl J,Vaarala Outi,Wilkinson Tom American journal of respiratory and critical care medicine Viral infections are major drivers of exacerbations and clinical burden in patients with asthma and chronic obstructive pulmonary disease (COPD). IFN-β is a key component of the innate immune response to viral infection. To date, studies of inhaled IFN-β treatment have not demonstrated a significant effect on asthma exacerbations. The dynamics of exogenous IFN-β activity were investigated to inform on future clinical indications for this potential antiviral therapy. Monocyte-derived macrophages (MDMs), alveolar macrophages, and primary bronchial epithelial cells (PBECs) were isolated from healthy control subjects and patients with COPD and infected with influenza virus either prior to or after IFN-β stimulation. Infection levels were measured by the percentage of nucleoprotein 1-positive cells using flow cytometry. Viral RNA shedding and IFN-stimulated gene expression were measured by quantitative PCR. Production of inflammatory cytokines was measured using MSD. Adding IFN-β to MDMs, alveolar macrophages, and PBECs prior to, but not after, infection reduced the percentage of nucleoprotein 1-positive cells by 85, 56, and 66%, respectively ( < 0.05). Inhibition of infection lasted for 24 hours after removal of IFN-β and was maintained albeit reduced up to 1 week in MDMs and 72 hours in PBECs; this was similar between healthy control subjects and patients with COPD. IFN-β did not induce inflammatory cytokine production by MDMs or PBECs but reduced influenza-induced IL-1β production by PBECs. modeling of IFN-β dynamics highlights the potential for intermittent prophylactic doses of exogenous IFN-β to modulate viral infection. This provides important insights to aid the future design of clinical trials of IFN-β in asthma and COPD. 10.1164/rccm.201901-0214OC
    MHC Class II-restricted antigen presentation by plasmacytoid dendritic cells drives proatherogenic T cell immunity. Sage Andrew P,Murphy Deirdre,Maffia Pasquale,Masters Leanne M,Sabir Suleman R,Baker Lauren L,Cambrook Helen,Finigan Alison J,Ait-Oufella Hafid,Grassia Gianluca,Harrison James E,Ludewig Burkhard,Reith Walter,Hansson Göran K,Reizis Boris,Hugues Stéphanie,Mallat Ziad Circulation BACKGROUND:Plasmacytoid dendritic cells (pDCs) bridge innate and adaptive immune responses and are important regulators of immuno-inflammatory diseases. However, their role in atherosclerosis remains elusive. METHODS AND RESULTS:Here, we used genetic approaches to investigate the role of pDCs in atherosclerosis. Selective pDC deficiency in vivo was achieved using CD11c-Cre × Tcf4(-/flox) bone marrow transplanted into Ldlr(-/-) mice. Compared with control Ldlr(-/-) chimeric mice, CD11c-Cre × Tcf4(-/flox) mice had reduced atherosclerosis levels. To begin to understand the mechanisms by which pDCs regulate atherosclerosis, we studied chimeric Ldlr(-/-) mice with selective MHCII deficiency on pDCs. Significantly, these mice also developed reduced atherosclerosis compared with controls without reductions in pDC numbers or changes in conventional DCs. MHCII-deficient pDCs showed defective stimulation of apolipoprotein B100-specific CD4(+) T cells in response to native low-density lipoprotein, whereas production of interferon-α was not affected. Finally, the atheroprotective effect of selective MHCII deficiency in pDCs was associated with significant reductions of proatherogenic T cell-derived interferon-γ and lesional T cell infiltration, and was abrogated in CD4(+) T cell-depleted animals. CONCLUSIONS:This study supports a proatherogenic role for pDCs in murine atherosclerosis and identifies a critical role for MHCII-restricted antigen presentation by pDCs in driving proatherogenic T cell immunity. 10.1161/CIRCULATIONAHA.114.011090
    Lung Innate Lymphoid Cell Composition Is Altered in Primary Graft Dysfunction. Monticelli Laurel A,Diamond Joshua M,Saenz Steven A,Tait Wojno Elia D,Porteous Mary K,Cantu Edward,Artis David,Christie Jason D American journal of respiratory and critical care medicine Primary graft dysfunction (PGD) is the leading cause of early morbidity and mortality after lung transplantation, but the immunologic mechanisms are poorly understood. Innate lymphoid cells (ILC) are a heterogeneous family of immune cells regulating pathologic inflammation and beneficial tissue repair. However, whether changes in donor-derived lung ILC populations are associated with PGD development has never been examined. To determine whether PGD in chronic obstructive pulmonary disease or interstitial lung disease transplant recipients is associated with alterations in ILC subset composition within the allograft. We performed a single-center cohort study of lung transplantation patients with surgical biopsies of donor tissue taken before, and immediately after, allograft reperfusion. Donor immune cells from 18 patients were characterized phenotypically by flow cytometry for single-cell resolution of distinct ILC subsets. Changes in the percentage of ILC subsets with reperfusion or PGD (grade 3 within 72 h) were assessed. Allograft reperfusion resulted in significantly decreased frequencies of natural killer cells and a trend toward reduced ILC populations, regardless of diagnosis (interstitial lung disease or chronic obstructive pulmonary disease). Seven patients developed PGD (38.9%), and PGD development was associated with selective reduction of the ILC2 subset after reperfusion. Conversely, patients without PGD exhibited significantly higher ILC1 frequencies before reperfusion, accompanied by elevated ILC2 frequencies after allograft reperfusion. The composition of donor ILC subsets is altered after allograft reperfusion and is associated with PGD development, suggesting that ILCs may be involved in regulating lung injury in lung transplant recipients. 10.1164/rccm.201906-1113OC
    Phase II Study of Combination Obinutuzumab, Ibrutinib, and Venetoclax in Treatment-Naïve and Relapsed or Refractory Chronic Lymphocytic Leukemia. Rogers Kerry A,Huang Ying,Ruppert Amy S,Abruzzo Lynne V,Andersen Barbara L,Awan Farrukh T,Bhat Seema A,Dean Allison,Lucas Margaret,Banks Christin,Grantier Cara,Heerema Nyla A,Lozanski Gerard,Maddocks Kami J,Valentine Thomas R,Weiss David M,Jones Jeffrey A,Woyach Jennifer A,Byrd John C Journal of clinical oncology : official journal of the American Society of Clinical Oncology PURPOSE:The development of highly effective targeted agents for chronic lymphocytic leukemia offers the potential for fixed-duration combinations that achieve deep remissions without cytotoxic chemotherapy. PATIENTS AND METHODS:This phase II study tested a combination regimen of obinutuzumab, ibrutinib, and venetoclax for a total of 14 cycles in both patients with treatment-naïve (n = 25) and relapsed or refractory (n = 25) chronic lymphocytic leukemia to determine the response to therapy and safety. RESULTS:The primary end point was the rate of complete remission with undetectable minimal residual disease by flow cytometry in both the blood and bone marrow 2 months after completion of treatment, which was 28% in both groups. The overall response rate at that time was 84% in treatment-naïve patients and 88% in relapsed or refractory patients. At that time, 67% of treatment-naïve patients and 50% of relapsed or refractory patients had undetectable minimal residual disease in both the blood and marrow. At a median follow-up of 24.2 months in treatment-naïve patients and 21.5 months in relapsed or refractory patients, the median progression-free and overall survival times were not yet reached, with only 1 patient experiencing progression and 1 death. Neutropenia and thrombocytopenia were the most frequent adverse events, followed by hypertension. Grade 3 or 4 neutropenia was experienced by 66% of patients, with more events in the relapsed or refractory cohort. There was only 1 episode of neutropenic fever. A favorable impact on both perceived and objective cognitive performance during treatment was observed. CONCLUSION:The combination regimen of obinutuzumab, ibrutinib, and venetoclax offers time-limited treatment that results in deep remissions and is now being studied in phase III cooperative group trials. 10.1200/JCO.20.00491
    CCL17 Aggravates Myocardial Injury by Suppressing Recruitment of Regulatory T Cells. Circulation BACKGROUND:Recent studies have established that CCR2 (C-C chemokine receptor type 2) marks proinflammatory subsets of monocytes, macrophages, and dendritic cells that contribute to adverse left ventricle (LV) remodeling and heart failure progression. Elucidation of the effector mechanisms that mediate adverse effects of CCR2 monocytes, macrophages, and dendritic cells will yield important insights into therapeutic strategies to suppress myocardial inflammation. METHODS:We used mouse models of reperfused myocardial infarction, angiotensin II and phenylephrine infusion, and diphtheria toxin cardiomyocyte ablation to investigate CCL17 (C-C chemokine ligand 17). We used knockout mice, flow cytometry, RNA sequencing, biochemical assays, cell trafficking studies, and in vivo cell depletion to identify the cell types that generate CCL17, define signaling pathways that controlled its expression, delineate the functional importance of CCL17 in adverse LV remodeling and heart failure progression, and determine the mechanistic basis by which CCL17 exerts its effects. RESULTS:We demonstrated that CCL17 is expressed in CCR2 macrophages and cluster of differentiation 11b conventional dendritic cells after myocardial infarction, angiotensin II and phenylephrine infusion, and diphtheria toxin cardiomyocyte ablation. We clarified the transcriptional signature of CCL17 macrophages and dendritic cells and identified granulocyte-macrophage colony-stimulating factor (GM-CSF) signaling as a key regulator of CCL17 expression through cooperative activation of STAT5 (signal transducer and activator of transcription 5) and canonical NF-κB (nuclear factor κ-light-chain-enhancer of activated B cells) signaling. deletion resulted in reduced LV remodeling, decreased myocardial fibrosis and cardiomyocyte hypertrophy, and improved LV systolic function after myocardial infarction and angiotensin II and phenylephrine infusion. We observed increased abundance of regulatory T cells (Tregs) in the myocardium of injured knockout mice. CCL17 inhibited Treg recruitment through biased activation of CCR4. CCL17 activated Gq signaling and CCL22 (C-C chemokine ligand 22) activated both Gq and ARRB (β-arrestin) signaling downstream of CCR4. CCL17 competitively inhibited CCL22 stimulated ARRB signaling and Treg migration. We provide evidence that Tregs mediated the protective effects of deletion on myocardial inflammation and adverse LV remodeling. CONCLUSIONS:These findings identify CCL17 as a proinflammatory mediator of CCR2 macrophages and dendritic cells and suggest that inhibition of CCL17 may serve as an effective strategy to promote Treg recruitment and suppress myocardial inflammation. 10.1161/CIRCULATIONAHA.121.055888
    Chronic pancreatitis is associated with disease-specific regulatory T-cell responses. Schmitz-Winnenthal Hubertus,Pietsch Dong-Ho Kim,Schimmack Simon,Bonertz Andreas,Udonta Florian,Ge Yingzi,Galindo Luis,Specht Sebastian,Volk Christine,Zgraggen Kaspar,Koch Moritz,Büchler Markus W,Weitz Jürgen,Beckhove Philipp Gastroenterology BACKGROUND & AIMS:Chronic pancreatitis is characterized by alternating phases of acute inflammation and quiescent disease. Involvement of T-cell responses has been suggested, but pancreatitis-specific T cells have not been described. METHODS:We characterized T-cell responses against pancreatitis, pancreatic carcinoma-associated antigens, and tetanus toxoid in the bone marrow, blood, and/or pancreatitis lesions of patients with pancreatitis, pancreatic cancer, and healthy individuals. T cells were functionally characterized by antigen-dependent secretion of interferon (IFN)-gamma, interleukin (Il)-4, and IL-10, which indicate type 1, type 2, or regulatory T-cell responses, respectively. Regulatory T cells were characterized by multicolor flow cytometry. Isolated regulatory T cells were tested for their capacity to recognize pancreatitis-associated antigens and to suppress conventional T cells in an antigen-dependent manner. T cell-derived cytokines in tissue lesions were quantified by enzyme-linked immunosorbent assay. RESULTS:Chronic pancreatitis patients showed similar to pancreatic cancer patients and healthy individuals type 1 T-cell responses against tetanus toxoid; however, they exhibited strong IL-10-based T-cell responses against pancreatitis-associated but not pancreatic carcinoma-associated antigens. T cells from pancreatic cancer patients responded to pancreatic cancer-associated but not pancreatitis-associated antigens with IFN-gamma secretion. Pancreatitis-specific IL-10 responses were mediated by IL-10(+)IFN-gamma(-)FoxP3(+) regulatory T cells, which were expanded in the blood, bone marrow, and pancreatitis lesions and possessed the potential to suppress the proliferation of autologous conventional T cells in an antigen-specific manner. Pancreatitis lesions, in comparison with pancreatic carcinomas, contained increased concentrations of IL-10 and reduced levels of IFN-gamma, suggesting pancreatitis-specific activity of regulatory T cells in situ. CONCLUSIONS:Chronic pancreatitis is associated with disease-specific regulatory T-cell responses. 10.1053/j.gastro.2009.11.011
    IL23 induces IL23R recycling and amplifies innate receptor-induced signalling and cytokines in human macrophages, and the IBD-protective IL23R R381Q variant modulates these outcomes. Sun Rui,Hedl Matija,Abraham Clara Gut OBJECTIVE:The interleukin (IL)23 pathway contributes to IBD pathogenesis and is being actively studied as a therapeutic target in patients with IBD. Unexpected outcomes in these therapeutic trials have highlighted the importance of understanding the cell types and mechanisms through which IL23 regulates immune outcomes. How IL23 regulates macrophage outcomes and the consequences of the IL23R R381Q IBD-protective variant on macrophages are not well defined; macrophages are key players in IBD pathogenesis and inflammation. DESIGN:We analysed protein and RNA expression, signalling and localisation in human monocyte-derived macrophages (MDMs) through western blot, ELISA, real-time PCR, flow cytometry, immunoprecipitation and microscopy. RESULTS:IL23R was critical for optimal levels of pattern-recognition receptor (PRR)-induced signalling and cytokines in human MDMs. In contrast to the coreceptor IL12Rβ1, IL23 induced dynamic IL23R cell surface regulation and this required clathrin and dynamin-mediated endocytosis and endocytic recycling-dependent pathways; these pathways were essential for IL23R-mediated outcomes. The IBD-protective IL23R R381Q variant showed distinct outcomes. Relative to IL23R R381, HeLa cells expressing IL23R Q381 showed decreased IL23R recycling and reduced assembly of IL23R Q381 with Janus kinase/signal transducer and activator of transcription pathway members. In MDMs from IL23R Q381 carriers, IL23R accumulated in late endosomes and lysosomes on IL23 treatment and cells demonstrated decreased IL23R- and PRR-induced signalling and cytokines relative to IL23R R381 MDMs. CONCLUSION:Macrophage-mediated inflammatory pathways are key contributors to IBD pathogenesis, and we identify an autocrine/paracrine IL23 requirement in PRR-initiated human macrophage outcomes and in human intestinal myeloid cells, establish that IL23R undergoes ligand-induced recycling, define mechanisms regulating IL23R-induced signalling and determine how the IBD-protective IL23R R381Q variant modulates these processes. 10.1136/gutjnl-2018-316830
    Specific microRNAs are preferentially expressed by skin stem cells to balance self-renewal and early lineage commitment. Zhang Liang,Stokes Nicole,Polak Lisa,Fuchs Elaine Cell stem cell Increasing evidence suggests that microRNAs may play important roles in regulating self-renewal and differentiation in mammalian stem cells (SCs). Here, we explore this issue in skin. We first characterize microRNA expression profiles of skin SCs versus their committed proliferative progenies and identify a microRNA subset associating with "stemness." Of these, miR-125b is dramatically downregulated in early SC progeny. We engineer an inducible mice system and show that when miR-125b is sustained in SC progenies, tissue balance is reversibly skewed toward stemness at the expense of epidermal, oil-gland, and HF differentiation. Using gain- and loss-of-function in vitro, we further implicate miR-125b as a repressor of SC differentiation. In vivo, transcripts repressed upon miR-125b induction are enriched >700% for predicted miR-125b targets normally downregulated upon SC-lineage commitment. We verify some of these miR-125b targets, and show that Blimp1 and VDR in particular can account for many tissue imbalances we see when miR-125b is deregulated. 10.1016/j.stem.2011.01.014
    NFAT-induced histone acetylation relay switch promotes c-Myc-dependent growth in pancreatic cancer cells. Köenig Alexander,Linhart Thomas,Schlengemann Katrin,Reutlinger Kristina,Wegele Jessica,Adler Guido,Singh Garima,Hofmann Leonie,Kunsch Steffen,Büch Thomas,Schäfer Eva,Gress Thomas M,Fernandez-Zapico Martin E,Ellenrieder Volker Gastroenterology BACKGROUND & AIMS:Induction of immediate early transcription factors (ITF) represents the first transcriptional program controlling mitogen-stimulated cell cycle progression in cancer. Here, we examined the transcriptional mechanisms regulating the ITF protein c-Myc and its role in pancreatic cancer growth in vitro and in vivo. METHODS:Expression of ITF proteins was examined by reverse-transcription polymerase chain reaction and immunoblotting, and its implications in cell cycle progression and growth was determined by flow cytometry and [(3)H]-thymidine incorporation. Intracellular Ca(2+) concentrations, calcineurin activity, and cellular nuclear factor of activated T cells (NFAT) distribution were analyzed. Transcription factor complex formations and promoter regulation were examined by immunoprecipitations, reporter gene assays, and chromatin immunoprecipitation. Using a combination of RNA interference knockdown technology and xenograft models, we analyzed the significance for pancreatic cancer tumor growth. RESULTS:Serum promotes pancreatic cancer growth through induction of the proproliferative NFAT/c-Myc axis. Mechanistically, serum increases intracellular Ca(2+) concentrations and activates the calcineurin/NFAT pathway to induce c-Myc transcription. NFAT binds to a serum responsive element within the proximal promoter, initiates p300-dependent histone acetylation, and creates a local chromatin structure permissive for the inducible recruitment of Ets-like gene (ELK)-1, a protein required for maximal activation of the c-Myc promoter. The functional significance of this novel pathway was emphasized by impaired c-Myc expression, G1 arrest, and reduced tumor growth upon NFAT depletion in vitro and in vivo. CONCLUSIONS:Our study uncovers a novel mechanism regulating cell growth and identifies the NFAT/ELK complex as modulators of early stages of mitogen-stimulated proliferation in pancreatic cancer cells. 10.1053/j.gastro.2009.10.045
    A model to study the phenotypic changes of interstitial cells of Cajal in gastrointestinal diseases. Ro Seungil,Park Chanjae,Jin Jingling,Zheng Huili,Blair Peter J,Redelman Doug,Ward Sean M,Yan Wei,Sanders Kenton M Gastroenterology BACKGROUND & AIMS:Interstitial cells of Cajal (ICC) express the receptor tyrosine kinase, KIT, the receptor for stem cell factor. In the gastrointestinal (GI) tract, ICC are pacemaker cells that generate spontaneous electrical slow waves, and mediate inputs from motor neurons. Absence or loss of ICC are associated with GI motility disorders, including those consequent of diabetes. Studies of ICC have been hampered by the low density of these cells and difficulties in recognizing these cells in cell dispersions. METHODS:Kit(+/copGFP) mice harboring a copepod super green fluorescent protein (copGFP) complementary DNA, inserted at the Kit locus, were generated. copGFP(+) ICC from GI muscles were analyzed using confocal microscopy and flow cytometry. copGFP(+) ICC from the jejunum were purified by a fluorescence-activated cell sorter and validated by cell-specific markers. Kit(+/copGFP) mice were crossbred with diabetic Lep(+/ob) mice to generate compound Kit(+/copGFP);Lep(ob/ob) mutant mice. copGFP(+) ICC from compound transgenic mice were analyzed by confocal microscopy. RESULTS:copGFP in Kit(+/copGFP) mice colocalized with KIT immunofluorescence and thus was predominantly found in ICC. In other smooth muscles, mast cells were also labeled, but these cells were relatively rare in the murine GI tract. copGFP(+) cells from jejunal muscles were Kit(+) and free of contaminating cell-specific markers. Kit(+/copGFP);Lep(ob/ob) mice displayed ICC networks that were dramatically disrupted during the development of diabetes. CONCLUSIONS:Kit(+/copGFP) mice offer a powerful new model to study the function and genetic regulation of ICC phenotypes. Isolation of ICC from animal models will help determine the causes and responses of ICC to therapeutic agents. 10.1053/j.gastro.2009.11.007
    Targeting monocyte-intrinsic enhancer reprogramming improves immunotherapy efficacy in hepatocellular carcinoma. Liu Man,Zhou Jingying,Liu Xiaoyu,Feng Yu,Yang Weiqin,Wu Feng,Cheung Otto Ka-Wing,Sun Hanyong,Zeng Xuezhen,Tang Wenshu,Mok Myth T S,Wong John,Yeung Philip Chun,Lai Paul Bo San,Chen Zhiwei,Jin Hongchuan,Chen Jie,Chan Stephen Lam,Chan Anthony W H,To Ka Fai,Sung Joseph J Y,Chen Minhu,Cheng Alfred Sze-Lok Gut OBJECTIVE:Hepatocellular carcinoma (HCC), mostly developed in fibrotic/cirrhotic liver, exhibits relatively low responsiveness to immune checkpoint blockade (ICB) therapy. As myeloid-derived suppressor cell (MDSC) is pivotal for immunosuppression, we investigated its role and regulation in the fibrotic microenvironment with an aim of developing mechanism-based combination immunotherapy. DESIGN:Functional significance of MDSCs was evaluated by flow cytometry using two orthotopic HCC models in fibrotic liver setting via carbon tetrachloride or high-fat high-carbohydrate diet and verified by clinical specimens. Mechanistic studies were conducted in human hepatic stellate cell (HSC)-peripheral blood mononuclear cell culture systems and fibrotic-HCC patient-derived MDSCs. The efficacy of single or combined therapy with anti-programmed death-1-ligand-1 (anti-PD-L1) and a clinically trialled BET bromodomain inhibitor i-BET762 was determined. RESULTS:Accumulation of monocytic MDSCs (M-MDSCs), but not polymorphonuclear MDSCs, in fibrotic livers significantly correlated with reduced tumour-infiltrating lymphocytes (TILs) and increased tumorigenicity in both mouse models. In human HCCs, the tumour-surrounding fibrotic livers were markedly enriched with M-MDSC, with its surrogate marker CD33 significantly associated with aggressive tumour phenotypes and poor survival rates. Mechanistically, activated HSCs induced monocyte-intrinsic p38 MAPK signalling to trigger enhancer reprogramming for M-MDSC development and immunosuppression. Treatment with p38 MAPK inhibitor abrogated HSC-M-MDSC crosstalk to prevent HCC growth. Concomitant with patient-derived M-MDSC suppression by i-BET762, combined treatment with anti-PD-L1 synergistically enhanced TILs, resulting in tumour eradication and prolonged survival in the fibrotic-HCC mouse model. CONCLUSION:Our results signify how non-tumour-intrinsic properties in the desmoplastic microenvironment can be exploited to reinstate immunosurveillance, providing readily translatable combination strategies to empower HCC immunotherapy. 10.1136/gutjnl-2018-317257
    Non-classical monocyte homing to the gut via α4β7 integrin mediates macrophage-dependent intestinal wound healing. Schleier Lena,Wiendl Maximilian,Heidbreder Karin,Binder Marie-Theres,Atreya Raja,Rath Timo,Becker Emily,Schulz-Kuhnt Anja,Stahl Annette,Schulze Lisa Lou,Ullrich Karen,Merz Simon F,Bornemann Lea,Gunzer Matthias,Watson Alastair J M,Neufert Clemens,Atreya Imke,Neurath Markus F,Zundler Sebastian Gut OBJECTIVE:To study the role of α4β7 integrin for gut homing of monocytes and to explore the biological consequences of therapeutic α4β7 inhibition with regard to intestinal wound healing. DESIGN:We studied the expression of homing markers on monocyte subsets in the peripheral blood and on macrophage subsets in the gut of patients with IBD and controls with flow cytometry and immunohistochemistry. Integrin function was addressed with dynamic adhesion assays and in vivo gut homing assays. In vivo wound healing was studied in mice deficient for or depleted of α4β7 integrin. RESULTS:Classical and non-classical monocytes were clearly dichotomous regarding homing marker expression including relevant expression of α4β7 integrin on human and mouse non-classical monocytes but not on classical monocytes. Monocyte-expressed α4β7 integrin was functionally important for dynamic adhesion to mucosal vascular addressin cell adhesion molecule 1 and in vivo gut homing. Impaired α4β7-dependent gut homing was associated with reduced (effect size about 20%) and delayed wound healing and suppressed perilesional presence of wound healing macrophages. Non-classical monocytes in the peripheral blood were increased in patients with IBD under clinical treatment with vedolizumab. CONCLUSION:In addition to reported effects on lymphocytes, anti-α4β7 therapy in IBD also targets non-classical monocytes. Impaired gut homing of such monocytes might lead to a reduction of wound healing macrophages and could potentially explain increased rates of postoperative complications in vedolizumab-treated patients, which have been observed in some studies. 10.1136/gutjnl-2018-316772
    Histone chaperone FACT complex mediates oxidative stress response to promote liver cancer progression. Shen Jialing,Chen Mengnuo,Lee Derek,Law Cheuk-Ting,Wei Lai,Tsang Felice Ho-Ching,Chin Don Wai-Ching,Cheng Carol Lai-Hung,Lee Joyce Man-Fong,Ng Irene Oi-Lin,Wong Carmen Chak-Lui,Wong Chun-Ming Gut OBJECTIVE:Facilitates Chromatin Transcription (FACT) complex is a histone chaperone participating in DNA repair-related and transcription-related chromatin dynamics. In this study, we investigated its oncogenic functions, underlying mechanisms and therapeutic implications in human hepatocellular carcinoma (HCC). DESIGN:We obtained HCC and its corresponding non-tumorous liver samples from 16 patients and identified FACT complex as the most upregulated histone chaperone by RNA-Seq. We further used CRISPR-based gene activation and knockout systems to demonstrate the functions of FACT complex in HCC growth and metastasis. Functional roles and mechanistic insights of FACT complex in oxidative stress response were investigated by ChIP assay, flow cytometry, gene expression assays and 4sU-DRB transcription elongation assay. Therapeutic effect of FACT complex inhibitor, Curaxin, was tested in both in vitro and in vivo models. RESULTS:We showed that FACT complex was remarkably upregulated in HCC and contributed to HCC progression. Importantly, we unprecedentedly revealed an indispensable role of FACT complex in NRF2-driven oxidative stress response. Oxidative stress prevented NRF2 and FACT complex from KEAP1-mediated protein ubiquitination and degradation. Stabilised NRF2 and FACT complex form a positive feedback loop; NRF2 transcriptionally activates the FACT complex, while FACT complex promotes the transcription elongation of NRF2 and its downstream antioxidant genes through facilitating rapid nucleosome disassembly for the passage of RNA polymerase. Therapeutically, Curaxin effectively suppressed HCC growth and sensitised HCC cell to sorafenib. CONCLUSION:In conclusion, our findings demonstrated that FACT complex is essential for the expeditious HCC oxidative stress response and is a potential therapeutic target for HCC treatment. 10.1136/gutjnl-2019-318668
    Light-dependent single-cell heterogeneity in the chloroplast redox state regulates cell fate in a marine diatom. Mizrachi Avia,Graff van Creveld Shiri,Shapiro Orr H,Rosenwasser Shilo,Vardi Assaf eLife Diatoms are photosynthetic microorganisms of great ecological and biogeochemical importance, forming vast blooms in aquatic ecosystems. However, we are still lacking fundamental understanding of how individual cells sense and respond to diverse stress conditions, and what acclimation strategies are employed during bloom dynamics. We investigated cellular responses to environmental stress at the single-cell level using the redox sensor roGFP targeted to various organelles in the diatom . We detected cell-to-cell variability using flow cytometry cell sorting and a microfluidics system for live imaging of oxidation dynamics. Chloroplast-targeted roGFP exhibited a light-dependent, bi-stable oxidation pattern in response to HO and high light, revealing distinct subpopulations of sensitive oxidized cells and resilient reduced cells. Early oxidation in the chloroplast preceded commitment to cell death, and can be used for sensing stress cues and regulating cell fate. We propose that light-dependent metabolic heterogeneity regulates diatoms' sensitivity to environmental stressors in the ocean. 10.7554/eLife.47732
    Evaluating cell-surface expression and measuring activation of mammalian odorant receptors in heterologous cells. Zhuang Hanyi,Matsunami Hiroaki Nature protocols A fundamental question in olfaction is which odorant receptors (ORs) are activated by a given odorant. A major roadblock to investigating odorant-OR relationships in mammals has been the inability to express ORs in heterologous cells suitable for screening active ligands for ORs. The discovery of the receptor-transporting protein family has facilitated the effective cell-surface expression of ORs in heterologous cells. The establishment of a robust heterologous expression system for mammalian ORs facilitates the high-throughput 'deorphanization' of these receptors by matching them to their cognate ligands. This protocol details the method used for evaluating the cell-surface expression and measuring the functional activation of ORs of transiently expressed mammalian ORs in HEK293T cells. The stages of OR cell-surface expression include cell culture preparation, transfer of cells, transfection, immunocytochemistry or flow cytometry, odorant stimulation and luciferase assay. This protocol can be completed in a period of 3 d from the transfer of cells to cell-surface expression detection and/or measurement of functional activation. 10.1038/nprot.2008.120
    Structure-Dependent Biodistribution of Liposomal Spherical Nucleic Acids. Ferrer Jennifer R,Sinegra Andrew J,Ivancic David,Yeap Xin Yi,Qiu Longhui,Wang Jiao-Jing,Zhang Zheng Jenny,Wertheim Jason A,Mirkin Chad A ACS nano Spherical nucleic acids (SNAs) are a class of nanomaterials with a structure defined by a radial distribution of densely packed, short DNA or RNA sequences around a nanoparticle core. This structure allows SNAs to rapidly enter mammalian cells, protects the displayed oligonucleotides from nuclease degradation, and enables co-delivery of other drug cargoes. Here, we investigate the biodistribution of liposomal spherical nucleic acid (LSNA) conjugates, SNA architectures formed from liposome templates and DNA modified with hydrophobic end groups (tails). We compared linear DNA with two types of LSNAs that differ only by the affinity of the modified DNA sequence for the liposome template. We use single-stranded DNA (ssDNA) terminated with either a low-affinity cholesterol tail (CHOL-LSNA) or a high-affinity diacylglycerol lipid tail (DPPE-LSNA). Both LSNA formulations, independent of DNA conjugation, reduce the inflammatory cytokine response to intravenously administered DNA. The difference in the affinity for the liposome template significantly affects DNA biodistribution. DNA from CHOL-LSNAs accumulates in greater amounts in the lungs than DNA from DPPE-LSNAs. In contrast, DNA from DPPE-LSNAs exhibits greater accumulation in the kidneys. Flow cytometry and fluorescence microscopy of tissue sections indicate that different cell populations-immune and nonimmune-sequester the DNA depending upon the chemical makeup of the LSNA. Taken together, these data suggest that the chemical structure of the LSNAs represents an opportunity to direct the biodistribution of nucleic acids to major tissues outside of the liver. 10.1021/acsnano.9b07254
    Atl1 regulates choice between global genome and transcription-coupled repair of O(6)-alkylguanines. Latypov Vitaly F,Tubbs Julie L,Watson Amanda J,Marriott Andrew S,McGown Gail,Thorncroft Mary,Wilkinson Oliver J,Senthong Pattama,Butt Amna,Arvai Andrew S,Millington Christopher L,Povey Andrew C,Williams David M,Santibanez-Koref Mauro F,Tainer John A,Margison Geoffrey P Molecular cell Nucleotide excision repair (NER) has long been known to remove DNA lesions induced by chemical carcinogens, and the molecular mechanism has been partially elucidated. Here we demonstrate that in Schizosaccharomyces pombe a DNA recognition protein, alkyltransferase-like 1 (Atl1), can play a pivotal role in selecting a specific NER pathway, depending on the nature of the DNA modification. The relative ease of dissociation of Atl1 from DNA containing small O(6)-alkylguanines allows accurate completion of global genome repair (GGR), whereas strong Atl1 binding to bulky O(6)-alkylguanines blocks GGR, stalls the transcription machinery, and diverts the damage to transcription-coupled repair. Our findings redraw the initial stages of the NER process in those organisms that express an alkyltransferase-like gene and raise the question of whether or not O(6)-alkylguanine lesions that are poor substrates for the alkyltransferase proteins in higher eukaryotes might, by analogy, signal such lesions for repair by NER. 10.1016/j.molcel.2012.04.028
    Expression noise and acetylation profiles distinguish HDAC functions. Weinberger Leehee,Voichek Yoav,Tirosh Itay,Hornung Gil,Amit Ido,Barkai Naama Molecular cell Gene expression shows a significant variation (noise) between genetically identical cells. Noise depends on the gene expression process regulated by the chromatin environment. We screened for chromatin factors that modulate noise in S. cerevisiae and analyzed the results using a theoretical model that infers regulatory mechanisms from the noise versus mean relationship. Distinct activities of the Rpd3(L) and Set3 histone deacetylase complexes were predicted. Both HDACs repressed expression. Yet, Rpd3(L)C decreased the frequency of transcriptional bursts, while Set3C decreased the burst size, as did H2B monoubiquitination (ubH2B). We mapped the acetylation of H3 lysine 9 (H3K9ac) upon deletion of multiple subunits of Set3C and Rpd3(L)C and of ubH2B effectors. ubH2B and Set3C appear to function in the same pathway to reduce the probability that an elongating PolII produces a functional transcript (PolII processivity), while Rpd3(L)C likely represses gene expression at a step preceding elongation. 10.1016/j.molcel.2012.05.008
    Orthogonal control of expression mean and variance by epigenetic features at different genomic loci. Dey Siddharth S,Foley Jonathan E,Limsirichai Prajit,Schaffer David V,Arkin Adam P Molecular systems biology While gene expression noise has been shown to drive dramatic phenotypic variations, the molecular basis for this variability in mammalian systems is not well understood. Gene expression has been shown to be regulated by promoter architecture and the associated chromatin environment. However, the exact contribution of these two factors in regulating expression noise has not been explored. Using a dual-reporter lentiviral model system, we deconvolved the influence of the promoter sequence to systematically study the contribution of the chromatin environment at different genomic locations in regulating expression noise. By integrating a large-scale analysis to quantify mRNA levels by smFISH and protein levels by flow cytometry in single cells, we found that mean expression and noise are uncorrelated across genomic locations. Furthermore, we showed that this independence could be explained by the orthogonal control of mean expression by the transcript burst size and noise by the burst frequency. Finally, we showed that genomic locations displaying higher expression noise are associated with more repressed chromatin, thereby indicating the contribution of the chromatin environment in regulating expression noise. 10.15252/msb.20145704
    Relationship of SARS-CoV-2-specific CD4 response to COVID-19 severity and impact of HIV-1 and tuberculosis coinfection. Riou Catherine,du Bruyn Elsa,Stek Cari,Daroowala Remy,Goliath Rene T,Abrahams Fatima,Said-Hartley Qonita,Allwood Brian W,Hsiao Nei-Yuan,Wilkinson Katalin A,Arlehamn Cecilia S Lindestam,Sette Alessandro,Wasserman Sean,Wilkinson Robert J, The Journal of clinical investigation T cells are involved in control of coronavirus disease 2019 (COVID-19), but limited knowledge is available on the relationship between antigen-specific T cell response and disease severity. Here, we used flow cytometry to assess the magnitude, function, and phenotype of SARS coronavirus 2-specific (SARS-CoV-2-specific) CD4+ T cells in 95 hospitalized COVID-19 patients, 38 of them being HIV-1 and/or tuberculosis (TB) coinfected, and 38 non-COVID-19 patients. We showed that SARS-CoV-2-specific CD4+ T cell attributes, rather than magnitude, were associated with disease severity, with severe disease being characterized by poor polyfunctional potential, reduced proliferation capacity, and enhanced HLA-DR expression. Moreover, HIV-1 and TB coinfection skewed the SARS-CoV-2 T cell response. HIV-1-mediated CD4+ T cell depletion associated with suboptimal T cell and humoral immune responses to SARS-CoV-2, and a decrease in the polyfunctional capacity of SARS-CoV-2-specific CD4+ T cells was observed in COVID-19 patients with active TB. Our results also revealed that COVID-19 patients displayed reduced frequency of Mycobacterium tuberculosis-specific CD4+ T cells, with possible implications for TB disease progression. These results corroborate the important role of SARS-CoV-2-specific T cells in COVID-19 pathogenesis and support the concept of altered T cell functions in patients with severe disease. 10.1172/JCI149125
    Platelet microparticles enhance the vasoregenerative potential of angiogenic early outgrowth cells after vascular injury. Mause Sebastian F,Ritzel Elisabeth,Liehn Elisa A,Hristov Mihail,Bidzhekov Kiril,Müller-Newen Gerhard,Soehnlein Oliver,Weber Christian Circulation BACKGROUND:Angiogenic early outgrowth cells (EOCs) have been reported to contribute to endothelial regeneration and to limit neointima formation after vascular injury. Vascular pathologies comprise platelet activation and concomitant generation of platelet microparticles (PMPs). We hypothesized that PMPs may interact with EOCs in the context of vascular injury and modulate their regenerative potential. METHODS AND RESULTS:Using flow cytometry, confocal microscopy, and scanning electron microscopy, we demonstrated the binding of thrombin/collagen-induced PMPs to EOCs with subsequent membrane assimilation and incorporation. This interaction promoted phenotypic alterations of EOCs with increased expression of endothelial cell markers and transfer of the chemokine receptor CXCR4 to EOCs with enhanced responsiveness to its ligand CXCL12/SDF-1alpha. In addition, PMPs augmented the adhesion of EOCs to extracellular matrix components and to the injured vessel wall and accelerated cytoskeletal reorganization and migration of EOCs. PMPs induced changes in the EOC secretome toward a more proangiogenic profile and amplified the EOC-mediated induction of proliferation, migration, and capillary tube formation by mature endothelial cells. Compared with untreated EOCs, the injection of PMP-treated EOCs resulted in accelerated reendothelialization after arterial denudation injury in athymic nude mice, whereas the EOC-mediated reduction of neointima formation remained unchanged. CONCLUSIONS:Our data provide evidence that PMPs can boost the potential of EOCs to restore endothelial integrity after vascular injury. Major mechanisms involve the enhancement of EOC recruitment, migration, differentiation, and release of proangiogenic factors. 10.1161/CIRCULATIONAHA.109.909473
    Nasal Pneumococcal Density Is Associated with Microaspiration and Heightened Human Alveolar Macrophage Responsiveness to Bacterial Pathogens. Mitsi Elena,Carniel Beatriz,Reiné Jesús,Rylance Jamie,Zaidi Seher,Soares-Schanoski Alessandra,Connor Victoria,Collins Andrea M,Schlitzer Andreas,Nikolaou Elissavet,Solórzano Carla,Pojar Sherin,Hill Helen,Hyder-Wright Angela D,Jambo Kondwani C,Oggioni Marco R,De Ste Croix Megan,Gordon Stephen B,Jochems Simon P,Ferreira Daniela M American journal of respiratory and critical care medicine Pneumococcal pneumonia remains a global health problem. Colonization of the nasopharynx with (Spn), although a prerequisite of infection, is the main source of exposure and immunological boosting in children and adults. However, our knowledge of how nasal colonization impacts on the lung cells, especially on the predominant alveolar macrophage (AM) population, is limited. Using a controlled human infection model to achieve nasal colonization with 6B serotype, we investigated the effect of Spn colonization on lung cells. We collected BAL from healthy pneumococcal-challenged participants aged 18-49 years. Confocal microscopy and molecular and classical microbiology were used to investigate microaspiration and pneumococcal presence in the lower airways. AM opsonophagocytic capacity was assessed by functional assays , whereas flow cytometry and transcriptomic analysis were used to assess further changes on the lung cellular populations. AMs from Spn-colonized individuals exhibited increased opsonophagocytosis to pneumococcus (11.4% median increase) for approximately 3 months after experimental pneumococcal colonization. AMs also had increased responses against other bacterial pathogens. Pneumococcal DNA detected in the BAL samples of Spn-colonized individuals were positively correlated with nasal pneumococcal density ( = 0.71;  = 0.029). Similarly, AM-heightened opsonophagocytic capacity was correlated with nasopharyngeal pneumococcal density ( = 0.61,  = 0.025). Our findings demonstrate that nasal colonization with pneumococcus and microaspiration prime AMs, leading to brisker responsiveness to both pneumococcus and unrelated bacterial pathogens. The relative abundance of AMs in the alveolar spaces, alongside their potential for nonspecific protection, render them an attractive target for novel vaccines. 10.1164/rccm.201903-0607OC
    miR-15b and miR-16 are implicated in activation of the rat hepatic stellate cell: An essential role for apoptosis. Guo Can-Jie,Pan Qin,Li Ding-Guo,Sun Hua,Liu Bo-Wei Journal of hepatology BACKGROUND/AIMS:To reveal the microRNA (miRNA) expression profile and related roles in rat HSCs during activation. METHODS:miRNA expression profiling was analyzed in quiescent and in culture-activated HSCs by microarray. The differentially expressed miRNAs, as verified by RT-PCR, were subjected to gene ontology (GO) analysis. Furthermore, the effects of miR-16 and miR-15b on the apoptosis of activated HSCs were investigated by Hoechst 33258, TUNEL staining and annexin-V/PI labeling flow cytometry. The underlying mechanism related to Bcl-2 and caspases was assessed. RESULTS:The upregulated and downregulated miRNAs in activated HSCs were 12 miRNAs and 9 miRNAs, respectively. The differential expression of miR-16, -15b, -122, -138, -143, and -140 was validated. High-enrichment GOs containing apoptosis-related targeted genes and miRNA-gene networks characterized by Bcl-2, which was targeted by the miR-15/16 family, uncovered the critical role of miR-16 and miR-15b in apoptosis. Restoring the intracellular miRNAs by miR-16 and miR-15b administration greatly reduced Bcl-2, and increased the expression of caspases 3, 8, and 9. Significantly elevated rates of apoptosis were then induced in activated HSCs. CONCLUSIONS:The activation of HSCs relate to 21 miRNAs. Among these, mir-15b and miR-16 may be essential for apoptosis by targeting Bcl-2 and the caspase signaling pathway. 10.1016/j.jhep.2008.11.025
    Recombineering in Corynebacterium glutamicum combined with optical nanosensors: a general strategy for fast producer strain generation. Binder Stephan,Siedler Solvej,Marienhagen Jan,Bott Michael,Eggeling Lothar Nucleic acids research Recombineering in bacteria is a powerful technique for genome reconstruction, but until now, it was not generally applicable for development of small-molecule producers because of the inconspicuous phenotype of most compounds of biotechnological relevance. Here, we establish recombineering for Corynebacterium glutamicum using RecT of prophage Rac and combine this with our recently developed nanosensor technology, which enables the detection and isolation of productive mutants at the single-cell level via fluorescence-activated cell sorting (FACS). We call this new technology RecFACS, which we use for genomic site-directed saturation mutagenesis without relying on pre-constructed libraries to directly isolate L-lysine-producing cells. A mixture of 19 different oligonucleotides was used targeting codon 81 in murE of the wild-type, at a locus where one single mutation is known to cause L-lysine production. Using RecFACS, productive mutants were screened and isolated. Sequencing revealed 12 different amino acid exchanges in the targeted murE codon, which caused different L-lysine production titers. Apart from introducing a rapid genome construction technology for C. glutamicum, the present work demonstrates that RecFACS is suitable to simply create producers as well as genetic diversity in one single step, thus establishing a new general concept in synthetic biology. 10.1093/nar/gkt312
    DPY30 regulates pathways in cellular senescence through ID protein expression. Simboeck Elisabeth,Gutierrez Arantxa,Cozzuto Luca,Beringer Malte,Caizzi Livia,Keyes William M,Di Croce Luciano The EMBO journal Cellular senescence is an intrinsic defense mechanism to various cellular stresses: while still metabolically active, senescent cells stop dividing and enter a proliferation arrest. Here, we identify DPY30, a member of all mammalian histone H3K4 histone methyltransferases (HMTases), as a key regulator of the proliferation potential of human primary cells. Following depletion of DPY30, cells show a severe proliferation defect and display a senescent phenotype, including a flattened and enlarged morphology, elevated level of reactive oxygen species (ROS), increased SA-β-galactosidase activity, and formation of senescence-associated heterochromatin foci (SAHFs). While DPY30 depletion leads to a reduced level of H3K4me3-marked active chromatin, we observed a concomitant activation of CDK inhibitors, including p16INK4a, independent of H3K4me3. ChIP experiments show that key regulators of cell-cycle progression, including ID proteins, are under direct control of DPY30. Because ID proteins are negative regulators of the transcription factors ETS1/2, depletion of DPY30 leads to the transcriptional activation of p16INK4a by ETS1/2 and thus to a senescent-like phenotype. Ectoptic re-introduction of ID protein expression can partially rescue the senescence-like phenotype induced by DPY30 depletion. Thus, our data indicate that DPY30 controls proliferation by regulating ID proteins expression, which in turn lead to senescence bypass. 10.1038/emboj.2013.159
    Langerhans cells are generated by two distinct PU.1-dependent transcriptional networks. Chopin Michaël,Seillet Cyril,Chevrier Stéphane,Wu Li,Wang Hongsheng,Morse Herbert C,Belz Gabrielle T,Nutt Stephen L The Journal of experimental medicine Langerhans cells (LCs) are the unique dendritic cells found in the epidermis. While a great deal of attention has focused on defining the developmental origins of LCs, reports addressing the transcriptional network ruling their differentiation remain sparse. We addressed the function of a group of key DC transcription factors-PU.1, ID2, IRF4, and IRF8-in the establishment of the LC network. We show that although steady-state LC homeostasis depends on PU.1 and ID2, the latter is dispensable for bone marrow-derived LCs. PU.1 controls LC differentiation by regulating the expression of the critical TGF-β responsive transcription factor RUNX3. PU.1 directly binds to the Runx3 regulatory elements in a TGF-β-dependent manner, whereas ectopic expression of RUNX3 rescued LC differentiation in the absence of PU.1 and promoted LC differentiation from PU.1-sufficient progenitors. These findings highlight the dual molecular network underlying LC differentiation, and show the central role of PU.1 in these processes. 10.1084/jem.20130930
    Nfil3-independent lineage maintenance and antiviral response of natural killer cells. Firth Matthew A,Madera Sharline,Beaulieu Aimee M,Gasteiger Georg,Castillo Eliseo F,Schluns Kimberly S,Kubo Masato,Rothman Paul B,Vivier Eric,Sun Joseph C The Journal of experimental medicine Development of the natural killer (NK) cell lineage is dependent on the transcription factor Nfil3 (or E4BP4), which is thought to act downstream of IL-15 signaling. Nfil3-deficient mice lack NK cells, whereas other lymphocyte lineages (B, T, and NKT cells) remain largely intact. We report the appearance of Ly49H-expressing NK cells in Nfil3(-/-) mice infected with mouse cytomegalovirus (MCMV) or recombinant viruses expressing the viral m157 glycoprotein. Nfil3(-/-) NK cells at the peak of antigen-driven expansion were functionally similar to NK cells from infected wild-type mice with respect to IFN-γ production and cytotoxicity, and could comparably produce long-lived memory NK cells that persisted in lymphoid and nonlymphoid tissues for >60 d. We demonstrate that generation and maintenance of NK cell memory is an Nfil3-independent but IL-15-dependent process. Furthermore, specific ablation of Nfil3 in either immature NK cells in the bone marrow or mature peripheral NK cells had no observable effect on NK cell lineage maintenance or homeostasis. Thus, expression of Nfil3 is crucial only early in the development of NK cells, and signals through activating receptors and proinflammatory cytokines during viral infection can bypass the requirement for Nfil3, promoting the proliferation and long-term survival of virus-specific NK cells. 10.1084/jem.20130417
    Ikaros is absolutely required for pre-B cell differentiation by attenuating IL-7 signals. Heizmann Beate,Kastner Philippe,Chan Susan The Journal of experimental medicine Pre-B cell receptor (pre-BCR) signaling and migration from IL-7-rich environments cooperate to drive pre-B cell differentiation via transcriptional programs that remain unclear. We show that the Ikaros transcription factor is required for the differentiation of large pre-B to small pre-B cells. Mice deleted for Ikaros in pro/pre-B cells show a complete block of differentiation at the fraction C' stage, and Ikaros-null pre-B cells cannot differentiate upon withdrawal of IL-7 in vitro. Restoration of Ikaros function rescues pre-B cell differentiation in vitro and in vivo and depends on DNA binding. Ikaros is required for the down-regulation of the pre-BCR, Igκ germline transcription, and Ig L chain recombination. Furthermore, Ikaros antagonizes the IL-7-dependent regulation of >3,000 genes, many of which are up- or down-regulated between fractions C' and D. Affected genes include those important for survival, metabolism, B cell signaling, and function, as well as transcriptional regulators like Ebf1, Pax5, and the Foxo1 family. Our data thus identify Ikaros as a central regulator of IL-7 signaling and pre-B cell development. 10.1084/jem.20131735
    Chemotherapy activates cancer-associated fibroblasts to maintain colorectal cancer-initiating cells by IL-17A. Lotti Fiorenza,Jarrar Awad M,Pai Rish K,Hitomi Masahiro,Lathia Justin,Mace Adam,Gantt Gerald A,Sukhdeo Kumar,DeVecchio Jennifer,Vasanji Amit,Leahy Patrick,Hjelmeland Anita B,Kalady Matthew F,Rich Jeremy N The Journal of experimental medicine Many solid cancers display cellular hierarchies with self-renewing, tumorigenic stemlike cells, or cancer-initiating cells (CICs) at the apex. Whereas CICs often exhibit relative resistance to conventional cancer therapies, they also receive critical maintenance cues from supportive stromal elements that also respond to cytotoxic therapies. To interrogate the interplay between chemotherapy and CICs, we investigated cellular heterogeneity in human colorectal cancers. Colorectal CICs were resistant to conventional chemotherapy in cell-autonomous assays, but CIC chemoresistance was also increased by cancer-associated fibroblasts (CAFs). Comparative analysis of matched colorectal cancer specimens from patients before and after cytotoxic treatment revealed a significant increase in CAFs. Chemotherapy-treated human CAFs promoted CIC self-renewal and in vivo tumor growth associated with increased secretion of specific cytokines and chemokines, including interleukin-17A (IL-17A). Exogenous IL-17A increased CIC self-renewal and invasion, and targeting IL-17A signaling impaired CIC growth. Notably, IL-17A was overexpressed by colorectal CAFs in response to chemotherapy with expression validated directly in patient-derived specimens without culture. These data suggest that chemotherapy induces remodeling of the tumor microenvironment to support the tumor cellular hierarchy through secreted factors. Incorporating simultaneous disruption of CIC mechanisms and interplay with the tumor microenvironment could optimize therapeutic targeting of cancer. 10.1084/jem.20131195
    A role for IL-25 and IL-33-driven type-2 innate lymphoid cells in atopic dermatitis. Salimi Maryam,Barlow Jillian L,Saunders Sean P,Xue Luzheng,Gutowska-Owsiak Danuta,Wang Xinwen,Huang Li-Chieh,Johnson David,Scanlon Seth T,McKenzie Andrew N J,Fallon Padraic G,Ogg Graham S The Journal of experimental medicine Type 2 innate lymphoid cells (ILC2s, nuocytes, NHC) require RORA and GATA3 for their development. We show that human ILC2s express skin homing receptors and infiltrate the skin after allergen challenge, where they produce the type 2 cytokines IL-5 and IL-13. Skin-derived ILC2s express the IL-33 receptor ST2, which is up-regulated during activation, and are enriched in lesional skin biopsies from atopic patients. Signaling via IL-33 induces type 2 cytokine and amphiregulin expression, and increases ILC2 migration. Furthermore, we demonstrate that E-cadherin ligation on human ILC2 dramatically inhibits IL-5 and IL-13 production. Interestingly, down-regulation of E-cadherin is characteristic of filaggrin insufficiency, a cardinal feature of atopic dermatitis (AD). ILC2 may contribute to increases in type 2 cytokine production in the absence of the suppressive E-cadherin ligation through this novel mechanism of barrier sensing. Using Rag1(-/-) and RORα-deficient mice, we confirm that ILC2s are present in mouse skin and promote AD-like inflammation. IL-25 and IL-33 are the predominant ILC2-inducing cytokines in this model. The presence of ILC2s in skin, and their production of type 2 cytokines in response to IL-33, identifies a role for ILC2s in the pathogenesis of cutaneous atopic disease. 10.1084/jem.20130351
    IRF4 controls the positioning of mature B cells in the lymphoid microenvironments by regulating NOTCH2 expression and activity. Simonetti Giorgia,Carette Amanda,Silva Kathryn,Wang Haowei,De Silva Nilushi S,Heise Nicole,Siebel Christian W,Shlomchik Mark J,Klein Ulf The Journal of experimental medicine The transcription factor interferon regulatory factor-4 (IRF4) is expressed in B cells at most developmental stages. In antigen-activated B cells, IRF4 controls germinal center formation, class-switch recombination, and the generation of plasma cells. Here we describe a novel function for IRF4 in the homeostasis of mature B cells. Inducible deletion of irf4 specifically in B cells in vivo led to the aberrant accumulation of irf4-deleted follicular B cells in the marginal zone (MZ) area. IRF4-deficient B cells showed elevated protein expression and activation of NOTCH2, a transmembrane receptor and transcriptional regulator known to be required for MZ B cell development. Administration of a NOTCH2-inhibitory antibody abolished nuclear translocation of NOTCH2 in B cells within 12 h and caused a rapid and progressive disintegration of the MZ that was virtually complete 48 h after injection. The disappearance of the MZ was accompanied by a transient increase of MZ-like B cells in the blood rather than increased B cell apoptosis, demonstrating that continued NOTCH2 activation is critical for the retention of B cells in the MZ. Our results suggest that IRF4 controls the positioning of mature B cells in the lymphoid microenvironments by regulating NOTCH2 expression. These findings may have implications for the understanding of B cell malignancies with dysregulated IRF4 and NOTCH2 activity. 10.1084/jem.20131026
    IL-9-mediated survival of type 2 innate lymphoid cells promotes damage control in helminth-induced lung inflammation. Turner Jan-Eric,Morrison Peter J,Wilhelm Christoph,Wilson Mark,Ahlfors Helena,Renauld Jean-Christophe,Panzer Ulf,Helmby Helena,Stockinger Brigitta The Journal of experimental medicine IL-9 fate reporter mice established type 2 innate lymphoid cells (ILC2s) as major producers of this cytokine in vivo. Here we focus on the role of IL-9 and ILC2s during the lung stage of infection with Nippostrongylus brasiliensis, which results in substantial tissue damage. IL-9 receptor (IL-9R)-deficient mice displayed reduced numbers of ILC2s in the lung after infection, resulting in impaired IL-5, IL-13, and amphiregulin levels, despite undiminished numbers of Th2 cells. As a consequence, the restoration of tissue integrity and lung function was strongly impaired in the absence of IL-9 signaling. ILC2s, in contrast to Th2 cells, expressed high levels of the IL-9R, and IL-9 signaling was crucial for the survival of activated ILC2s in vitro. Furthermore, ILC2s in the lungs of infected mice required the IL-9R to up-regulate the antiapoptotic protein BCL-3 in vivo. This highlights a unique role for IL-9 as an autocrine amplifier of ILC2 function, promoting tissue repair in the recovery phase after helminth-induced lung inflammation. 10.1084/jem.20130071
    Gata2 cis-element is required for hematopoietic stem cell generation in the mammalian embryo. Gao Xin,Johnson Kirby D,Chang Yuan-I,Boyer Meghan E,Dewey Colin N,Zhang Jing,Bresnick Emery H The Journal of experimental medicine The generation of hematopoietic stem cells (HSCs) from hemogenic endothelium within the aorta, gonad, mesonephros (AGM) region of the mammalian embryo is crucial for development of the adult hematopoietic system. We described a deletion of a Gata2 cis-element (+9.5) that depletes fetal liver HSCs, is lethal at E13-14 of embryogenesis, and is mutated in an immunodeficiency that progresses to myelodysplasia/leukemia. Here, we demonstrate that the +9.5 element enhances Gata2 expression and is required to generate long-term repopulating HSCs in the AGM. Deletion of the +9.5 element abrogated the capacity of hemogenic endothelium to generate HSC-containing clusters in the aorta. Genomic analyses indicated that the +9.5 element regulated a rich ensemble of genes that control hemogenic endothelium and HSCs, as well as genes not implicated in hematopoiesis. These results reveal a mechanism that controls stem cell emergence from hemogenic endothelium to establish the adult hematopoietic system. 10.1084/jem.20130733
    Gata2 is required for HSC generation and survival. de Pater Emma,Kaimakis Polynikis,Vink Chris S,Yokomizo Tomomasa,Yamada-Inagawa Tomoko,van der Linden Reinier,Kartalaei Parham Solaimani,Camper Sally A,Speck Nancy,Dzierzak Elaine The Journal of experimental medicine Knowledge of the key transcription factors that drive hematopoietic stem cell (HSC) generation is of particular importance for current hematopoietic regenerative approaches and reprogramming strategies. Whereas GATA2 has long been implicated as a hematopoietic transcription factor and its dysregulated expression is associated with human immunodeficiency syndromes and vascular integrity, it is as yet unknown how GATA2 functions in the generation of HSCs. HSCs are generated from endothelial cells of the major embryonic vasculature (aorta, vitelline, and umbilical arteries) and are found in intra-aortic hematopoietic clusters. In this study, we find that GATA2 function is essential for the generation of HSCs during the stage of endothelial-to-hematopoietic cell transition. Specific deletion of Gata2 in Vec (Vascular Endothelial Cadherin)-expressing endothelial cells results in a deficiency of long-term repopulating HSCs and intra-aortic cluster cells. By specific deletion of Gata2 in Vav-expressing hematopoietic cells (after HSC generation), we further show that GATA2 is essential for HSC survival. This is in contrast to the known activity of the RUNX1 transcription factor, which functions only in the generation of HSCs, and highlights the unique requirement for GATA2 function in HSCs throughout all developmental stages. 10.1084/jem.20130751
    MHC class II-dependent B cell APC function is required for induction of CNS autoimmunity independent of myelin-specific antibodies. Molnarfi Nicolas,Schulze-Topphoff Ulf,Weber Martin S,Patarroyo Juan C,Prod'homme Thomas,Varrin-Doyer Michel,Shetty Aparna,Linington Christopher,Slavin Anthony J,Hidalgo Juan,Jenne Dieter E,Wekerle Hartmut,Sobel Raymond A,Bernard Claude C A,Shlomchik Mark J,Zamvil Scott S The Journal of experimental medicine Whether B cells serve as antigen-presenting cells (APCs) for activation of pathogenic T cells in the multiple sclerosis model experimental autoimmune encephalomyelitis (EAE) is unclear. To evaluate their role as APCs, we engineered mice selectively deficient in MHC II on B cells (B-MHC II(-/-)), and to distinguish this function from antibody production, we created transgenic (Tg) mice that express the myelin oligodendrocyte glycoprotein (MOG)-specific B cell receptor (BCR; IgH(MOG-mem)) but cannot secrete antibodies. B-MHC II(-/-) mice were resistant to EAE induced by recombinant human MOG (rhMOG), a T cell- and B cell-dependent autoantigen, and exhibited diminished Th1 and Th17 responses, suggesting a role for B cell APC function. In comparison, selective B cell IL-6 deficiency reduced EAE susceptibility and Th17 responses alone. Administration of MOG-specific antibodies only partially restored EAE susceptibility in B-MHC II(-/-) mice. In the absence of antibodies, IgH(MOG-mem) mice, but not mice expressing a BCR of irrelevant specificity, were fully susceptible to acute rhMOG-induced EAE, also demonstrating the importance of BCR specificity. Spontaneous opticospinal EAE and meningeal follicle-like structures were observed in IgH(MOG-mem) mice crossed with MOG-specific TCR Tg mice. Thus, B cells provide a critical cellular function in pathogenesis of central nervous system autoimmunity independent of their humoral involvement, findings which may be relevant to B cell-targeted therapies. 10.1084/jem.20130699
    Mitochondrial respiratory capacity is a critical regulator of CD8+ T cell memory development. van der Windt Gerritje J W,Everts Bart,Chang Chih-Hao,Curtis Jonathan D,Freitas Tori C,Amiel Eyal,Pearce Edward J,Pearce Erika L Immunity CD8(+) T cells undergo major metabolic changes upon activation, but how metabolism influences the establishment of long-lived memory T cells after infection remains a key question. We have shown here that CD8(+) memory T cells, but not CD8(+) T effector (Teff) cells, possessed substantial mitochondrial spare respiratory capacity (SRC). SRC is the extra capacity available in cells to produce energy in response to increased stress or work and as such is associated with cellular survival. We found that interleukin-15 (IL-15), a cytokine critical for CD8(+) memory T cells, regulated SRC and oxidative metabolism by promoting mitochondrial biogenesis and expression of carnitine palmitoyl transferase (CPT1a), a metabolic enzyme that controls the rate-limiting step to mitochondrial fatty acid oxidation (FAO). These results show how cytokines control the bioenergetic stability of memory T cells after infection by regulating mitochondrial metabolism. 10.1016/j.immuni.2011.12.007
    The transcription factors T-bet and Eomes control key checkpoints of natural killer cell maturation. Gordon Scott M,Chaix Julie,Rupp Levi J,Wu Junmin,Madera Sharline,Sun Joseph C,Lindsten Tullia,Reiner Steven L Immunity Natural killer (NK) cells play critical roles defending against tumors and pathogens. We show that mice lacking both transcription factors Eomesodermin (Eomes) and T-bet failed to develop NK cells. Developmental stability of immature NK cells constitutively expressing the death ligand TRAIL depended on T-bet. Conversely, maturation characterized by loss of constitutive TRAIL expression and induction of Ly49 receptor diversity and integrin CD49b (DX5(+)) required Eomes. Mature NK cells from which Eomes was deleted reverted to phenotypic immaturity if T-bet was present or downregulated NK lineage antigens if T-bet was absent, despite retaining expression of Ly49 receptors. Fetal and adult hepatic hematopoiesis restricted Eomes expression and limited NK development to the T-bet-dependent, immature stage, whereas medullary hematopoiesis permitted Eomes-dependent NK maturation in adult mice. These findings reveal two sequential, genetically separable checkpoints of NK cell maturation, the progression of which is metered largely by the anatomic localization of hematopoiesis. 10.1016/j.immuni.2011.11.016
    Characterization and functional analyses of hepatic mesothelial cells in mouse liver development. Onitsuka Izumi,Tanaka Minoru,Miyajima Atsushi Gastroenterology BACKGROUND & AIMS:At the onset of liver development, cardiac mesoderm, septum transversum mesenchyme, and endothelial cells are involved in the specification and/or proliferation of hepatoblasts. After this initial stage, however, it is unclear which cells support the proliferation and differentiation of hepatocytes. Here we characterized the nature of mouse hepatic mesothelial cells (MCs) and investigated their role in organogenesis. METHODS:Using anti-podocalyxin-like protein 1 (PCLP1) and anti-mesothelin antibodies, we characterized MCs during liver development by immunohistochemistry, flow cytometry, and gene expression analysis. The possible role of MCs in hepatogenesis was investigated by in vitro culture and analysis of Wilms' tumor 1 homologue (WT1) knockout mice. RESULTS:PCLP1 was highly expressed in immature MCs, covering the surface of lobes. PCLP1 expression in MCs was down-regulated along with development, whereas mesothelin expression was up-regulated, indicating that these molecules distinguished developmental stages of MCs. The proliferation potential of MCs was high in the fetus and declined after birth. Fetal MCs expressed various growth factors and strongly enhanced the expansion of fetal hepatocytes in vitro, whereas differentiated MCs exhibited less growth factor expression, and differentiated MCs failed to enhance hepatocyte proliferation in vitro. In WT1-deficient embryos, hepatocyte proliferation was impaired due to defective MCs. CONCLUSIONS:The mesothelium is not only an inert protective sheet covering the parenchyma but also changes its characteristics dynamically during development and plays an active role in organogenesis by promoting expansion of parenchymal cells. 10.1053/j.gastro.2009.12.059
    Tracheobronchial transplantation with a stem-cell-seeded bioartificial nanocomposite: a proof-of-concept study. Jungebluth Philipp,Alici Evren,Baiguera Silvia,Blomberg Pontus,Bozóky Béla,Crowley Claire,Einarsson Oskar,Gudbjartsson Tomas,Le Guyader Sylvie,Henriksson Gert,Hermanson Ola,Juto Jan Erik,Leidner Bertil,Lilja Tobias,Liska Jan,Luedde Tom,Lundin Vanessa,Moll Guido,Roderburg Christoph,Strömblad Staffan,Sutlu Tolga,Watz Emma,Seifalian Alexander,Macchiarini Paolo Lancet (London, England) BACKGROUND:Tracheal tumours can be surgically resected but most are an inoperable size at the time of diagnosis; therefore, new therapeutic options are needed. We report the clinical transplantation of the tracheobronchial airway with a stem-cell-seeded bioartificial nanocomposite. METHODS:A 36-year-old male patient, previously treated with debulking surgery and radiation therapy, presented with recurrent primary cancer of the distal trachea and main bronchi. After complete tumour resection, the airway was replaced with a tailored bioartificial nanocomposite previously seeded with autologous bone-marrow mononuclear cells via a bioreactor for 36 h. Postoperative granulocyte colony-stimulating factor filgrastim (10 μg/kg) and epoetin beta (40,000 UI) were given over 14 days. We undertook flow cytometry, scanning electron microscopy, confocal microscopy epigenetics, multiplex, miRNA, and gene expression analyses. FINDINGS:We noted an extracellular matrix-like coating and proliferating cells including a CD105+ subpopulation in the scaffold after the reseeding and bioreactor process. There were no major complications, and the patient was asymptomatic and tumour free 5 months after transplantation. The bioartificial nanocomposite has patent anastomoses, lined with a vascularised neomucosa, and was partly covered by nearly healthy epithelium. Postoperatively, we detected a mobilisation of peripheral cells displaying increased mesenchymal stromal cell phenotype, and upregulation of epoetin receptors, antiapoptotic genes, and miR-34 and miR-449 biomarkers. These findings, together with increased levels of regenerative-associated plasma factors, strongly suggest stem-cell homing and cell-mediated wound repair, extracellular matrix remodelling, and neovascularisation of the graft. INTERPRETATION:Tailor-made bioartificial scaffolds can be used to replace complex airway defects. The bioreactor reseeding process and pharmacological-induced site-specific and graft-specific regeneration and tissue protection are key factors for successful clinical outcome. FUNDING:European Commission, Knut and Alice Wallenberg Foundation, Swedish Research Council, StratRegen, Vinnova Foundation, Radiumhemmet, Clinigene EU Network of Excellence, Swedish Cancer Society, Centre for Biosciences (The Live Cell imaging Unit), and UCL Business. 10.1016/S0140-6736(11)61715-7
    Serum amyloid A promotes lung neutrophilia by increasing IL-17A levels in the mucosa and γδ T cells. Anthony Desiree,Seow Huei Jiunn,Uddin Mohib,Thompson Michelle,Dousha Lovisa,Vlahos Ross,Irving Louis B,Levy Bruce D,Anderson Gary P,Bozinovski Steven American journal of respiratory and critical care medicine RATIONALE:Neutrophilic inflammation is an important pathologic feature of chronic obstructive pulmonary disease (COPD) and infectious exacerbations of COPD. Serum amyloid A (SAA) promotes neutrophilic inflammation by its interaction with lung mucosal ALX/FPR2 receptors. However, little is known about how this endogenous mediator regulates IL-17A immunity. OBJECTIVES:To determine whether SAA causes neutrophilic inflammation by IL-17A-dependent mechanisms. METHODS:The relationship between SAA and neutrophils was investigated in lung sections from patients with COPD and a chronic mouse model of SAA exposure. A neutralizing antibody to IL-17A was used to block SAA responses in vivo, and a cell-sorting strategy was used to identify cellular sources. MEASUREMENTS AND MAIN RESULTS:SAA mRNA expression was positively associated with tissue neutrophils in COPD (P < 0.05). SAA predominately promoted expression of the TH17 polarizing cytokine IL-6, which was opposed by 15-epi-lipoxin A4, a counter-regulatory mediator, and ALX/FPR2 ligand. SAA-induced inflammation was markedly reduced by a neutralizing antibody to IL-17A in vivo. Cellular sources of IL-17A induced by SAA include CD4(+) T cells, γδ T cells, and an Epcam(+)CD45(-) population enriched for epithelial cells. SAA promotes expression of IL-17A in γδ T cells and this innate cell proportionally expressed higher levels of IL-17A transcript than CD4(+) T cells or epithelial cells. CONCLUSIONS:The SAA-IL-17A axis represents an important innate defense network that may underlie persistent neutrophilic airway inflammation in COPD and modulating the ALX/FPR2 receptor represents a novel approach to targeting aberrant IL-17A-mediated lung immunity. 10.1164/rccm.201211-2139OC
    RAE1ε ligand expressed on pancreatic islets recruits NKG2D receptor-expressing cytotoxic T cells independent of T cell receptor recognition. Markiewicz Mary A,Wise Erica L,Buchwald Zachary S,Pinto Amelia K,Zafirova Biljana,Polic Bojan,Shaw Andrey S Immunity The mechanisms by which cytotoxic T lymphocytes (CTLs) enter and are retained in nonlymphoid tissue are not well characterized. With a transgenic mouse expressing the NKG2D ligand retinoic acid early transcript 1ε (RAE1ε) in β-islet cells of the pancreas, we found that RAE1 expression was sufficient to induce the recruitment of adoptively transferred CTLs to islets. This was dependent on NKG2D expression by the CTLs and independent of antigen recognition. Surprisingly, the recruitment of CTLs resulted in the subsequent recruitment of a large number of endogenous lymphocytes. Whereas transgenic mice did not develop diabetes, RAE1 expression was sufficient to induce insulitis in older, unmanipulated transgenic mice that was enhanced by viral infection and pancreatic inflammation. These results demonstrate that the expression of an NKG2D ligand in islets is sufficient to recruit CTLs regardless of their antigen specificity and to induce insulitis. 10.1016/j.immuni.2011.11.014
    Anti-TNF treatment blocks the induction of T cell-dependent humoral responses. Salinas Gabriela Franco,De Rycke Leen,Barendregt Barbara,Paramarta Jacqueline E,Hreggvidsdottir Hulda,Cantaert Tineke,van der Burg Mirjam,Tak Paul P,Baeten Dominique Annals of the rheumatic diseases OBJECTIVE:Experimental and human data suggest that tumour necrosis factor (TNF) blockade may affect B cell responses, in particular the induction of T cell-dependent (TD) humoral immunity. This study aimed to assess this hypothesis directly in patients with arthritis by analysing longitudinally the effect of TNF blockade on B cell activation and the maturation of humoral responses against TD and T cell-independent vaccines. MATERIALS AND METHODS:Peripheral blood samples were obtained from 56 spondyloarthritis patients before and after treatment with either non-steroidal anti-inflammatory drug (NSAID) alone or TNF blockers and analysed for B cell activation, plasma cell differentiation, germinal centre versus extra-follicular B cell maturation, and somatic hypermutation. Vaccine responses to hepatitis B and Streptococcus pneumoniae were measured by ELISA. RESULTS:TNF blockade augmented B cell activation as reflected by the expression of early activation markers, CD40, and costimulatory molecules, without affecting differentiation towards plasmablasts. This was associated with a specific increase of the unswitched fraction of circulating memory B cells and a decreased level of somatic hypermutation in anti-TNF treated patients, indicating an impairment of the germinal centre-dependent B cell maturation. In agreement with these findings, TNF blockade profoundly suppressed the response to the TD vaccination against hepatitis B, whereas the T cell-independent response against pneumococcal polysaccharides was only modestly affected. CONCLUSIONS:These data indicate that TNF blockade severely impedes the induction of primary TD humoral responses, probably by interfering with the germinal centre reaction. 10.1136/annrheumdis-2011-201270
    T-REX on-demand redox targeting in live cells. Parvez Saba,Long Marcus J C,Lin Hong-Yu,Zhao Yi,Haegele Joseph A,Pham Vanha N,Lee Dustin K,Aye Yimon Nature protocols This protocol describes targetable reactive electrophiles and oxidants (T-REX)-a live-cell-based tool designed to (i) interrogate the consequences of specific and time-resolved redox events, and (ii) screen for bona fide redox-sensor targets. A small-molecule toolset comprising photocaged precursors to specific reactive redox signals is constructed such that these inert precursors specifically and irreversibly tag any HaloTag-fused protein of interest (POI) in mammalian and Escherichia coli cells. Syntheses of the alkyne-functionalized endogenous reactive signal 4-hydroxynonenal (HNE(alkyne)) and the HaloTag-targetable photocaged precursor to HNE(alkyne) (also known as Ht-PreHNE or HtPHA) are described. Low-energy light prompts photo-uncaging (t <1-2 min) and target-specific modification. The targeted modification of the POI enables precisely timed and spatially controlled redox events with no off-target modification. Two independent pathways are described, along with a simple setup to functionally validate known targets or discover novel sensors. T-REX sidesteps mixed responses caused by uncontrolled whole-cell swamping with reactive signals. Modification and downstream response can be analyzed by in-gel fluorescence, proteomics, qRT-PCR, immunofluorescence, fluorescence resonance energy transfer (FRET)-based and dual-luciferase reporters, or flow cytometry assays. T-REX targeting takes 4 h from initial probe treatment. Analysis of targeted redox responses takes an additional 4-24 h, depending on the nature of the pathway and the type of readouts used. 10.1038/nprot.2016.114
    Association of Ribonuclease T2 Gene Polymorphisms With Decreased Expression and Clinical Characteristics of Severity in Crohn's Disease. Gonsky Rivkah,Fleshner Phillip,Deem Richard L,Biener-Ramanujan Eva,Li Dalin,Potdar Alka A,Bilsborough Janine,Yang Shaohong,McGovern Dermot P B,Targan Stephan R Gastroenterology BACKGROUND & AIMS:Variants in the tumor necrosis factor superfamily member 15 gene (TNFSF15, also called TL1A) have been associated with risk for inflammatory bowel disease (IBD). TL1A affects expression of multiple cytokines to promote mucosal inflammation. Little is known about the TL1A-response pathways that regulate cytokine expression. We investigated T-cell gene expression patterns to determine the mechanisms by which TL1A regulates cytokine production, and whether these associate with outcomes of patients with Crohn's disease (CD). METHODS:Peripheral T cells isolated from normal donors were cultured with TL1A. We performed gene expression profile analysis by RNA sequencing of subsets of interferon gamma (IFNG)-producing and non-producing cells purified by flow cytometry. Unsupervised hierarchical clustering analysis was used to identify gene expression differences between these subsets. Ribonuclease T2 gene (RNASET2) expression and methylation were assessed by quantitative trait loci analyses. Clinical characteristics of patients (complications, resistance to therapy, and recurrence time) were associated with single nucleotide polymorphisms in RNASET2. We performed motif screening to identify polymorphisms that disrupt transcription factor binding sites. Levels of RNASET2 were knocked down with small interfering RNA in CD4 T cells and the effect on protein expression was determined by proteomic analysis and cytokine production. Cell aggregation was measured by flow cytometry. RESULTS:We identified 764 genes with at least a 2-fold difference in TL1A-mediated expression between IFNG-secreting and non-secreting T cells (P < 1 × 10). Many of these genes were located near IBD susceptibility variants. RNASET2 was the only IBD risk-associated gene with >5-fold down-regulation in the IFNG-secreting subset. RNASET2 disease risk variants were associated with decreased expression in peripheral and mucosal tissues and DNA hypermethylation in CD patients requiring surgical intervention. RNASET2 disease risk variants were associated in CD patients with more complicated disease or resistance to therapy, defined in part by failed response to treatment, increased length of intestinal resection, shorter time to repeat surgery, and high Rutgeerts score (>2) in postoperative endoscopy. The RNASET2 variant rs2149092 was predicted to disrupt a consensus binding site for the transcription factor ETS within an enhancer region. Expression of RNASET2 correlated with expression of ETS. RNASET2 knockdown in T cells increased expression of IFNG and intercellular adhesion molecule 1 (ICAM1) and induced T-cell aggregation. A blocking antibody against (ILFA1), disrupting the lymphocyte function-associated antigen 1-intercellular adhesion molecule 1 interaction, reduced T-cell production of IFNG. CONCLUSIONS:We identified decreased expression of RNASET2 as a component of TL1A-mediated increase in production of IFNG and as a potential biomarker for patients with severe CD. Further study of the role of RNASET2 in regulating mucosal inflammation may lead to development of novel therapeutic targets. 10.1053/j.gastro.2017.04.002
    The vigorous immune microenvironment of microsatellite instable colon cancer is balanced by multiple counter-inhibitory checkpoints. Llosa Nicolas J,Cruise Michael,Tam Ada,Wicks Elizabeth C,Hechenbleikner Elizabeth M,Taube Janis M,Blosser Richard L,Fan Hongni,Wang Hao,Luber Brandon S,Zhang Ming,Papadopoulos Nickolas,Kinzler Kenneth W,Vogelstein Bert,Sears Cynthia L,Anders Robert A,Pardoll Drew M,Housseau Franck Cancer discovery UNLABELLED:We examined the immune microenvironment of primary colorectal cancer using immunohistochemistry, laser capture microdissection/qRT-PCR, flow cytometry, and functional analysis of tumor-infiltrating lymphocytes. A subset of colorectal cancer displayed high infiltration with activated CD8(+) cytotoxic T lymphocyte (CTL) as well as activated Th1 cells characterized by IFNγ production and the Th1 transcription factor TBET. Parallel analysis of tumor genotypes revealed that virtually all of the tumors with this active Th1/CTL microenvironment had defects in mismatch repair, as evidenced by microsatellite instability (MSI). Counterbalancing this active Th1/CTL microenvironment, MSI tumors selectively demonstrated highly upregulated expression of multiple immune checkpoints, including five-PD-1, PD-L1, CTLA-4, LAG-3, and IDO-currently being targeted clinically with inhibitors. These findings link tumor genotype with the immune microenvironment, and explain why MSI tumors are not naturally eliminated despite a hostile Th1/CTL microenvironment. They further suggest that blockade of specific checkpoints may be selectively efficacious in the MSI subset of colorectal cancer. SIGNIFICANCE:The findings reported in this article are the first to demonstrate a link between a genetically defined subtype of cancer and its corresponding expression of immune checkpoints in the tumor microenvironment. The mismatch repair-defective subset of colorectal cancer selectively upregulates at least five checkpoint molecules that are targets of inhibitors currently being clinically tested. 10.1158/2159-8290.CD-14-0863
    Cardiac Resident Macrophage-Derived Legumain Improves Cardiac Repair by Promoting Clearance and Degradation of Apoptotic Cardiomyocytes After Myocardial Infarction. Circulation BACKGROUND:Cardiac resident macrophages are self-maintaining and originate from embryonic hematopoiesis. After myocardial infarction, cardiac resident macrophages are responsible for the efficient clearance and degradation of apoptotic cardiomyocytes (efferocytosis). This process is required for inflammation resolution and tissue repair; however, the underlying molecular mechanisms remain unknown. Therefore, we aimed to identify the mechanisms of the continued clearance and degradation of phagolysosomal cargo by cardiac resident macrophages during myocardial infarction. METHODS:Multiple transgenic mice such as Lgmn, Lgmn; LysM, Lgmn; Cx3cr1, Lgmn; Lyve, and cardiac macrophage Lgmn overexpression by adenovirus gene transfer were used to determine the functional significance of Lgmn in myocardial infarction. Immune cell filtration and inflammation were examined by flow cytometry and quantitative real-time polymerase chain reaction. Moreover, legumain (Lgmn) expression was analyzed by immunohistochemistry and quantitative real-time polymerase chain reaction in the cardiac tissues of patients with ischemic cardiomyopathy and healthy control subjects. RESULTS:We identified as a gene specifically expressed by cardiac resident macrophages. Lgmn deficiency resulted in a considerable exacerbation in cardiac function, accompanied by the accumulation of apoptotic cardiomyocytes and a reduced index of in vivo efferocytosis in the border area. It also led to decreased cytosolic calcium attributable to defective intracellular calcium mobilization. Furthermore, the formation of LC3-II-dependent phagosome around secondary-encountered apoptotic cardiomyocytes was disabled. In addition, Lgmn deficiency increased infiltration of MHC-II CCR2 macrophages and the enhanced recruitment of MHC-II CCR2 monocytes with downregulation of the anti-inflammatory mediators, interleukin-10, and transforming growth factor-β and upregulationof the proinflammatory mediators interleukin-1β, tumor necrosis factor-α, interleukin-6, and interferon-γ. CONCLUSIONS:Our results directly link efferocytosis to wound healing in the heart and identify Lgmn as a significant link between acute inflammation resolution and organ function. 10.1161/CIRCULATIONAHA.121.057549
    TRPM4 cation channel mediates axonal and neuronal degeneration in experimental autoimmune encephalomyelitis and multiple sclerosis. Schattling Benjamin,Steinbach Karin,Thies Edda,Kruse Martin,Menigoz Aurélie,Ufer Friederike,Flockerzi Veit,Brück Wolfgang,Pongs Olaf,Vennekens Rudi,Kneussel Matthias,Freichel Marc,Merkler Doron,Friese Manuel A Nature medicine In multiple sclerosis, an inflammatory disease of the central nervous system (CNS), axonal and neuronal loss are major causes for irreversible neurological disability. However, which molecules contribute to axonal and neuronal injury under inflammatory conditions remains largely unknown. Here we show that the transient receptor potential melastatin 4 (TRPM4) cation channel is crucial in this process. TRPM4 is expressed in mouse and human neuronal somata, but it is also expressed in axons in inflammatory CNS lesions in experimental autoimmune encephalomyelitis (EAE) in mice and in human multiple sclerosis tissue. Deficiency or pharmacological inhibition of TRPM4 using the antidiabetic drug glibenclamide resulted in reduced axonal and neuronal degeneration and attenuated clinical disease scores in EAE, but this occurred without altering EAE-relevant immune function. Furthermore, Trpm4(-/-) mouse neurons were protected against inflammatory effector mechanisms such as excitotoxic stress and energy deficiency in vitro. Electrophysiological recordings revealed TRPM4-dependent neuronal ion influx and oncotic cell swelling upon excitotoxic stimulation. Therefore, interference with TRPM4 could translate into a new neuroprotective treatment strategy. 10.1038/nm.3015
    Inhibition of IL-12/IL-23 signaling reduces Alzheimer's disease-like pathology and cognitive decline. Vom Berg Johannes,Prokop Stefan,Miller Kelly R,Obst Juliane,Kälin Roland E,Lopategui-Cabezas Ileana,Wegner Anja,Mair Florian,Schipke Carola G,Peters Oliver,Winter York,Becher Burkhard,Heppner Frank L Nature medicine The pathology of Alzheimer's disease has an inflammatory component that is characterized by upregulation of proinflammatory cytokines, particularly in response to amyloid-β (Aβ). Using the APPPS1 Alzheimer's disease mouse model, we found increased production of the common interleukin-12 (IL-12) and IL-23 subunit p40 by microglia. Genetic ablation of the IL-12/IL-23 signaling molecules p40, p35 or p19, in which deficiency of p40 or its receptor complex had the strongest effect, resulted in decreased cerebral amyloid load. Although deletion of IL-12/IL-23 signaling from the radiation-resistant glial compartment of the brain was most efficient in mitigating cerebral amyloidosis, peripheral administration of a neutralizing p40-specific antibody likewise resulted in a reduction of cerebral amyloid load in APPPS1 mice. Furthermore, intracerebroventricular delivery of antibodies to p40 significantly reduced the concentration of soluble Aβ species and reversed cognitive deficits in aged APPPS1 mice. The concentration of p40 was also increased in the cerebrospinal fluid of subjects with Alzheimer's disease, which suggests that inhibition of the IL-12/IL-23 pathway may attenuate Alzheimer's disease pathology and cognitive deficits. 10.1038/nm.2965
    OX40 stimulation and PD-L1 blockade synergistically augment HBV-specific CD4 T cells in patients with HBeAg-negative infection. Jacobi Felix Johannes,Wild Katharina,Smits Maike,Zoldan Katharina,Csernalabics Benedikt,Flecken Tobias,Lang Julia,Ehrenmann Philipp,Emmerich Florian,Hofmann Maike,Thimme Robert,Neumann-Haefelin Christoph,Boettler Tobias Journal of hepatology BACKGROUND & AIMS:Current antiviral therapies lack the potential to eliminate persistent hepatitis B virus (HBV) infection. HBV-specific T cells are crucial for HBV control and have recently been shown to be protective in patients following discontinuation of antiviral therapy. Thus, T cell-based approaches may greatly improve the therapeutic landscape of HBV infection. We aimed to augment HBV-specific CD4 T cells from chronically infected patients by targeting different immunological pathways. METHODS:Expression of various co-stimulatory and inhibitory receptors on HBV- and influenza-specific CD4 T cells was analyzed directly ex vivo by MHC class II-tetramers. Patients infected with HBV genotype D were screened for CD4 T cell responses by IFN-γ ELISpot and intracellular cytokine staining following stimulation with overlapping peptides (OLPs) spanning the HBV-polyprotein. Stimulation with recombinant IL-7, an agonistic OX40-antibody or blockade of PD-L1 was performed in antigen-specific in vitro cultures. Cytokine secretion and expression of transcription factors were analyzed by flow cytometry. Responses targeting influenza, Epstein-Barr virus and tetanus toxoid served as controls. RESULTS:Tetramer-staining revealed that the IL-7 receptor-alpha (CD127), OX40 and PD-1 constitute possible therapeutic targets as they were all strongly expressed on HBV-specific CD4 T cells ex vivo. The HBV-specific CD4 T cell responses identified by OLP screening targeted predominantly the HBV-polymerase and core proteins. Combined OX40 stimulation and PD-L1 blockade significantly augmented IFN-γ and IL-21 producing HBV-specific CD4 T cells in vitro, suggesting active T helper type 1 cell and follicular T helper cell programs. Indeed, transcription factors T-bet and Bcl6 were strongly expressed in cytokine-producing cells. CONCLUSIONS:Combined OX40 stimulation and PD-L1 blockade augmented secretion of the helper T cell signature cytokines IFN-γ and IL-21, suggesting that immunotherapeutic approaches can improve HBV-specific CD4 T cell responses. LAY SUMMARY:CD4 T cells are important in controlling viral infections but are impaired in the context of chronic hepatitis B virus (HBV) infection. Therapeutic approaches to cure chronic HBV infection are highly likely to require an immune-stimulatory component. This study demonstrates that HBV-specific CD4 T cells can be functionally augmented by combined stimulation of the co-stimulatory molecule OX40 and blockade of the inhibitory PD-1 pathway. 10.1016/j.jhep.2019.02.016
    Maintenance of hematopoietic stem cells through regulation of Wnt and mTOR pathways. Huang Jian,Nguyen-McCarty Michelle,Hexner Elizabeth O,Danet-Desnoyers Gwenn,Klein Peter S Nature medicine Hematopoietic stem cell (HSC) self renewal and lineage commitment depend on complex interactions with the microenvironment. The ability to maintain or expand HSCs for clinical applications or basic research has been substantially limited because these interactions are not well defined. Recent evidence suggests that HSCs reside in a low-perfusion, reduced-nutrient niche and that nutrient-sensing pathways contribute to HSC homeostasis. Here we report that suppression of the mTOR pathway, an established nutrient sensor, combined with activation of canonical Wnt-β-catenin signaling, allows for the ex vivo maintenance of human and mouse long-term HSCs under cytokine-free conditions. We also show that the combination of two clinically approved medications that together activate Wnt-β-catenin and inhibit mTOR signaling increases the number (but not the proportion) of long-term HSCs in vivo. 10.1038/nm.2984
    NK cells impede glioblastoma virotherapy through NKp30 and NKp46 natural cytotoxicity receptors. Alvarez-Breckenridge Christopher A,Yu Jianhua,Price Richard,Wojton Jeffrey,Pradarelli Jason,Mao Hsiaoyin,Wei Min,Wang Yan,He Shun,Hardcastle Jayson,Fernandez Soledad A,Kaur Balveen,Lawler Sean E,Vivier Eric,Mandelboim Ofer,Moretta Alessandro,Caligiuri Michael A,Chiocca E Antonio Nature medicine The role of the immune response to oncolytic Herpes simplex viral (oHSV) therapy for glioblastoma is controversial because it might enhance or inhibit efficacy. We found that within hours of oHSV infection of glioblastomas in mice, activated natural killer (NK) cells are recruited to the site of infection. This response substantially diminished the efficacy of glioblastoma virotherapy. oHSV-activated NK cells coordinated macrophage and microglia activation within tumors. In vitro, human NK cells preferentially lysed oHSV-infected human glioblastoma cell lines. This enhanced killing depended on the NK cell natural cytotoxicity receptors (NCRs) NKp30 and NKp46, whose ligands are upregulated in oHSV-infected glioblastoma cells. We found that HSV titers and oHSV efficacy are increased in Ncr1(-/-) mice and a Ncr1(-/-) NK cell adoptive transfer model of glioma, respectively. These results demonstrate that glioblastoma virotherapy is limited partially by an antiviral NK cell response involving specific NCRs, uncovering new potential targets to enhance cancer virotherapy. 10.1038/nm.3013
    C5a receptor targeting in neointima formation after arterial injury in atherosclerosis-prone mice. Shagdarsuren Erdenechimeg,Bidzhekov Kiril,Mause Sebastian F,Simsekyilmaz Sakine,Polakowski Thomas,Hawlisch Heiko,Gessner J Engelbert,Zernecke Alma,Weber Christian Circulation BACKGROUND:Receptor binding of complement C5a leads to proinflammatory activation of many cell types, but the role of receptor-mediated action during arterial remodeling after injury has not been studied. In the present study, we examined the contribution of the C5a receptor (C5aR) to neointima formation in apolipoprotein E-deficient mice employing a C5aR antagonist (C5aRA) and a C5aR-blocking monoclonal antibody. METHODS AND RESULTS:Mice fed an atherogenic diet were subjected to wire-induced endothelial denudation of the carotid artery and treated with C5aRA and anti-C5aR-blocking monoclonal antibody or vehicle control. Compared with controls, neointima formation was significantly reduced in mice receiving C5aRA or anti-C5aR-blocking monoclonal antibody for 1 week but not for 3 weeks, attributable to an increased content of vascular smooth muscle cells, whereas a marked decrease in monocyte and neutrophil content was associated with reduced vascular cell adhesion molecule-1. As assessed by immunohistochemistry, reverse transcription polymerase chain reaction, and flow cytometry, C5aR was expressed in lesional and cultured vascular smooth muscle cells, upregulated by injury or tumor necrosis factor-alpha, and reduced by C5aRA. Plasma levels and neointimal plasminogen activator inhibitor-1 peaked 1 week after injury and were downregulated in C5aRA-treated mice. In vitro, C5a induced plasminogen activator inhibitor-1 expression in endothelial cells and vascular smooth muscle cells in a C5aRA-dependent manner, possibly accounting for higher vascular smooth muscle cell immigration. CONCLUSIONS:One-week treatment with C5aRA or anti-C5aR-blocking monoclonal antibody limited neointimal hyperplasia and inflammatory cell content and was associated with reduced vascular cell adhesion molecule-1 expression. However, treatment for 3 weeks failed to reduce but rather stabilized plaques, likely by reducing vascular plasminogen activator inhibitor-1 and increasing vascular smooth muscle cell migration. 10.1161/CIRCULATIONAHA.110.954370
    Defective immune responses in mice lacking LUBAC-mediated linear ubiquitination in B cells. Sasaki Yoshiteru,Sano Soichi,Nakahara Masaki,Murata Shigeo,Kometani Kohei,Aiba Yuichi,Sakamoto Shinji,Watanabe Yoshihiro,Tanaka Keiji,Kurosaki Tomohiro,Iwai Kazuhiro The EMBO journal The linear ubiquitin chain assembly complex (LUBAC) plays a crucial role in activating the canonical NF-κB pathway, which is important for B-cell development and function. Here, we describe a mouse model (B-HOIP(Δlinear)) in which the linear polyubiquitination activity of LUBAC is specifically ablated in B cells. Canonical NF-κB and ERK activation, mediated by the tumour necrosis factor (TNF) receptor superfamily receptors CD40 and TACI, was impaired in B cells from B-HOIP(Δlinear) mice due to defective activation of the IKK complex; however, B-cell receptor (BCR)-mediated activation of the NF-κB and ERK pathways was unaffected. B-HOIP(Δlinear) mice show impaired B1-cell development and defective antibody responses to thymus-dependent and thymus-independent II antigens. Taken together, these data suggest that LUBAC-mediated linear polyubiquitination is essential for B-cell development and activation, possibly via canonical NF-κB and ERK activation induced by the TNF receptor superfamily, but not by the BCR. 10.1038/emboj.2013.184
    Initiation of MLL-rearranged AML is dependent on C/EBPα. Ohlsson Ewa,Hasemann Marie Sigurd,Willer Anton,Lauridsen Felicia Kathrine Bratt,Rapin Nicolas,Jendholm Johan,Porse Bo Torben The Journal of experimental medicine MLL-fusion proteins are potent inducers of oncogenic transformation, and their expression is considered to be the main oncogenic driving force in ∼10% of human acute myeloid leukemia (AML) patients. These oncogenic fusion proteins are responsible for the initiation of a downstream transcriptional program leading to the expression of factors such as MEIS1 and HOXA9, which in turn can replace MLL-fusion proteins in overexpression experiments. To what extent MLL fusion proteins act on their own during tumor initiation, or if they collaborate with other transcriptional regulators, is unclear. Here, we have compared gene expression profiles from human MLL-rearranged AML to normal progenitors and identified the myeloid tumor suppressor C/EBPα as a putative collaborator in MLL-rearranged AML. Interestingly, we find that deletion of Cebpa rendered murine hematopoietic progenitors completely resistant to MLL-ENL-induced leukemic transformation, whereas C/EBPα was dispensable in already established AMLs. Furthermore, we show that Cebpa-deficient granulocytic-monocytic progenitors were equally resistant to transformation and that C/EBPα collaborates with MLL-ENL in the induction of a transcriptional program, which is also apparent in human AML. Thus, our studies demonstrate a key role of C/EBPα in MLL fusion-driven transformation and find that it sharply demarcates tumor initiation and maintenance. 10.1084/jem.20130932
    CCR7 provides localized access to IL-2 and defines homeostatically distinct regulatory T cell subsets. Smigiel Kate S,Richards Elizabeth,Srivastava Shivani,Thomas Kerri R,Dudda Jan C,Klonowski Kimberly D,Campbell Daniel J The Journal of experimental medicine Immune tolerance and activation depend on precise control over the number and function of immunosuppressive Foxp3(+) regulatory T (T reg) cells, and the importance of IL-2 in maintaining tolerance and preventing autoimmunity is clear. However, the homeostatic requirement for IL-2 among specific populations of peripheral T reg cells remains poorly understood. We show that IL-2 selectively maintains a population of quiescent CD44(lo)CD62L(hi) T reg cells that gain access to paracrine IL-2 produced in the T cell zones of secondary lymphoid tissues due to their expression of the chemokine receptor CCR7. In contrast, CD44(hi)CD62L(lo)CCR7(lo) T reg cells that populate nonlymphoid tissues do not access IL-2-prevalent regions in vivo and are insensitive to IL-2 blockade; instead, their maintenance depends on continued signaling through the co-stimulatory receptor ICOS (inducible co-stimulator). Thus, we define a fundamental homeostatic subdivision in T reg cell populations based on their localization and provide an integrated framework for understanding how T reg cell abundance and function are controlled by unique signals in different tissue environments. 10.1084/jem.20131142
    Pro-inflammatory human Th17 cells selectively express P-glycoprotein and are refractory to glucocorticoids. Ramesh Radha,Kozhaya Lina,McKevitt Kelly,Djuretic Ivana M,Carlson Thaddeus J,Quintero Maria A,McCauley Jacob L,Abreu Maria T,Unutmaz Derya,Sundrud Mark S The Journal of experimental medicine IL-17A-expressing CD4(+) T cells (Th17 cells) are generally regarded as key effectors of autoimmune inflammation. However, not all Th17 cells are pro-inflammatory. Pathogenic Th17 cells that induce autoimmunity in mice are distinguished from nonpathogenic Th17 cells by a unique transcriptional signature, including high Il23r expression, and these cells require Il23r for their inflammatory function. In contrast, defining features of human pro-inflammatory Th17 cells are unknown. We show that pro-inflammatory human Th17 cells are restricted to a subset of CCR6(+)CXCR3(hi)CCR4(lo)CCR10(-)CD161(+) cells that transiently express c-Kit and stably express P-glycoprotein (P-gp)/multi-drug resistance type 1 (MDR1). In contrast to MDR1(-) Th1 or Th17 cells, MDR1(+) Th17 cells produce both Th17 (IL-17A, IL-17F, and IL-22) and Th1 (IFN-γ) cytokines upon TCR stimulation and do not express IL-10 or other anti-inflammatory molecules. These cells also display a transcriptional signature akin to pathogenic mouse Th17 cells and show heightened functional responses to IL-23 stimulation. In vivo, MDR1(+) Th17 cells are enriched and activated in the gut of Crohn's disease patients. Furthermore, MDR1(+) Th17 cells are refractory to several glucocorticoids used to treat clinical autoimmune disease. Thus, MDR1(+) Th17 cells may be important mediators of chronic inflammation, particularly in clinical settings of steroid resistant inflammatory disease. 10.1084/jem.20130301
    Single-Cell Analysis Reveals Heterogeneity of High Endothelial Venules and Different Regulation of Genes Controlling Lymphocyte Entry to Lymph Nodes. Veerman Krystle,Tardiveau Claire,Martins Frédéric,Coudert Juliette,Girard Jean-Philippe Cell reports High-endothelial venules (HEVs) are specialized blood vessels allowing recirculation of naive lymphocytes through lymphoid organs. Here, using full-length, single-cell RNA sequencing, RNA fluorescence in situ hybridization (FISH), flow cytometry, and immunohistofluorescence, we reveal the heterogeneity of HEVs in adult mouse peripheral lymph nodes (PLNs) under conditions of homeostasis, antigenic stimulation, and after inhibition of lymphotoxin-β receptor (LTβR) signaling. We demonstrate that HEV endothelial cells are in an activated state during homeostasis, and we identify the genes characteristic of the differentiated HEV phenotype. We show that LTβR signaling regulates many HEV genes and pathways in resting PLNs and that immune stimulation induces a global and temporary inflammatory phenotype in HEVs without compromising their ability to recruit naive lymphocytes. Most importantly, we uncover differences in the regulation of genes controlling lymphocyte trafficking, Glycam1, Fut7, Gcnt1, Chst4, B3gnt3, and Ccl21a, that have implications for HEV function and regulation in health and disease. 10.1016/j.celrep.2019.02.042
    EndoU is a novel regulator of AICD during peripheral B cell selection. Poe Jonathan C,Kountikov Evgueni I,Lykken Jacquelyn M,Natarajan Abirami,Marchuk Douglas A,Tedder Thomas F The Journal of experimental medicine Balanced transmembrane signals maintain a competent peripheral B cell pool limited in self-reactive B cells that may produce pathogenic autoantibodies. To identify molecules regulating peripheral B cell survival and tolerance to self-antigens (Ags), a gene modifier screen was performed with B cells from CD22-deficient C57BL/6 (CD22(-/-[B6])) mice that undergo activation-induced cell death (AICD) and fail to up-regulate c-Myc expression after B cell Ag receptor ligation. Likewise, lysozyme auto-Ag-specific B cells in Ig(Tg) hen egg lysozyme (HEL) transgenic mice inhabit the spleen but undergo AICD after auto-Ag encounter. This gene modifier screen identified EndoU, a single-stranded RNA-binding protein of ancient origin, as a major regulator of B cell survival in both models. EndoU gene disruption prevents AICD and normalizes c-Myc expression. These findings reveal that EndoU is a critical regulator of an unexpected and novel RNA-dependent pathway controlling peripheral B cell survival and Ag responsiveness that may contribute to peripheral B cell tolerance. 10.1084/jem.20130648
    Local BLyS production by T follicular cells mediates retention of high affinity B cells during affinity maturation. Goenka Radhika,Matthews Andrew H,Zhang Bochao,O'Neill Patrick J,Scholz Jean L,Migone Thi-Sau,Leonard Warren J,Stohl William,Hershberg Uri,Cancro Michael P The Journal of experimental medicine We have assessed the role of B lymphocyte stimulator (BLyS) and its receptors in the germinal center (GC) reaction and affinity maturation. Despite ample BLyS retention on B cells in follicular (FO) regions, the GC microenvironment lacks substantial BLyS. This reflects IL-21-mediated down-regulation of the BLyS receptor TACI (transmembrane activator and calcium modulator and cyclophilin ligand interactor) on GC B cells, thus limiting their capacity for BLyS binding and retention. Within the GC, FO helper T cells (TFH cells) provide a local source of BLyS. Whereas T cell-derived BLyS is dispensable for normal GC cellularity and somatic hypermutation, it is required for the efficient selection of high affinity GC B cell clones. These findings suggest that during affinity maturation, high affinity clones rely on TFH-derived BLyS for their persistence. 10.1084/jem.20130505
    TRAF3 regulates the effector function of regulatory T cells and humoral immune responses. Chang Jae-Hoon,Hu Hongbo,Jin Jin,Puebla-Osorio Nahum,Xiao Yichuan,Gilbert Brian E,Brink Robert,Ullrich Stephen E,Sun Shao-Cong The Journal of experimental medicine Regulatory T cells (Treg cells) control different aspects of immune responses, but how the effector functions of Treg cells are regulated is incompletely understood. Here we identified TNF receptor-associated factor 3 (TRAF3) as a regulator of Treg cell function. Treg cell-specific ablation of TRAF3 impaired CD4 T cell homeostasis, characterized by an increase in the Th1 type of effector/memory T cells. Moreover, the ablation of TRAF3 in Treg cells resulted in increased antigen-stimulated activation of follicular T helper cells (TFH cells), coupled with heightened formation of germinal centers and production of high-affinity IgG antibodies. Although the loss of TRAF3 did not reduce the overall frequency of Treg cells, it attenuated the antigen-stimulated production of follicular Treg cells (TFR cells). TRAF3 signaling in Treg cells was required to maintain high level expression of inducible co-stimulator (ICOS), which in turn was required for TFR cell generation and inhibition of antibody responses. These findings establish TRAF3 as a mediator of Treg cell function in the regulation of antibody responses and suggest a role for TRAF3 in mediating ICOS expression in Treg cells. 10.1084/jem.20131019
    Musashi-2 controls cell fate, lineage bias, and TGF-β signaling in HSCs. Park Sun-Mi,Deering Raquel P,Lu Yuheng,Tivnan Patrick,Lianoglou Steve,Al-Shahrour Fatima,Ebert Benjamin L,Hacohen Nir,Leslie Christina,Daley George Q,Lengner Christopher J,Kharas Michael G The Journal of experimental medicine Hematopoietic stem cells (HSCs) are maintained through the regulation of symmetric and asymmetric cell division. We report that conditional ablation of the RNA-binding protein Msi2 results in a failure of HSC maintenance and engraftment caused by a loss of quiescence and increased commitment divisions. Contrary to previous studies, we found that these phenotypes were independent of Numb. Global transcriptome profiling and RNA target analysis uncovered Msi2 interactions at multiple nodes within pathways that govern RNA translation, stem cell function, and TGF-β signaling. Msi2-null HSCs are insensitive to TGF-β-mediated expansion and have decreased signaling output, resulting in a loss of myeloid-restricted HSCs and myeloid reconstitution. Thus, Msi2 is an important regulator of the HSC translatome and balances HSC homeostasis and lineage bias. 10.1084/jem.20130736
    HIV-associated follicular bronchiolitis. Shipe Ryan,Lawrence Jason,Green Jeffery,Enfield Kyle American journal of respiratory and critical care medicine 10.1164/rccm.201206-1096IM
    Identification and characterisation of citrullinated antigen-specific B cells in peripheral blood of patients with rheumatoid arthritis. Kerkman Priscilla F,Fabre Emeline,van der Voort Ellen I H,Zaldumbide Arnaud,Rombouts Yoann,Rispens Theo,Wolbink Gertjan,Hoeben Rob C,Spits Hergen,Baeten Dominique L P,Huizinga Tom W J,Toes René E M,Scherer Hans U Annals of the rheumatic diseases OBJECTIVES:Immunity to citrullinated antigens is a hallmark of rheumatoid arthritis (RA). We set out to elucidate its biology by identifying and characterising citrullinated antigen-specific B cells in peripheral blood of patients with RA. METHODS:Differentially labelled streptavidin and extravidin tetramers were conjugated to biotinylated CCP2 or control antigens and used in flow cytometry to identify citrullinated antigen-specific B cells in peripheral blood. Tetramer-positive and tetramer-negative B cells were isolated by fluorescence activated cell sorting (FACS) followed by in vitro culture and analysis of culture supernatants for the presence of antibodies against citrullinated protein antigens (ACPA) by ELISA. Cells were phenotypically characterised by flow cytometry. RESULTS:By combining differentially labelled CCP2 tetramers, we successfully separated citrullinated antigen-specific B cells from non-specific background signals. Isolated tetramer-positive B cells, but not tetramer-negative cells, produced large amounts of ACPA upon in vitro stimulation. Phenotypic analyses revealed that citrullinated antigen-specific B cells displayed markers of class-switched memory B cells and plasmablasts, whereas only few cells displayed a naïve phenotype. The frequency of tetramer-positive cells was high (up to 1/500 memory B cells with a median of 1/12 500 total B cells) and correlated with ACPA serum titres and spontaneous ACPA production in culture. CONCLUSIONS:We developed a technology to identify and isolate citrullinated antigen-specific B cells from peripheral blood of patients with RA. Most cells have a memory phenotype, express IgA or IgG and are present in relatively high frequencies. These data pave the path for a direct and detailed molecular characterisation of ACPA-expressing B cells and could lead to the identification of novel therapeutic targets. 10.1136/annrheumdis-2014-207182
    Direct delivery of functional proteins and enzymes to the cytosol using nanoparticle-stabilized nanocapsules. Tang Rui,Kim Chang Soo,Solfiell David J,Rana Subinoy,Mout Rubul,Velázquez-Delgado Elih M,Chompoosor Apiwat,Jeong Youngdo,Yan Bo,Zhu Zheng-Jiang,Kim Chaekyu,Hardy Jeanne A,Rotello Vincent M ACS nano Intracellular protein delivery is an important tool for both therapeutic and fundamental applications. Effective protein delivery faces two major challenges: efficient cellular uptake and avoiding endosomal sequestration. We report here a general strategy for direct delivery of functional proteins to the cytosol using nanoparticle-stabilized capsules (NPSCs). These NPSCs are formed and stabilized through supramolecular interactions between the nanoparticle, the protein cargo, and the fatty acid capsule interior. The NPSCs are ~130 nm in diameter and feature low toxicity and excellent stability in serum. The effectiveness of these NPSCs as therapeutic protein carriers was demonstrated through the delivery of fully functional caspase-3 to HeLa cells with concomitant apoptosis. Analogous delivery of green fluorescent protein (GFP) confirmed cytosolic delivery as well as intracellular targeting of the delivered protein, demonstrating the utility of the system for both therapeutic and imaging applications. 10.1021/nn402753y
    Development of poly(β-amino ester)-based biodegradable nanoparticles for nonviral delivery of minicircle DNA. Keeney Michael,Ong Sang-Ging,Padilla Amanda,Yao Zhenyu,Goodman Stuart,Wu Joseph C,Yang Fan ACS nano Gene therapy provides a powerful tool for regulating cellular processes and tissue repair. Minicircle (MC) DNA are supercoiled DNA molecules free of bacterial plasmid backbone elements and have been reported to enhance prolonged gene expression compared to conventional plasmids. Despite the great promise of MC DNA for gene therapy, methods for safe and efficient MC DNA delivery remain lacking. To overcome this bottleneck, here we report the development of a poly(β-amino ester) (PBAE)-based, biodegradable nanoparticulate platform for efficient delivery of MC DNA driven by a Ubc promoter in vitro and in vivo. By synthesizing and screening a small library of 18 PBAE polymers with different backbone and end-group chemistry, we identified lead cationic PBAE structures that can complex with minicircle DNA to form nanoparticles, and delivery efficiency can be further modulated by tuning PBAE chemistry. Using human embryonic kidney 293 cells and mouse embryonic fibroblasts as model cell types, we identified a few PBAE polymers that allow efficient MC delivery at levels that are comparable or even surpassing Lipofectamine 2000. The biodegradable nature of PBAE-based nanoparticles facilitates in vivo applications and clinical translation. When injected via intraperitoneal route in vivo, MC alone resulted in high transgene expression, and a lead PBAE/MC nanoparticle formulation achieved a further 2-fold increase in protein expression compared to MC alone. Together, our results highlight the promise of PBAE-based nanoparticles as promising nonviral gene carriers for MC delivery, which may provide a valuable tool for broad applications of MC DNA-based gene therapy. 10.1021/nn402657d
    The intestinal stem cell signature identifies colorectal cancer stem cells and predicts disease relapse. Merlos-Suárez Anna,Barriga Francisco M,Jung Peter,Iglesias Mar,Céspedes María Virtudes,Rossell David,Sevillano Marta,Hernando-Momblona Xavier,da Silva-Diz Victoria,Muñoz Purificación,Clevers Hans,Sancho Elena,Mangues Ramón,Batlle Eduard Cell stem cell A frequent complication in colorectal cancer (CRC) is regeneration of the tumor after therapy. Here, we report that a gene signature specific for adult intestinal stem cells (ISCs) predicts disease relapse in CRC patients. ISCs are marked by high expression of the EphB2 receptor, which becomes gradually silenced as cells differentiate. Using EphB2 and the ISC marker Lgr5, we have FACS-purified and profiled mouse ISCs, crypt proliferative progenitors, and late transient amplifying cells to define a gene program specific for normal ISCs. Furthermore, we discovered that ISC-specific genes identify a stem-like cell population positioned at the bottom of tumor structures reminiscent of crypts. EphB2 sorted ISC-like tumor cells display robust tumor-initiating capacity in immunodeficient mice as well as long-term self-renewal potential. Taken together, our data suggest that the ISC program defines a cancer stem cell niche within colorectal tumors and plays a central role in CRC relapse. 10.1016/j.stem.2011.02.020
    Cross-genotype AR3-specific neutralizing antibodies confer long-term protection in injecting drug users after HCV clearance. Merat Sabrina J,Bru Camille,van de Berg Dorien,Molenkamp Richard,Tarr Alexander W,Koekkoek Sylvie,Kootstra Neeltje A,Prins Maria,Ball Jonathan K,Bakker Arjen Q,de Jong Menno D,Spits Hergen,Beaumont Tim,Schinkel Janke Journal of hepatology BACKGROUND & AIMS:In order to design an effective vaccine against hepatitis C virus (HCV) infection, it is necessary to understand immune protection. A number of broadly reactive neutralizing antibodies have been isolated from B cells of HCV-infected patients. However, it remains unclear whether B cells producing such antibodies contribute to HCV clearance and long-term immune protection against HCV. METHODS:We analysed the B cell repertoire of 13 injecting drug users from the Amsterdam Cohort Study, who were followed up for a median of 17.5 years after primary infection. Individuals were classified into 2 groups based on the outcome of HCV infection: 5 who became chronically infected either after primary infection or after reinfection, and 8 who were HCV RNA negative following spontaneous clearance of ≥1 HCV infection(s). From each individual, 10,000 CD27+IgG+B cells, collected 0.75 year after HCV infection, were cultured to characterize the antibody repertoire. RESULTS:Using a multiplex flow cytometry-based assay to study the antibody binding to E1E2 from genotype 1 to 6, we found that a high frequency of cross-genotype antibodies was associated with spontaneous clearance of 1 or multiple infections (p = 0.03). Epitope specificity of these cross-genotype antibodies was determined by alanine mutant scanning in 4 individuals who were HCV RNA negative following spontaneous clearance of 1 or multiple infections. Interestingly, the cross-genotype antibodies were mainly antigenic region 3 (AR3)-specific and showed cross-neutralizing activity against HCV. In addition to AR3 antibodies, 3 individuals developed antibodies recognizing antigenic region 4, of which 1 monoclonal antibody showed cross-neutralizing capacity. CONCLUSIONS:Together, these data suggest that a strong B cell response producing cross-genotype and neutralizing antibodies, especially targeting AR3, contributes to HCV clearance and long-term immune protection against HCV. LAY SUMMARY:Although effective treatments against hepatitis C virus (HCV) are available, 500,000 people die from liver disease caused by HCV each year and approximately 1.75 million people are newly infected. This could be prevented by a vaccine. To design a vaccine against HCV, more insight into the role of antibodies in the protection against HCV infection is needed. In a cohort of injecting drug users, we found that antibodies interfering with virus cell entry, and recognizing multiple HCV genotypes, conferred long-term protection against chronic HCV infection. 10.1016/j.jhep.2019.02.013
    A minimal ubiquitous chromatin opening element (UCOE) effectively prevents silencing of juxtaposed heterologous promoters by epigenetic remodeling in multipotent and pluripotent stem cells. Müller-Kuller Uta,Ackermann Mania,Kolodziej Stephan,Brendel Christian,Fritsch Jessica,Lachmann Nico,Kunkel Hana,Lausen Jörn,Schambach Axel,Moritz Thomas,Grez Manuel Nucleic acids research Epigenetic silencing of transgene expression represents a major obstacle for the efficient genetic modification of multipotent and pluripotent stem cells. We and others have demonstrated that a 1.5 kb methylation-free CpG island from the human HNRPA2B1-CBX3 housekeeping genes (A2UCOE) effectively prevents transgene silencing and variegation in cell lines, multipotent and pluripotent stem cells, and their differentiated progeny. However, the bidirectional promoter activity of this element may disturb expression of neighboring genes. Furthermore, the epigenetic basis underlying the anti-silencing effect of the UCOE on juxtaposed promoters has been only partially explored. In this study we removed the HNRPA2B1 moiety from the A2UCOE and demonstrate efficient anti-silencing properties also for a minimal 0.7 kb element containing merely the CBX3 promoter. This DNA element largely prevents silencing of viral and tissue-specific promoters in multipotent and pluripotent stem cells. The protective activity of CBX3 was associated with reduced promoter CpG-methylation, decreased levels of repressive and increased levels of active histone marks. Moreover, the anti-silencing effect of CBX3 was locally restricted and when linked to tissue-specific promoters did not activate transcription in off target cells. Thus, CBX3 is a highly attractive element for sustained, tissue-specific and copy-number dependent transgene expression in vitro and in vivo. 10.1093/nar/gkv019
    Multi-kilobase homozygous targeted gene replacement in human induced pluripotent stem cells. Byrne Susan M,Ortiz Luis,Mali Prashant,Aach John,Church George M Nucleic acids research Sequence-specific nucleases such as TALEN and the CRISPR/Cas9 system have so far been used to disrupt, correct or insert transgenes at precise locations in mammalian genomes. We demonstrate efficient 'knock-in' targeted replacement of multi-kilobase genes in human induced pluripotent stem cells (iPSC). Using a model system replacing endogenous human genes with their mouse counterpart, we performed a comprehensive study of targeting vector design parameters for homologous recombination. A 2.7 kilobase (kb) homozygous gene replacement was achieved in up to 11% of iPSC without selection. The optimal homology arm length was around 2 kb, with homology length being especially critical on the arm not adjacent to the cut site. Homologous sequence inside the cut sites was detrimental to targeting efficiency, consistent with a synthesis-dependent strand annealing (SDSA) mechanism. Using two nuclease sites, we observed a high degree of gene excisions and inversions, which sometimes occurred more frequently than indel mutations. While homozygous deletions of 86 kb were achieved with up to 8% frequency, deletion frequencies were not solely a function of nuclease activity and deletion size. Our results analyzing the optimal parameters for targeting vector design will inform future gene targeting efforts involving multi-kilobase gene segments, particularly in human iPSC. 10.1093/nar/gku1246
    Use of a high affinity DNA ligand in flow cytometry. Davis K A,Abrams B,Lin Y,Jayasena S D Nucleic acids research To investigate the feasibility of using oligonucleotides in flow cytometry we describe a model system consisting of human neutrophil elastase (HNE) coated on 3.3 micro beads and a high affinity DNA ligand for HNE isolated by in vitro selection (SELEX). In this system the fluoresceinated DNA ligand was equally effective as an anti- HNE antibody in detecting HNE on beads. The location on and the chemistry of attachment of fluorescein to the DNA ligand is critical for the sensitivity of detection. DNA constructs in which fluorescein was conjugated via an ethylene glycol tether to either the 5'-end or near the 3'-end gave much higher signals than did probes with fluorescein directly conjugated to either end. Second-step staining with strepavidin-conjugated phycoerythrin was accomplished using a biotinylated DNA ligand in the initial staining of HNE beads. These data suggest that instead of, or in addition to, antibodies high affinity oligonucleotide probes can be useful in diagnostic applications based on flow cytometry. 10.1093/nar/24.4.702
    Dissection of regenerating T-Cell responses against tuberculosis in HIV-infected adults sensitized by Mycobacterium tuberculosis. Wilkinson Katalin A,Seldon Ronnett,Meintjes Graeme,Rangaka Molebogeng X,Hanekom Willem A,Maartens Gary,Wilkinson Robert J American journal of respiratory and critical care medicine RATIONALE:Combination antiretroviral treatment (cART) reduces the risk of tuberculosis in HIV-infected people. Therefore a novel approach to gain insight into protection against tuberculosis is to analyze the T cells that expand in people sensitized by Mycobacterium tuberculosis (MTB) during cART. OBJECTIVES:To longitudinally analyze CD4 T-cell subsets during the first year of cART, from the time of starting cART (Day 0), in 19 HIV-infected, MTB-sensitized adults. METHODS:Peripheral blood mononuclear cells were obtained on Day 0, Weeks 2, 4, 12, 24, 36, and 48 of cART and were stimulated with purified protein derivative (PPD) followed by flow cytometry to analyze surface markers and intracellular cytokines. MEASUREMENTS AND MAIN RESULTS:CD4(+) T cells significantly increased during follow-up and the viral load fell to undetectable levels in each patient, indicating successful immune restoration. Central memory CD27(+)CD45RA(-) and CD27(+)CCR5(-) CD4(+) cells expanded by 12 weeks (P < 0.02) followed by naive CD27(+)CD45RA(+) cells at 36 weeks (P = 0.02). Terminally differentiated effector CD4(+)CD27(-)CCR7(-) cells decreased by 12 weeks (P = 0.02), paralleled by a proportional decline of PPD-specific CD4(+)IFN-gamma(+) cells (P = 0.02). However, the absolute numbers of PPD-specific IFN-gamma-producing cells, determined by enzyme-linked immunospot assay, increased (P = 0.02). CONCLUSIONS:Rapid effector responses are often measured when evaluating immunity. We show that although cART is associated with an absolute increase in effector function, the proportional response decreased and the strongest correlate of increased cART-mediated immunity in this study was the central memory response. 10.1164/rccm.200904-0568OC
    Lineage-specific regulation of inducible and constitutive mast cells in allergic airway inflammation. Derakhshan Tahereh,Samuchiwal Sachin K,Hallen Nils,Bankova Lora G,Boyce Joshua A,Barrett Nora A,Austen K Frank,Dwyer Daniel F The Journal of experimental medicine Murine mast cells (MCs) contain two lineages: inducible bone marrow-derived mucosal MCs (MMCs) and constitutive embryonic-derived connective tissue MCs (CTMCs). Here, we use RNA sequencing, flow cytometry, and genetic deletion in two allergic lung inflammation models to define these two lineages. We found that inducible MCs, marked by β7 integrin expression, are highly distinct from airway CTMCs at rest and during inflammation and unaffected by targeted CTMC deletion. β7High MCs expand and mature during lung inflammation as part of a TGF-β-inducible transcriptional program that includes the MMC-associated proteases Mcpt1 and Mcpt2, the basophil-associated protease Mcpt8, granule components, and the epithelial-binding αE integrin. In vitro studies using bone marrow-derived MCs (BMMCs) identified a requirement for SCF in this this TGF-β-mediated development and found that epithelial cells directly elicit TGF-β-dependent BMMC up-regulation of mMCP-1 and αE integrin. Thus, our findings characterize the expansion of a distinct inducible MC subset in C57BL/6 mice and highlight the potential for epithelium to direct MMC development. 10.1084/jem.20200321
    A Human Lung Challenge Model to Evaluate the Safety and Immunogenicity of PPD and Live Bacillus Calmette-Guérin. Davids Malika,Pooran Anil,Hermann Clemens,Mottay Lynelle,Thompson Fawziyah,Cardenas Jacob,Gu Jinghua,Koeuth Thearith,Meldau Richard,Limberis Jason,Gina Phindile,Srivastava Shashikant,Calder Bridget,Esmail Aliasgar,Tomasicchio Michele,Blackburn Jonathan,Gumbo Tawanda,Dheda Keertan American journal of respiratory and critical care medicine A human model to better understand tuberculosis immunopathogenesis and facilitate vaccine development is urgently needed. We evaluated the feasibility, safety, and immunogenicity of live bacillus Calmette-Guérin (BCG) in a lung-oriented controlled human infection model. We recruited 106 healthy South African participants with varying degrees of tuberculosis susceptibility. Live BCG, sterile PPD, and saline were bronchoscopically instilled into separate lung segments ( = 65). A control group ( = 34) underwent a single bronchoscopy without challenge. The primary outcome was safety. Cellular and antibody immune signatures were identified in BAL before and 3 days after challenge using flow cytometry, ELISA, RNA sequencing, and mass spectrometry. The frequency of adverse events was low (9.4%;  = 10), similar in the challenge versus control groups ( = 0.8), and all adverse events were mild and managed conservatively in an outpatient setting. The optimal PPD and BCG dose was 0.5 TU and 10 cfu, respectively, based on changes in BAL cellular profiles ( = 0.02) and antibody responses ( = 0.01) at incremental doses before versus after challenge. At 10 versus 10 cfu BCG, there was a significant increase in number of differentially expressed genes (367 vs. 3;  < 0.001) and dysregulated proteins (64 vs. 0;  < 0.001). Immune responses were highly setting specific ( vs. ) and compartment specific (BAL vs. blood) and localized to the challenged lung segments. A lung-oriented mycobacterial controlled human infection model using live BCG and PPD is feasible and safe. These data inform the study of tuberculosis immunopathogenesis and strategies for evaluation and development of tuberculosis vaccine candidates. 10.1164/rccm.201908-1580OC
    Autoantibodies against IL-17A, IL-17F, and IL-22 in patients with chronic mucocutaneous candidiasis and autoimmune polyendocrine syndrome type I. Puel Anne,Döffinger Rainer,Natividad Angels,Chrabieh Maya,Barcenas-Morales Gabriela,Picard Capucine,Cobat Aurélie,Ouachée-Chardin Marie,Toulon Antoine,Bustamante Jacinta,Al-Muhsen Saleh,Al-Owain Mohammed,Arkwright Peter D,Costigan Colm,McConnell Vivienne,Cant Andrew J,Abinun Mario,Polak Michel,Bougnères Pierre-François,Kumararatne Dinakantha,Marodi László,Nahum Amit,Roifman Chaim,Blanche Stéphane,Fischer Alain,Bodemer Christine,Abel Laurent,Lilic Desa,Casanova Jean-Laurent The Journal of experimental medicine Most patients with autoimmune polyendocrine syndrome type I (APS-I) display chronic mucocutaneous candidiasis (CMC). We hypothesized that this CMC might result from autoimmunity to interleukin (IL)-17 cytokines. We found high titers of autoantibodies (auto-Abs) against IL-17A, IL-17F, and/or IL-22 in the sera of all 33 patients tested, as detected by multiplex particle-based flow cytometry. The auto-Abs against IL-17A, IL-17F, and IL-22 were specific in the five patients tested, as shown by Western blotting. The auto-Abs against IL-17A were neutralizing in the only patient tested, as shown by bioassays of IL-17A activity. None of the 37 healthy controls and none of the 103 patients with other autoimmune disorders tested had such auto-Abs. None of the patients with APS-I had auto-Abs against cytokines previously shown to cause other well-defined clinical syndromes in other patients (IL-6, interferon [IFN]-gamma, or granulocyte/macrophage colony-stimulating factor) or against other cytokines (IL-1beta, IL-10, IL-12, IL-18, IL-21, IL-23, IL-26, IFN-beta, tumor necrosis factor [alpha], or transforming growth factor beta). These findings suggest that auto-Abs against IL-17A, IL-17F, and IL-22 may cause CMC in patients with APS-I. 10.1084/jem.20091983
    Donor myocardial infarction impairs the therapeutic potential of bone marrow cells by an interleukin-1-mediated inflammatory response. Wang Xiaoyin,Takagawa Junya,Lam Viola C,Haddad Daniel J,Tobler Diana L,Mok Pamela Y,Zhang Yan,Clifford Brian T,Pinnamaneni Kranthi,Saini Shereen A,Su Robert,Bartel Maya J,Sievers Richard E,Carbone Larry,Kogan Scott,Yeghiazarians Yerem,Hermiston Michelle,Springer Matthew L Science translational medicine Delivery of bone marrow cells (BMCs) to the heart has substantially improved cardiac function in most rodent models of myocardial infarction (MI), but clinical trials of BMC therapy have led to only modest improvements. Rodent models typically involve intramyocardial injection of BMCs from distinct donor individuals who are healthy. In contrast, autologous BMCs from individuals after MI are used for clinical trials. Using BMCs from donor mice after MI, we discovered that recent MI impaired BMC therapeutic efficacy. MI led to myocardial inflammation and an increased inflammatory state in the bone marrow, changing the BMC composition and reducing their efficacy. Injection of a general anti-inflammatory drug or a specific interleukin-1 inhibitor to donor mice after MI prevented this impairment. Our findings offer an explanation of why human trials have not matched the success of rodent experiments and suggest potential strategies to improve the success of clinical autologous BMC therapy. 10.1126/scitranslmed.3002814
    In situ bone tissue engineering via ultrasound-mediated gene delivery to endogenous progenitor cells in mini-pigs. Bez Maxim,Sheyn Dmitriy,Tawackoli Wafa,Avalos Pablo,Shapiro Galina,Giaconi Joseph C,Da Xiaoyu,David Shiran Ben,Gavrity Jayne,Awad Hani A,Bae Hyun W,Ley Eric J,Kremen Thomas J,Gazit Zulma,Ferrara Katherine W,Pelled Gadi,Gazit Dan Science translational medicine More than 2 million bone-grafting procedures are performed each year using autografts or allografts. However, both options carry disadvantages, and there remains a clear medical need for the development of new therapies for massive bone loss and fracture nonunions. We hypothesized that localized ultrasound-mediated, microbubble-enhanced therapeutic gene delivery to endogenous stem cells would induce efficient bone regeneration and fracture repair. To test this hypothesis, we surgically created a critical-sized bone fracture in the tibiae of Yucatán mini-pigs, a clinically relevant large animal model. A collagen scaffold was implanted in the fracture to facilitate recruitment of endogenous mesenchymal stem/progenitor cells (MSCs) into the fracture site. Two weeks later, transcutaneous ultrasound-mediated reporter gene delivery successfully transfected 40% of cells at the fracture site, and flow cytometry showed that 80% of the transfected cells expressed MSC markers. Human (-) plasmid DNA was delivered using ultrasound in the same animal model, leading to transient expression and secretion of BMP-6 localized to the fracture area. Micro-computed tomography and biomechanical analyses showed that ultrasound-mediated gene delivery led to complete radiographic and functional fracture healing in all animals 6 weeks after treatment, whereas nonunion was evident in control animals. Collectively, these findings demonstrate that ultrasound-mediated gene delivery to endogenous mesenchymal progenitor cells can effectively treat nonhealing bone fractures in large animals, thereby addressing a major orthopedic unmet need and offering new possibilities for clinical translation. 10.1126/scitranslmed.aal3128
    Augmented Reduced-Intensity Regimen Does Not Improve Postallogeneic Transplant Outcomes in Acute Myeloid Leukemia. Craddock Charles,Jackson Aimee,Loke Justin,Siddique Shamyla,Hodgkinson Andrea,Mason John,Andrew Georgia,Nagra Sandeep,Malladi Ram,Peniket Andrew,Gilleece Maria,Salim Rahuman,Tholouli Eleni,Potter Victoria,Crawley Charles,Wheatley Keith,Protheroe Rachel,Vyas Paresh,Hunter Ann,Parker Anne,Wilson Keith,Pavlu Jiri,Byrne Jenny,Dillon Richard,Khan Naeem,McCarthy Nicholas,Freeman Sylvie D Journal of clinical oncology : official journal of the American Society of Clinical Oncology PURPOSE:Reduced-intensity conditioning (RIC) regimens have extended the curative potential of allogeneic stem-cell transplantation to older adults with high-risk acute myeloid leukemia (AML) and myelodysplasia (MDS) but are associated with a high risk of disease relapse. Strategies to reduce recurrence are urgently required. Registry data have demonstrated improved outcomes using a sequential transplant regimen, fludarabine/amsacrine/cytarabine-busulphan (FLAMSA-Bu), but the impact of this intensified conditioning regimen has not been studied in randomized trials. PATIENTS AND METHODS:Two hundred forty-four patients (median age, 59 years) with high-risk AML (n = 164) or MDS (n = 80) were randomly assigned 1:1 to a fludarabine-based RIC regimen or FLAMSA-Bu. Pretransplant measurable residual disease (MRD) was monitored by flow cytometry (MFC-MRD) and correlated with outcome. RESULTS:There was no difference in 2-year overall survival (hazard ratio 1.05 [85% CI, 0.80 to 1.38] = .81) or cumulative incidence of relapse (CIR) (hazard ratio 0.94 [95%CI, 0.60 to 1.46] = .81) between the control and FLAMSA-Bu arms. Detectable pretransplant MFC-MRD was associated with an increased CIR (2-year CIR 41.0% 20.0%, = .01) in the overall trial cohort with a comparable prognostic impact when measured by an unsupervised analysis approach. There was no evidence of interaction between MRD status and conditioning regimen intensity for relapse or survival. Acquisition of full donor T-cell chimerism at 3 months abrogated the adverse impact of pretransplant MRD on CIR and overall survival. CONCLUSION:The intensified RIC conditioning regimen, FLAMSA-Bu, did not improve outcomes in adults transplanted for high-risk AML or MDS regardless of pretransplant MRD status. Our data instead support the exploration of interventions with the ability to accelerate acquisition of full donor T-cell chimerism as a tractable strategy to improve outcomes in patients allografted for AML. 10.1200/JCO.20.02308
    SIRPA is a specific cell-surface marker for isolating cardiomyocytes derived from human pluripotent stem cells. Dubois Nicole C,Craft April M,Sharma Parveen,Elliott David A,Stanley Edouard G,Elefanty Andrew G,Gramolini Anthony,Keller Gordon Nature biotechnology To identify cell-surface markers specific to human cardiomyocytes, we screened cardiovascular cell populations derived from human embryonic stem cells (hESCs) against a panel of 370 known CD antibodies. This screen identified the signal-regulatory protein alpha (SIRPA) as a marker expressed specifically on cardiomyocytes derived from hESCs and human induced pluripotent stem cells (hiPSCs), and PECAM, THY1, PDGFRB and ITGA1 as markers of the nonmyocyte population. Cell sorting with an antibody against SIRPA allowed for the enrichment of cardiac precursors and cardiomyocytes from hESC/hiPSC differentiation cultures, yielding populations of up to 98% cardiac troponin T-positive cells. When plated in culture, SIRPA-positive cells were contracting and could be maintained over extended periods of time. These findings provide a simple method for isolating populations of cardiomyocytes from human pluripotent stem cell cultures, and thereby establish a readily adaptable technology for generating large numbers of enriched cardiomyocytes for therapeutic applications. 10.1038/nbt.2005
    Innate type 2 immunity is associated with eosinophilic pleural effusion in primary spontaneous pneumothorax. Kwon Bo-In,Hong Seokchan,Shin Kihyuk,Choi Eun-Hye,Hwang Jung-Joo,Lee Seung-Hyo American journal of respiratory and critical care medicine RATIONALE:Eosinophilic pleural effusion (EPE) is characterized by greater than 10% eosinophilia and is frequently associated with air and/or blood in the pleural cavity. Primary spontaneous pneumothorax (PSP), defined as the spontaneous presence of air in the pleural space, is one of the most common causes of EPE. Recent studies have shown that type 2 immune responses play important roles in eosinophilic airway inflammation resulting in pleural pathology. OBJECTIVES:To determine the predominant immune responses associated with PSP in humans, and to examine whether IL-33, thymic stromal lymphopoietin (TSLP), or type 2 innate lymphoid cell (ILC2)-mediated immune responses are associated factors. METHODS:Eosinophil-associated cytokines were measured in the pleural fluid of patients with PSP and control subjects. Th2 cell and ILC2 responses in the pleural cavity and peripheral blood were also evaluated by in vitro restimulation and intracellular cytokine staining of T cells and ILC2s in patients with PSP (n = 62) and control subjects (n = 33). IL-33-mediated IL-5 production by ILC2s was also evaluated. MEASUREMENTS AND MAIN RESULTS:Significantly higher concentrations of IL-5 and eotaxin-3 were detected in the pleural fluid of patients with PSP, in addition to significantly higher concentrations of IL-33 and TSLP. Although IL-5 production was induced by IL-33 treatment of ILC2s, other Th2 cell-mediated immune responses were not detected. CONCLUSIONS:Our results indicate that innate immune responses characterized by the production of IL-33, TSLP, and IL-5 are associated with the development of EPE in PSP by an ILC2-dependent and Th2-independent mechanism. 10.1164/rccm.201302-0295OC
    Increased frequency of myeloid-derived suppressor cells during active tuberculosis and after recent mycobacterium tuberculosis infection suppresses T-cell function. du Plessis Nelita,Loebenberg Laurianne,Kriel Magdalena,von Groote-Bidlingmaier Florian,Ribechini Eliana,Loxton Andre G,van Helden Paul D,Lutz Manfred B,Walzl Gerhard American journal of respiratory and critical care medicine RATIONALE:Inadequacy of T-cell responses may result in the development of tuberculosis (TB). Myeloid-derived suppressor cells (MDSCs) have been described as suppressors of T-cell function in cancer biology and recently in several infectious diseases. OBJECTIVES:To explore the presence and role of MDSCs in TB. METHODS:We analyzed surface markers of MDSCs in peripheral blood and at the site of disease in TB cases and in patients with lung cancer, and in peripheral blood of asymptomatic tuberculin skin test-positive individuals with recent (household) or remote exposure to Mycobacterium tuberculosis (M.tb) and in uninfected healthy control subjects. To evaluate the suppressive capacity of MDSCs, cells of household contacts infected with M.tb and TB cases were isolated and cocultured with CD3(+) T cells. MEASUREMENTS AND MAIN RESULTS:Our results demonstrate an increased presence of MDSCs after recent M.tb infection and disease. We confirm their suppression of CD4(+) T-cell function, including reduced cytokine responses and inhibition of CD4(+) T-cell proliferation. Only MDSCs from TB cases reduced T-cell activation, altered T-cell trafficking, and suppressed CD8(+) T-cell functions. M.tb-expanded MDSCs were associated with significantly higher IL-1β, IL-6, IL-8, granulocyte colony-stimulating factor, and monocyte chemotactic protein-1, and reduced granulocyte-macrophage colony-stimulating factor and macrophage inflammatory protein-1 beta levels in coculture. CONCLUSIONS:These data reveal that innate MDSCs are induced not only during active TB at similar levels as found in cancer, but also in healthy individuals after recent exposure to M.tb. These cells diminish protective T-cell responses and may contribute to the inability of hosts to eradicate the infection and add to the subsequent development of TB disease. 10.1164/rccm.201302-0249OC
    Early T-cell precursor leukaemia: a subtype of very high-risk acute lymphoblastic leukaemia. Coustan-Smith Elaine,Mullighan Charles G,Onciu Mihaela,Behm Frederick G,Raimondi Susana C,Pei Deqing,Cheng Cheng,Su Xiaoping,Rubnitz Jeffrey E,Basso Giuseppe,Biondi Andrea,Pui Ching-Hon,Downing James R,Campana Dario The Lancet. Oncology BACKGROUND:About a fifth of children with acute T-lymphoblastic leukaemia (T-ALL) succumb to the disease, suggesting an unrecognised biological heterogeneity that might contribute to drug resistance. We postulated that T-ALL originating from early T-cell precursors (ETPs), a recently defined subset of thymocytes that retain stem-cell-like features, would respond poorly to lymphoid-cell-directed therapy. We studied leukaemic cells, collected at diagnosis, to identify cases with ETP features and determine their clinical outcome. METHODS:Leukaemic cells from 239 patients with T-ALL enrolled at St Jude Children's Research Hospital (n=139) and in the Italian national study Associazione Italiana Ematologia Oncologia Pediatrica (AIEOP) ALL-2000 (n=100) were assessed by gene-expression profiling, flow cytometry, and single nucleotide polymorphism array analysis. Probabilities of survival and treatment failure were calculated for subgroups considered to have ETP-ALL or typical T-ALL. FINDINGS:30 patients (12.6%) had leukaemic lymphoblasts with an ETP-related gene-expression signature or its associated distinctive immunophenotype (CD1a(-), CD8(-), CD5(weak) with stem-cell or myeloid markers). Cases of ETP-ALL showed increased genomic instability, in terms of number and size of gene lesions, compared with those with typical T-ALL. Patients with this form of leukaemia had high risk of remission failure or haematological relapse (72% [95% CI 40-100] at 10 years vs 10% [4-16] at 10 years for patients with typical T-ALL treated at St Jude Children's Research Hospital; and 57% [25-89] at 2 years vs 14% [6-22] at 2 years for patients treated in the AIEOP trial). INTERPRETATION:ETP-ALL is a distinct, previously unrecognised, pathobiological entity that confers a poor prognosis with use of standard intensive chemotherapy. Its early recognition, by use of the gene expression and immunophenotypic criteria outlined here, is essential for the development of an effective clinical management strategy. FUNDING:US National Cancer Institute, Cariplo Foundation, Citta della Speranza Foundation, Italian Association for Cancer Research (AIRC), Italian Ministry for University and Research, and American Lebanese Syrian Associated Charities (ALSAC). 10.1016/S1470-2045(08)70314-0
    Detection of DNA sequences in nuclei in suspension by in situ hybridization and dual beam flow cytometry. Trask B,van den Engh G,Landegent J,in de Wal N J,van der Ploeg M Science (New York, N.Y.) This report describes the fluorescence hybridization of DNA sequence probes to interphase nuclei in suspension and the quantification of bound probe by dual beam flow cytometry. Nuclear proteins were first cross-linked with dimethylsuberimidate to prevent disintegration of the nuclei during denaturation and hybridization. To demonstrate that in situ hybridization can be performed in suspension, stabilized mouse thymocyte nuclei were hybridized with a probe for mouse satellite DNA sequences. The DNA probes were labeled with 2-acetylaminofluorene. After hybridization, an indirect immunofluorescent labeling procedure was used to visualize the target sequences. With dual beam flow cytometry, both the amount of hybridized probe and the DNA content of individual nuclei were determined. Thus, the specificity of DNA hybridization can be combined with the speed and quantitative analysis provided by flow cytometry. 10.1126/science.2416058
    KLRG1+ natural killer cells exert a novel antifibrotic function in chronic hepatitis B. Wijaya Ratna S,Read Scott A,Schibeci Stephen,Eslam Mohammed,Azardaryany Mahmoud K,El-Khobar Korri,van der Poorten David,Lin Rita,Yuen Lawrence,Lam Vincent,George Jacob,Douglas Mark W,Ahlenstiel Golo Journal of hepatology BACKGROUND & AIMS:Natural killer (NK) cells are known to exert strong antiviral activity. Killer cell lectin-like receptor subfamily G member 1 (KLRG1) is expressed by terminally differentiated NK cells and KLRG1-expressing lymphocytes are known to expand following chronic viral infections. We aimed to elucidate the previously unknown role of KLRG1 in the pathogenesis of chronic hepatitis B (CHB). METHODS:KLRG1+ NK cells were taken from the blood and liver of healthy individuals and patients with CHB. The phenotype and function of these cells was assessed using flow cytometry and in vitro stimulation. RESULTS:Patients with CHB had a higher frequency of KLRG1+ NK cells compared to healthy controls (blood 13.4 vs. 2.3%, p <0.0001 and liver 23.4 vs. 2.6%, p <0.01). KLRG1+ NK cells were less responsive to K562 and cytokine stimulation, but demonstrated enhanced cytotoxicity (9.0 vs. 4.8%, p <0.05) and IFN-γ release (8.0 vs. 1.5%, p <0.05) via antibody dependent cellular cytotoxicity compared to their KLRG1- counterparts. KLRG1+ NK cells possessed a mature phenotype, demonstrating stronger cytolytic activity and IFN-γ secretion against hepatic stellate cells (HSCs) than KLRG1- NK cells. Moreover, KLRG1+ NK cells more effectively induced primary HSC apoptosis in a TRAIL-dependent manner. Increased KLRG1+ NK cell frequency in the liver and blood was associated with lower fibrosis stage (F0/F1) in patients with CHB. Finally, the expression of CD44, degranulation and IFN-γ production were all increased in KLRG1+ NK cells following stimulation with osteopontin, the CD44 ligand, suggesting that HSC-derived osteopontin may cause KLRG1+ NK cell activation. CONCLUSIONS:KLRG1+ NK cells likely play an antifibrotic role during the natural course of CHB infection. Harnessing this antifibrotic function may provide a novel therapeutic approach to treat liver fibrosis in patients with CHB. LAY SUMMARY:Individuals that are chronically infected with hepatitis B virus (HBV) possess an increased number of immune cells, called natural killer (NK) cells expressing the surface marker KLRG1 in the blood and liver. Here, we demonstrate that these specific NK cells are able to kill activated stellate cells in the liver. Because activated stellate cells contribute to liver scarring, i.e. fibrosis, and subsequent liver dysfunction in individuals with chronic HBV infection, KLRG1+ NK cells are a novel immune cell type that can limit liver scarring. 10.1016/j.jhep.2019.03.012
    miR-371-3 expression predicts neural differentiation propensity in human pluripotent stem cells. Kim Hyesoo,Lee Gabsang,Ganat Yosif,Papapetrou Eirini P,Lipchina Inna,Socci Nicholas D,Sadelain Michel,Studer Lorenz Cell stem cell The use of pluripotent stem cells in regenerative medicine and disease modeling is complicated by the variation in differentiation properties between lines. In this study, we characterized 13 human embryonic stem cell (hESC) and 26 human induced pluripotent stem cell (hiPSC) lines to identify markers that predict neural differentiation behavior. At a general level, markers previously known to distinguish mouse ESCs from epiblast stem cells (EPI-SCs) correlated with neural differentiation behavior. More specifically, quantitative analysis of miR-371-3 expression prospectively identified hESC and hiPSC lines with differential neurogenic differentiation propensity and in vivo dopamine neuron engraftment potential. Transient KLF4 transduction increased miR-371-3 expression and altered neurogenic behavior and pluripotency marker expression. Conversely, suppression of miR-371-3 expression in KLF4-transduced cells rescued neural differentiation propensity. miR-371-3 expression level therefore appears to have both a predictive and a functional role in determining human pluripotent stem cell neurogenic differentiation behavior. 10.1016/j.stem.2011.04.002
    A rapid, quantitative and inexpensive method for detecting apoptosis by flow cytometry in transiently transfected cells. Lamm G M,Steinlein P,Cotten M,Christofori G Nucleic acids research We describe a rapid and quantitative flow cytometric method for determining the apoptotic or anti-apoptotic potential of a gene in various cell types. A plasmid carrying green fluorescent protein (GFP) is co-transfected with an expression vector encoding the gene of interest. Subsequently cells are stained with propidium iodide and, utilising flow cytometry, transfected, GFP-expressing single cells are detected and apoptotic cells in this population are identified by their DNA content of <2 N. The method detects apoptosis as reliably as established methods using in situ nick-end labelling but is faster, easier and less expensive. 10.1093/nar/25.23.4855
    Inferring tumor progression from genomic heterogeneity. Navin Nicholas,Krasnitz Alexander,Rodgers Linda,Cook Kerry,Meth Jennifer,Kendall Jude,Riggs Michael,Eberling Yvonne,Troge Jennifer,Grubor Vladimir,Levy Dan,Lundin Pär,Månér Susanne,Zetterberg Anders,Hicks James,Wigler Michael Genome research Cancer progression in humans is difficult to infer because we do not routinely sample patients at multiple stages of their disease. However, heterogeneous breast tumors provide a unique opportunity to study human tumor progression because they still contain evidence of early and intermediate subpopulations in the form of the phylogenetic relationships. We have developed a method we call Sector-Ploidy-Profiling (SPP) to study the clonal composition of breast tumors. SPP involves macro-dissecting tumors, flow-sorting genomic subpopulations by DNA content, and profiling genomes using comparative genomic hybridization (CGH). Breast carcinomas display two classes of genomic structural variation: (1) monogenomic and (2) polygenomic. Monogenomic tumors appear to contain a single major clonal subpopulation with a highly stable chromosome structure. Polygenomic tumors contain multiple clonal tumor subpopulations, which may occupy the same sectors, or separate anatomic locations. In polygenomic tumors, we show that heterogeneity can be ascribed to a few clonal subpopulations, rather than a series of gradual intermediates. By comparing multiple subpopulations from different anatomic locations, we have inferred pathways of cancer progression and the organization of tumor growth. 10.1101/gr.099622.109
    A Reaction-Based Fluorescent Probe for Imaging of Formaldehyde in Living Cells. Roth Aaron,Li Hao,Anorma Chelsea,Chan Jefferson Journal of the American Chemical Society Formaldehyde (FA), in the 0.2-0.4 mM range, is produced and maintained endogenously via enzymatic pathways. At these levels, FA can promote cell proliferation as well as mediate memory formation. Once elevated, FA stress is known to induce cognitive impairments, memory loss, and neurodegeneration owing to its potent DNA and protein cross-linking mechanisms. Optical imaging is a powerful noninvasive approach used to study FA in living systems; however, biocompatible chemical probes for FA are currently lacking. Herein, we report the design, synthesis, and biological evaluation of Formaldehyde Probe 1 (FP1), a new fluorescent indicator based on the 2-aza-Cope sigmatropic rearrangement. The remarkable sensitivity, selectivity, and photostability of FP1 has enabled us to visualize FA in live HEK293TN and Neuroscreen-1 cells. We envision that FP1 will find widespread applications in the study of FA associated with normal and pathological processes. 10.1021/jacs.5b05339
    Measurable residual disease-guided treatment with azacitidine to prevent haematological relapse in patients with myelodysplastic syndrome and acute myeloid leukaemia (RELAZA2): an open-label, multicentre, phase 2 trial. Platzbecker Uwe,Middeke Jan Moritz,Sockel Katja,Herbst Regina,Wolf Dominik,Baldus Claudia D,Oelschlägel Uta,Mütherig Anke,Fransecky Lars,Noppeney Richard,Bug Gesine,Götze Katharina S,Krämer Alwin,Bochtler Tilmann,Stelljes Matthias,Groth Christoph,Schubert Antje,Mende Marika,Stölzel Friedrich,Borkmann Christine,Kubasch Anne Sophie,von Bonin Malte,Serve Hubert,Hänel Mathias,Dührsen Ulrich,Schetelig Johannes,Röllig Christoph,Kramer Michael,Ehninger Gerhard,Bornhäuser Martin,Thiede Christian The Lancet. Oncology BACKGROUND:Monitoring of measurable residual disease (MRD) in patients with advanced myelodysplastic syndromes (MDS) or acute myeloid leukaemia (AML) who achieve a morphological complete remission can predict haematological relapse. In this prospective study, we aimed to determine whether MRD-guided pre-emptive treatment with azacitidine could prevent relapse in these patients. METHODS:The relapse prevention with azacitidine (RELAZA2) study is an open-label, multicentre, phase 2 trial done at nine university health centres in Germany. Patients aged 18 years or older with advanced MDS or AML, who had achieved a complete remission after conventional chemotherapy or allogeneic haemopoietic stem-cell transplantation, were prospectively screened for MRD during 24 months from baseline by either quantitative PCR for mutant NPM1, leukaemia-specific fusion genes (DEK-NUP214, RUNX1-RUNX1T1, CBFb-MYH11), or analysis of donor-chimaerism in flow cytometry-sorted CD34-positive cells in patients who received allogeneic haemopoietic stem-cell transplantation. MRD-positive patients in confirmed complete remission received azacitidine 75 mg/m per day subcutaneously on days 1-7 of a 29-day cycle for 24 cycles. After six cycles, MRD status was reassessed and patients with major responses (MRD negativity) were eligible for a treatment de-escalation. The primary endpoint was the proportion of patients who were relapse-free and alive 6 months after the start of pre-emptive treatment. Analyses were done per protocol. This trial is registered with ClincialTrials.gov, number NCT01462578, and finished recruitment on Aug 21, 2018. FINDINGS:Between Oct 10, 2011, and Aug 20, 2015, we screened 198 patients with advanced MDS (n=26) or AML (n=172), of whom 60 (30%) developed MRD during the 24-month screening period and 53 (88%) were eligible to start study treatment. 6 months after initiation of azacitidine, 31 (58%, 95% CI 44-72) of 53 patients were relapse-free and alive (p<0·0001; one-sided binomial test for null hypothesis p≤0·3). With a median follow-up of 13 months (IQR 8·5-22·8) after the start of MRD-guided treatment, relapse-free survival at 12 months was 46% (95% CI 32-59) in the 53 patients who were MRD-positive and received azacitidine. In MRD-negative patients, 12-month relapse-free survival was 88% (95% CI 82-94; hazard ratio 6·6 [95% CI 3·7-11·8], p<0·0001). The most common (grade 3-4) adverse event was neutropenia, occurring in 45 (85%) of 53 patients. One patient with neutropenia died because of an infection considered possibly related to study treatment. INTERPRETATION:Pre-emptive therapy with azacitidine can prevent or substantially delay haematological relapse in MRD-positive patients with MDS or AML who are at high risk of relapse. Our study also suggests that continuous MRD negativity during regular MRD monitoring might be prognostic for patient outcomes. FUNDING:Celgene Pharma, José Carreras Leukaemia Foundation, National Center for Tumor Diseases (NCT), and German Cancer Consortium (DKTK) Foundation. 10.1016/S1470-2045(18)30580-1
    Replication fork integrity and intra-S phase checkpoint suppress gene amplification. Kondratova Anna,Watanabe Takaaki,Marotta Michael,Cannon Matthew,Segall Anca M,Serre David,Tanaka Hisashi Nucleic acids research Gene amplification is a phenotype-causing form of chromosome instability and is initiated by DNA double-strand breaks (DSBs). Cells with mutant p53 lose G1/S checkpoint and are permissive to gene amplification. In this study we show that mammalian cells become proficient for spontaneous gene amplification when the function of the DSB repair protein complex MRN (Mre11/Rad50/Nbs1) is impaired. Cells with impaired MRN complex experienced severe replication stress and gained substrates for gene amplification during replication, as evidenced by the increase of replication-associated single-stranded breaks that were converted to DSBs most likely through replication fork reversal. Impaired MRN complex directly compromised ATM/ATR-mediated checkpoints and allowed cells to progress through cell cycle in the presence of DSBs. Such compromised intra-S phase checkpoints promoted gene amplification independently from mutant p53. Finally, cells adapted to endogenous replication stress by globally suppressing genes for DNA replication and cell cycle progression. Our results indicate that the MRN complex suppresses gene amplification by stabilizing replication forks and by securing DNA damage response to replication-associated DSBs. 10.1093/nar/gkv084
    Single molecule analysis of Trypanosoma brucei DNA replication dynamics. Calderano Simone Guedes,Drosopoulos William C,Quaresma Marina Mônaco,Marques Catarina A,Kosiyatrakul Settapong,McCulloch Richard,Schildkraut Carl L,Elias Maria Carolina Nucleic acids research Eukaryotic genome duplication relies on origins of replication, distributed over multiple chromosomes, to initiate DNA replication. A recent genome-wide analysis of Trypanosoma brucei, the etiological agent of sleeping sickness, localized its replication origins to the boundaries of multigenic transcription units. To better understand genomic replication in this organism, we examined replication by single molecule analysis of replicated DNA. We determined the average speed of replication forks of procyclic and bloodstream form cells and we found that T. brucei DNA replication rate is similar to rates seen in other eukaryotes. We also analyzed the replication dynamics of a central region of chromosome 1 in procyclic forms. We present evidence for replication terminating within the central part of the chromosome and thus emanating from both sides, suggesting a previously unmapped origin toward the 5' extremity of chromosome 1. Also, termination is not at a fixed location in chromosome 1, but is rather variable. Importantly, we found a replication origin located near an ORC1/CDC6 binding site that is detected after replicative stress induced by hydroxyurea treatment, suggesting it may be a dormant origin activated in response to replicative stress. Collectively, our findings support the existence of more replication origins in T. brucei than previously appreciated. 10.1093/nar/gku1389
    Dynamic Escherichia coli SeqA complexes organize the newly replicated DNA at a considerable distance from the replisome. Helgesen Emily,Fossum-Raunehaug Solveig,Sætre Frank,Schink Kay Oliver,Skarstad Kirsten Nucleic acids research The Escherichia coli SeqA protein binds to newly replicated, hemimethylated DNA behind replication forks and forms structures consisting of several hundred SeqA molecules bound to about 100 kb of DNA. It has been suggested that SeqA structures either direct the new sister DNA molecules away from each other or constitute a spacer that keeps the sisters together. We have developed an image analysis script that automatically measures the distance between neighboring foci in cells. Using this tool as well as direct stochastic optical reconstruction microscopy (dSTORM) we find that in cells with fluorescently tagged SeqA and replisome the sister SeqA structures were situated close together (less than about 30 nm apart) and relatively far from the replisome (on average 200-300 nm). The results support the idea that newly replicated sister molecules are kept together behind the fork and suggest the existence of a stretch of DNA between the replisome and SeqA which enjoys added stabilization. This could be important in facilitating DNA transactions such as recombination, mismatch repair and topoisomerase activity. In slowly growing cells without ongoing replication forks the SeqA protein was found to reside at the fully methylated origins prior to initiation of replication. 10.1093/nar/gkv146
    IgE⁺ memory B cells and plasma cells generated through a germinal-center pathway. Talay Oezcan,Yan Donghong,Brightbill Hans D,Straney Elizabeth E M,Zhou Meijuan,Ladi Ena,Lee Wyne P,Egen Jackson G,Austin Cary D,Xu Min,Wu Lawren C Nature immunology Immunoglobulin E (IgE) antibodies are pathogenic in asthma and allergic diseases, but the in vivo biology of IgE-producing (IgE(+)) cells is poorly understood. A model of the differentiation of IgE(+) B cells proposes that IgE(+) cells develop through a germinal-center IgG1(+) intermediate and that IgE memory resides in the compartment of IgG1(+) memory B cells. Here we have used a reporter mouse expressing green fluorescent protein associated with membrane IgE transcripts (IgE-GFP) to assess in vivo IgE responses. In contrast to the IgG1-centered model of IgE switching and memory, we found that IgE(+) cells developed through a germinal-center IgE(+) intermediate to form IgE(+) memory B cells and plasma cells. Our studies delineate a new model for the in vivo biology of IgE switching and memory. 10.1038/ni.2256
    Diabetes impairs hematopoietic stem cell mobilization by altering niche function. Ferraro Francesca,Lymperi Stefania,Méndez-Ferrer Simón,Saez Borja,Spencer Joel A,Yeap Beow Y,Masselli Elena,Graiani Gallia,Prezioso Lucia,Rizzini Elisa Lodi,Mangoni Marcellina,Rizzoli Vittorio,Sykes Stephen M,Lin Charles P,Frenette Paul S,Quaini Federico,Scadden David T Science translational medicine Success with transplantation of autologous hematopoietic stem and progenitor cells (HSPCs) in patients depends on adequate collection of these cells after mobilization from the bone marrow niche by the cytokine granulocyte colony-stimulating factor (G-CSF). However, some patients fail to achieve sufficient HSPC mobilization. Retrospective analysis of bone marrow transplant patient records revealed that diabetes correlated with poor mobilization of CD34+ HSPCs. In mouse models of type 1 and type 2 diabetes (streptozotocin-induced and db/db mice, respectively), we found impaired egress of murine HSPCs from the bone marrow after G-CSF treatment. Furthermore, HSPCs were aberrantly localized in the marrow niche of the diabetic mice, and abnormalities in the number and function of sympathetic nerve termini were associated with this mislocalization. Aberrant responses to β-adrenergic stimulation of the bone marrow included an inability of marrow mesenchymal stem cells expressing the marker nestin to down-modulate the chemokine CXCL12 in response to G-CSF treatment (mesenchymal stem cells are reported to be critical for HSPC mobilization). The HSPC mobilization defect was rescued by direct pharmacological inhibition of the interaction of CXCL12 with its receptor CXCR4 using the drug AMD3100. These data suggest that there are diabetes-induced changes in bone marrow physiology and microanatomy and point to a potential intervention to overcome poor HSPC mobilization in diabetic patients. 10.1126/scitranslmed.3002191
    Interferon-Gamma-Producing CD8 Tissue Resident Memory T Cells Are a Targetable Hallmark of Immune Checkpoint Inhibitor-Colitis. Sasson Sarah C,Slevin Stephanie M,Cheung Vincent T F,Nassiri Isar,Olsson-Brown Anna,Fryer Eve,Ferreira Ricardo C,Trzupek Dominik,Gupta Tarun,Al-Hillawi Lulia,Issaias Mari-Lenna,Easton Alistair,Campo Leticia,FitzPatrick Michael E B,Adams Joss,Chitnis Meenali,Protheroe Andrew,Tuthill Mark,Coupe Nicholas,Simmons Alison,Payne Miranda,Middleton Mark R,Travis Simon P L, ,Fairfax Benjamin P,Klenerman Paul,Brain Oliver Gastroenterology BACKGROUND & AIMS:The pathogenesis of immune checkpoint inhibitor (ICI)-colitis remains incompletely understood. We sought to identify key cellular drivers of ICI-colitis and their similarities to idiopathic ulcerative colitis, and to determine potential novel therapeutic targets. METHODS:We used a cross-sectional approach to study patients with ICI-colitis, those receiving ICI without the development of colitis, idiopathic ulcerative colitis, and healthy controls. A subset of patients with ICI-colitis were studied longitudinally. We applied a range of methods, including multiparameter and spectral flow cytometry, spectral immunofluorescence microscopy, targeted gene panels, and bulk and single-cell RNA sequencing. RESULTS:We demonstrate CD8 tissue resident memory T (T) cells are the dominant activated T cell subset in ICI-colitis. The pattern of gastrointestinal immunopathology is distinct from ulcerative colitis at both the immune and epithelial-signaling levels. CD8 T cell activation correlates with clinical and endoscopic ICI-colitis severity. Single-cell RNA sequencing analysis confirms activated CD8 T cells express high levels of transcripts for checkpoint inhibitors and interferon-gamma in ICI-colitis. We demonstrate similar findings in both anti-CTLA-4/PD-1 combination therapy and in anti-PD-1 inhibitor-associated colitis. On the basis of our data, we successfully targeted this pathway in a patient with refractory ICI-colitis, using the JAK inhibitor tofacitinib. CONCLUSIONS:Interferon gamma-producing CD8 T cells are a pathological hallmark of ICI-colitis and a novel target for therapy. 10.1053/j.gastro.2021.06.025
    Itk-mediated integration of T cell receptor and cytokine signaling regulates the balance between Th17 and regulatory T cells. Gomez-Rodriguez Julio,Wohlfert Elizabeth A,Handon Robin,Meylan Françoise,Wu Julie Z,Anderson Stacie M,Kirby Martha R,Belkaid Yasmine,Schwartzberg Pamela L The Journal of experimental medicine A proper balance between Th17 and T regulatory cells (Treg cells) is critical for generating protective immune responses while minimizing autoimmunity. We show that the Tec family kinase Itk (IL2-inducible T cell kinase), a component of T cell receptor (TCR) signaling pathways, influences this balance by regulating cross talk between TCR and cytokine signaling. Under both Th17 and Treg cell differentiation conditions, Itk(-/-) CD4(+) T cells develop higher percentages of functional FoxP3(+) cells, associated with increased sensitivity to IL-2. Itk(-/-) CD4(+) T cells also preferentially develop into Treg cells in vivo. We find that Itk-deficient T cells exhibit reduced TCR-induced phosphorylation of mammalian target of rapamycin (mTOR) targets, accompanied by downstream metabolic alterations. Surprisingly, Itk(-/-) cells also exhibit reduced IL-2-induced mTOR activation, despite increased STAT5 phosphorylation. We demonstrate that in wild-type CD4(+) T cells, TCR stimulation leads to a dose-dependent repression of Pten. However, at low TCR stimulation or in the absence of Itk, Pten is not effectively repressed, thereby uncoupling STAT5 phosphorylation and phosphoinositide-3-kinase (PI3K) pathways. Moreover, Itk-deficient CD4(+) T cells show impaired TCR-mediated induction of Myc and miR-19b, known repressors of Pten. Our results demonstrate that Itk helps orchestrate positive feedback loops integrating multiple T cell signaling pathways, suggesting Itk as a potential target for altering the balance between Th17 and Treg cells. 10.1084/jem.20131459
    Follicular regulatory T cells control humoral autoimmunity via NFAT2-regulated CXCR5 expression. Vaeth Martin,Müller Gerd,Stauss Dennis,Dietz Lena,Klein-Hessling Stefan,Serfling Edgar,Lipp Martin,Berberich Ingolf,Berberich-Siebelt Friederike The Journal of experimental medicine Maturation of high-affinity B lymphocytes is precisely controlled during the germinal center reaction. This is dependent on CD4(+)CXCR5(+) follicular helper T cells (TFH) and inhibited by CD4(+)CXCR5(+)Foxp3(+) follicular regulatory T cells (TFR). Because NFAT2 was found to be highly expressed and activated in follicular T cells, we addressed its function herein. Unexpectedly, ablation of NFAT2 in T cells caused an augmented GC reaction upon immunization. Consistently, however, TFR cells were clearly reduced in the follicular T cell population due to impaired homing to B cell follicles. This was TFR-intrinsic because only in these cells NFAT2 was essential to up-regulate CXCR5. The physiological relevance for humoral (auto-)immunity was corroborated by exacerbated lupuslike disease in the presence of NFAT2-deficient TFR cells. 10.1084/jem.20130604
    The protein tyrosine phosphatase PTP1B is a negative regulator of CD40 and BAFF-R signaling and controls B cell autoimmunity. Medgyesi David,Hobeika Elias,Biesen Robert,Kollert Florian,Taddeo Adriano,Voll Reinhard E,Hiepe Falk,Reth Michael The Journal of experimental medicine Tyrosine phosphorylation of signaling molecules that mediate B cell activation in response to various stimuli is tightly regulated by protein tyrosine phosphatases (PTPs). PTP1B is a ubiquitously expressed tyrosine phosphatase with well-characterized functions in metabolic signaling pathways. We show here that PTP1B negatively regulates CD40, B cell activating factor receptor (BAFF-R), and TLR4 signaling in B cells. Specifically, PTP1B counteracts p38 mitogen-activated protein kinase (MAPK) activation by directly dephosphorylating Tyr(182) of this kinase. Mice with a B cell-specific PTP1B deficiency show increased T cell-dependent immune responses and elevated total serum IgG. Furthermore, aged animals develop systemic autoimmunity with elevated serum anti-dsDNA, spontaneous germinal centers in the spleen, and deposition of IgG immune complexes and C3 in the kidney. In a clinical setting, we observed that B cells of rheumatoid arthritis patients have significantly reduced PTP1B expression. Our data suggest that PTP1B plays an important role in the control of B cell activation and the maintenance of immunological tolerance. 10.1084/jem.20131196
    Stromal control of cystine metabolism promotes cancer cell survival in chronic lymphocytic leukaemia. Zhang Wan,Trachootham Dunyaporn,Liu Jinyun,Chen Gang,Pelicano Helene,Garcia-Prieto Celia,Lu Weiqin,Burger Jan A,Croce Carlo M,Plunkett William,Keating Michael J,Huang Peng Nature cell biology Tissue stromal cells interact with leukaemia cells and profoundly affect their viability and drug sensitivity. Here we show a biochemical mechanism by which bone marrow stromal cells modulate the redox status of chronic lymphocytic leukaemia (CLL) cells and promote cellular survival and drug resistance. Primary CLL cells from patients exhibit a limited ability to transport cystine for glutathione (GSH) synthesis owing to a low expression level of Xc-transporter. In contrast, bone marrow stromal cells effectively import cystine and convert it to cysteine, which is then released into the microenvironment for uptake by CLL cells to promote GSH synthesis. The elevated level of GSH enhances leukaemia cell survival and protects them from drug-induced cytotoxicity. Furthermore, disabling this protective mechanism significantly sensitizes CLL cells to drug treatment in the stromal environment. This stromal-leukaemia interaction is critical for CLL cell survival and represents a key biochemical pathway for effectively targeting leukaemia cells to overcome drug resistance in vivo. 10.1038/ncb2432
    The earliest thymic T cell progenitors sustain B cell and myeloid lineage potential. Luc Sidinh,Luis Tiago C,Boukarabila Hanane,Macaulay Iain C,Buza-Vidas Natalija,Bouriez-Jones Tiphaine,Lutteropp Michael,Woll Petter S,Loughran Stephen J,Mead Adam J,Hultquist Anne,Brown John,Mizukami Takuo,Matsuoka Sahoko,Ferry Helen,Anderson Kristina,Duarte Sara,Atkinson Deborah,Soneji Shamit,Domanski Aniela,Farley Alison,Sanjuan-Pla Alejandra,Carella Cintia,Patient Roger,de Bruijn Marella,Enver Tariq,Nerlov Claus,Blackburn Clare,Godin Isabelle,Jacobsen Sten Eirik W Nature immunology The stepwise commitment from hematopoietic stem cells in the bone marrow to T lymphocyte-restricted progenitors in the thymus represents a paradigm for understanding the requirement for distinct extrinsic cues during different stages of lineage restriction from multipotent to lineage-restricted progenitors. However, the commitment stage at which progenitors migrate from the bone marrow to the thymus remains unclear. Here we provide functional and molecular evidence at the single-cell level that the earliest progenitors in the neonatal thymus had combined granulocyte-monocyte, T lymphocyte and B lymphocyte lineage potential but not megakaryocyte-erythroid lineage potential. These potentials were identical to those of candidate thymus-seeding progenitors in the bone marrow, which were closely related at the molecular level. Our findings establish the distinct lineage-restriction stage at which the T cell lineage-commitment process transits from the bone marrow to the remote thymus. 10.1038/ni.2255
    The kinase Btk negatively regulates the production of reactive oxygen species and stimulation-induced apoptosis in human neutrophils. Honda Fumiko,Kano Hirotsugu,Kanegane Hirokazu,Nonoyama Shigeaki,Kim Eun-Sung,Lee Sang-Kyou,Takagi Masatoshi,Mizutani Shuki,Morio Tomohiro Nature immunology The function of the kinase Btk in neutrophil activation is largely unexplored. Here we found that Btk-deficient neutrophils had more production of reactive oxygen species (ROS) after engagement of Toll-like receptors (TLRs) or receptors for tumor-necrosis factor (TNF), which was associated with more apoptosis and was reversed by transduction of recombinant Btk. Btk-deficient neutrophils in the resting state showed hyperphosphorylation and activation of phosphatidylinositol-3-OH kinase (PI(3)K) and protein tyrosine kinases (PTKs) and were in a 'primed' state with plasma membrane-associated GTPase Rac2. In the absence of Btk, the adaptor Mal was associated with PI(3)K and PTKs at the plasma membrane, whereas in control resting neutrophils, Btk interacted with and confined Mal in the cytoplasm. Our data identify Btk as a critical gatekeeper of neutrophil responses. 10.1038/ni.2234
    Early window of diabetes determinism in NOD mice, dependent on the complement receptor CRIg, identified by noninvasive imaging. Fu Wenxian,Wojtkiewicz Gregory,Weissleder Ralph,Benoist Christophe,Mathis Diane Nature immunology All juvenile mice of the nonobese diabetic (NOD) strain develop insulitis, but there is considerable variation in their progression to diabetes. Here we used a strategy based on magnetic resonance imaging (MRI) of magnetic nanoparticles to noninvasively visualize local effects of pancreatic-islet inflammation to predict the onset of diabetes in NOD mice. MRI signals acquired during a narrow early time window allowed us to sort mice into groups that would progress to clinical disease or not and to estimate the time to diabetes development. We exploited this approach to identify previously unknown molecular and cellular elements correlated with disease protection, including the complement receptor of the immunoglobulin superfamily (CRIg), which marked a subset of macrophages associated with diabetes resistance. Administration of a fusion of CRIg and the Fc portion of immunoglobulin resulted in lower MRI signals and diabetes incidence. In addition to identifying regulators of disease progression, we show here that diabetes is set at an early age in NOD mice. 10.1038/ni.2233
    Prediction of recurrence and progression in primary superficial bladder cancer with DNA flow cytometry. Saracino G A,Ditonno P,Disabato G,Traficante A,Battaglia M,Lucivero G,Selvaggi F P European urology Intravesical chemotherapy has been well established as an effective therapy for recurrent superficial bladder tumors. We investigated the role of flow cytometry as a predictor of tumor recurrence/progression after intravesical chemotherapy. Flow cytometric analysis of nuclear DNA ploidy pattern was performed on 'cold cup' biopsy samples of 52 patients with primary superficial bladder cancer. Cell suspensions, retrieved after mechanical fragmentation, were stained with propidium iodide and examined on FACScan flow cytometer. Clinical follow-up ranged from 3 to 57 months with a median of 20 months. Of the 52 patients, 24 aneuploid and 28 diploid tumors were observed. The degree of ploidy in relation to histological grade showed an increasing frequency of aneuploid pattern in grades 2 and 3 but with no statistical significance. 17.8% of diploid tumors versus 54.1% of aneuploid tumors recurred (p less than 0.05). 12.5% of the aneuploid tumors progressed. No progression among diploid tumors was observed. Of the 52 patients examined, 35 (16 aneuploid and 19 diploid) were treated, after TUR, with intravesical prophylactic therapy. Epirubicin in 24, mitomycin C in 4 and recombinant interferon alpha 2a in 7 were used. 50% of aneuploid tumors versus 10.5% of diploid tumors recurred (p less than 0.05). Strong predictors of response to intravesical prophylaxis of recurrence were G1 grade and diploid DNA content.
    High-dimensional single-cell analysis predicts response to anti-PD-1 immunotherapy. Nature medicine Immune-checkpoint blockade has revolutionized cancer therapy. In particular, inhibition of programmed cell death protein 1 (PD-1) has been found to be effective for the treatment of metastatic melanoma and other cancers. Despite a dramatic increase in progression-free survival, a large proportion of patients do not show durable responses. Therefore, predictive biomarkers of a clinical response are urgently needed. Here we used high-dimensional single-cell mass cytometry and a bioinformatics pipeline for the in-depth characterization of the immune cell subsets in the peripheral blood of patients with stage IV melanoma before and after 12 weeks of anti-PD-1 immunotherapy. During therapy, we observed a clear response to immunotherapy in the T cell compartment. However, before commencing therapy, a strong predictor of progression-free and overall survival in response to anti-PD-1 immunotherapy was the frequency of CD14CD16HLA-DR monocytes. We confirmed this by conventional flow cytometry in an independent, blinded validation cohort, and we propose that the frequency of monocytes in PBMCs may serve in clinical decision support. 10.1038/nm.4466
    T-bet and Eomes instruct the development of two distinct natural killer cell lineages in the liver and in the bone marrow. Daussy Cécile,Faure Fabrice,Mayol Katia,Viel Sébastien,Gasteiger Georg,Charrier Emily,Bienvenu Jacques,Henry Thomas,Debien Emilie,Hasan Uzma A,Marvel Jacqueline,Yoh Keigyou,Takahashi Satoru,Prinz Immo,de Bernard Simon,Buffat Laurent,Walzer Thierry The Journal of experimental medicine Trail(+)DX5(-)Eomes(-) natural killer (NK) cells arise in the mouse fetal liver and persist in the adult liver. Their relationships with Trail(-)DX5(+) NK cells remain controversial. We generated a novel Eomes-GFP reporter murine model to address this question. We found that Eomes(-) NK cells are not precursors of classical Eomes(+) NK cells but rather constitute a distinct lineage of innate lymphoid cells. Eomes(-) NK cells are strictly dependent on both T-bet and IL-15, similarly to NKT cells. We observed that, in the liver, expression of T-bet in progenitors represses Eomes expression and the development of Eomes(+) NK cells. Reciprocally, the bone marrow (BM) microenvironment restricts T-bet expression in developing NK cells. Ectopic expression of T-bet forces the development of Eomes(-) NK cells, demonstrating that repression of T-bet is essential for the development of Eomes(+) NK cells. Gene profile analyses show that Eomes(-) NK cells share part of their transcriptional program with NKT cells, including genes involved in liver homing and NK cell receptors. Moreover, Eomes(-) NK cells produce a broad range of cytokines, including IL-2 and TNF in vitro and in vivo, during immune responses against vaccinia virus. Thus, mutually exclusive expression of T-bet and Eomes drives the development of different NK cell lineages with complementary functions. 10.1084/jem.20131560
    Transgenic kallikrein 5 mice reproduce major cutaneous and systemic hallmarks of Netherton syndrome. Furio Laetitia,de Veer Simon,Jaillet Madeleine,Briot Anais,Robin Aurelie,Deraison Celine,Hovnanian Alain The Journal of experimental medicine Netherton syndrome (NS) is a severe genetic skin disease in which absence of a key protease inhibitor causes congenital exfoliative erythroderma, eczematous-like lesions, and atopic manifestations. Several proteases are overactive in NS, including kallikrein-related peptidase (KLK) 5, KLK7, and elastase-2 (ELA2), which are suggested to be part of a proteolytic cascade initiated by KLK5. To address the role of KLK5 in NS, we have generated a new transgenic murine model expressing human KLK5 in the granular layer of the epidermis (Tg-KLK5). Transgene expression resulted in increased proteolytic activity attributable to KLK5 and its downstream targets KLK7, KLK14, and ELA2. Tg-KLK5 mice developed an exfoliative erythroderma with scaling, growth delay, and hair abnormalities. The skin barrier was defective and the stratum corneum was detached through desmosomal cleavage. Importantly, Tg-KLK5 mice displayed cutaneous and systemic hallmarks of severe inflammation and allergy with pruritus. The skin showed enhanced expression of inflammatory cytokines and chemokines, infiltration of immune cells, and markers of Th2/Th17/Th22 T cell responses. Moreover, serum IgE and Tslp levels were elevated. Our study identifies KLK5 as an important contributor to the NS proteolytic cascade and provides a new and viable model for the evaluation of future targeted therapies for NS or related diseases such as atopic dermatitis. 10.1084/jem.20131797
    Impaired IL-10-dependent induction of tolerogenic dendritic cells by CD4+CD25hiCD127lo/- natural regulatory T cells in human allergic asthma. Nguyen Khoa D,Vanichsarn Christopher,Nadeau Kari C American journal of respiratory and critical care medicine RATIONALE:Tolerogenic dendritic cells and natural regulatory T cells have been implicated in the process of infectious tolerance in human allergic asthma. However, the significance of the influence of natural regulatory T cells on tolerogenic dendritic cells in the disease has not been investigated. OBJECTIVES:We aimed to characterize the mechanism of induction of the tolerogenic phenotype in circulating blood dendritic cells by allergic asthmatic natural regulatory T cells. METHODS:The study was performed in a cohort of 21 subjects with allergic asthma, 21 healthy control subjects, and 21 subjects with nonallergic asthma. We cultured blood dendritic cells with natural regulatory T cells to study the induction of tolerogenic dendritic cells. Flow cytometry and proliferation assays were employed to analyze phenotype and function of dendritic cells as well as IL-10 production from natural regulatory T cells. MEASUREMENTS AND MAIN RESULTS:Dendritic cells cultured with natural regulatory T cells up-regulated IL-10, down-regulated costimulatory molecules, and stimulated the proliferation of CD4(+)CD25(-) effector T cells less potently. Allergic asthmatic natural regulatory T cells were significantly less efficient in inducing this tolerogenic phenotype of dendritic cells compared with healthy control and nonallergic asthmatic counterparts. Furthermore, this defective function of natural regulatory T cells was associated with their decreased IL-10 expression, disease severity, and could be reversed by oral corticosteroid therapy. CONCLUSIONS:These results provided the first evidences of impaired induction of tolerogenic dendritic cells mediated by natural regulatory T cells in human allergic asthma. 10.1164/rccm.200905-0761OC
    Anti-TNFα agents curb platelet activation in patients with rheumatoid arthritis. Manfredi Angelo A,Baldini Mattia,Camera Marina,Baldissera Elena,Brambilla Marta,Peretti Giuseppe,Maseri Attilio,Rovere-Querini Patrizia,Tremoli Elena,Sabbadini Maria Grazia,Maugeri Norma Annals of the rheumatic diseases BACKGROUND:Cardiovascular disease is important in rheumatoid arthritis (RA). Tissue factor (TF) is expressed upon platelet activation and initiates coagulation. Anti-tumour necrosis factor-α (TNFα) agents seem to decrease RA-associated cardiovascular events. We investigated whether (1) TNFα activates human platelets and (2) TNFα pharmacological blockade modulates the platelet-leucocyte reciprocal activation in RA. DESIGN:The expression of platelet TNFα receptors has been assessed by flow cytometry and immunogold electron microscopy. Platelet and leucocyte activation has been assessed also in the presence of antibodies against the TNFα receptors 1 and 2 and of infliximab. TF expression, binding to fibrinogen and phosphatidylserine exposure, has been assessed by flow cytometry, TF activity by coagulation time and by endogenous thrombin generation. Markers of platelet and leucocyte activation have been assessed in 161 subjects: 42 patients with RA, 12 with osteoarthritis, 37 age-matched and sex-matched patients with chronic stable angina and 70 age-matched and sex-matched healthy subjects. RESULTS:TNFα elicited the platelet activation and the expression of TF, which in turn prompted thrombin generation and clot formation. Inhibition of the TNFα-induced activation restricted platelet ability to activate leucocytes and to induce leucocyte TF. TNFα inhibition did not influence platelet activation induced by collagen, ADP or thrombin receptor activating peptide-6. Platelets of patients with RA were more activated than those of controls. Activation was reduced in patients treated with TNFα inhibitors. CONCLUSIONS:TNFα-dependent pathways control platelet activation and TF expression in RA. Further studies will verify whether the protective effect of TNFα inhibitors on cardiovascular events involves their ability to modulate platelet function. 10.1136/annrheumdis-2015-208442
    Tissue-infiltrating neutrophils represent the main source of IL-23 in the colon of patients with IBD. Kvedaraite Egle,Lourda Magda,Ideström Maja,Chen Puran,Olsson-Åkefeldt Selma,Forkel Marianne,Gavhed Désirée,Lindforss Ulrik,Mjösberg Jenny,Henter Jan-Inge,Svensson Mattias Gut OBJECTIVE:In IBD, interleukin-23 (IL-23) and its receptor (IL-23R) are implicated in disease initiation and progression. Novel insight into which cells produce IL-23 at the site of inflammation at an early stage of IBD will promote the development of new tools for diagnosis, treatment and patient monitoring. We examined the cellular source of IL-23 in colon tissue of untreated newly diagnosed paediatric patients with IBD. DESIGN:Colon tissues from IBD and non-IBD patients were analysed by quantitative real-time PCR (qPCR), immunofluorescence confocal microscopy and flow cytometry after appropriate sample preparation. Blood samples from IBD and non-IBD patients and healthy controls were analysed using flow cytometry and qPCR. RESULTS:We discovered that tissue-infiltrating neutrophils were the main source of IL-23 in the colon of paediatric patients with IBD, while IL-23(+) human leucocyte antigen-DR(+) or IL-23(+)CD14(+) cells were scarce or non-detectable, respectively. The colonic IL-23(+) neutrophils expressed C-X-C motif (CXC)R1 and CXCR2, receptors for the CXC ligand 8 (CXCL8) chemokine family, and a corresponding CXCR1(+)CXCR2(+)IL-23(+)subpopulation of neutrophils was also identified in the blood of both patients with IBD and healthy individuals. However, CXCL8-family chemokines were only elevated in colon tissue from patients with IBD. CONCLUSIONS:This study provides the first evidence of CXCR1(+)CXCR2(+)IL-23-producing neutrophils that infiltrate and accumulate in inflamed colon tissue of patients with IBD. Thus, this novel source of IL-23 may play a key role in disease progression and will be important to take into consideration in the development of future strategies to monitor, treat and prevent IBD. 10.1136/gutjnl-2014-309014
    Cyclic RGD-linked polymeric micelles for targeted delivery of platinum anticancer drugs to glioblastoma through the blood-brain tumor barrier. Miura Yutaka,Takenaka Tomoya,Toh Kazuko,Wu Shourong,Nishihara Hiroshi,Kano Mitsunobu R,Ino Yasushi,Nomoto Takahiro,Matsumoto Yu,Koyama Hiroyuki,Cabral Horacio,Nishiyama Nobuhiro,Kataoka Kazunori ACS nano Ligand-mediated drug delivery systems have enormous potential for improving the efficacy of cancer treatment. In particular, Arg-Gly-Asp peptides are promising ligand molecules for targeting αvβ3/αvβ5 integrins, which are overexpressed in angiogenic sites and tumors, such as intractable human glioblastoma (U87MG). We here achieved highly efficient drug delivery to U87MG tumors by using a platinum anticancer drug-incorporating polymeric micelle (PM) with cyclic Arg-Gly-Asp (cRGD) ligand molecules. Intravital confocal laser scanning microscopy revealed that the cRGD-linked polymeric micelles (cRGD/m) accumulated rapidly and had high permeability from vessels into the tumor parenchyma compared with the PM having nontargeted ligand, "cyclic-Arg-Ala-Asp" (cRAD). As both cRGD/m- and cRAD-linked polymeric micelles have similar characteristics, including their size, surface charge, and the amount of incorporated drugs, it is likely that the selective and accelerated accumulation of cRGD/m into tumors occurred via an active internalization pathway, possibly transcytosis, thereby producing significant antitumor effects in an orthotopic mouse model of U87MG human glioblastoma. 10.1021/nn402662d
    DNA probes for monitoring dynamic and transient molecular encounters on live cell membranes. You Mingxu,Lyu Yifan,Han Da,Qiu Liping,Liu Qiaoling,Chen Tao,Sam Wu Cuichen,Peng Lu,Zhang Liqin,Bao Gang,Tan Weihong Nature nanotechnology Cells interact with the extracellular environment through molecules expressed on the membrane. Disruption of these membrane-bound interactions (or encounters) can result in disease progression. Advances in super-resolution microscopy have allowed membrane encounters to be examined, however, these methods cannot image entire membranes and cannot provide information on the dynamic interactions between membrane-bound molecules. Here, we show a novel DNA probe that can transduce transient membrane encounter events into readable cumulative fluorescence signals. The probe, which translocates from one anchor site to another, mimicking motor proteins, is realized through a toehold-mediated DNA strand displacement reaction. Using this probe, we successfully monitored rapid encounter events of membrane lipid domains using flow cytometry and fluorescence microscopy. Our results show a preference for encounters within the same lipid domains. 10.1038/nnano.2017.23
    Ae2a,b-deficient mice develop antimitochondrial antibodies and other features resembling primary biliary cirrhosis. Salas January T,Banales Jesús M,Sarvide Sarai,Recalde Sergio,Ferrer Alex,Uriarte Iker,Oude Elferink Ronald P J,Prieto Jesús,Medina Juan F Gastroenterology BACKGROUND & AIMS:Cl(-)/HCO(3)(-) anion exchanger 2 (AE2) is involved in intracellular pH (pH(i)) regulation and transepithelial acid-base transport, including secretin-stimulated biliary bicarbonate excretion. AE2 gene expression was found to be reduced in liver biopsy specimens and blood mononuclear cells from patients with primary biliary cirrhosis (PBC), a disease characterized by chronic nonsuppurative cholangitis associated with antimitochondrial antibodies (AMA) and other autoimmune phenomena. In mice with widespread Ae2 gene disruption, we previously reported altered spermiogenesis and reduced gastric acid secretion. We now describe the hepatobiliary and immunologic changes observed in these Ae2(a.b)-deficient mice. METHODS:In this murine model, splenocyte pH(i) and T-cell populations were studied by flow cytometry. CD3-stimulated cytokine secretion was estimated using cytokine arrays. AMA were evaluated by immunoblotting and proteomics. Hepatobiliary changes were assessed by immunohistopathology, flow cytometry, and serum biochemistry. Cholangiocyte gene expression was analyzed by real-time polymerase chain reaction. RESULTS:Ae2(a,b)(-/-) mice exhibit splenomegaly, elevated pH(i) in splenocytes, increased production of interleukin-12p70 and interferon gamma, expanded CD8(+) T-cell population, and under represented CD4(+)FoxP3(+)/regulatory T cells. Most Ae2(a,b)(-/-) mice tested positively for AMA, showing increased serum levels of immunoglobulin M and G, and liver-specific alkaline phosphatase. About one third of Ae2(a,b)(-/-) mice had extensive portal inflammation with CD8(+) and CD4(+) T lymphocytes surrounding damaged bile ducts. Cholangiocytes isolated from Ae2(a,b)(-/-) mice showed gene expression changes compatible with oxidative stress and increased antigen presentation. CONCLUSIONS:Ae2 deficiency alters pH(i) homeostasis in immunocytes and gene expression profile in cholangiocytes, leading to immunologic and hepatobiliary changes that resemble PBC. 10.1053/j.gastro.2008.02.020
    Myeloperoxidase-rich Ly-6C+ myeloid cells infiltrate allografts and contribute to an imaging signature of organ rejection in mice. Swirski Filip K,Wildgruber Moritz,Ueno Takuya,Figueiredo Jose-Luiz,Panizzi Peter,Iwamoto Yoshiko,Zhang Elizabeth,Stone James R,Rodriguez Elisenda,Chen John W,Pittet Mikael J,Weissleder Ralph,Nahrendorf Matthias The Journal of clinical investigation Rates of graft rejection are high among recipients of heart transplants. The onset and progression of clinically significant heart transplant rejection are currently monitored by serial biopsy, but this approach is highly invasive and lacks sensitivity. Here, we have developed what we believe to be a new technique to measure organ rejection noninvasively that involves the exploration of tissue-infiltrating leukocytes as biomarker sources for diagnostic imaging. Specifically, we profiled the myeloid response in a murine model of heart transplantation with the aim of defining and validating an imaging signature of graft rejection. Ly-6Chi monocytes, which promote inflammation, accumulated progressively in allografts but only transiently in isografts. Ly-6Clo monocytes, which help resolve inflammation, did not accumulate, although they composed the majority of the few remaining monocytes in isografts. The persistence of Ly-6Chi monocytes in allografts prompted us to screen for a Ly-6Chi monocyte-associated imaging marker. Low-density array data revealed that Ly-6Chi monocytes express 10-fold higher levels of myeloperoxidase (MPO) than Ly-6Clo monocytes. Noninvasive magnetic resonance imaging of MPO with an MPO-activatable Gd-chelate revealed a spatially defined T1-weighted signal in rejected allografts but not in isografts or MPO-deficient allograft recipients. Flow cytometry, enzymography, and histology validated the approach by mapping MPO activity to Ly-6Chi monocytes and neutrophils. Thus, MPO imaging represents a potential alternative to the current invasive clinical standard by which transplants are monitored. 10.1172/JCI42304
    Mesenchymal Stem/Stromal Cell Engulfment Reveals Metastatic Advantage in Breast Cancer. Chen Yu-Chih,Gonzalez Maria E,Burman Boris,Zhao Xintao,Anwar Talha,Tran Mai,Medhora Natasha,Hiziroglu Ayse B,Lee Woncheol,Cheng Yu-Heng,Choi Yehyun,Yoon Euisik,Kleer Celina G Cell reports Twenty percent of breast cancer (BC) patients develop distant metastasis for which there is no cure. Mesenchymal stem/stromal cells (MSCs) in the tumor microenvironment were shown to stimulate metastasis, but the mechanisms are unclear. Here, we identified and quantified cancer cells engulfing stromal cells in clinical samples of BC metastasis by dual immunostaining for EZH2 and ALDH1 expression. Using flow cytometry and a microfluidic single-cell paring and retrieval platform, we show that MSC engulfment capacity is associated with BC cell metastatic potential and generates cells with mesenchymal-like, invasion, and stem cell traits. Whole-transcriptome analyses of selectively retrieved engulfing BC cells identify a gene signature of MSC engulfment consisting of WNT5A, MSR1, ELMO1, IL1RL2, ZPLD1, and SIRPB1. These results delineate a mechanism by which MSCs in the tumor microenvironment promote metastasis and provide a microfluidic platform with the potential to predict BC metastasis in clinical samples. 10.1016/j.celrep.2019.05.084
    Membrane Crossing and Membranotropic Activity of Cell-Penetrating Peptides: Dangerous Liaisons? Walrant Astrid,Cardon Sébastien,Burlina Fabienne,Sagan Sandrine Accounts of chemical research Living organisms have to maintain a stable balance in molecules and ions in the changing environment in which they are living, a process known as homeostasis. At the level of cells, the plasma membrane has a major role in homeostasis, since this hydrophobic film prevents passive diffusion of large and hydrophilic molecules between the extracellular and intracellular milieu. Living organisms have evolved with highly sophisticated transport systems to control exchanges across this barrier: import of nutrients and fuel essential for their survival; recognition of chemical or physical messengers allowing information interchanges with surrounding cells. Besides specialized proteins, endocytosis mechanisms at the level of the lipid bilayer can transport molecules from the outside across the cell membrane, in an energy-dependent manner. The cell membrane is highly heterogeneous in its molecular composition (tens of different lipids, proteins, polysaccharides, and combinations of these) and dynamic with bending, deformation, and elastic properties that depend on the local composition of membrane domains. Many viruses, microorganisms, and toxins exploit the plasma membrane to enter into cells. Chemists develop strategies to target the plasma membrane with molecules capable of circumventing this hydrophobic barrier, in particular to transport and deliver nonpermeable drugs in cells for biotechnological or pharmaceutical purposes. Drug delivery systems are numerous and include lipid-, sugar-, protein-, and peptide-based delivery systems, since these biomolecules generally have good biocompatibility, biodegradability, environmental sustainability, cost effectiveness, and availability. Among those, cell-penetrating peptides (CPPs), reported for the first time in the early 1990s, are attracting major interest not only as potential drug delivery systems but also at the level of fundamental research. It was indeed demonstrated very early that these peptides, which generally correspond to highly cationic sequences, can still cross the cell membrane at 4 °C, a temperature at which all active transport and endocytosis pathways are totally inhibited. Therefore, how these charged hydrophilic peptides cross the hydrophobic membrane barrier is of utmost interest as a pure basic and physicochemical question. In this Account, we focus on cationic cell-penetrating peptides (CPPs) and the way they cross cell membranes. We summarize the history of this field that emerged around 20 years ago. CPPs were indeed first identified as protein-transduction domains from the human immunodeficiency virus (HIV) TAT protein and the Antennapedia homeoprotein, a transcription factor from Drosophila. We highlight our contribution to the elucidation of CPP internalization pathways, in particular translocation, which implies perturbation and reorganization of the lipid bilayer, and endocytosis depending on sulfated glycosaminoglycans. We show a particular role of Trp (indole side chain) and Arg (guanidinium side chain), which are essential amino acids for CPP internalization. Interactions with the cell-surface are not only Coulombic; H-bonds and hydrophobic interactions contribute also significantly to CPP entry. The capacity of CPPs to cross cell membrane is not related to their strength of membrane binding. Finally, we present optimized methods based on mass spectrometry and fluorescence spectroscopy that allow unequivocal quantification of CPPs inside cells or bound to the outer leaflet of the membrane, and discuss some limitations of the technique of flow cytometry that we have recently highlighted. 10.1021/acs.accounts.7b00455
    H3K9 methyltransferase G9a negatively regulates UHRF1 transcription during leukemia cell differentiation. Kim Kee-Beom,Son Hye-Ju,Choi Sulji,Hahm Ja Young,Jung Hyeonsoo,Baek Hee Jo,Kook Hoon,Hahn Yoonsoo,Kook Hyun,Seo Sang-Beom Nucleic acids research Histone H3K9 methyltransferase (HMTase) G9a-mediated transcriptional repression is a major epigenetic silencing mechanism. UHRF1 (ubiquitin-like with PHD and ring finger domains 1) binds to hemimethylated DNA and plays an essential role in the maintenance of DNA methylation. Here, we provide evidence that UHRF1 is transcriptionally downregulated by H3K9 HMTase G9a. We found that increased expression of G9a along with transcription factor YY1 specifically represses UHRF1 transcription during TPA-mediated leukemia cell differentiation. Using ChIP analysis, we found that UHRF1 was among the transcriptionally silenced genes during leukemia cell differentiation. Using a DNA methylation profiling array, we discovered that the UHRF1 promoter was hypomethylated in samples from leukemia patients, further supporting its overexpression and oncogenic activity. Finally, we showed that G9a regulates UHRF1-mediated H3K23 ubiquitination and proper DNA replication maintenance. Therefore, we propose that H3K9 HMTase G9a is a specific epigenetic regulator of UHRF1. 10.1093/nar/gkv183
    Long-Term Follow-Up of CD19-CAR T-Cell Therapy in Children and Young Adults With B-ALL. Journal of clinical oncology : official journal of the American Society of Clinical Oncology PURPOSE:CD19 chimeric antigen receptor (CD19-CAR) T cells induce high response rates in children and young adults (CAYAs) with B-cell acute lymphoblastic leukemia (B-ALL), but relapse rates are high. The role for allogeneic hematopoietic stem-cell transplant (alloHSCT) following CD19-CAR T-cell therapy to improve long-term outcomes in CAYAs has not been examined. METHODS:We conducted a phase I trial of autologous CD19.28ζ-CAR T cells in CAYAs with relapsed or refractory B-ALL. Response and long-term clinical outcomes were assessed in relation to disease and treatment variables. RESULTS:Fifty CAYAs with B-ALL were treated (median age, 13.5 years; range, 4.3-30.4). Thirty-one (62.0%) patients achieved a complete remission (CR), 28 (90.3%) of whom were minimal residual disease-negative by flow cytometry. Utilization of fludarabine/cyclophosphamide-based lymphodepletion was associated with improved CR rates (29/42, 69%) compared with non-fludarabine/cyclophosphamide-based lymphodepletion (2/8, 25%; = .041). With median follow-up of 4.8 years, median overall survival was 10.5 months (95% CI, 6.3 to 29.2 months). Twenty-one of 28 (75.0%) patients achieving a minimal residual disease-negative CR proceeded to alloHSCT. For those proceeding to alloHSCT, median overall survival was 70.2 months (95% CI, 10.4 months to not estimable). The cumulative incidence of relapse after alloHSCT was 9.5% (95% CI, 1.5 to 26.8) at 24 months; 5-year EFS following alloHSCT was 61.9% (95% CI, 38.1 to 78.8). CONCLUSION:We provide the longest follow-up in CAYAs with B-ALL after CD19-CAR T-cell therapy reported to date and demonstrate that sequential therapy with CD19.28ζ-CAR T cells followed by alloHSCT can mediate durable disease control in a sizable fraction of CAYAs with relapsed or refractory B-ALL (ClinicalTrials.gov identifier: NCT01593696). 10.1200/JCO.20.02262
    Interferon-γ-dependent immune responses contribute to the pathogenesis of sclerosing cholangitis in mice. Ravichandran Gevitha,Neumann Katrin,Berkhout Laura K,Weidemann Sören,Langeneckert Annika E,Schwinge Dorothee,Poch Tobias,Huber Samuel,Schiller Birgit,Hess Leonard U,Ziegler Annerose E,Oldhafer Karl J,Barikbin Roja,Schramm Christoph,Altfeld Marcus,Tiegs Gisa Journal of hepatology BACKGROUND AND AIMS:Primary sclerosing cholangitis (PSC) is an idiopathic, chronic cholestatic liver disorder characterized by biliary inflammation and fibrosis. Increased numbers of intrahepatic interferon-γ- (IFNγ) producing lymphocytes have been documented in patients with PSC, yet their functional role remains to be determined. METHODS:Liver tissue samples were collected from patients with PSC. The contribution of lymphocytes to liver pathology was assessed in Mdr2 x Rag1 mice, which lack T and B cells, and following depletion of CD90.2 or natural killer (NK)p46 cells in Mdr2 mice. Liver pathology was also determined in Mdr2 x Ifng mice and following anti-IFNγ antibody treatment of Mdr2 mice. Immune cell composition was analysed by multi-colour flow cytometry. Liver injury and fibrosis were determined by standard assays. RESULTS:Patients with PSC showed increased IFNγ serum levels and elevated numbers of hepatic CD56 NK cells. In Mdr2 mice, hepatic CD8 T cells and NK cells were the primary source of IFNγ. Depletion of CD90.2 cells reduced hepatic Ifng expression, NK cell cytotoxicity and liver injury similar to Mdr2 x Rag1 mice. Depletion of NK cells resulted in reduced CD8 T cell cytotoxicity and liver fibrosis. The complete absence of IFNγ in Mdr2x Ifng mice reduced NK cell and CD8 T cell frequencies expressing the cytotoxic effector molecules granzyme B and TRAIL and prevented liver fibrosis. The antifibrotic effect of IFNγ was also observed upon antibody-dependent neutralisation in Mdr2 mice. CONCLUSION:IFNγ changed the phenotype of hepatic CD8 T cells and NK cells towards increased cytotoxicity and its absence attenuated liver fibrosis in chronic sclerosing cholangitis. Therefore, unravelling the immunopathogenesis of PSC with a particular focus on IFNγ might help to develop novel treatment options. LAY SUMMARY:Primary sclerosing cholangitis (PSC) is a chronic cholestatic liver disease characterized by biliary inflammation and fibrosis, whose current medical treatment is hardly effective. We observed an increased interferon (IFN)-γ response in patients with PSC and in a mouse model of sclerosing cholangitis. IFNγ changed the phenotype of hepatic CD8 T lymphocytes and NK cells towards increased cytotoxicity, and its absence decreased liver cell death, reduced frequencies of inflammatory macrophages in the liver and attenuated liver fibrosis. Therefore, IFNγ-dependent immune responses may disclose checkpoints for future therapeutic intervention strategies in sclerosing cholangitis. 10.1016/j.jhep.2019.05.023
    Reduction of immunosuppressive tumor microenvironment in cholangiocarcinoma by ex vivo targeting immune checkpoint molecules. Zhou Guoying,Sprengers Dave,Mancham Shanta,Erkens Remco,Boor Patrick P C,van Beek Adriaan A,Doukas Michail,Noordam Lisanne,Campos Carrascosa Lucia,de Ruiter Valeska,van Leeuwen Roelof W F,Polak Wojciech G,de Jonge Jeroen,Groot Koerkamp Bas,van Rosmalen Belle,van Gulik Thomas M,Verheij Joanne,IJzermans Jan N M,Bruno Marco J,Kwekkeboom Jaap Journal of hepatology BACKGROUND & AIMS:Cholangiocarcinoma is an aggressive hepatobiliary malignancy originating from biliary tract epithelium. Whether cholangiocarcinoma is responsive to immune checkpoint antibody therapy is unknown, and knowledge of its tumor immune microenvironment is limited. We aimed to characterize tumor-infiltrating lymphocytes (TILs) in cholangiocarcinoma and assess functional effects of targeting checkpoint molecules on TILs. METHODS:We isolated TILs from resected tumors of patients with cholangiocarcinoma and investigated their compositions compared with their counterparts in tumor-free liver (TFL) tissues and blood, by flow cytometry and immunohistochemistry. We measured expression of immune co-stimulatory and co-inhibitory molecules on TILs, and determined whether targeting these molecules improved ex vivo functions of TILs. RESULTS:Proportions of cytotoxic T cells and natural killer cells were decreased, whereas regulatory T cells were increased in tumors compared with TFL. While regulatory T cells accumulated in tumors, the majority of cytotoxic and helper T cells were sequestered at tumor margins, and natural killer cells were excluded from the tumors. The co-stimulatory receptor GITR and co-inhibitory receptors PD1 and CTLA4 were over-expressed on tumor-infiltrating T cells compared with T cells in TFL and blood. Antagonistic targeting of PD1 or CTLA4 or agonistic targeting of GITR enhanced effector molecule production and T cell proliferation in ex vivo stimulation of TILs derived from cholangiocarcinoma. The inter-individual variations in TIL responses to checkpoint treatments were correlated with differences in TIL immune phenotype. CONCLUSIONS:Decreased numbers of cytotoxic immune cells and increased numbers of suppressor T cells that over-express co-inhibitory receptors suggest that the tumor microenvironment in cholangiocarcinoma is immunosuppressive. Targeting GITR, PD1 or CTLA4 enhances effector functions of tumor-infiltrating T cells, indicating that these molecules are potential immunotherapeutic targets for patients with cholangiocarcinoma. LAY SUMMARY:The defense functions of immune cells are suppressed in cholangiocarcinoma tumors. Stimulating or blocking "immune checkpoint" molecules expressed on tumor-infiltrating T cells can enhance the defense functions of these cells. Therefore, these molecules may be promising targets for therapeutic stimulation of immune cells to eradicate the tumors and prevent cancer recurrence in patients with cholangiocarcinoma. 10.1016/j.jhep.2019.05.026
    Gold nanocrystal labeling allows low-density lipoprotein imaging from the subcellular to macroscopic level. Allijn Iris E,Leong Wei,Tang Jun,Gianella Anita,Mieszawska Aneta J,Fay Francois,Ma Ge,Russell Stewart,Callo Catherine B,Gordon Ronald E,Korkmaz Emine,Post Jan Andries,Zhao Yiming,Gerritsen Hans C,Thran Axel,Proksa Roland,Daerr Heiner,Storm Gert,Fuster Valentin,Fisher Edward A,Fayad Zahi A,Mulder Willem J M,Cormode David P ACS nano Low-density lipoprotein (LDL) plays a critical role in cholesterol transport and is closely linked to the progression of several diseases. This motivates the development of methods to study LDL behavior from the microscopic to whole-body level. We have developed an approach to efficiently load LDL with a range of diagnostically active nanocrystals or hydrophobic agents. We performed focused experiments on LDL labeled with gold nanocrystals (Au-LDL). The labeling procedure had minimal effect on LDL size, morphology, or composition. Biological function was found to be maintained from both in vitro and in vivo experiments. Tumor-bearing mice were injected intravenously with LDL, DiR-LDL, Au-LDL, or a gold-loaded nanoemulsion. LDL accumulation in the tumors was detected with whole-body imaging methods, such as computed tomography (CT), spectral CT, and fluorescence imaging. Cellular localization was studied with transmission electron microscopy and fluorescence techniques. This LDL labeling procedure should permit the study of lipoprotein biointeractions in unprecedented detail. 10.1021/nn403258w
    CRISPR/Cas9 β-globin gene targeting in human haematopoietic stem cells. Dever Daniel P,Bak Rasmus O,Reinisch Andreas,Camarena Joab,Washington Gabriel,Nicolas Carmencita E,Pavel-Dinu Mara,Saxena Nivi,Wilkens Alec B,Mantri Sruthi,Uchida Nobuko,Hendel Ayal,Narla Anupama,Majeti Ravindra,Weinberg Kenneth I,Porteus Matthew H Nature The β-haemoglobinopathies, such as sickle cell disease and β-thalassaemia, are caused by mutations in the β-globin (HBB) gene and affect millions of people worldwide. Ex vivo gene correction in patient-derived haematopoietic stem cells followed by autologous transplantation could be used to cure β-haemoglobinopathies. Here we present a CRISPR/Cas9 gene-editing system that combines Cas9 ribonucleoproteins and adeno-associated viral vector delivery of a homologous donor to achieve homologous recombination at the HBB gene in haematopoietic stem cells. Notably, we devise an enrichment model to purify a population of haematopoietic stem and progenitor cells with more than 90% targeted integration. We also show efficient correction of the Glu6Val mutation responsible for sickle cell disease by using patient-derived stem and progenitor cells that, after differentiation into erythrocytes, express adult β-globin (HbA) messenger RNA, which confirms intact transcriptional regulation of edited HBB alleles. Collectively, these preclinical studies outline a CRISPR-based methodology for targeting haematopoietic stem cells by homologous recombination at the HBB locus to advance the development of next-generation therapies for β-haemoglobinopathies. 10.1038/nature20134
    Lung dendritic cell expression of maturation molecules increases with worsening chronic obstructive pulmonary disease. Freeman Christine M,Martinez Fernando J,Han MeiLan K,Ames Theresa M,Chensue Stephen W,Todt Jill C,Arenberg Douglas A,Meldrum Catherine A,Getty Christi,McCloskey Lisa,Curtis Jeffrey L American journal of respiratory and critical care medicine RATIONALE:Dendritic cells (DCs) have not been well studied in chronic obstructive pulmonary disease (COPD), yet their integral role in activating and differentiating T cells makes them potential participants in COPD pathogenesis. OBJECTIVES:To determine the expression of maturation molecules by individual DC subsets in relationship to COPD stage and to expression of the acute activation marker CD69 by lung CD4(+) T cells. METHODS:We nonenzymatically released lung leukocytes from human surgical specimens (n = 42) and used flow cytometry to identify three DC subsets (mDC1, mDC2, and pDC) and to measure their expression of three costimulatory molecules (CD40, CD80 and CD86) and of CD83, the definitive marker of DC maturation. Spearman nonparametric correlation analysis was used to identify significant correlations between expression of DC maturation molecules and COPD severity. MEASUREMENTS AND MAIN RESULTS:Expression of CD40 by mDC1 and mDC2 and of CD86 by mDC2 was high regardless of GOLD stage, but CD80 and CD83 on these two DC subsets increased with disease progression. pDC also showed significant increases in expression of CD40 and CD80. Expression of all but one of the DC molecules that increased with COPD severity also correlated with CD69 expression on lung CD4(+) T cells from the same patients, with the exception of CD83 on mDC2. CONCLUSIONS:This cross-sectional study implies that COPD progression is associated with significant increases in costimulatory molecule expression by multiple lung DC subsets. Interactions with lung DCs may contribute to the immunophenotype of CD4(+) T cells in advanced COPD. Clinical trial registered with www.clinicaltrials.gov (NCT00281229). 10.1164/rccm.200904-0552OC
    Condition-specific genetic interaction maps reveal crosstalk between the cAMP/PKA and the HOG MAPK pathways in the activation of the general stress response. Gutin Jenia,Sadeh Amit,Rahat Ayelet,Aharoni Amir,Friedman Nir Molecular systems biology Cells must quickly respond and efficiently adapt to environmental changes. The yeast Saccharomyces cerevisiae has multiple pathways that respond to specific environmental insults, as well as a generic stress response program. The later is regulated by two transcription factors, Msn2 and Msn4, that integrate information from upstream pathways to produce fast, tunable, and robust response to different environmental changes. To understand this integration, we employed a systematic approach to genetically dissect the contribution of various cellular pathways to Msn2/4 regulation under a range of stress and growth conditions. We established a high-throughput liquid handling and automated flow cytometry system and measured GFP levels in 68 single-knockout and 1,566 double-knockout strains that carry an HSP12-GFP allele as a reporter for Msn2/4 activity. Based on the expression of this Msn2/4 reporter in five different conditions, we identified numerous genetic and epistatic interactions between different components in the network upstream to Msn2/4. Our analysis gains new insights into the functional specialization of the RAS paralogs in the repression of stress response and identifies a three-way crosstalk between the Mediator complex, the HOG MAPK pathway, and the cAMP/PKA pathway. 10.15252/msb.20156451
    Mechanism for epigenetic variegation of gene expression at yeast telomeric heterochromatin. Kitada Tasuku,Kuryan Benjamin G,Tran Nancy Nga Huynh,Song Chunying,Xue Yong,Carey Michael,Grunstein Michael Genes & development Yeast contains heterochromatin at telomeres and the silent mating-type loci (HML/HMR). Genes positioned within the telomeric heterochromatin of Saccharomyces cerevisiae switch stochastically between epigenetically bistable ON and OFF expression states. Important aspects of the mechanism of variegated gene expression, including the chromatin structure of the natural ON state and the mechanism by which it is maintained, are unknown. To address this issue, we developed approaches to select cells in the ON and OFF states. We found by chromatin immunoprecipitation (ChIP) that natural ON telomeres are associated with Rap1 binding and, surprisingly, also contain known characteristics of OFF telomeres, including significant amounts of Sir3 and H4K16 deacetylated nucleosomes. Moreover, we found that H3K79 methylation (H3K79me), H3K4me, and H3K36me, which are depleted from OFF telomeres, are enriched at ON telomeres. We demonstrate in vitro that H3K79me, but not H3K4me or H3K36me, disrupts transcriptional silencing. Importantly, H3K79me does not significantly reduce Sir complex binding in vivo or in vitro. Finally, we show that maintenance of H3K79me at ON telomeres is dependent on transcription. Therefore, although Sir proteins are required for silencing, we propose that epigenetic variegation of telomeric gene expression is due to the bistable enrichment/depletion of H3K79me and not the fluctuation in the amount of Sir protein binding to nucleosomes. 10.1101/gad.201095.112
    Analysis of classical neutrophils and polymorphonuclear myeloid-derived suppressor cells in cancer patients and tumor-bearing mice. Veglia Filippo,Hashimoto Ayumi,Dweep Harsh,Sanseviero Emilio,De Leo Alessandra,Tcyganov Evgenii,Kossenkov Andrew,Mulligan Charles,Nam Brian,Masters Gregory,Patel Jaymala,Bhargava Vipul,Wilkinson Patrick,Smirnov Denis,Sepulveda Manuel A,Singhal Sunil,Eruslanov Evgeniy B,Cristescu Razvan,Loboda Andrey,Nefedova Yulia,Gabrilovich Dmitry I The Journal of experimental medicine In this study, using single-cell RNA-seq, cell mass spectrometry, flow cytometry, and functional analysis, we characterized the heterogeneity of polymorphonuclear neutrophils (PMNs) in cancer. We describe three populations of PMNs in tumor-bearing mice: classical PMNs, polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs), and activated PMN-MDSCs with potent immune suppressive activity. In spleens of mice, PMN-MDSCs gradually replaced PMNs during tumor progression. Activated PMN-MDSCs were found only in tumors, where they were present at the very early stages of the disease. These populations of PMNs in mice could be separated based on the expression of CD14. In peripheral blood of cancer patients, we identified two distinct populations of PMNs with characteristics of classical PMNs and PMN-MDSCs. The gene signature of tumor PMN-MDSCs was similar to that in mouse activated PMN-MDSCs and was closely associated with negative clinical outcome in cancer patients. Thus, we provide evidence that PMN-MDSCs are a distinct population of PMNs with unique features and potential for selective targeting opportunities. 10.1084/jem.20201803
    The biology of tumor growth in the non-Hodgkin's lymphomas. A dual parameter flow cytometry study of 220 cases. Shackney S E,Levine A M,Fisher R I,Nichols P,Jaffe E,Schuette W H,Simon R,Smith C A,Occhipinti S J,Parker J W The Journal of clinical investigation Dual parameter flow cytometry studies (cell DNA content and electronic cell volume) were performed in 220 cases of non-Hodgkin's lymphoma. All cases were characterized as B or T cell malignancies, based on immunologic surface marker characteristics. Aneuploidy by flow cytometry was more common among the B cell lymphomas than among the T cell lymphomas, and was most common among the large B cell lymphomas and B cell lymphomas of intermediate size. Ploidy index distributions showed a prominent hyperdiploid peak, as well as tumor cell populations with near-tetraploid DNA contents. In serial studies, a decrease in ploidy index was observed in association with clinical and histologic transformation in one case. The highest S fractions were observed among the large and intermediate B cell lymphomas and among the aggressive T cell lymphomas. In clinical samples consisting of mixtures of diploid and aneuploid populations, the data on the aneuploid components could often be separated from other components of the mixture in multiparameter studies on the basis of the larger electronic cell volumes of the aneuploid cells. In each case, the aneuploid large cell component almost invariably had a higher S fraction than the residual component(s) of the mixture. Overall, the data are consistent with a model of clonal selection and clonal evolution in the lymphomas in which early cytogenetic abnormalities that involve little or no change in total cell DNA content are followed by cell tetraploidization that is associated with cytogenetic instability and chromosome loss over the course of time. 10.1172/JCI111306
    Flow cytometry and Zollinger-Ellison syndrome: relationship to clinical course. Metz D C,Kuchnio M,Fraker D L,Venzon D J,Jaffe G,Jensen R T,Stetler-Stevenson M Gastroenterology BACKGROUND:With successful means of controlling gastric acid secretion in patients with Zollinger-Ellison syndrome, the gastrinoma itself is becoming the major determinant of long-term survival. No methods have yet been described to predict which tumors will have more malignant courses thereby indicating which patients should undergo aggressive surgery or antitumor therapy. Because DNA analysis, using flow cytometry, has proved helpful in this regard in other tumors, the current study was designed to evaluate its utility in gastrinoma patients. METHODS:Flow cytometry was performed on 81 paraffin-embedded gastrinoma specimens from 59 patients. Results were compared with preoperative patient characteristics, findings at surgery, and postoperative follow up. RESULTS:Tumors were diploid in 54% of patients, near diploid in 15%, pure tetraploid in 0%, nontetraploid aneuploid in 25%, and multiple stem line aneuploid in 5%. All patients with multiple stem line aneuploid tumors had wide-spread metastases whereas all patients with nontetraploid aneuploid tumors had localized or regional disease. Median S phase percentage was 3.6. S phase percentages were higher in patients with widespread metastatic disease than in patients with localized or regional disease. Disease extent also correlated closely with fasting serum gastrin level. After removing this variable with logistic regression analysis, the significant correlation between disease extent and DNA analysis persisted. CONCLUSIONS:DNA analysis of gastrinoma tissue specimens correlates independently with the extent of disease and may be useful in planning therapeutic strategies for patients with Zollinger-Ellison syndrome. 10.1016/0016-5085(93)90898-m
    Interrogating the repertoire: broadening the scope of peptide-MHC multimer analysis. Davis Mark M,Altman John D,Newell Evan W Nature reviews. Immunology Labelling antigen-specific T cells with peptide-MHC multimers has provided an invaluable way to monitor T cell-mediated immune responses. A number of recent developments in this technology have made these multimers much easier to make and use in large numbers. Furthermore, enrichment techniques have provided a greatly increased sensitivity that allows the analysis of the naive T cell repertoire directly. Thus, we can expect a flood of new information to emerge in the coming years. 10.1038/nri3020
    A high-throughput approach to identify genomic variants of bacterial metabolite producers at the single-cell level. Binder Stephan,Schendzielorz Georg,Stäbler Norma,Krumbach Karin,Hoffmann Kristina,Bott Michael,Eggeling Lothar Genome biology We present a novel method for visualizing intracellular metabolite concentrations within single cells of Escherichia coli and Corynebacterium glutamicum that expedites the screening process of producers. It is based on transcription factors and we used it to isolate new L-lysine producing mutants of C. glutamicum from a large library of mutagenized cells using fluorescence-activated cell sorting (FACS). This high-throughput method fills the gap between existing high-throughput methods for mutant generation and genome analysis. The technology has diverse applications in the analysis of producer populations and screening of mutant libraries that carry mutations in plasmids or genomes. 10.1186/gb-2012-13-5-r40
    TLR9 signalling in HCV-associated atypical memory B cells triggers Th1 and rheumatoid factor autoantibody responses. Comarmond Cloé,Lorin Valérie,Marques Cindy,Maciejewski-Duval Anna,Joher Nizar,Planchais Cyril,Touzot Maxime,Biard Lucie,Hieu Thierry,Quiniou Valentin,Desbois Anne-Claire,Rosenzwajg Michelle,Klatzmann David,Cacoub Patrice,Mouquet Hugo,Saadoun David Journal of hepatology BACKGROUND & AIMS:Hepatitis C virus (HCV) infection contributes to the development of autoimmune disorders such as cryoglobulinaemia vasculitis (CV). However, it remains unclear why only some individuals with HCV develop HCV-associated CV (HCV-CV). HCV-CV is characterized by the expansion of anergic CD19CD27CD21 atypical memory B cells (AtMs). Herein, we report the mechanisms by which AtMs participate in HCV-associated autoimmunity. METHODS:The phenotype and function of peripheral AtMs were studied by multicolour flow cytometry and co-culture assays with effector T cells and regulatory T cells in 20 patients with HCV-CV, 10 chronicallyHCV-infected patients without CV and 8 healthy donors. We performed gene expression profile analysis of AtMs stimulated or not by TLR9. Immunoglobulin gene repertoire and antibody reactivity profiles of AtM-expressing IgM antibodies were analysed following single B cell FACS sorting and expression-cloning of monoclonal antibodies. RESULTS:The TbetCD11cCD27CD21 AtM population is expanded in patients with HCV-CV compared to HCV controls without CV. TLR9 activation of AtMs induces a specific transcriptional signature centred on TNFα overexpression, and an enhanced secretion of TNFα and rheumatoid factor-type IgMs in patients with HCV-CV. AtMs stimulated through TLR9 promote type 1 effector T cell activation and reduce the proliferation of CD4CD25CD127FoxP3 regulatory T cells. AtM expansions display intraclonal diversity with immunoglobulin features of antigen-driven maturation. AtM-derived IgM monoclonal antibodies do not react against ubiquitous autoantigens or HCV antigens including NS3 and E2 proteins. Rather, AtM-derived antibodies possess rheumatoid factor activity and target unique epitopes on the human IgG-Fc region. CONCLUSION:Our data strongly suggest a central role for TLR9 activation of AtMs in driving HCV-CV autoimmunity through rheumatoid factor production and type 1 T cell responses. LAY SUMMARY:B cells are best known for their capacity to produce antibodies, which often play a deleterious role in the development of autoimmune diseases. During chronic hepatitis C, self-reactive B cells proliferate and can be responsible for autoimmune symptoms (arthritis, purpura, neuropathy, renal disease) and/or lymphoma. Direct-acting antiviral therapy clears the hepatitis C virus and eliminates deleterious B cells. 10.1016/j.jhep.2019.06.029
    Platelet secretion and hemostasis require syntaxin-binding protein STXBP5. Ye Shaojing,Huang Yunjie,Joshi Smita,Zhang Jinchao,Yang Fanmuyi,Zhang Guoying,Smyth Susan S,Li Zhenyu,Takai Yoshimi,Whiteheart Sidney W The Journal of clinical investigation Genome-wide association studies (GWAS) have linked genes encoding several soluble NSF attachment protein receptor (SNARE) regulators to cardiovascular disease risk factors. Because these regulatory proteins may directly affect platelet secretion, we used SNARE-containing complexes to affinity purify potential regulators from human platelet extracts. Syntaxin-binding protein 5 (STXBP5; also known as tomosyn-1) was identified by mass spectrometry, and its expression in isolated platelets was confirmed by RT-PCR analysis. Coimmunoprecipitation studies showed that STXBP5 interacts with core secretion machinery complexes, such as syntaxin-11/SNAP23 heterodimers, and fractionation studies suggested that STXBP5 also interacts with the platelet cytoskeleton. Platelets from Stxbp5 KO mice had normal expression of other key secretory components; however, stimulation-dependent secretion from each of the 3 granule types was markedly defective. Secretion defects in STXBP5-deficient platelets were confirmed via lumi-aggregometry and FACS analysis for P-selectin and LAMP-1 exposure. Interestingly, STXBP5-deficient platelets had altered granule cargo levels, despite having normal morphology and granule numbers. Consistent with secretion and cargo deficiencies, Stxbp5 KO mice showed dramatic bleeding in the tail transection model and defective hemostasis in the FeCl3-induced carotid injury model. Transplantation experiments indicated that these defects were due to loss of STXBP5 in BM-derived cells. Our data demonstrate that STXBP5 is required for normal arterial hemostasis, due to its contributions to platelet granule cargo packaging and secretion. 10.1172/JCI75572
    Papillon-Lefèvre syndrome patient reveals species-dependent requirements for neutrophil defenses. Sørensen Ole E,Clemmensen Stine N,Dahl Sara L,Østergaard Ole,Heegaard Niels H,Glenthøj Andreas,Nielsen Finn Cilius,Borregaard Niels The Journal of clinical investigation Papillon-Lefèvre syndrome (PLS) results from mutations that inactivate cysteine protease cathepsin C (CTSC), which processes a variety of serine proteases considered essential for antimicrobial defense. Despite serine protease-deficient immune cell populations, PLS patients do not exhibit marked immunodeficiency. Here, we characterized a 24-year-old woman who had suffered from severe juvenile periodontal disease, but was otherwise healthy, and identified a homozygous missense mutation in CTSC indicative of PLS. Proteome analysis of patient neutrophil granules revealed that several proteins that normally localize to azurophil granules, including the major serine proteases, elastase, cathepsin G, and proteinase 3, were absent. Accordingly, neutrophils from this patient were incapable of producing neutrophil extracellular traps (NETs) in response to ROS and were unable to process endogenous cathelicidin hCAP-18 into the antibacterial peptide LL-37 in response to ionomycin. In immature myeloid cells from patient bone marrow, biosynthesis of CTSC and neutrophil serine proteases appeared normal along with initial processing and sorting to cellular storage. In contrast, these proteins were completely absent in mature neutrophils, indicating that CTSC mutation promotes protease degradation in more mature hematopoietic subsets, but does not affect protease production in progenitor cells. Together, these data indicate CTSC protects serine proteases from degradation in mature immune cells and suggest that neutrophil serine proteases are dispensable for human immunoprotection. 10.1172/JCI76009
    Intrathymic programming of effector fates in three molecularly distinct γδ T cell subtypes. Narayan Kavitha,Sylvia Katelyn E,Malhotra Nidhi,Yin Catherine C,Martens Gregory,Vallerskog Therese,Kornfeld Hardy,Xiong Na,Cohen Nadia R,Brenner Michael B,Berg Leslie J,Kang Joonsoo, Nature immunology Innate γδ T cells function in the early phase of immune responses. Although innate γδ T cells have often been studied as one homogenous population, they can be functionally classified into effector subsets on the basis of the production of signature cytokines, analogous to adaptive helper T cell subsets. However, unlike the function of adaptive T cells, γδ effector T cell function correlates with genomically encoded T cell antigen receptor (TCR) chains, which suggests that clonal TCR selection is not the main determinant of the differentiation of γδ effector cells. A high-resolution transcriptome analysis of all emergent γδ thymocyte subsets segregated on the basis of use of the TCR γ-chain or δ-chain indicated the existence of three separate subtypes of γδ effector cells in the thymus. The immature γδ subsets were distinguished by unique transcription-factor modules that program effector function. 10.1038/ni.2247
    Nonclassical MHC class Ib-restricted cytotoxic T cells monitor antigen processing in the endoplasmic reticulum. Nagarajan Niranjana A,Gonzalez Federico,Shastri Nilabh Nature immunology The aminopeptidase ERAAP is essential for trimming peptides presented by major histocompatibility complex (MHC) class I molecules. Inhibition of ERAAP by cytomegalovirus results in evasion of the immune response by this virus, and polymorphisms in ERAAP are associated with autoimmune disorders. How normal ERAAP function is monitored is unknown. We found that inhibition of ERAAP rapidly induced presentation of the peptide FYAEATPML (FL9) by the MHC class Ib molecule Qa-1(b). Antigen-experienced T cells specific for the Qa-1(b)-FL9 complex were frequent in naive mice. Wild-type mice immunized with ERAAP-deficient cells mounted a potent CD8(+) T cell response specific for Qa-1(b)-FL9. MHC class Ib-restricted cytolytic effector cells specifically eliminated ERAAP-deficient cells in vitro and in vivo. Thus, nonclassical Qa-1(b)-peptide complexes direct cytotoxic T cells to targets with defective antigen processing in the endoplasmic reticulum. 10.1038/ni.2282
    TGF-β and retinoic acid induce the microRNA miR-10a, which targets Bcl-6 and constrains the plasticity of helper T cells. Takahashi Hayato,Kanno Tomohiko,Nakayamada Shingo,Hirahara Kiyoshi,Sciumè Giuseppe,Muljo Stefan A,Kuchen Stefan,Casellas Rafael,Wei Lai,Kanno Yuka,O'Shea John J Nature immunology Distinct CD4(+) T cell subsets are critical for host defense and immunoregulation. Although these subsets can act as terminally differentiated lineages, they have been increasingly noted to demonstrated plasticity. MicroRNAs are factors that control T cell stability and plasticity. Here we report that naturally occurring regulatory T cells (T(reg) cells) had high expression of the microRNA miR-10a and that miR-10a was induced by retinoic acid and transforming growth factor-β (TGF-β) in inducible T(reg) cells. By simultaneously targeting the transcriptional repressor Bcl-6 and the corepressor Ncor2, miR-10a attenuated the phenotypic conversion of inducible T(reg) cells into follicular helper T cells. We also found that miR-10a limited differentiation into the T(H)17 subset of helper T cells and therefore represents a factor that can fine-tune the plasticity and fate of helper T cells. 10.1038/ni.2286
    Helicobacter pylori gamma-glutamyl transpeptidase is a pathogenic factor in the development of peptic ulcer disease. Gong Min,Ling Samantha Shi Min,Lui Sook Yin,Yeoh Khay Guan,Ho Bow Gastroenterology BACKGROUND & AIMS:gamma-Glutamyl transpeptidase (GGT) has been reported to be a virulence factor of Helicobacter pylori associated with bacterial colonization and cell apoptosis. But its mechanism of pathogenesis is not firmly established. This study aims to examine its role in H pylori-mediated infection. METHODS:Various H pylori isogenic mutants were constructed by a polymerase chain reaction (PCR) approach. H pylori native GGT protein (HP-nGGT) was purified with ion-exchange and gel-filtration chromatography. Generation of H2O2 was measured with fluorimetric analysis, whereas nuclear factor-kappaB (NF-kappaB) activation was determined by luciferase assay and Western blot. Cytokine production was examined by enzyme-linked immunoabsorbent assay and real-time PCR. DNA damage was assessed with comet assay and flow cytometry. The GGT activity of 98 H pylori isolates was analyzed by an enzymatic assay. RESULTS:Purified HP-nGGT generated H2O2 in primary gastric epithelial cells and AGS gastric cancer cells, resulting in the activation of NF-kappaB and up-regulation of interleukin-8 (IL-8) production. In addition, HP-nGGT caused an increase in the level of 8-OH-dG, indicative of oxidative DNA damage. In contrast, Deltaggt showed significantly reduced levels of H2O2 generation, IL-8 production, and DNA damage in cells compared with the wild type (P<.05). The clinical importance of GGT was indicated by significantly higher (P<.001) activity in H pylori isolates obtained from patients with peptic ulcer disease (n=54) than isolates from patients with nonulcer dyspepsia (n=44). CONCLUSION:Our findings provide evidence that GGT is a pathogenic factor associated with H pylori-induced peptic ulcer disease. 10.1053/j.gastro.2010.03.050
    CSF-1-dependant donor-derived macrophages mediate chronic graft-versus-host disease. Alexander Kylie A,Flynn Ryan,Lineburg Katie E,Kuns Rachel D,Teal Bianca E,Olver Stuart D,Lor Mary,Raffelt Neil C,Koyama Motoko,Leveque Lucie,Le Texier Laetitia,Melino Michelle,Markey Kate A,Varelias Antiopi,Engwerda Christian,Serody Jonathan S,Janela Baptiste,Ginhoux Florent,Clouston Andrew D,Blazar Bruce R,Hill Geoffrey R,MacDonald Kelli P A The Journal of clinical investigation Chronic GVHD (cGVHD) is the major cause of late, nonrelapse death following stem cell transplantation and characteristically develops in organs such as skin and lung. Here, we used multiple murine models of cGVHD to investigate the contribution of macrophage populations in the development of cGVHD. Using an established IL-17-dependent sclerodermatous cGVHD model, we confirmed that macrophages infiltrating the skin are derived from donor bone marrow (F4/80+CSF-1R+CD206+iNOS-). Cutaneous cGVHD developed in a CSF-1/CSF-1R-dependent manner, as treatment of recipients after transplantation with CSF-1 exacerbated macrophage infiltration and cutaneous pathology. Additionally, recipients of grafts from Csf1r-/- mice had substantially less macrophage infiltration and cutaneous pathology as compared with those receiving wild-type grafts. Neither CCL2/CCR2 nor GM-CSF/GM-CSFR signaling pathways were required for macrophage infiltration or development of cGVHD. In a different cGVHD model, in which bronchiolitis obliterans is a prominent manifestation, F4/80+ macrophage infiltration was similarly noted in the lungs of recipients after transplantation, and lung cGVHD was also IL-17 and CSF-1/CSF-1R dependent. Importantly, depletion of macrophages using an anti-CSF-1R mAb markedly reduced cutaneous and pulmonary cGVHD. Taken together, these data indicate that donor macrophages mediate the development of cGVHD and suggest that targeting CSF-1 signaling after transplantation may prevent and treat cGVHD. 10.1172/JCI75935
    Mast cells aggravate sepsis by inhibiting peritoneal macrophage phagocytosis. Dahdah Albert,Gautier Gregory,Attout Tarik,Fiore Frédéric,Lebourdais Emeline,Msallam Rasha,Daëron Marc,Monteiro Renato C,Benhamou Marc,Charles Nicolas,Davoust Jean,Blank Ulrich,Malissen Bernard,Launay Pierre The Journal of clinical investigation Controlling the overwhelming inflammatory reaction associated with polymicrobial sepsis remains a prevalent clinical challenge with few treatment options. In septic peritonitis, blood neutrophils and monocytes are rapidly recruited into the peritoneal cavity to control infection, but the role of resident sentinel cells during the early phase of infection is less clear. In particular, the influence of mast cells on other tissue-resident cells remains poorly understood. Here, we developed a mouse model that allows both visualization and conditional ablation of mast cells and basophils to investigate the role of mast cells in severe septic peritonitis. Specific depletion of mast cells led to increased survival rates in mice with acute sepsis. Furthermore, we determined that mast cells impair the phagocytic action of resident macrophages, thereby allowing local and systemic bacterial proliferation. Mast cells did not influence local recruitment of neutrophils and monocytes or the release of inflammatory cytokines. Phagocytosis inhibition by mast cells involved their ability to release prestored IL-4 within 15 minutes after bacterial encounter, and treatment with an IL-4-neutralizing antibody prevented this inhibitory effect and improved survival of septic mice. Our study uncovers a local crosstalk between mast cells and macrophages during the early phase of sepsis development that aggravates the outcome of severe bacterial infection. 10.1172/JCI75212
    Proteinase 3-dependent caspase-3 cleavage modulates neutrophil death and inflammation. Loison Fabien,Zhu Haiyan,Karatepe Kutay,Kasorn Anongnard,Liu Peng,Ye Keqiang,Zhou Jiaxi,Cao Shannan,Gong Haiyan,Jenne Dieter E,Remold-O'Donnell Eileen,Xu Yuanfu,Luo Hongbo R The Journal of clinical investigation Caspase-3-mediated spontaneous death in neutrophils is a prototype of programmed cell death and is critical for modulating physiopathological inflammatory responses; however, the underlying regulatory pathways remain ill defined. Here we determined that in aging neutrophils, the cleavage and activation of caspase-3 is independent of the canonical caspase-8- or caspase-9-mediated pathway. Instead, caspase-3 activation was mediated by serine protease proteinase 3 (PR3), which is present in the cytosol of aging neutrophils. Specifically, PR3 cleaved procaspase-3 at a site upstream of the canonical caspase-9 cleavage site. In mature neutrophils, PR3 was sequestered in granules and released during aging via lysosomal membrane permeabilization (LMP), leading to procaspase-3 cleavage and apoptosis. Pharmacological inhibition or knockdown of PR3 delayed neutrophil death in vitro and consistently delayed neutrophil death and augmented neutrophil accumulation at sites of inflammation in a murine model of peritonitis. Adoptive transfer of both WT and PR3-deficient neutrophils revealed that the delayed death of neutrophils lacking PR3 is due to an altered intrinsic apoptosis/survival pathway, rather than the inflammatory microenvironment. The presence of the suicide protease inhibitor SERPINB1 counterbalanced the protease activity of PR3 in aging neutrophils, and deletion of Serpinb1 accelerated neutrophil death. Taken together, our results reveal that PR3-mediated caspase-3 activation controls neutrophil spontaneous death. 10.1172/JCI76246
    A lymphatic defect causes ocular hypertension and glaucoma in mice. Thomson Benjamin R,Heinen Stefan,Jeansson Marie,Ghosh Asish K,Fatima Anees,Sung Hoon-Ki,Onay Tuncer,Chen Hui,Yamaguchi Shinji,Economides Aris N,Flenniken Ann,Gale Nicholas W,Hong Young-Kwon,Fawzi Amani,Liu Xiaorong,Kume Tsutomu,Quaggin Susan E The Journal of clinical investigation Glaucoma is a leading cause of blindness, afflicting more than 60 million people worldwide. Increased intraocular pressure (IOP) due to impaired aqueous humor drainage is a major risk factor for the development of glaucoma. Here, we demonstrated that genetic disruption of the angiopoietin/TIE2 (ANGPT/TIE2) signaling pathway results in high IOP, buphthalmos, and classic features of glaucoma, including retinal ganglion degeneration and vision loss. Eyes from mice with induced deletion of Angpt1 and Angpt2 (A1A2Flox(WB) mice) lacked drainage pathways in the corneal limbus, including Schlemm's canal and lymphatic capillaries, which share expression of the PROX1, VEGFR3, and FOXC family of transcription factors. VEGFR3 and FOXCs have been linked to lymphatic disorders in patients, and FOXC1 has been linked to glaucoma. In contrast to blood endothelium, in which ANGPT2 is an antagonist of ANGPT1, we have shown that both ligands cooperate to regulate TIE2 in the lymphatic network of the eye. While A1A2Flox(WB) mice developed high IOP and glaucoma, expression of ANGPT1 or ANGPT2 alone was sufficient for ocular drainage. Furthermore, we demonstrated that loss of FOXC2 from lymphatics results in TIE2 downregulation, suggesting a mechanism for ocular defects in patients with FOXC mutations. These data reveal a pathogenetic and molecular basis for glaucoma and demonstrate the importance of angiopoietin ligand cooperation in the lymphatic endothelium. 10.1172/JCI77162
    Deep sequencing reveals stepwise mutation acquisition in paroxysmal nocturnal hemoglobinuria. Shen Wenyi,Clemente Michael J,Hosono Naoko,Yoshida Kenichi,Przychodzen Bartlomiej,Yoshizato Tetsuichi,Shiraishi Yuichi,Miyano Satoru,Ogawa Seishi,Maciejewski Jaroslaw P,Makishima Hideki The Journal of clinical investigation Paroxysmal nocturnal hemoglobinuria (PNH) is a nonmalignant clonal disease of hematopoietic stem cells that is associated with hemolysis, marrow failure, and thrombophilia. PNH has been considered a monogenic disease that results from somatic mutations in the gene encoding PIGA, which is required for biosynthesis of glycosylphosphatidylinisotol-anchored (GPI-anchored) proteins. The loss of certain GPI-anchored proteins is hypothesized to provide the mutant clone with an extrinsic growth advantage, but some features of PNH argue that there are intrinsic drivers of clonal expansion. Here, we performed whole-exome sequencing of paired PNH+ and PNH- fractions on samples taken from 12 patients as well as targeted deep sequencing of an additional 36 PNH patients. We identified additional somatic mutations that resulted in a complex hierarchical clonal architecture, similar to that observed in myeloid neoplasms. In addition to mutations in PIGA, mutations were found in genes known to be involved in myeloid neoplasm pathogenesis, including TET2, SUZ12, U2AF1, and JAK2. Clonal analysis indicated that these additional mutations arose either as a subclone within the PIGA-mutant population, or prior to PIGA mutation. Together, our data indicate that in addition to PIGA mutations, accessory genetic events are frequent in PNH, suggesting a stepwise clonal evolution derived from a singular stem cell clone. 10.1172/JCI74747
    B7-H1-expressing antigen-presenting cells mediate polarization of protumorigenic Th22 subsets. Kuang Dong-Ming,Xiao Xiao,Zhao Qiyi,Chen Min-Min,Li Xue-Feng,Liu Rui-Xian,Wei Yuan,Ouyang Fang-Zhu,Chen Dong-Ping,Wu Yan,Lao Xiang-Ming,Deng Hong,Zheng Limin The Journal of clinical investigation Classical IL-22-producing T helper cells (Th22 cells) mediate inflammatory responses independently of IFN-γ and IL-17; however, nonclassical Th22 cells have been recently identified and coexpress IFN-γ and/or IL-17 along with IL-22. Little is known about how classical and nonclassical Th22 subsets in human diseases are regulated. Here, we used samples of human blood, normal and peritumoral liver, and hepatocellular carcinoma (HCC) to delineate the phenotype, distribution, generation, and functional relevance of various Th22 subsets. Three nonclassical Th22 subsets constituted the majority of all Th22 cells in human liver and HCC tissues, although the classical Th22 subset was predominant in blood. Monocytes activated by TLR2 and TLR4 agonists served as the antigen-presenting cells (APCs) that most efficiently triggered the expansion of nonclassical Th22 subsets from memory T cells and classical Th22 subsets from naive T cells. Moreover, B7-H1-expressing monocytes skewed Th22 polarization away from IFN-γ and toward IL-17 through interaction with programmed death 1 (PD-1), an effect that can create favorable conditions for in vivo aggressive cancer growth and angiogenesis. Our results provide insight into the selective modulation of Th22 subsets and suggest that strategies to influence functional activities of inflammatory cells may benefit anticancer therapy. 10.1172/JCI74381
    Innate Lymphoid Cells Promote Recovery of Ventricular Function After Myocardial Infarction. Journal of the American College of Cardiology BACKGROUND:Innate lymphoid cells type 2 (ILC2s) play critical homeostatic functions in peripheral tissues. ILC2s reside in perivascular niches and limit atherosclerosis development. OBJECTIVES:ILC2s also reside in the pericardium but their role in postischemic injury is unknown. METHODS:We examined the role of ILC2 in a mouse model of myocardial infarction (MI), and compared mice with or without genetic deletion of ILC2. We determined infarct size using histology and heart function using echocardiography. We assessed cardiac ILC2 using flow cytometry and RNA sequencing. Based on these data, we devised a therapeutic strategy to activate ILC2 in mice with acute MI, using exogenous interleukin (IL)-2. We also assessed the ability of low-dose IL-2 to activate ILC2 in a double-blind randomized clinical trial of patients with acute coronary syndromes (ACS). RESULTS:We found that ILC2 levels were increased in pericardial adipose tissue after experimental MI, and genetic ablation of ILC2 impeded the recovery of heart function. RNA sequencing revealed distinct transcript signatures in ILC2, and pointed to IL-2 axis as a major upstream regulator. Treatment of T-cell-deficient mice with IL-2 (to activate ILC2) significantly improved the recovery of heart function post-MI. Administration of low-dose IL-2 to patients with ACS led to activation of circulating ILC2, with significant increase in circulating IL-5, a prototypic ILC2-derived cytokine. CONCLUSIONS:ILC2s promote cardiac healing and improve the recovery of heart function after MI in mice. Activation of ILC2 using low-dose IL-2 could be a novel therapeutic strategy to promote a reparative response after MI. 10.1016/j.jacc.2021.07.018
    Type I interferons directly inhibit regulatory T cells to allow optimal antiviral T cell responses during acute LCMV infection. Srivastava Shivani,Koch Meghan A,Pepper Marion,Campbell Daniel J The Journal of experimental medicine Regulatory T (T reg) cells play an essential role in preventing autoimmunity but can also impair clearance of foreign pathogens. Paradoxically, signals known to promote T reg cell function are abundant during infection and could inappropriately enhance T reg cell activity. How T reg cell function is restrained during infection to allow the generation of effective antiviral responses remains largely unclear. We demonstrate that the potent antiviral type I interferons (IFNs) directly inhibit co-stimulation-dependent T reg cell activation and proliferation, both in vitro and in vivo during acute infection with lymphocytic choriomeningitis virus (LCMV). Loss of the type I IFN receptor specifically in T reg cells results in functional impairment of virus-specific CD8(+) and CD4(+) T cells and inefficient viral clearance. Together, these data demonstrate that inhibition of T reg cells by IFNs is necessary for the generation of optimal antiviral T cell responses during acute LCMV infection. 10.1084/jem.20131556
    Identification of a human splenic marginal zone B cell precursor with NOTCH2-dependent differentiation properties. Descatoire Marc,Weller Sandra,Irtan Sabine,Sarnacki Sabine,Feuillard Jean,Storck Sébastien,Guiochon-Mantel Anne,Bouligand Jérôme,Morali Alain,Cohen Joseph,Jacquemin Emmanuel,Iascone Maria,Bole-Feysot Christine,Cagnard Nicolas,Weill Jean-Claude,Reynaud Claude-Agnès The Journal of experimental medicine Mouse splenic marginal zone precursors (MZPs) differentiate into marginal zone B (MZB) cells under a signaling pathway involving Notch2 and its ligand, delta-like 1 ligand (Dll1). We report the identification of an MZP subset in the spleen of young children. These MZPs differentiate into MZ-like B cells in vitro in the presence of OP9 cells expressing human DLL1, as demonstrated by the up-regulation of classical MZB cell markers. A set of diagnostic genes discriminating IgM(+)IgD(+)CD27(+) blood and splenic MZB cells from switched B cells was identified (up-regulation of SOX7, down-regulation of TOX, COCH, and HOPX), and their expression during the induction assay mirrored the one of MZB cells. Moreover, Alagille patients with a NOTCH2 haploinsufficiency display a marked reduction of IgM(+)IgD(+)CD27(+) B cells in blood, whereas their switched memory B cells are not affected. Altogether, these results argue in favor of the existence of a rodent-like MZB cell lineage in humans. 10.1084/jem.20132203
    Type I IFN suppresses Cxcr2 driven neutrophil recruitment into the sensory ganglia during viral infection. Stock Angus T,Smith Jeffrey M,Carbone Francis R The Journal of experimental medicine Infection induces the expression of inflammatory chemokines that recruit immune cells to the site of inflammation. Whereas tissues such as the intestine and skin express unique chemokines during homeostasis, whether different tissues express distinct chemokine profiles during inflammation remains unclear. With this in mind, we performed a comprehensive screen of the chemokines expressed by two tissues (skin and sensory ganglia) infected with a common viral pathogen (herpes simplex virus type 1). After infection, the skin and ganglia showed marked differences in their expression of the family of Cxcr2 chemokine ligands. Specifically, Cxcl1/2/3, which in turn controlled neutrophil recruitment, was up-regulated in the skin but absent from the ganglia. Within the ganglia, Cxcl2 expression and subsequent neutrophil recruitment was inhibited by type I interferon (IFN). Using a combination of bone marrow chimeras and intracellular chemokine staining, we show that type I IFN acted by directly suppressing Cxcl2 expression by monocytes, abrogating their ability to recruit neutrophils to the ganglia. Overall, our findings describe a novel role for IFN in the direct, and selective, inhibition of Cxcr2 chemokine ligands, which results in the inhibition of neutrophil recruitment to neuronal tissue. 10.1084/jem.20132183
    Leucine-rich repeat containing 8A (LRRC8A) is essential for T lymphocyte development and function. Kumar Lalit,Chou Janet,Yee Christina S K,Borzutzky Arturo,Vollmann Elisabeth H,von Andrian Ulrich H,Park Shin-Young,Hollander Georg,Manis John P,Poliani P Luigi,Geha Raif S The Journal of experimental medicine Lrrc8a is a ubiquitously expressed gene that encodes a leucine-rich repeat (LRR)-containing protein detected at higher levels on the surface of thymocytes than on other immune cells. We generated Lrrc8a(-/-) mice to investigate the role of LRRC8A in lymphocyte development and function. Lrrc8a(-/-) mice had increased prenatal and postnatal mortality, growth retardation, and multiple tissue abnormalities. Lrrc8a(-/-) mice displayed a modest block in B cell development but intact intrinsic B cell function. In contrast, both Lrrc8a(-/-) mice and Lrrc8a(-/-)→Rag2(-/-) bone marrow chimeras exhibited a severe cell-intrinsic block in early thymic development, with decreased proliferation and increased apoptosis of thymocytes, and impaired peripheral T cell function. Thymic epithelial cells expressed an LRRC8A ligand that was critical for double-negative to double-positive thymocyte differentiation and survival in vitro. LRRC8A constitutively associated with the GRB2-GAB2 complex and lymphocyte-specific protein tyrosine kinase (LCK) in thymocytes. LRRC8A ligation activated AKT via the LCK-ZAP-70-GAB2-PI3K pathway, and AKT phosphorylation was markedly reduced in the thymus of Lrrc8a(-/-) mice. These findings reveal an essential role for LRRC8A in T cell development, survival, and function. 10.1084/jem.20131379
    MyD88-dependent interplay between myeloid and endothelial cells in the initiation and progression of obesity-associated inflammatory diseases. Yu Minjia,Zhou Hao,Zhao Junjie,Xiao Nengming,Roychowdhury Sanjoy,Schmitt David,Hu Bingqing,Ransohoff Richard M,Harding Clifford V,Hise Amy G,Hazen Stanley L,DeFranco Anthony L,Fox Paul L,Morton Richard E,Dicorleto Paul E,Febbraio Maria,Nagy Laura E,Smith Jonathan D,Wang Jian-an,Li Xiaoxia The Journal of experimental medicine Low-grade systemic inflammation is often associated with metabolic syndrome, which plays a critical role in the development of the obesity-associated inflammatory diseases, including insulin resistance and atherosclerosis. Here, we investigate how Toll-like receptor-MyD88 signaling in myeloid and endothelial cells coordinately participates in the initiation and progression of high fat diet-induced systemic inflammation and metabolic inflammatory diseases. MyD88 deficiency in myeloid cells inhibits macrophage recruitment to adipose tissue and their switch to an M1-like phenotype. This is accompanied by substantially reduced diet-induced systemic inflammation, insulin resistance, and atherosclerosis. MyD88 deficiency in endothelial cells results in a moderate reduction in diet-induced adipose macrophage infiltration and M1 polarization, selective insulin sensitivity in adipose tissue, and amelioration of spontaneous atherosclerosis. Both in vivo and ex vivo studies suggest that MyD88-dependent GM-CSF production from the endothelial cells might play a critical role in the initiation of obesity-associated inflammation and development of atherosclerosis by priming the monocytes in the adipose and arterial tissues to differentiate into M1-like inflammatory macrophages. Collectively, these results implicate a critical MyD88-dependent interplay between myeloid and endothelial cells in the initiation and progression of obesity-associated inflammatory diseases. 10.1084/jem.20131314
    Lack of Siglec-7 expression identifies a dysfunctional natural killer cell subset associated with liver inflammation and fibrosis in chronic HCV infection. Varchetta Stefania,Mele Dalila,Lombardi Andrea,Oliviero Barbara,Mantovani Stefania,Tinelli Carmine,Spreafico Marta,Prati Daniele,Ludovisi Serena,Ferraioli Giovanna,Filice Carlo,Aghemo Alessio,Lampertico Pietro,Facchetti Floriana,Bernuzzi Francesca,Invernizzi Pietro,Mondelli Mario U Gut OBJECTIVE:Sialic-acid-binding immunoglobulin-like lectin-7 (Siglec-7) is a natural killer (NK) cell inhibitory receptor associated with NK phenotypic and functional abnormalities in HIV-1 infection. We investigated the significance of NK-expressed and serum soluble Siglec-7 in relation to NK functional ability and parameters of liver necroinflammation and fibrosis in chronic HCV infection. DESIGN:NK-expressed and serum Siglec-7 were evaluated in 130 and 166 HCV-infected individuals by flow cytometry and ELISA, respectively. NK cell degranulation and cytokine secretion were determined by flow cytometry. 65 patients with chronic HBV infection, 84 with chronic biliary disorders and 168 healthy donors served as controls. RESULTS:Expression of Siglec-7 was significantly decreased on NK cells from HCV-infected and HBV-infected patients and, conversely, serum Siglec-7 was significantly increased in these patients compared with controls. The frequency of Siglec-7pos NK cells was significantly higher at baseline in sustained virological responders to pegylated interferon-α/ribavirin treatment than in non-responders. Activating receptor expression was significantly higher in Siglec-7pos NK cells and was associated with increased degranulation and cytokine secretion compared with Siglec-7 cells. In chronic HCV infection, there was an inverse correlation between Siglec-7 expression and serum aminotransferases, γ-glutamyl transpeptidase, liver stiffness, aspartate aminotransferase to platelet ratio index and fibrosis-4 scores, and a positive correlation between serum Siglec-7 and the same clinical parameters, including histological staging. CONCLUSIONS:These findings identify Siglec-7 NK cells as a dysfunctional subpopulation associated with severe liver disease in chronic HCV infection. 10.1136/gutjnl-2015-310327
    Distinct, strict requirements for Gfi-1b in adult bone marrow red cell and platelet generation. Foudi Adlen,Kramer Daniel J,Qin Jinzhong,Ye Denise,Behlich Anna-Sophie,Mordecai Scott,Preffer Frederic I,Amzallag Arnaud,Ramaswamy Sridhar,Hochedlinger Konrad,Orkin Stuart H,Hock Hanno The Journal of experimental medicine The zinc finger transcriptional repressor Gfi-1b is essential for erythroid and megakaryocytic development in the embryo. Its roles in the maintenance of bone marrow erythropoiesis and thrombopoiesis have not been defined. We investigated Gfi-1b's adult functions using a loxP-flanked Gfi-1b allele in combination with a novel doxycycline-inducible Cre transgene that efficiently mediates recombination in the bone marrow. We reveal strict, lineage-intrinsic requirements for continuous adult Gfi-1b expression at two distinct critical stages of erythropoiesis and megakaryopoiesis. Induced disruption of Gfi-1b was lethal within 3 wk with severely reduced hemoglobin levels and platelet counts. The erythroid lineage was arrested early in bipotential progenitors, which did not give rise to mature erythroid cells in vitro or in vivo. Yet Gfi-1b(-/-) progenitors had initiated the erythroid program as they expressed many lineage-restricted genes, including Klf1/Eklf and Erythropoietin receptor. In contrast, the megakaryocytic lineage developed beyond the progenitor stage in Gfi-1b's absence and was arrested at the promegakaryocyte stage, after nuclear polyploidization, but before cytoplasmic maturation. Genome-wide analyses revealed that Gfi-1b directly regulates a wide spectrum of megakaryocytic and erythroid genes, predominantly repressing their expression. Together our study establishes Gfi-1b as a master transcriptional repressor of adult erythropoiesis and thrombopoiesis. 10.1084/jem.20131065
    The BTB-ZF transcription factor Zbtb20 is driven by Irf4 to promote plasma cell differentiation and longevity. Chevrier Stéphane,Emslie Dianne,Shi Wei,Kratina Tobias,Wellard Cameron,Karnowski Alexander,Erikci Erdem,Smyth Gordon K,Chowdhury Kamal,Tarlinton David,Corcoran Lynn M The Journal of experimental medicine The transcriptional network regulating antibody-secreting cell (ASC) differentiation has been extensively studied, but our current understanding is limited. The mechanisms of action of known "master" regulators are still unclear, while the participation of new factors is being revealed. Here, we identify Zbtb20, a Bcl6 homologue, as a novel regulator of late B cell development. Within the B cell lineage, Zbtb20 is specifically expressed in B1 and germinal center B cells and peaks in long-lived bone marrow (BM) ASCs. Unlike Bcl6, an inhibitor of ASC differentiation, ectopic Zbtb20 expression in primary B cells facilitates terminal B cell differentiation to ASCs. In plasma cell lines, Zbtb20 induces cell survival and blocks cell cycle progression. Immunized Zbtb20-deficient mice exhibit curtailed humoral responses and accelerated loss of antigen-specific plasma cells, specifically from the BM pool. Strikingly, Zbtb20 induction does not require Blimp1 but depends directly on Irf4, acting at a newly identified Zbtb20 promoter in ASCs. These results identify Zbtb20 as an important player in late B cell differentiation and provide new insights into this complex process. 10.1084/jem.20131831
    Enhancement of an anti-tumor immune response by transient blockade of central T cell tolerance. Khan Imran S,Mouchess Maria L,Zhu Meng-Lei,Conley Bridget,Fasano Kayla J,Hou Yafei,Fong Lawrence,Su Maureen A,Anderson Mark S The Journal of experimental medicine Thymic central tolerance is a critical process that prevents autoimmunity but also presents a challenge to the generation of anti-tumor immune responses. Medullary thymic epithelial cells (mTECs) eliminate self-reactive T cells by displaying a diverse repertoire of tissue-specific antigens (TSAs) that are also shared by tumors. Therefore, while protecting against autoimmunity, mTECs simultaneously limit the generation of tumor-specific effector T cells by expressing tumor self-antigens. This ectopic expression of TSAs largely depends on autoimmune regulator (Aire), which is expressed in mature mTECs. Thus, therapies to deplete Aire-expressing mTECs represent an attractive strategy to increase the pool of tumor-specific effector T cells. Recent work has implicated the TNF family members RANK and RANK-Ligand (RANKL) in the development of Aire-expressing mTECs. We show that in vivo RANKL blockade selectively and transiently depletes Aire and TSA expression in the thymus to create a window of defective negative selection. Furthermore, we demonstrate that RANKL blockade can rescue melanoma-specific T cells from thymic deletion and that persistence of these tumor-specific effector T cells promoted increased host survival in response to tumor challenge. These results indicate that modulating central tolerance through RANKL can alter thymic output and potentially provide therapeutic benefit by enhancing anti-tumor immunity. 10.1084/jem.20131889
    Improved HSC reconstitution and protection from inflammatory stress and chemotherapy in mice lacking granzyme B. Carnevalli Larissa S,Scognamiglio Roberta,Cabezas-Wallscheid Nina,Rahmig Susann,Laurenti Elisa,Masuda Kohei,Jöckel Lars,Kuck Andrea,Sujer Stefanie,Polykratis Apostolos,Erlacher Miriam,Pasparakis Manolis,Essers Marieke A G,Trumpp Andreas The Journal of experimental medicine The serine protease granzyme B (GzmB) is stored in the granules of cytotoxic T and NK cells and facilitates immune-mediated destruction of virus-infected cells. In this study, we use genetic tools to report novel roles for GzmB as an important regulator of hematopoietic stem cell (HSC) function in response to stress. HSCs lacking the GzmB gene show improved bone marrow (BM) reconstitution associated with increased HSC proliferation and mitochondrial activity. In addition, recipients deficient in GzmB support superior engraftment of wild-type HSCs compared with hosts with normal BM niches. Stimulation of mice with lipopolysaccharide strongly induced GzmB protein expression in HSCs, which was mediated by the TLR4-TRIF-p65 NF-κB pathway. This is associated with increased cell death and GzmB secretion into the BM environment, suggesting an extracellular role of GzmB in modulating HSC niches. Moreover, treatment with the chemotherapeutic agent 5-fluorouracil (5-FU) also induces GzmB production in HSCs. In this situation GzmB is not secreted, but instead causes cell-autonomous apoptosis. Accordingly, GzmB-deficient mice are more resistant to serial 5-FU treatments. Collectively, these results identify GzmB as a negative regulator of HSC function that is induced by stress and chemotherapy in both HSCs and their niches. Blockade of GzmB production may help to improve hematopoiesis in various situations of BM stress. 10.1084/jem.20131072
    PD-L1 is a novel direct target of HIF-1α, and its blockade under hypoxia enhanced MDSC-mediated T cell activation. Noman Muhammad Zaeem,Desantis Giacomo,Janji Bassam,Hasmim Meriem,Karray Saoussen,Dessen Philippe,Bronte Vincenzo,Chouaib Salem The Journal of experimental medicine Tumor-infiltrating myeloid cells such as myeloid-derived suppressor cells (MDSCs) and tumor-associated macrophages (TAMs) form an important component of the hypoxic tumor microenvironment. Here, we investigated the influence of hypoxia on immune checkpoint receptors (programmed death [PD]-1 and CTLA-4) and their respective ligands (PD-1 ligand 1 [PD-L1], PD-L2, CD80, and CD86) on MDSCs. We demonstrate that MDSCs at the tumor site show a differential expression of PD-L1 as compared with MDSCs from peripheral lymphoid organ (spleen). Hypoxia caused a rapid, dramatic, and selective up-regulation of PD-L1 on splenic MDSCs in tumor-bearing mice. This was not limited to MDSCs, as hypoxia also significantly increased the expression of PD-L1 on macrophages, dendritic cells, and tumor cells. Furthermore, PD-L1 up-regulation under hypoxia was dependent on hypoxia-inducible factor-1α (HIF-1α) but not HIF-2α. Chromatin immunoprecipitation and luciferase reporter assay revealed direct binding of HIF-1α to a transcriptionally active hypoxia-response element (HRE) in the PD-L1 proximal promoter. Blockade of PD-L1 under hypoxia enhanced MDSC-mediated T cell activation and was accompanied by the down-regulation of MDSCs IL-6 and IL-10. Finally, neutralizing antibodies against IL-10 under hypoxia significantly abrogated the suppressive activity of MDSCs. Simultaneous blockade of PD-L1 along with inhibition of HIF-1α may thus represent a novel approach for cancer immunotherapy. 10.1084/jem.20131916
    Role of cell cycle on the cellular uptake and dilution of nanoparticles in a cell population. Kim Jong Ah,Åberg Christoffer,Salvati Anna,Dawson Kenneth A Nature nanotechnology Nanoparticles are considered a primary vehicle for targeted therapies because they can pass biological barriers and enter and distribute within cells by energy-dependent pathways. So far, most studies have shown that nanoparticle properties, such as size and surface, can influence how cells internalize nanoparticles. Here, we show that uptake of nanoparticles by cells is also influenced by their cell cycle phase. Although cells in different phases of the cell cycle were found to internalize nanoparticles at similar rates, after 24 h the concentration of nanoparticles in the cells could be ranked according to the different phases: G2/M > S > G0/G1. Nanoparticles that are internalized by cells are not exported from cells but are split between daughter cells when the parent cell divides. Our results suggest that future studies on nanoparticle uptake should consider the cell cycle, because, in a cell population, the dose of internalized nanoparticles in each cell varies as the cell advances through the cell cycle. 10.1038/nnano.2011.191
    Cloaking Silica Nanoparticles with Functional Protein Coatings for Reduced Complement Activation and Cellular Uptake. Park Jae Hyeon,Jackman Joshua A,Ferhan Abdul Rahim,Belling Jason N,Mokrzecka Natalia,Weiss Paul S,Cho Nam-Joon ACS nano Silica-coated nanoparticles are widely used in biomedical applications such as theranostics, imaging, and drug delivery. While silica-coated nanoparticles are biocompatible, experimental evidence shows that they can trigger innate immune reactions, and a broader understanding of what types of reactions are caused and how to mitigate them is needed. Herein, we investigated how the noncovalent surface functionalization of silica nanoparticles with purified proteins can inhibit nanoparticle-induced complement activation and macrophage uptake, two of the most clinically relevant innate immune reactions related to nanomedicines. Silica nanoparticles were tested alone and after coating with bovine serum albumin, human serum albumin, fibrinogen, complement factor H (FH), or immunoglobulin G (IgG) proteins. Enzyme-linked immunosorbent assays measuring the generation of various complement activation products indicated that silica nanoparticles induce complement activation the alternative pathway. All protein coatings other than IgG protected against complement activation to varying extents. Most proteins acted as steric blockers to inhibit complement protein deposition on the nanoparticle surface, while FH coatings were biologically active and inhibited a key step in the amplification loop of complement activation, as confirmed by Western blot analysis. Flow cytometry and fluorescence microscopy experiments further revealed that complement activation-inhibiting protein coatings blunted macrophage uptake as well. Taken together, our findings demonstrate a simple and effective way to coat silica nanoparticles with purified protein coatings in order to mitigate innate immune reactions. Such methods are readily scalable and might constitute a useful strategy for improving the immunological safety profile of silica and silica-coated nanoparticles as well as other types of inorganic nanoparticles. 10.1021/acsnano.0c05097
    Clonal deletion and the fate of autoreactive thymocytes that survive negative selection. Pobezinsky Leonid A,Angelov Georgi S,Tai Xuguang,Jeurling Susanna,Van Laethem François,Feigenbaum Lionel,Park Jung-Hyun,Singer Alfred Nature immunology Clonal deletion of autoreactive thymocytes is important for self-tolerance, but the intrathymic signals that induce clonal deletion have not been clearly identified. We now report that clonal deletion during negative selection required CD28-mediated costimulation of autoreactive thymocytes at the CD4(+)CD8(lo) intermediate stage of differentiation. Autoreactive thymocytes were prevented from undergoing clonal deletion by either a lack of CD28 costimulation or transgenic overexpression of the antiapoptotic factors Bcl-2 or Mcl-1, with surviving thymocytes differentiating into anergic CD4(-)CD8(-) double-negative thymocytes positive for the T cell antigen receptor αβ subtype (TCRαβ) that 'preferentially' migrated to the intestine, where they re-expressed CD8α and were sequestered as CD8αα(+) intraepithelial lymphocytes (IELs). Our study identifies costimulation by CD28 as the intrathymic signal required for clonal deletion and identifies CD8αα(+) IELs as the developmental fate of autoreactive thymocytes that survive negative selection. 10.1038/ni.2292
    FOXP3 expression in blood, synovial fluid and synovial tissue during inflammatory arthritis and intra-articular corticosteroid treatment. Raghavan S,Cao D,Widhe M,Roth K,Herrath J,Engström M,Roncador G,Banham A H,Trollmo C,Catrina A I,Malmström V Annals of the rheumatic diseases OBJECTIVE:To analyse the distribution of FOXP3+CD25+CD4+ regulatory T cells (Treg) in peripheral blood, synovial fluid and tissue of patients with rheumatic disease during relapse and after local treatment. METHODS:FOXP3 expression was assessed by flow cytometry, immunohistochemistry, immunofluorescence and real-time polymerase chain reaction (RT-PCR). The functional suppressive capacity of Treg was analysed after co-culture with effector CD4+CD25- T cells through assessment of proliferation and cytokine secretion. RESULTS:It was shown that FOXP3 protein and mRNA expression in synovial fluid T cells was not confined solely to CD25(bright) T cells as seen in blood, but included CD25(intermediate) and even CD25(neg) T cells. Indeed, synovial fluid CD25(high) T cells showed similar suppressive capacity as CD25(bright) T cells, indicating the presence of functional Treg in T cells with lower intensity of CD25. In synovial tissue, FOXP3+ cells were present in low numbers within T-cell infiltrates and decreased further after intra-articular glucocorticosteroid administration, in parallel with the general reduction in inflammation. CONCLUSIONS:Identification of synovial fluid FOXP3+ Treg with varying intensities of CD25 opens up possibilities for thorough characterisation of this important T-cell subset in the inflammatory compartment. However, only scarce synovial membrane expression of FOXP3 was found even in the absence of overt inflammation, suggesting that the synovial membrane is a site that would benefit therapeutically from Treg expansion. 10.1136/ard.2008.100768
    Leonard Herzenberg (1931-2013). Roederer Mario Nature 10.1038/504034a
    GPR120 Inhibits Colitis Through Regulation of CD4 T Cell Interleukin 10 Production. Yang Wenjing,Liu Han,Xu Leiqi,Yu Tianming,Zhao Xiaojing,Yao Suxia,Zhao Qihong,Barnes Sean,Cohn Steven M,Dann Sara M,Zhang Hongjie,Zuo Xiuli,Li Yanqing,Cong Yingzi Gastroenterology BACKGROUND & AIMS:G protein-coupled receptor (GPR) 120 has been implicated in regulating metabolic syndromes with anti-inflammatory function. However, the role of GPR120 in intestinal inflammation is unknown. Here, we investigated whether and how GPR120 regulates CD4 T cell function to inhibit colitis development. METHODS:Dextran sodium sulfate (DSS)-induced colitis model, Citrobacter rodentium infection model, and CD4 T cell adoptive transfer model were used to analyze the role of GPR120 in regulating colitis development. The effect of GPR120 on CD4 T cell functions was analyzed by RNA sequencing, flow cytometry, and Seahorse metabolic assays. Mice were administered GPR120 agonist for investigating the potential of GPR120 agonist in preventing and treating colitis. RESULTS:Deficiency of GPR120 in CD4 T cells resulted in more severe colitis in mice upon dextran sodium sulfate insult and enteric infection. Transfer of GPR120-deficient CD4CD45Rb T cells induced more severe colitis in Rag mice with lower intestinal interleukin (IL) 10CD4 T cells. Treatment with the GPR120 agonist CpdA promoted CD4 T cell production of IL10 by up-regulating Blimp1 and enhancing glycolysis, which was regulated by mTOR. GPR120 agonist-treated wild-type, but not IL10-deficient and Blimp1-deficient, T helper 1 cells induced less severe colitis. Furthermore, oral administration of GPR120 agonist protected mice from intestinal inflammation in both prevention and treatment schemes. Gpr120 expression was positively correlated with Il10 expression in the human colonic mucosa, including patients with inflammatory bowel diseases. CONCLUSIONS:Our findings show the role of GPR120 in regulating intestinal CD4 T cell production of IL10 to inhibit colitis development, which identifies GPR120 as a potential therapeutic target for treating inflammatory bowel diseases. 10.1053/j.gastro.2021.09.018
    Delivering nanoparticles to lungs while avoiding liver and spleen through adsorption on red blood cells. Anselmo Aaron C,Gupta Vivek,Zern Blaine J,Pan Daniel,Zakrewsky Michael,Muzykantov Vladimir,Mitragotri Samir ACS nano Nanoparticulate drug delivery systems are one of the most widely investigated approaches for developing novel therapies for a variety of diseases. However, rapid clearance and poor targeting limit their clinical utility. Here, we describe an approach to harness the flexibility, circulation, and vascular mobility of red blood cells (RBCs) to simultaneously overcome these limitations (cellular hitchhiking). A noncovalent attachment of nanoparticles to RBCs simultaneously increases their level in blood over a 24 h period and allows transient accumulation in the lungs, while reducing their uptake by liver and spleen. RBC-adsorbed nanoparticles exhibited ∼3-fold increase in blood persistence and ∼7-fold higher accumulation in lungs. RBC-adsorbed nanoparticles improved lung/liver and lung/spleen nanoparticle accumulation by over 15-fold and 10-fold, respectively. Accumulation in lungs is attributed to mechanical transfer of particles from the RBC surface to lung endothelium. Independent tracing of both nanoparticles and RBCs in vivo confirmed that RBCs themselves do not accumulate in lungs. Attachment of anti-ICAM-1 antibody to the exposed surface of NPs that were attached to RBCs led to further increase in lung targeting and retention over 24 h. Cellular hitchhiking onto RBCs provides a new platform for improving the blood pharmacokinetics and vascular delivery of nanoparticles while simultaneously avoiding uptake by liver and spleen, thus opening the door for new applications. 10.1021/nn404853z
    Natural variation in the parameters of innate immune cells is preferentially driven by genetic factors. Patin Etienne,Hasan Milena,Bergstedt Jacob,Rouilly Vincent,Libri Valentina,Urrutia Alejandra,Alanio Cécile,Scepanovic Petar,Hammer Christian,Jönsson Friederike,Beitz Benoît,Quach Hélène,Lim Yoong Wearn,Hunkapiller Julie,Zepeda Magge,Green Cherie,Piasecka Barbara,Leloup Claire,Rogge Lars,Huetz François,Peguillet Isabelle,Lantz Olivier,Fontes Magnus,Di Santo James P,Thomas Stéphanie,Fellay Jacques,Duffy Darragh,Quintana-Murci Lluís,Albert Matthew L, Nature immunology The quantification and characterization of circulating immune cells provide key indicators of human health and disease. To identify the relative effects of environmental and genetic factors on variation in the parameters of innate and adaptive immune cells in homeostatic conditions, we combined standardized flow cytometry of blood leukocytes and genome-wide DNA genotyping of 1,000 healthy, unrelated people of Western European ancestry. We found that smoking, together with age, sex and latent infection with cytomegalovirus, were the main non-genetic factors that affected variation in parameters of human immune cells. Genome-wide association studies of 166 immunophenotypes identified 15 loci that showed enrichment for disease-associated variants. Finally, we demonstrated that the parameters of innate cells were more strongly controlled by genetic variation than were those of adaptive cells, which were driven by mainly environmental exposure. Our data establish a resource that will generate new hypotheses in immunology and highlight the role of innate immunity in susceptibility to common autoimmune diseases. 10.1038/s41590-018-0049-7
    Telomere analysis by fluorescence in situ hybridization and flow cytometry. Hultdin M,Grönlund E,Norrback K,Eriksson-Lindström E,Just T,Roos G Nucleic acids research Determination of telomere length is traditionally performed by Southern blotting and densitometry, giving a mean telomere restriction fragment (TRF) value for the total cell population studied. Fluorescence in situ hybridization (FISH) of telomere repeats has been used to calculate telomere length, a method called quantitative (Q)-FISH. We here present a quantitative flow cytometric approach, Q-FISHFCM, for evaluation of telomere length distribution in individual cells based on in situ hybridization using a fluorescein-labeled peptide nucleic acid (PNA) (CCCTAA)3probe and DNA staining with propidium iodide. A simple and rapid protocol with results within 30 h was developed giving high reproducibility. One important feature of the protocol was the use of an internal cell line control, giving an automatic compensation for potential differences in the hybridization steps. This protocol was tested successfully on cell lines and clinical samples from bone marrow, blood, lymph nodes and tonsils. A significant correlation was found between Southern blotting and Q-FISHFCMtelomere length values ( P = 0.002). The mean sub-telomeric DNA length of the tested cell lines and clinical samples was estimated to be 3.2 kbp. With the Q-FISHFCMmethod the fluorescence signal could be determined in different cell cycle phases, indicating that in human cells the vast majority of telomeric DNA is replicated early in S phase. 10.1093/nar/26.16.3651