An array of 60,000 antibodies for proteome-scale antibody generation and target discovery.
Wang Zhaohui,Li Yang,Hou Bing,Pronobis Mira I,Wang Mingqiao,Wang Yuemeng,Cheng Guangcun,Weng Weining,Wang Yiqiang,Tang Yanfang,Xu Xuefan,Pan Rong,Lin Fei,Wang Nan,Chen Ziqing,Wang Shiwei,Ma Luyan Zulie,Li Yangrui,Huang Dongliang,Jiang Li,Wang Zhiqiang,Zeng Wenfang,Zhang Ying,Du Xuemei,Lin Ying,Li Zhiqing,Xia Qingyou,Geng Jing,Dai Huaping,Yu Yuan,Zhao Xiao-Dong,Yuan Zheng,Yan Jian,Nie Qinghua,Zhang Xiquan,Wang Kun,Chen Fulin,Zhang Qin,Zhu Yuxian,Zheng Susan,Poss Kenneth D,Tao Sheng-Ce,Meng Xun
Antibodies are essential for elucidating gene function. However, affordable technology for proteome-scale antibody generation does not exist. To address this, we developed Proteome Epitope Tag Antibody Library (PETAL) and its array. PETAL consists of 62,208 monoclonal antibodies (mAbs) against 15,199 peptides from diverse proteomes. PETAL harbors binders for a great multitude of proteins in nature due to antibody multispecificity, an intrinsic antibody feature. Distinctive combinations of 10,000 to 20,000 mAbs were found to target specific proteomes by array screening. Phenotype-specific mAb-protein pairs were found for maize and zebrafish samples. Immunofluorescence and flow cytometry mAbs for membrane proteins and chromatin immunoprecipitation-sequencing mAbs for transcription factors were identified from respective proteome-binding PETAL mAbs. Differential screening of cell surface proteomes of tumor and normal tissues identified internalizing tumor antigens for antibody-drug conjugates. By finding high-affinity mAbs at a fraction of current time and cost, PETAL enables proteome-scale antibody generation and target discovery.
Blocking integrin αβ-mediated CD4 T cell recruitment to the intestine and liver protects mice from western diet-induced non-alcoholic steatohepatitis.
Rai Ravi P,Liu Yunshan,Iyer Smita S,Liu Silvia,Gupta Biki,Desai Chirayu,Kumar Pradeep,Smith Tekla,Singhi Aatur D,Nusrat Asma,Parkos Charles A,Monga Satdarshan P,Czaja Mark J,Anania Frank A,Raeman Reben
Journal of hepatology
BACKGROUND & AIMS:The heterodimeric integrin receptor αβ regulates CD4 T cell recruitment to inflamed tissues, but its role in the pathogenesis of non-alcoholic steatohepatitis (NASH) is unknown. Herein, we examined the role of αβ-mediated recruitment of CD4 T cells to the intestine and liver in NASH. METHODS:Male littermate F11r (control) and junctional adhesion molecule A knockout F11r mice were fed a normal diet or a western diet (WD) for 8 weeks. Liver and intestinal tissues were analyzed by histology, quantitative reverse transcription PCR (qRT-PCR), 16s rRNA sequencing and flow cytometry. Colonic mucosa-associated microbiota were analyzed using 16s rRNA sequencing. Liver biopsies from patients with NASH were analyzed by confocal imaging and qRT-PCR. RESULTS:WD-fed knockout mice developed NASH and had increased hepatic and intestinal αβ CD4 T cells relative to control mice who developed mild hepatic steatosis. The increase in αβ CD4 T cells was associated with markedly higher expression of the αβ ligand mucosal addressin cell adhesion molecule 1 (MAdCAM-1) in the colonic mucosa and livers of WD-fed knockout mice. Elevated MAdCAM-1 expression correlated with increased mucosa-associated Proteobacteria in the WD-fed knockout mice. Antibiotics reduced MAdCAM-1 expression indicating that the diet-altered microbiota promoted colonic and hepatic MAdCAM-1 expression. αβ blockade in WD-fed knockout mice significantly decreased αβ CD4 T cell recruitment to the intestine and liver, attenuated hepatic inflammation and fibrosis, and improved metabolic indices. MAdCAM-1 blockade also reduced hepatic inflammation and fibrosis in WD-fed knockout mice. Hepatic MAdCAM-1 expression was elevated in patients with NASH and correlated with higher expression of α and β integrins. CONCLUSIONS:These findings establish αβ/MAdCAM-1 as a critical axis regulating NASH development through colonic and hepatic CD4 T cell recruitment. LAY SUMMARY:Non-alcoholic steatohepatitis (NASH) is an advanced and progressive form of non-alcoholic fatty liver disease (NAFLD), and despite its growing incidence no therapies currently exist to halt NAFLD progression. Herein, we show that blocking integrin receptor αβ-mediated recruitment of CD4 T cells to the intestine and liver not only attenuates hepatic inflammation and fibrosis, but also improves metabolic derangements associated with NASH. These findings provide evidence for the potential therapeutic application of αβ antibody in the treatment of human NASH.
Targeting CD70 with cusatuzumab eliminates acute myeloid leukemia stem cells in patients treated with hypomethylating agents.
Riether Carsten,Pabst Thomas,Höpner Sabine,Bacher Ulrike,Hinterbrandner Magdalena,Banz Yara,Müller Rouven,Manz Markus G,Gharib Walid H,Francisco David,Bruggmann Remy,van Rompaey Luc,Moshir Mahan,Delahaye Tim,Gandini Domenica,Erzeel Ellen,Hultberg Anna,Fung Samson,de Haard Hans,Leupin Nicolas,Ochsenbein Adrian F
Acute myeloid leukemia (AML) is driven by leukemia stem cells (LSCs) that resist conventional chemotherapy and are the major cause of relapse. Hypomethylating agents (HMAs) are the standard of care in the treatment of older or unfit patients with AML, but responses are modest and not durable. Here we demonstrate that LSCs upregulate the tumor necrosis factor family ligand CD70 in response to HMA treatment resulting in increased CD70/CD27 signaling. Blocking CD70/CD27 signaling and targeting CD70-expressing LSCs with cusatuzumab, a human αCD70 monoclonal antibody with enhanced antibody-dependent cellular cytotoxicity activity, eliminated LSCs in vitro and in xenotransplantation experiments. Based on these preclinical results, we performed a phase 1/2 trial in previously untreated older patients with AML with a single dose of cusatuzumab monotherapy followed by a combination therapy with the HMA azacitidine ( NCT03030612 ). We report results from the phase 1 dose escalation part of the clinical trial. Hematological responses in the 12 patients enrolled included 8 complete remission, 2 complete remission with incomplete blood count recovery and 2 partial remission with 4 patients achieving minimal residual disease negativity by flow cytometry at <10. Median time to response was 3.3 months. Median progression-free survival was not reached yet at the time of the data cutoff. No dose-limiting toxicities were reported and the maximum tolerated dose of cusatuzumab was not reached. Importantly, cusatuzumab treatment substantially reduced LSCs and triggered gene signatures related to myeloid differentiation and apoptosis.
Dysregulation of the IL-13 receptor system: a novel pathomechanism in pulmonary arterial hypertension.
Hecker Matthias,Zaslona Zbigniew,Kwapiszewska Grazyna,Niess Gero,Zakrzewicz Anna,Hergenreider Eduard,Wilhelm Jochen,Marsh Leigh M,Sedding Daniel,Klepetko Walter,Lohmeyer Jürgen,Dimmeler Stefanie,Seeger Werner,Weissmann Norbert,Schermuly Ralph T,Kneidinger Nikolaus,Eickelberg Oliver,Morty Rory E
American journal of respiratory and critical care medicine
RATIONALE:Idiopathic pulmonary arterial hypertension (IPAH) is characterized by medial hypertrophy due to pulmonary artery smooth muscle cell (paSMC) hyperplasia. Inflammation is proposed to play a role in vessel remodeling associated with IPAH. IL-13 is emerging as a regulator of tissue remodeling; however, the contribution of the IL-13 system to IPAH has not been assessed. OBJECTIVES:The objective of this study was to assess the possible contribution of the IL-13 system to IPAH. METHODS:Expression and localization of IL-13, and IL-13 receptors IL-4R, IL-13Rα1, and IL-13Rα2 were assessed by real-time reverse transcription-polymerase chain reaction, immunohistochemistry, and flow cytometry in lung tissue, paSMC, and microdissected vascular lesions from patients with IPAH, and in lung tissue from rodents with hypoxia- or monocrotaline-induced pulmonary hypertension. A whole-genome microarray analysis was used to study IL-13-regulated genes in paSMC. MEASUREMENTS AND MAIN RESULTS:Pulmonary expression of the IL-13 decoy receptor IL-13Rα2 was up-regulated relative to that of the IL-13 signaling receptors IL-4R and IL-13Rα1 in patients with IPAH and in two animal models of IPAH. IL-13, signaling via STAT3 and STAT6, suppressed proliferation of paSMC by promoting G(0)/G(1) arrest. Whole-genome microarrays revealed that IL-13 suppressed endothelin-1 production by paSMC, suggesting that IL-13 controlled paSMC growth by regulating endothelin production. Ectopic expression of the il13ra2 gene resulted in partial loss of paSMC growth control by IL-13 and blunted IL-13 suppression of endothelin-1 production by paSMC, whereas small-interfering RNA knockdown of il13ra2 gene expression had the opposite effects. CONCLUSIONS:The IL-13 system is a novel regulator of paSMC growth. Dysregulation of IL-13 receptor expression in IPAH may partially underlie smooth muscle hypertrophy associated with pathological vascular remodeling in IPAH.
Impact of red blood cell transfusion on platelet activation and aggregation in healthy volunteers: results of the TRANSFUSION study.
Silvain Johanne,Pena Ana,Cayla Guillaume,Brieger David,Bellemain-Appaix Anne,Chastre Thomas,Vignalou Jean-Baptiste,Beygui Farzin,Barthelemy Olivier,Collet Jean-Philippe,Montalescot Gilles
European heart journal
AIMS:The underlying mechanisms leading to recurrent ischaemic events or mortality after red blood cell (RBC) transfusion in anaemic acute coronary syndrome patients are poorly understood. The aim of this paper is to determine whether RBC transfusion increases platelet activation and aggregation. METHODS AND RESULTS:In vitro transfusions (n = 45) were performed by the addition of RBCs obtained from transfusion packs to fresh whole blood provided by healthy volunteers. Residual platelet aggregation (RPA) and maximal platelet aggregation (MPA) were assessed before and after in vitro transfusion using light transmission aggregometry performed with four different agonists. Flow cytometry was used for the measurement of P-selectin expression and vasodilatator-stimulated phosphoprotein (VASP) platelet reactivity index (PRI). To control for the effect of haemoconcentration, the same experiments were repeated after hematocrit adjustment using volunteer's platelet poor plasma. Transfusion increased platelet aggregation as measured by RPA with ADP 5 µM (57.7 ± 25 vs. 65.7 ± 24%; P = 0.03) or Collagen 2 µg/mL (59.4 ± 28 vs. 69.7 ± 24%; P = 0.03). There were no significant differences with Arachidonic Acid 1.25 mM or Epinephrine 20 µM and results were similar when MPA was considered. Platelet activation was also increased by transfusion as confirmed by an elevation of P-selectin expression induced by 20 µM ADP (12.2 ± 18 vs. 23.9 ± 18%; P = 0.002) or 50 µM ADP (15.4 ± 18.6 vs.26.8 ± 21.2%; P = 0.004) and an increase in VASP PRI (77.8 ± 6 vs. 81.9 ± 3%; P = 0.03). These effects were all independent of hematocrit. CONCLUSION:Red blood cell transfusion increases platelet activation and aggregation in vitro in healthy volunteers. This effect might be mediated through the P2Y(12) activation pathway.
JAK selectivity and the implications for clinical inhibition of pharmacodynamic cytokine signalling by filgotinib, upadacitinib, tofacitinib and baricitinib.
Traves Paqui G,Murray Bernard,Campigotto Federico,Galien René,Meng Amy,Di Paolo Julie A
Annals of the rheumatic diseases
OBJECTIVE:Janus kinase inhibitors (JAKinibs) are efficacious in rheumatoid arthritis (RA) with variable reported rates of adverse events, potentially related to differential JAK family member selectivity. Filgotinib was compared with baricitinib, tofacitinib and upadacitinib to elucidate the pharmacological basis underlying its clinical efficacy and safety. METHODS:In vitro JAKinib inhibition of signal transducer and activator of transcription phosphorylation (pSTAT) was measured by flow cytometry in peripheral blood mononuclear cells and whole blood from healthy donors and patients with RA following cytokine stimulation of distinct JAK/STAT pathways. The average daily pSTAT and time above 50% inhibition were calculated at clinical plasma drug exposures in immune cells. The translation of these measures was evaluated in ex vivo-stimulated assays in phase 1 healthy volunteers. RESULTS:JAKinib potencies depended on cytokine stimulus, pSTAT readout and cell type. JAK1-dependent pathways (interferon (IFN)α/pSTAT5, interleukin (IL)-6/pSTAT1) were among the most potently inhibited by all JAKinibs in healthy and RA blood, with filgotinib exhibiting the greatest selectivity for JAK1 pathways. Filgotinib (200 mg once daily) had calculated average daily target inhibition for IFNα/pSTAT5 and IL-6/pSTAT1 that was equivalent to tofacitinib (5 mg two times per day), upadacitinib (15 mg once daily) and baricitinib (4 mg once daily), with the least average daily inhibition for the JAK2-dependent and JAK3-dependent pathways including IL-2, IL-15, IL-4 (JAK1/JAK3), IFNγ (JAK1/JAK2), granulocyte colony stimulating factor, IL-12, IL-23 (JAK2/tyrosine kinase 2) and granulocyte-macrophage colony-stimulating factor (JAK2/JAK2). Ex vivo pharmacodynamic data from phase 1 healthy volunteers clinically confirmed JAK1 selectivity of filgotinib. CONCLUSION:Filgotinib inhibited JAK1-mediated signalling similarly to other JAKinibs, but with less inhibition of JAK2-dependent and JAK3-dependent pathways, providing a mechanistic rationale for its apparently differentiated efficacy:safety profile.
Heparin-binding, hemagglutinin-specific IFN-gamma synthesis at the site of infection during active tuberculosis in humans.
Place Sammy,Verscheure Virginie,de San Nour,Hougardy Jean-Michel,Schepers Kinda,Dirix Violette,Dediste Anne,Michel Olivier,Drowart Annie,Allard Sabine D,Doherty T Mark,Lecher Sophie,Locht Camille,Mascart Françoise
American journal of respiratory and critical care medicine
RATIONALE:Tuberculosis (TB) remains a major cause of mortality. A better understanding of the immune responses to mycobacterial antigens may be helpful to develop improved vaccines and diagnostics. OBJECTIVES:The mycobacterial antigen heparin-binding hemagglutinin (HBHA) induces strong IFN-γ responses by circulating lymphocytes from subjects latently infected with Mycobacterium tuberculosis, and low responses associated with CD4(+) regulatory T (Treg) cells in patients with TB. Here, we investigated HBHA-specific IFN-γ responses at the site of the TB disease. METHODS:Bronchoalveolar lavages, pleural fluids, and blood were prospectively collected from 61 patients with a possible diagnosis of pulmonary or pleural TB. HBHA-specific IFN-γ production was analyzed by flow cytometry and ELISA. The suppressive effect of pleural Treg cells was investigated by depletion experiments. MEASUREMENTS AND MAIN RESULTS:The percentages of HBHA-induced IFN-γ(+) alveolar and pleural lymphocytes were higher for pulmonary (P < 0.0001) and for pleural (P < 0.01) TB than for non-TB controls. Local CD4(+) and CD8(+) T cells produced the HBHA-specific IFN-γ. This local secretion was not suppressed by Treg lymphocytes, contrasting with previously reported data on circulating lymphocytes. CONCLUSIONS:Patients with TB display differential effector and regulatory T-cell responses to HBHA in local and circulating lymphocytes with a predominant effector CD4(+) and CD8(+) response locally, compared with a predominant Treg response among circulating lymphocytes. These findings may be helpful for the design of new vaccines against TB, and the detection of HBHA-specific T cells at the site of the infection may be a promising tool for the rapid diagnosis of active TB.
Stochastic asymmetric repartition of lytic machinery in dividing CD8 T cells generates heterogeneous killing behavior.
Lafouresse Fanny,Jugele Romain,Müller Sabina,Doineau Marine,Duplan-Eche Valérie,Espinosa Eric,Puisségur Marie-Pierre,Gadat Sébastien,Valitutti Salvatore
Cytotoxic immune cells are endowed with a high degree of heterogeneity in their lytic function, but how this heterogeneity is generated is still an open question. We therefore investigated if human CD8 T cells could segregate their lytic components during telophase, using imaging flow cytometry, confocal microscopy, and live-cell imaging. We show that CD107a-intracellular vesicles, perforin, and granzyme B unevenly segregate in a constant fraction of telophasic cells during each division round. Mathematical modeling posits that unequal lytic molecule inheritance by daughter cells results from the random distribution of lytic granules on the two sides of the cleavage furrow. Finally, we establish that the level of lytic compartment in individual cytotoxic T lymphocyte (CTL) dictates CTL killing capacity.
Density of Conjugated Antibody Determines the Extent of Fc Receptor Dependent Capture of Nanoparticles by Liver Sinusoidal Endothelial Cells.
Kappel Cinja,Seidl Christine,Medina-Montano Carolina,Schinnerer Meike,Alberg Irina,Leps Christian,Sohl Julian,Hartmann Ann-Kathrin,Fichter Michael,Kuske Michael,Schunke Jenny,Kuhn Gabor,Tubbe Ingrid,Paßlick David,Hobernik Dominika,Bent Rebekka,Haas Katharina,Montermann Evelyn,Walzer Kerstin,Diken Mustafa,Schmidt Manfred,Zentel Rudolf,Nuhn Lutz,Schild Hansjörg,Tenzer Stefan,Mailänder Volker,Barz Matthias,Bros Matthias,Grabbe Stephan
Despite considerable progress in the design of multifunctionalized nanoparticles (NPs) that selectively target specific cell types, their systemic application often results in unwanted liver accumulation. The exact mechanisms for this general observation are still unclear. Here we asked whether the number of cell-targeting antibodies per NP determines the extent of NP liver accumulation and also addressed the mechanisms by which antibody-coated NPs are retained in the liver. We used polysarcosine-based peptobrushes (PBs), which in an unmodified form remain in the circulation for >24 h due to the absence of a protein corona formation and low unspecific cell binding, and conjugated them with specific average numbers (2, 6, and 12) of antibodies specific for the dendritic cell (DC) surface receptor, DEC205. We assessed the time-dependent biodistribution of PB-antibody conjugates by imaging and flow cytometry. We observed that PB-antibody conjugates were trapped in the liver and that the extent of liver accumulation strongly increased with the number of attached antibodies. PB-antibody conjugates were selectively captured in the liver Fc receptors (FcR) on liver sinusoidal endothelial cells, since systemic administration of FcR-blocking agents or the use of F(ab') fragments prevented liver accumulation. Cumulatively, our study demonstrates that liver endothelial cells play a yet scarcely acknowledged role in liver entrapment of antibody-coated NPs and that low antibody numbers on NPs and the use of F(ab') antibody fragments are both sufficient for cell type-specific targeting of secondary lymphoid organs and necessary to minimize unwanted liver accumulation.
Gut microbiota metabolism of dietary fiber influences allergic airway disease and hematopoiesis.
Trompette Aurélien,Gollwitzer Eva S,Yadava Koshika,Sichelstiel Anke K,Sprenger Norbert,Ngom-Bru Catherine,Blanchard Carine,Junt Tobias,Nicod Laurent P,Harris Nicola L,Marsland Benjamin J
Metabolites from intestinal microbiota are key determinants of host-microbe mutualism and, consequently, the health or disease of the intestinal tract. However, whether such host-microbe crosstalk influences inflammation in peripheral tissues, such as the lung, is poorly understood. We found that dietary fermentable fiber content changed the composition of the gut and lung microbiota, in particular by altering the ratio of Firmicutes to Bacteroidetes. The gut microbiota metabolized the fiber, consequently increasing the concentration of circulating short-chain fatty acids (SCFAs). Mice fed a high-fiber diet had increased circulating levels of SCFAs and were protected against allergic inflammation in the lung, whereas a low-fiber diet decreased levels of SCFAs and increased allergic airway disease. Treatment of mice with the SCFA propionate led to alterations in bone marrow hematopoiesis that were characterized by enhanced generation of macrophage and dendritic cell (DC) precursors and subsequent seeding of the lungs by DCs with high phagocytic capacity but an impaired ability to promote T helper type 2 (TH2) cell effector function. The effects of propionate on allergic inflammation were dependent on G protein-coupled receptor 41 (GPR41, also called free fatty acid receptor 3 or FFAR3), but not GPR43 (also called free fatty acid receptor 2 or FFAR2). Our results show that dietary fermentable fiber and SCFAs can shape the immunological environment in the lung and influence the severity of allergic inflammation.
Mfge8 promotes obesity by mediating the uptake of dietary fats and serum fatty acids.
Khalifeh-Soltani Amin,McKleroy William,Sakuma Stephen,Cheung Yuk Yin,Tharp Kevin,Qiu Yifu,Turner Scott M,Chawla Ajay,Stahl Andreas,Atabai Kamran
Fatty acids are integral mediators of energy storage, membrane formation and cell signaling. The pathways that orchestrate uptake of fatty acids remain incompletely understood. Expression of the integrin ligand Mfge8 is increased in human obesity and in mice on a high-fat diet, but its role in obesity is unknown. We show here that Mfge8 promotes the absorption of dietary triglycerides and the cellular uptake of fatty acid and that Mfge8-deficient (Mfge8(-/-)) mice are protected from diet-induced obesity, steatohepatitis and insulin resistance. Mechanistically, we found that Mfge8 coordinates fatty acid uptake through αvβ3 integrin- and αvβ5 integrin-dependent phosphorylation of Akt by phosphatidylinositide-3 kinase and mTOR complex 2, leading to translocation of Cd36 and Fatp1 from cytoplasmic vesicles to the cell surface. Collectively, our results imply a role for Mfge8 in regulating the absorption and storage of dietary fats, as well as in the development of obesity and its complications.
Bacterial symbiont subpopulations have different roles in a deep-sea symbiosis.
Hinzke Tjorven,Kleiner Manuel,Meister Mareike,Schlüter Rabea,Hentschker Christian,Pané-Farré Jan,Hildebrandt Petra,Felbeck Horst,Sievert Stefan M,Bonn Florian,Völker Uwe,Becher Dörte,Schweder Thomas,Markert Stephanie
The hydrothermal vent tubeworm hosts a single 16S rRNA phylotype of intracellular sulfur-oxidizing symbionts, which vary considerably in cell morphology and exhibit a remarkable degree of physiological diversity and redundancy, even in the same host. To elucidate whether multiple metabolic routes are employed in the same cells or rather in distinct symbiont subpopulations, we enriched symbionts according to cell size by density gradient centrifugation. Metaproteomic analysis, microscopy, and flow cytometry strongly suggest that symbiont cells of different sizes represent metabolically dissimilar stages of a physiological differentiation process: While small symbionts actively divide and may establish cellular symbiont-host interaction, large symbionts apparently do not divide, but still replicate DNA, leading to DNA endoreduplication. Moreover, in large symbionts, carbon fixation and biomass production seem to be metabolic priorities. We propose that this division of labor between smaller and larger symbionts benefits the productivity of the symbiosis as a whole.
Peripheral prepositioning and local CXCL9 chemokine-mediated guidance orchestrate rapid memory CD8+ T cell responses in the lymph node.
Kastenmüller Wolfgang,Brandes Marlene,Wang Ze,Herz Jasmin,Egen Jackson G,Germain Ronald N
After an infection, the immune system generates long-lived memory lymphocytes whose increased frequency and altered state of differentiation enhance host defense against reinfection. Recently, the spatial distribution of memory cells was found to contribute to their protective function. Effector memory CD8+ T cells reside in peripheral tissue sites of initial pathogen encounter, in apparent anticipation of reinfection. Here we show that within lymph nodes (LNs), memory CD8+ T cells were concentrated near peripheral entry portals of lymph-borne pathogens, promoting rapid engagement of infected sentinel macrophages. A feed-forward CXCL9-dependent circuit provided additional chemotactic cues that further increase local memory cell density. Memory CD8+ T cells also produced effector responses to local cytokine triggers, but their dynamic behavior differed from that seen after antigen recognition. These data reveal the distinct localization and dynamic behavior of naive versus memory T cells within LNs and how these differences contribute to host defense.
Luminal bacteria recruit CD103+ dendritic cells into the intestinal epithelium to sample bacterial antigens for presentation.
Farache Julia,Koren Idan,Milo Idan,Gurevich Irina,Kim Ki-Wook,Zigmond Ehud,Furtado Glaucia C,Lira Sergio A,Shakhar Guy
CD103+ dendritic cells (DCs) carry bacteria from the small intestine and can present antigens to T cells. Yet they have not been recorded sampling luminal bacteria or presenting bacterial antigens in mesentery lymph nodes. We used 2-photon microscopy in live Cx3cr1(+/gfp) ×Cd11c-YFP mice to study these processes. At steady state, sparse CD103+ DCs occupied the epithelium. They patrolled among enterocytes while extending dendrites toward the lumen, likely using tight-junction proteins to penetrate the epithelium. Challenge with Salmonella triggered chemokine- and toll-like receptor (TLR)-dependent recruitment of additional DCs from the lamina propria (LP). The DCs efficiently phagocytosed the bacteria using intraepithelial dendrites. Noninvasive bacteria were similarly sampled. In contrast, CD103+ DCs sampled soluble luminal antigen inefficiently. In mice harboring CD103+ DCs, antigen-specific CD8 T cells were subsequently activated in MLNs. Intestinal CD103+ DCs are therefore equipped with unique mechanisms to independently complete the processes of uptake, transportation, and presentation of bacterial antigens.
Inflammatory monocytes recruited to allergic skin acquire an anti-inflammatory M2 phenotype via basophil-derived interleukin-4.
Egawa Mayumi,Mukai Kaori,Yoshikawa Soichiro,Iki Misako,Mukaida Naofumi,Kawano Yohei,Minegishi Yoshiyuki,Karasuyama Hajime
Monocytes and macrophages are important effectors and regulators of inflammation, and both can be divided into distinct subsets based on their phenotypes. The developmental and functional relationship between individual subsets of monocytes and those of macrophages has not been fully elucidated, although Ly6C(+)CCR2(+) inflammatory and Ly6C(-)CCR2(-) resident monocytes are generally thought to differentiate into M1 (classically activated) and M2 (alternatively activated) macrophages, respectively. Here we show that inflammatory monocytes recruited to allergic skin acquired an M2-like phenotype in response to basophil-derived interleukin-4 (IL-4) and exerted an anti-inflammatory function. CCR2-deficient mice unexpectedly displayed an exacerbation rather than alleviation of allergic inflammation, in spite of impaired recruitment of inflammatory monocytes to skin lesions. Adoptive transfer of inflammatory monocytes from wild-type but not IL-4 receptor-deficient mice dampened the exacerbated inflammation in CCR2-deficient mice. Thus, inflammatory monocytes can be converted from being proinflammatory to anti-inflammatory under the influence of basophils in allergic reactions.
The BAFF receptor transduces survival signals by co-opting the B cell receptor signaling pathway.
Schweighoffer Edina,Vanes Lesley,Nys Josquin,Cantrell Doreen,McCleary Scott,Smithers Nicholas,Tybulewicz Victor L J
Follicular B cell survival requires signaling from BAFFR, a receptor for BAFF and the B cell antigen receptor (BCR). This "tonic" BCR survival signal is distinct from that induced by antigen binding and may be ligand-independent. We show that inducible inactivation of the Syk tyrosine kinase, a key signal transducer from the BCR following antigen binding, resulted in the death of most follicular B cells because Syk-deficient cells were unable to survive in response to BAFF. Genetic rescue studies demonstrated that Syk transduces BAFFR survival signals via ERK and PI3 kinase. Surprisingly, BAFFR signaling directly induced phosphorylation of both Syk and the BCR-associated Igα signaling subunit, and this Syk phosphorylation required the BCR. We conclude that the BCR and Igα may be required for B cell survival because they function as adaptor proteins in a BAFFR signaling pathway leading to activation of Syk, demonstrating previously unrecognized crosstalk between the two receptors.
Tumor necrosis factor-α blocks differentiation and enhances suppressive activity of immature myeloid cells during chronic inflammation.
Sade-Feldman Moshe,Kanterman Julia,Ish-Shalom Eliran,Elnekave Mazal,Horwitz Elad,Baniyash Michal
Elevated concentrations of tumor necrosis factor-α (TNF-α) are detected in pathologies characterized by chronic inflammation. Whether TNF-α plays a role in manipulating the host's immune system toward generating an immunosuppressive milieu, typical of ongoing chronic inflammation, is unclear. Here we showed that TNF-α exhibited a dual function during chronic inflammation: arresting differentiation of immature myeloid-derived suppressor cells (MDSCs) primarily via the S100A8 and S100A9 inflammatory proteins and their corresponding receptor (RAGE) and augmenting MDSC suppressive activity. These functions led to in vivo T and NK cell dysfunction accompanied by T cell antigen receptor ζ chain downregulation. Furthermore, administration of etanercept (TNF-α antagonist) during early chronic inflammatory stages reduced MDSCs' suppressive activity and enhanced their maturation into dendritic cells and macrophages, resulting in the restoration of in vivo immune functions and recovery of ζ chain expression. Thus, TNF has a fundamental role in promoting an immunosuppressive environment generated during chronic inflammation.
Persistent antigen and germinal center B cells sustain T follicular helper cell responses and phenotype.
Baumjohann Dirk,Preite Silvia,Reboldi Andrea,Ronchi Francesca,Ansel K Mark,Lanzavecchia Antonio,Sallusto Federica
T follicular helper (Tfh) cells provide help to B cells and are crucial for establishment of germinal center (GC) reactions, including production of high-affinity antibodies and generation of memory B cells and long-lived plasma cells. Here we report that the magnitude of the Tfh cell response was dictated by the amount of antigen and directly correlated with the magnitude of the GC B cell response. In addition, maintenance of the Tfh cell phenotype required sustained antigenic stimulation by GC B cells. In lymphopenic conditions, a strong and prolonged Tfh cell response led to bystander B cell activation, hypergammaglobulinemia, and production of poly- and self-reactive antibodies. These data demonstrate that antigen dose determines the size and duration of the Tfh cell response and GC reaction, highlight the transient nature of the Tfh cell phenotype, and suggest a link between overstimulation of Tfh cells and the development of dysregulated humoral immune responses.
Distinct roles for neutrophils and dendritic cells in inflammation and autoimmunity in motheaten mice.
Abram Clare L,Roberge Gray L,Pao Lily I,Neel Benjamin G,Lowell Clifford A
The motheaten mouse has long served as a paradigm for complex autoimmune and inflammatory disease. Null mutations in Ptpn6, which encodes the nonreceptor protein-tyrosine phosphatase Shp1, cause the motheaten phenotype. However, Shp1 regulates multiple signaling pathways in different hematopoietic cell types, so the cellular and molecular mechanism of autoimmunity and inflammation in the motheaten mouse has remained unclear. By using floxed Ptpn6 mice, we dissected the contribution of innate immune cells to the motheaten phenotype. Ptpn6 deletion in neutrophils resulted in cutaneous inflammation, but not autoimmunity, providing an animal model of human neutrophilic dermatoses. By contrast, dendritic cell deletion caused severe autoimmunity, without inflammation. Genetic and biochemical analysis showed that inflammation was caused by enhanced neutrophil integrin signaling through Src-family and Syk kinases, whereas autoimmunity resulted from exaggerated MyD88-dependent signaling in dendritic cells. Our data demonstrate that disruption of distinct Shp1-regulated pathways in different cell types combine to cause motheaten disease.
HIV-1 persistence in CD4+ T cells with stem cell-like properties.
Buzon Maria J,Sun Hong,Li Chun,Shaw Amy,Seiss Katherine,Ouyang Zhengyu,Martin-Gayo Enrique,Leng Jin,Henrich Timothy J,Li Jonathan Z,Pereyra Florencia,Zurakowski Ryan,Walker Bruce D,Rosenberg Eric S,Yu Xu G,Lichterfeld Mathias
Cellular HIV-1 reservoirs that persist despite antiretroviral treatment are incompletely defined. We show that during suppressive antiretroviral therapy, CD4(+) T memory stem cells (TSCM cells) harbor high per-cell levels of HIV-1 DNA and make increasing contributions to the total viral CD4(+) T cell reservoir over time. Moreover, we conducted phylogenetic studies that suggested long-term persistence of viral quasispecies in CD4(+) TSCM cells. Thus, HIV-1 may exploit the stem cell characteristics of cellular immune memory to promote long-term viral persistence.
Coordinated actions of SLX1-SLX4 and MUS81-EME1 for Holliday junction resolution in human cells.
Wyatt Haley D M,Sarbajna Shriparna,Matos Joao,West Stephen C
Holliday junctions (HJs) are four-way DNA intermediates that form during homologous recombination, and their efficient resolution is essential for chromosome segregation. Here, we show that three structure-selective endonucleases, namely SLX1-SLX4, MUS81-EME1, and GEN1, define two pathways of HJ resolution in human cells. One pathway is mediated by GEN1, whereas SLX1-SLX4 and MUS81-EME1 provide a second and genetically distinct pathway (SLX-MUS). Cells depleted for SLX-MUS or GEN1 pathway proteins exhibit severe defects in chromosome segregation and reduced survival. In response to CDK-mediated phosphorylation, SLX1-SLX4 and MUS81-EME1 associate at the G2/M transition to form a stable SLX-MUS holoenzyme, which can be reconstituted in vitro. Biochemical studies show that SLX-MUS is a HJ resolvase that coordinates the active sites of two distinct endonucleases during HJ resolution. This cleavage reaction is more efficient and orchestrated than that mediated by SLX1-SLX4 alone, which exhibits a potent nickase activity that acts promiscuously upon DNA secondary structures.
The chemokine receptor CCR9 is required for the T-cell-mediated regulation of chronic ileitis in mice.
Wermers Joshua D,McNamee Eoin N,Wurbel Marc-André,Jedlicka Paul,Rivera-Nieves Jesús
BACKGROUND & AIMS:A balance between effector and regulatory T-cell (Treg) responses is required to maintain intestinal homeostasis. To regulate immunity, T cells migrate to the intestine using a combination of adhesion molecules and chemokine receptors. However, it is not known whether the migration pathways of effector cells and Tregs are distinct or shared. We sought to determine whether interaction between the chemokine receptor 9 (CCR9) and its ligand, chemokine ligand 25 (CCL25), allows effectors or Tregs to localize to chronically inflamed small intestine. METHODS:By using a mouse model that develops Crohn's-like ileitis (tumor necrosis factor Δadenosine uracyl-rich element [TNFΔARE] mice) we examined the role of CCL25-CCR9 interactions for effector and Treg traffic using flow cytometry, quantitative reverse-transcription polymerase chain reaction, immunohistochemistry, immunoneutralization, and proliferation analyses. RESULTS:In TNFΔARE mice, expression of CCL25 and the frequency of CCR9-expressing lymphocytes increased during late-stage disease. In the absence of CCR9, TNFΔARE mice developed exacerbated disease, compared with their CCR9-sufficient counterparts, which coincided with a deficiency of CD4(+)/CD25(+)/forkhead box P3(+) and CD8(+)/CD103(+) Tregs within the intestinal lamina propria and mesenteric lymph nodes. Furthermore, the CD8(+)/CCR9(+) subset decreased the proliferation of CD4(+) T cells in vitro. Administration of a monoclonal antibody against CCR9 to TNFΔARE mice exacerbated ileitis in vivo, confirming the regulatory role of CD8(+)/CCR9(+) cells. CONCLUSIONS:Signaling of the chemokine CCL25 through its receptor CCR9 induces Tregs to migrate to the intestine. These findings raise concerns about the development of reagents to disrupt this pathway for the treatment of patients with Crohn's disease.
A glioblastoma neurosphere line with alternative lengthening of telomeres.
Heaphy Christopher M,Schreck Karisa C,Raabe Eric,Mao Xing Gang,An Ping,Chu Qian,Poh Weijie,Jiao Yuchen,Rodriguez Fausto J,Odia Yazmin,Meeker Alan K,Eberhart Charles G
Single-Cell Transcriptome Analysis of Colon Cancer Cell Response to 5-Fluorouracil-Induced DNA Damage.
Park Sung Rye,Namkoong Sim,Friesen Leon,Cho Chun-Seok,Zhang Zac Zezhi,Chen Yu-Chih,Yoon Euisik,Kim Chang H,Kwak Hojoong,Kang Hyun Min,Lee Jun Hee
DNA damage often induces heterogeneous cell-fate responses, such as cell-cycle arrest and apoptosis. Through single-cell RNA sequencing (scRNA-seq), we characterize the transcriptome response of cultured colon cancer cell lines to 5-fluorouracil (5FU)-induced DNA damage. After 5FU treatment, a single population of colon cancer cells adopts three distinct transcriptome phenotypes, which correspond to diversified cell-fate responses: apoptosis, cell-cycle checkpoint, and stress resistance. Although some genes are regulated uniformly across all groups of cells, many genes showed group-specific expression patterns mediating DNA damage responses specific to the corresponding cell fate. Some of these observations are reproduced at the protein level by flow cytometry and are replicated in cells treated with other 5FU-unrelated genotoxic drugs, camptothecin and etoposide. This work provides a resource for understanding heterogeneous DNA damage responses involving fractional killing and chemoresistance, which are among the major challenges in current cancer chemotherapy.
In vivo labeling reveals continuous trafficking of TCF-1+ T cells between tumor and lymphoid tissue.
The Journal of experimental medicine
Improving the efficacy of immune checkpoint therapies will require a better understanding of how immune cells are recruited and sustained in tumors. Here, we used the photoconversion of the tumor immune cell compartment to identify newly entering lymphocytes, determine how they change over time, and investigate their egress from the tumor. Combining single-cell transcriptomics and flow cytometry, we found that while a diverse mix of CD8 T cell subsets enter the tumor, all CD8 T cells retained within this environment for more than 72 h developed an exhausted phenotype, revealing the rapid establishment of this program. Rather than forming tumor-resident populations, non-effector subsets, which express TCF-1 and include memory and stem-like cells, were continuously recruited into the tumor, but this recruitment was balanced by concurrent egress to the tumor-draining lymph node. Thus, the TCF-1+ CD8 T cell niche in tumors is highly dynamic, with the circulation of cells between the tumor and peripheral lymphoid tissue to bridge systemic and intratumoral responses.
The cytokines IL-21 and GM-CSF have opposing regulatory roles in the apoptosis of conventional dendritic cells.
Wan Chi-Keung,Oh Jangsuk,Li Peng,West Erin E,Wong Elizabeth A,Andraski Allison B,Spolski Rosanne,Yu Zu-Xi,He Jianping,Kelsall Brian L,Leonard Warren J
Interleukin-21 (IL-21) has broad actions on T and B cells, but its actions in innate immunity are poorly understood. Here we show that IL-21 induced apoptosis of conventional dendritic cells (cDCs) via STAT3 and Bim, and this was inhibited by granulocyte-macrophage colony-stimulating factor (GM-CSF). ChIP-Seq analysis revealed genome-wide binding competition between GM-CSF-induced STAT5 and IL-21-induced STAT3. Expression of IL-21 in vivo decreased cDC numbers, and this was prevented by GM-CSF. Moreover, repetitive α-galactosylceramide injection of mice induced IL-21 but decreased GM-CSF production by natural killer T (NKT) cells, correlating with decreased cDC numbers. Furthermore, adoptive transfer of wild-type CD4+ T cells caused more severe colitis with increased DCs and interferon-γ (IFN-γ)-producing CD4+ T cells in Il21r(-/-)Rag2(-/-) mice (which lack T cells and have IL-21-unresponsive DCs) than in Rag2(-/-) mice. Thus, IL-21 and GM-CSF exhibit cross-regulatory actions on gene regulation and apoptosis, regulating cDC numbers and thereby the magnitude of the immune response.
Signals via the adaptor MyD88 in B cells and DCs make distinct and synergistic contributions to immune activation and tissue damage in lupus.
Teichmann Lino L,Schenten Dominik,Medzhitov Ruslan,Kashgarian Michael,Shlomchik Mark J
Detection of self nucleic acids by Toll-like receptors (TLR) preciptates autoimmune diseases, including systemic lupus erythematosus (SLE). It remains unknown how TLR signals in specific cell types contribute to distinct manifestations of SLE. Here, we demonstrate that formation of anti-nuclear antibodies in MRL.Fas(lpr) mice entirely depends on the TLR signaling adaptor MyD88 in B cells. Further, MyD88 deficiency in B cells ameliorated nephritis, including antibody-independent interstitial T cell infiltrates, suggesting that nucleic acid-specific B cells activate nephrotoxic T cells. Surprisingly, MyD88 deletion in dendritic cells (DCs) did not affect nephritis, despite the importance of DCs in renal inflammation. In contrast, MyD88 in DCs was critical for dermatitis, revealing a separate pathogenetic mechanism. DC-expressed MyD88 promoted interferon-α production by plasmacytoid DCs, which was associated with Death domain-associated protein 6 upregulation and B lymphopenia. Our findings thus reveal unique immunopathological consequences of MyD88 signaling in B cells and DCs in lupus.
The actin and tetraspanin networks organize receptor nanoclusters to regulate B cell receptor-mediated signaling.
Mattila Pieta K,Feest Christoph,Depoil David,Treanor Bebhinn,Montaner Beatriz,Otipoby Kevin L,Carter Robert,Justement Louis B,Bruckbauer Andreas,Batista Facundo D
A key role is emerging for the cytoskeleton in coordinating receptor signaling, although the underlying molecular requirements remain unclear. Here we show that cytoskeleton disruption triggered signaling requiring not only the B cell receptor (BCR), but also the coreceptor CD19 and tetraspanin CD81, thus providing a mechanism for signal amplification upon surface-bound antigen stimulation. By using superresolution microscopy, we demonstrated that endogenous IgM, IgD, and CD19 exhibited distinct nanoscale organization within the plasma membrane of primary B cells. Upon stimulation, we detect a local convergence of receptors, although their global organization was not dramatically altered. Thus, we postulate that cytoskeleton reorganization releases BCR nanoclusters, which can interact with CD19 held in place by the tetraspanin network. These results not only suggest that receptor compartmentalization regulates antigen-induced activation but also imply a potential role for CD19 in mediating ligand-independent "tonic" BCR signaling necessary for B cell survival.
High-affinity neoantigens correlate with better prognosis and trigger potent antihepatocellular carcinoma (HCC) activity by activating CD39CD8 T cells.
Liu Ting,Tan Jizhou,Wu Minhao,Fan Wenzhe,Wei Jialiang,Zhu Bowen,Guo Jian,Wang Shutong,Zhou Penghui,Zhang Hui,Shi Liangrong,Li Jiaping
OBJECTIVE:It remains controversial whether tumour mutational burden (TMB) or neoantigens are prognostic markers in hepatocellular carcinoma (HCC). This study aimed to define the function of TMB or neoantigens in antitumour immunotherapy. DESIGN:Neoantigens of patients (n=56) were analysed by pVAC tools with major histocompatibility complex-1 (MHC-I) algorithms based on whole exome sequencing and neoantigens with mutant type IC <50 nM were defined as high-affinity neoantigens (HANs). Patients were segregated into HAN-high/low groups by median of HAN value, and overall survival (OS) was analysed. Autologous organoid killing model was developed to clarify the antitumour activity of HANs. RESULTS:The value of HAN showed a better correlation with OS (=0.0199) than TMB (=0.7505) or neoantigens (=0.2297) in patients with HCC and positively correlated with the frequency of CD39CD8 tumour infiltrating lymphocytes (TILs). Furthermore, HAN-specific CD8 T cells were identified in CD39CD8 TILs, which showed better antitumour activity in HAN-high versus HAN-low group. In addition, more effective HAN peptides were identified in HAN-high versus HAN-low group. Besides, flow cytometry data showed that in fresh tumour, CD39PD-1CD8 TILs displayed an effector phenotype and stronger antitumour activity in HAN-high versus HAN-low group. More importantly, patients in HAN-high versus HAN-low group showed a better prognosis after anti-PD-1 therapy. CONCLUSIONS:Our study first demonstrates that HAN value positively correlates with better OS in patients with HCC. HANs trigger antitumour activity by activating tumour-reactive CD39CD8 T cells, and patients in HAN-high group benefited more from anti-PD-1 therapy than HAN-low group. These findings may provide a novel strategy for personalised antitumour therapies for HCC.
Association Between Expression Level of PD1 by Tumor-Infiltrating CD8 T Cells and Features of Hepatocellular Carcinoma.
Kim Hyung-Don,Song Gi-Won,Park Seongyeol,Jung Min Kyung,Kim Min Hwan,Kang Hyo Jeong,Yoo Changhoon,Yi Kijong,Kim Kyung Hwan,Eo Sukyeong,Moon Deok-Bog,Hong Seung-Mo,Ju Young Seok,Shin Eui-Cheol,Hwang Shin,Park Su-Hyung
BACKGROUND & AIMS:T-cell exhaustion, or an impaired capacity to secrete cytokines and proliferate with overexpression of immune checkpoint receptors, occurs during chronic viral infections but has also been observed in tumors, including hepatocellular carcinomas (HCCs). We investigated features of exhaustion in CD8 T cells isolated from HCC specimens. METHODS:We obtained HCC specimens, along with adjacent nontumor tissues and blood samples, from 90 patients who underwent surgical resection at Asan Medical Center (Seoul, Korea) from April 2016 through April 2018. Intrahepatic lymphocytes and tumor-infiltrating T cells were analyzed by flow cytometry. Tumor-infiltrating CD8 T cells were sorted by flow cytometry into populations based on expression level of programmed cell death 1 (PDCD1 or PD1): PD1-high, PD1-intermediate, and PD1-negative. Sorted cells were analyzed by RNA sequencing. Proliferation and production of interferon gamma (IFNG) and tumor necrosis factor (TNF) by CD8 T cells were measured in response to anti-CD3 and antibodies against immune checkpoint receptors including PD1, hepatitis A virus cellular receptor 2 (HAVCR2 or TIM3), lymphocyte activating 3 (LAG3), or isotype control. Tumor-associated antigen-specific CD8 T cells were identified using HLA-A*0201 dextramers. PDL1 expression on tumor tissue was assessed by immunohistochemistry. RESULTS:PD1-high, PD1-intermediate, and PD1-negative CD8 T cells from HCCs had distinct gene expression profiles. PD1-high cells expressed higher levels of genes that regulate T-cell exhaustion than PD1-intermediate cells. PD1-high cells expressed TIM3 and LAG3, and low proportions of TCF1, TBET/eomesodermin, and CD127. PD1-high cells produced the lowest amounts of IFNG and TNF upon anti-CD3 stimulation. Differences in the PD1 expression patterns of CD8 T cells led to the identification of 2 subgroups of HCCs: HCCs with a discrete population of PD1-high cells were more aggressive than HCCs without a discrete population of PD1-high cells. HCCs with a discrete population of PD1-high cells had higher levels of predictive biomarkers of response to anti-PD1 therapy. Incubation of CD8 T cells from HCCs with a discrete population of PD1-high cells with antibodies against PD1 and TIM3 or LAG3 further restored proliferation and production of IFNG and TNF in response to anti-CD3. CONCLUSIONS:We found HCC specimens to contain CD8 T cells that express different levels of PD1. HCCs with a discrete population of PD1-high CD8 T cells express TIM3 and/or LAG3 and produce low levels of IFNG and TNF in response to anti-CD3. Incubation of these cells with antibodies against PD1 and TIM3 or LAG3 further restore proliferation and production of cytokines; HCCs with a discrete population of PD1-high CD8 T cells might be more susceptible to combined immune checkpoint blockade-based therapies.
Conversion of human fibroblasts to angioblast-like progenitor cells.
Kurian Leo,Sancho-Martinez Ignacio,Nivet Emmanuel,Aguirre Aitor,Moon Krystal,Pendaries Caroline,Volle-Challier Cecile,Bono Francoise,Herbert Jean-Marc,Pulecio Julian,Xia Yun,Li Mo,Montserrat Nuria,Ruiz Sergio,Dubova Ilir,Rodriguez Concepcion,Denli Ahmet M,Boscolo Francesca S,Thiagarajan Rathi D,Gage Fred H,Loring Jeanne F,Laurent Louise C,Izpisua Belmonte Juan Carlos
Lineage conversion of one somatic cell type to another is an attractive approach for generating specific human cell types. Lineage conversion can be direct, in the absence of proliferation and multipotent progenitor generation, or indirect, by the generation of expandable multipotent progenitor states. We report the development of a reprogramming methodology in which cells transition through a plastic intermediate state, induced by brief exposure to reprogramming factors, followed by differentiation. We use this approach to convert human fibroblasts to mesodermal progenitor cells, including by non-integrative approaches. These progenitor cells demonstrated bipotent differentiation potential and could generate endothelial and smooth muscle lineages. Differentiated endothelial cells exhibited neo-angiogenesis and anastomosis in vivo. This methodology for indirect lineage conversion to angioblast-like cells adds to the armamentarium of reprogramming approaches aimed at the study and treatment of ischemic pathologies.
Genetic variants regulating immune cell levels in health and disease.
Orrù Valeria,Steri Maristella,Sole Gabriella,Sidore Carlo,Virdis Francesca,Dei Mariano,Lai Sandra,Zoledziewska Magdalena,Busonero Fabio,Mulas Antonella,Floris Matteo,Mentzen Wieslawa I,Urru Silvana A M,Olla Stefania,Marongiu Michele,Piras Maria G,Lobina Monia,Maschio Andrea,Pitzalis Maristella,Urru Maria F,Marcelli Marco,Cusano Roberto,Deidda Francesca,Serra Valentina,Oppo Manuela,Pilu Rosella,Reinier Frederic,Berutti Riccardo,Pireddu Luca,Zara Ilenia,Porcu Eleonora,Kwong Alan,Brennan Christine,Tarrier Brendan,Lyons Robert,Kang Hyun M,Uzzau Sergio,Atzeni Rossano,Valentini Maria,Firinu Davide,Leoni Lidia,Rotta Gianluca,Naitza Silvia,Angius Andrea,Congia Mauro,Whalen Michael B,Jones Chris M,Schlessinger David,Abecasis Gonçalo R,Fiorillo Edoardo,Sanna Serena,Cucca Francesco
The complex network of specialized cells and molecules in the immune system has evolved to defend against pathogens, but inadvertent immune system attacks on "self" result in autoimmune disease. Both genetic regulation of immune cell levels and their relationships with autoimmunity are largely undetermined. Here, we report genetic contributions to quantitative levels of 95 cell types encompassing 272 immune traits, in a cohort of 1,629 individuals from four clustered Sardinian villages. We first estimated trait heritability, showing that it can be substantial, accounting for up to 87% of the variance (mean 41%). Next, by assessing ∼8.2 million variants that we identified and confirmed in an extended set of 2,870 individuals, 23 independent variants at 13 loci associated with at least one trait. Notably, variants at three loci (HLA, IL2RA, and SH2B3/ATXN2) overlap with known autoimmune disease associations. These results connect specific cellular phenotypes to specific genetic variants, helping to explicate their involvement in disease.
Post-natal paucity of regulatory T cells and control of NK cell activation in experimental biliary atresia.
Miethke Alexander G,Saxena Vijay,Shivakumar Pranavkumar,Sabla Gregg E,Simmons Julia,Chougnet Claire A
Journal of hepatology
BACKGROUND & AIMS:Although recent studies have identified important roles for T and NK cells in the pathogenesis of biliary atresia (BA), the mechanisms by which susceptibility to bile duct injury is restricted to the neonatal period are unknown. METHODS:We characterised hepatic regulatory T cells (Tregs) by flow cytometry in two groups of neonatal mice challenged with rhesus rotavirus (RRV) at day 7 (no ductal injury) or day 1 of life (resulting in BA), determined the functional interaction with effector cells in co-culture assays, and examined the effect of adoptive transfer of CD4+ cells on the BA phenotype. RESULTS:While day 7 RRV infection increased hepatic Tregs (Foxp3+ CD4+ CD25+) by 10-fold within 3 days, no increase in Tregs occurred at this time point following infection on day 1. In vitro, Tregs effectively suppressed NK cell activation by hepatic dendritic cells and decreased the production of pro-inflammatory cytokines, including TNFalpha and IL-15, following RRV infection. In vivo, adoptive transfer of CD4+ cells prior to RRV inoculation led to increased survival, improved weight gain, decreased population of hepatic NK cells, and persistence of donor Tregs in the liver. CONCLUSIONS:(1) The liver is devoid of Tregs early after perinatal RRV infection; (2) Tregs suppress DC-dependent activation of naive NK cells in vitro, and Treg-containing CD4+ cells inhibit hepatic NK cell expansion in vivo. Thus, the post-natal absence of Tregs may be a key factor that allows hepatic DCs to act unopposed in NK cell activation during the initiation of neonatal bile duct injury.
Integrative molecular profiling of autoreactive CD4 T cells in autoimmune hepatitis.
Renand Amédée,Cervera-Marzal Iñaki,Gil Laurine,Dong Chuang,Garcia Alexandra,Kervagoret Erwan,Aublé Hélène,Habes Sarah,Chevalier Caroline,Vavasseur Fabienne,Clémenceau Béatrice,Cardon Anaïs,Judor Jean-Paul,Mosnier Jean-François,Tanné Florence,Laplaud David-Axel,Brouard Sophie,Gournay Jérôme,Milpied Pierre,Conchon Sophie
Journal of hepatology
BACKGROUND & AIMS:In most autoimmune disorders, crosstalk of B cells and CD4 T cells results in the accumulation of autoantibodies. In autoimmune hepatitis (AIH), the presence of anti-soluble liver antigen (SLA) autoantibodies is associated with reduced overall survival, but the associated autoreactive CD4 T cells have not yet been characterised. Herein, we isolated and deeply characterised SLA-specific CD4 T cells in patients with AIH. METHODS:We used brief ex vivo restimulation with overlapping SLA peptides to isolate and phenotype circulating SLA-specific CD4 T cells, and integrative single-cell RNA-seq (scRNA-seq) to characterise their transcriptome and T-cell receptor (TCR) repertoire. Autoreactive TCRs were cloned and used to identify dominant SLA-derived epitopes. SLA-specific CD4 T cells were tracked in peripheral blood through TCR sequencing to identify their phenotypic niche. We further characterised disease-associated peripheral blood T cells by high-content flow cytometry in 42 patients with AIH and 17 controls with non-alcoholic steatohepatitis. RESULTS:Autoreactive SLA-specific CD4 T cells were only detected in patients with anti-SLA autoantibodies and had a memory PD-1CXCR5CCR6CD27 phenotype. ScRNA-seq revealed their pro-inflammatory/B-helper profile. SLA and SLA contain dominant T-cell epitopes. Autoreactive TCR clonotypes were predominantly found in the memory PD-1CXCR5CD4 T cells, which were significantly increased in the blood of patients with AIH and supported B-cell differentiation through IL-21. Finally, we identified specific T-cell phenotypes linked to disease activity and IgG level during AIH. CONCLUSIONS:We provide a deep characterisation of rare circulating autoreactive CD4 T cells and identify their peripheral reservoir in AIH. We also propose a specific phenotype of autoreactive T cells related to AIH disease activity, which will be essential to track, delineate, and potentially target these pathogenic cells. LAY SUMMARY:One principal characteristic of autoimmune hepatitis (AIH), like for many other autoimmune diseases, is the accumulation of autoantibodies produced by B lymphocytes following their interaction with autoreactive CD4 T lymphocytes. In this study, we identified and characterised with high resolution these CD4 T cells. This will be essential to track, delineate, and potentially target them during AIH.
Vascular repair and regeneration in cardiometabolic diseases.
Hess David A,Verma Subodh,Bhatt Deepak,Bakbak Ehab,Terenzi Daniella C,Puar Pankaj,Cosentino Francesco
European heart journal
Chronic cardiometabolic assaults during type 2 diabetes (T2D) and obesity induce a progenitor cell imbalance in the circulation characterized by overproduction and release of pro-inflammatory monocytes and granulocytes from the bone marrow alongside aberrant differentiation and mobilization of pro-vascular progenitor cells that generate downstream progeny for the coordination of blood vessel repair. This imbalance can be detected in the peripheral blood of individuals with established T2D and severe obesity using multiparametric flow cytometry analyses to discern pro-inflammatory vs. pro-angiogenic progenitor cell subsets identified by high aldehyde dehydrogenase activity, a conserved progenitor cell protective function, combined with lineage-restricted cell surface marker analyses. Recent evidence suggests that progenitor cell imbalance can be reversed by treatment with pharmacological agents or surgical interventions that reduce hyperglycaemia or excess adiposity. In this state-of-the-art review, we present current strategies to assess the progression of pro-vascular regenerative cell depletion in peripheral blood samples of individuals with T2D and obesity and we summarize novel clinical data that intervention using sodium-glucose co-transporter 2 inhibition or gastric bypass surgery can efficiently restore cell-mediated vascular repair mechanisms associated with profound cardiovascular benefits in recent outcome trials. Collectively, this thesis generates a compelling argument for early intervention using current pharmacological agents to prevent or restore imbalanced circulating progenitor content and maintain vascular regenerative cell trafficking to sites of ischaemic damage. This conceptual advancement may lead to the design of novel therapeutic approaches to prevent or reverse the devastating cardiovascular comorbidities currently associated with T2D and obesity.
Genome-wide analysis of replication timing by next-generation sequencing with E/L Repli-seq.
Marchal Claire,Sasaki Takayo,Vera Daniel,Wilson Korey,Sima Jiao,Rivera-Mulia Juan Carlos,Trevilla-García Claudia,Nogues Coralin,Nafie Ebtesam,Gilbert David M
This protocol is an extension to: Nat. Protoc. 6, 870-895 (2014); doi:10.1038/nprot.2011.328; published online 02 June 2011Cycling cells duplicate their DNA content during S phase, following a defined program called replication timing (RT). Early- and late-replicating regions differ in terms of mutation rates, transcriptional activity, chromatin marks and subnuclear position. Moreover, RT is regulated during development and is altered in diseases. Here, we describe E/L Repli-seq, an extension of our Repli-chip protocol. E/L Repli-seq is a rapid, robust and relatively inexpensive protocol for analyzing RT by next-generation sequencing (NGS), allowing genome-wide assessment of how cellular processes are linked to RT. Briefly, cells are pulse-labeled with BrdU, and early and late S-phase fractions are sorted by flow cytometry. Labeled nascent DNA is immunoprecipitated from both fractions and sequenced. Data processing leads to a single bedGraph file containing the ratio of nascent DNA from early versus late S-phase fractions. The results are comparable to those of Repli-chip, with the additional benefits of genome-wide sequence information and an increased dynamic range. We also provide computational pipelines for downstream analyses, for parsing phased genomes using single-nucleotide polymorphisms (SNPs) to analyze RT allelic asynchrony, and for direct comparison to Repli-chip data. This protocol can be performed in up to 3 d before sequencing, and requires basic cellular and molecular biology skills, as well as a basic understanding of Unix and R.
Innate-like Gene Expression of Lung-Resident Memory CD8 T Cells during Experimental Human Influenza: A Clinical Study.
Paterson Suzanna,Kar Satwik,Ung Seng Kuong,Gardener Zoe,Bergstrom Emma,Ascough Stephanie,Kalyan Mohini,Zyla Joanna,Maertzdorf Jeroen,Mollenkopf Hans-Joachim,Weiner January,Jozwik Agnieszka,Jarvis Hannah,Jha Akhilesh,Nicholson Bradly P,Veldman Timothy,Woods Chris W,Mallia Patrick,Kon Onn Min,Kaufmann Stefan H E,Openshaw Peter J,Chiu Christopher
American journal of respiratory and critical care medicine
Suboptimal vaccine immunogenicity and antigenic mismatch, compounded by poor uptake, means that influenza remains a major global disease. T cells recognizing peptides derived from conserved viral proteins could enhance vaccine-induced cross-strain protection. To investigate the kinetics, phenotypes, and function of influenza virus-specific CD8 resident memory T (Trm) cells in the lower airway and infer the molecular pathways associated with their response to infection . Healthy volunteers, aged 18-55, were inoculated intranasally with influenza A/California/4/09(H1N1). Blood, upper airway, and (in a subgroup) lower airway samples were obtained throughout infection. Symptoms were assessed by using self-reported diaries, and the nasal viral load was assessed by using quantitative PCR. T-cell responses were analyzed by using a three-color FluoroSpot assay, flow cytometry with MHC I-peptide tetramers, and RNA sequencing, with candidate markers being confirmed by using the immunohistochemistry results for endobronchial biopsy specimens. After challenge, 57% of participants became infected. Preexisting influenza-specific CD8 T cells in blood correlated strongly with a reduced viral load, which peaked at Day 3. Influenza-specific CD8 T cells in BAL fluid were highly enriched and predominantly expressed the Trm markers CD69 and CD103. Comparison between preinfection CD8 T cells in BAL fluid and blood by using RNA sequencing revealed 3,928 differentially expressed genes, including all major Trm-cell markers. However, gene set enrichment analysis of BAL-fluid CD8 T cells showed primarily innate cell-related pathways and, during infection, included upregulation of innate chemokines (, , and ) that were also expressed by CD8 cells in bronchial tissues. CD8 Trm cells in the human lung display innate-like gene and protein expression that demonstrates blurred divisions between innate and adaptive immunity. Clinical study registered with www.clinicaltrials.gov (NCT02755948).
Generation and customization of biosynthetic excitable tissues for electrophysiological studies and cell-based therapies.
Nguyen Hung X,Kirkton Robert D,Bursac Nenad
We describe a two-stage protocol to generate electrically excitable and actively conducting cell networks with stable and customizable electrophysiological phenotypes. Using this method, we have engineered monoclonally derived excitable tissues as a robust and reproducible platform to investigate how specific ion channels and mutations affect action potential (AP) shape and conduction. In the first stage of the protocol, we combine computational modeling, site-directed mutagenesis, and electrophysiological techniques to derive optimal sets of mammalian and/or prokaryotic ion channels that produce specific AP shape and conduction characteristics. In the second stage of the protocol, selected ion channels are stably expressed in unexcitable human cells by means of viral or nonviral delivery, followed by flow cytometry or antibiotic selection to purify the desired phenotype. This protocol can be used with traditional heterologous expression systems or primary excitable cells, and application of this method to primary fibroblasts may enable an alternative approach to cardiac cell therapy. Compared with existing methods, this protocol generates a well-defined, relatively homogeneous electrophysiological phenotype of excitable cells that facilitates experimental and computational studies of AP conduction and can decrease arrhythmogenic risk upon cell transplantation. Although basic cell culture and molecular biology techniques are sufficient to generate excitable tissues using the described protocol, experience with patch-clamp techniques is required to characterize and optimize derived cell populations.
IRE1α induces thioredoxin-interacting protein to activate the NLRP3 inflammasome and promote programmed cell death under irremediable ER stress.
Lerner Alana G,Upton John-Paul,Praveen P V K,Ghosh Rajarshi,Nakagawa Yoshimi,Igbaria Aeid,Shen Sarah,Nguyen Vinh,Backes Bradley J,Heiman Myriam,Heintz Nathaniel,Greengard Paul,Hui Simon,Tang Qizhi,Trusina Ala,Oakes Scott A,Papa Feroz R
When unfolded proteins accumulate to irremediably high levels within the endoplasmic reticulum (ER), intracellular signaling pathways called the unfolded protein response (UPR) become hyperactivated to cause programmed cell death. We discovered that thioredoxin-interacting protein (TXNIP) is a critical node in this "terminal UPR." TXNIP becomes rapidly induced by IRE1α, an ER bifunctional kinase/endoribonuclease (RNase). Hyperactivated IRE1α increases TXNIP mRNA stability by reducing levels of a TXNIP destabilizing microRNA, miR-17. In turn, elevated TXNIP protein activates the NLRP3 inflammasome, causing procaspase-1 cleavage and interleukin 1β (IL-1β) secretion. Txnip gene deletion reduces pancreatic β cell death during ER stress and suppresses diabetes caused by proinsulin misfolding in the Akita mouse. Finally, small molecule IRE1α RNase inhibitors suppress TXNIP production to block IL-1β secretion. In summary, the IRE1α-TXNIP pathway is used in the terminal UPR to promote sterile inflammation and programmed cell death and may be targeted to develop effective treatments for cell degenerative diseases.
Immune parameters correlate with protection against ebola virus infection in rodents and nonhuman primates.
Wong Gary,Richardson Jason S,Pillet Stéphane,Patel Ami,Qiu Xiangguo,Alimonti Judie,Hogan Jeff,Zhang Yi,Takada Ayato,Feldmann Heinz,Kobinger Gary P
Science translational medicine
Ebola virus causes severe hemorrhagic fever in susceptible hosts. Currently, no licensed vaccines or treatments are available; however, several experimental vaccines have been successful in protecting rodents and nonhuman primates (NHPs) from the lethal Zaire ebolavirus (ZEBOV) infection. The objective of this study was to evaluate immune responses correlating with survival in these animals after lethal challenge with ZEBOV. Knockout mice with impaired ability to generate normal T and/or B cell responses were vaccinated and challenged with ZEBOV. Vaccine-induced protection in mice was mainly mediated by B cells and CD4(+) T cells. Vaccinated, outbred guinea pigs and NHPs demonstrated the highest correlation between survival and levels of total immunoglobulin G (IgG) specific to the ZEBOV glycoprotein (ZGP). These results highlight the relevance of total ZGP-specific IgG levels as a meaningful correlate of protection against ZEBOV exposure.
Embryonic and induced pluripotent stem cell staining and sorting with the live-cell fluorescence imaging probe CDy1.
Kang Nam-Young,Yun Seong-Wook,Ha Hyung-Ho,Park Sung-Jin,Chang Young-Tae
Detecting and isolating specific types of cells is crucial to understanding a variety of biological processes, including development, aging, regeneration and pathogenesis; this understanding, in turn, allows the use of cells for therapeutic purposes, for which stem cells have emerged recently as invaluable materials. The current methods of isolation and characterization of stem cells depend on cell morphology in culture or on immunostaining of specific markers. These methods are, however, time consuming and involve the use of antibodies that may often make the cells unsuitable for further study. We recently developed a fluorescent small molecule named CDy1 (compound of designation yellow 1) that selectively stains live embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs). This protocol describes detailed procedures for staining ESC and iPSC in live conditions and for fluorescence-activated cell sorting (FACS) of ESC using CDy1. Cell staining, image acquisition and FACS can be done within 6 h.
Malt1 protease inactivation efficiently dampens immune responses but causes spontaneous autoimmunity.
Jaworski Maike,Marsland Ben J,Gehrig Jasmine,Held Werner,Favre Stéphanie,Luther Sanjiv A,Perroud Mai,Golshayan Déla,Gaide Olivier,Thome Margot
The EMBO journal
The protease activity of the paracaspase Malt1 has recently gained interest as a drug target for immunomodulation and the treatment of diffuse large B-cell lymphomas. To address the consequences of Malt1 protease inactivation on the immune response in vivo, we generated knock-in mice expressing a catalytically inactive C472A mutant of Malt1 that conserves its scaffold function. Like Malt1-deficient mice, knock-in mice had strong defects in the activation of lymphocytes, NK and dendritic cells, and the development of B1 and marginal zone B cells and were completely protected against the induction of autoimmune encephalomyelitis. Malt1 inactivation also protected the mice from experimental induction of colitis. However, Malt1 knock-in mice but not Malt1-deficient mice spontaneously developed signs of autoimmune gastritis that correlated with an absence of Treg cells, an accumulation of T cells with an activated phenotype and high serum levels of IgE and IgG1. Thus, removal of the enzymatic activity of Malt1 efficiently dampens the immune response, but favors autoimmunity through impaired Treg development, which could be relevant for therapeutic Malt1-targeting strategies.
APC/C(Cdh1) controls CtIP stability during the cell cycle and in response to DNA damage.
Lafranchi Lorenzo,de Boer Harmen R,de Vries Elisabeth G E,Ong Shao-En,Sartori Alessandro A,van Vugt Marcel A T M
The EMBO journal
Human cells have evolved elaborate mechanisms for responding to DNA damage to maintain genome stability and prevent carcinogenesis. For instance, the cell cycle can be arrested at different stages to allow time for DNA repair. The APC/C(C) (dh1) ubiquitin ligase mainly regulates mitotic exit but is also implicated in the DNA damage-induced G2 arrest. However, it is currently unknown whether APC/C(C) (dh1) also contributes to DNA repair. Here, we show that Cdh1 depletion causes increased levels of genomic instability and enhanced sensitivity to DNA-damaging agents. Using an integrated proteomics and bioinformatics approach, we identify CtIP, a DNA-end resection factor, as a novel APC/C(C) (dh1) target. CtIP interacts with Cdh1 through a conserved KEN box, mutation of which impedes ubiquitylation and downregulation of CtIP both during G1 and after DNA damage in G2. Finally, we find that abrogating the CtIP-Cdh1 interaction results in delayed CtIP clearance from DNA damage foci, increased DNA-end resection, and reduced homologous recombination efficiency. Combined, our results highlight the impact of APC/C(C) (dh1) on the maintenance of genome integrity and show that this is, at least partially, achieved by controlling CtIP stability in a cell cycle- and DNA damage-dependent manner.
p38 MAPK signaling underlies a cell-autonomous loss of stem cell self-renewal in skeletal muscle of aged mice.
Bernet Jennifer D,Doles Jason D,Hall John K,Kelly Tanaka Kathleen,Carter Thomas A,Olwin Bradley B
Skeletal muscle aging results in a gradual loss of skeletal muscle mass, skeletal muscle function and regenerative capacity, which can lead to sarcopenia and increased mortality. Although the mechanisms underlying sarcopenia remain unclear, the skeletal muscle stem cell, or satellite cell, is required for muscle regeneration. Therefore, identification of signaling pathways affecting satellite cell function during aging may provide insights into therapeutic targets for combating sarcopenia. Here, we show that a cell-autonomous loss in self-renewal occurs via alterations in fibroblast growth factor receptor-1, p38α and p38β mitogen-activated protein kinase signaling in satellite cells from aged mice. We further demonstrate that pharmacological manipulation of these pathways can ameliorate age-associated self-renewal defects. Thus, our data highlight an age-associated deregulation of a satellite cell homeostatic network and reveal potential therapeutic opportunities for the treatment of progressive muscle wasting.
All-In-One Dendrimer-Based Lipid Nanoparticles Enable Precise HDR-Mediated Gene Editing In Vivo.
Farbiak Lukas,Cheng Qiang,Wei Tuo,Álvarez-Benedicto Ester,Johnson Lindsay T,Lee Sang,Siegwart Daniel J
Advanced materials (Deerfield Beach, Fla.)
Clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated (Cas) protein gene editing is poised to transform the treatment of genetic diseases. However, limited progress has been made toward precise editing of DNA via homology-directed repair (HDR) that requires careful orchestration of complex steps. Herein, dendrimer-based lipid nanoparticles (dLNPs) are engineered to co-encapsulate and deliver multiple components for in vivo HDR correction. BFP/GFP switchable HEK293 cells with a single Y66H amino acid mutation are employed to assess HDR-mediated gene editing following simultaneous, one-pot delivery of Cas9 mRNA, single-guide RNA, and donor DNA. Molar ratios of individual LNP components and weight ratios of the three nucleic acids are systematically optimized to increase HDR efficiency. Using flow cytometry, fluorescence imaging, and DNA sequencing to quantify editing, optimized 4A3-SC8 dLNPs edit >91% of all cells with 56% HDR efficiency in vitro and >20% HDR efficiency in xenograft tumors in vivo. Due to the all-in-one simplicity and high efficacy, the developed dLNPs offer a promising route toward the gene correction of disease-causing mutations.
IκΒα inhibits apoptosis at the outer mitochondrial membrane independently of NF-κB retention.
Pazarentzos Evangelos,Mahul-Mellier Anne-Laure,Datler Christoph,Chaisaklert Wanwisa,Hwang Ming-Shih,Kroon Jan,Qize Ding,Osborne Foy,Al-Rubaish Abdullah,Al-Ali Amein,Mazarakis Nicholas D,Aboagye Eric O,Grimm Stefan
The EMBO journal
IκBα resides in the cytosol where it retains the inducible transcription factor NF-κB. We show that IκBα also localises to the outer mitochondrial membrane (OMM) to inhibit apoptosis. This effect is especially pronounced in tumour cells with constitutively active NF-κB that accumulate high amounts of mitochondrial IκBα as a NF-κB target gene. 3T3 IκBα(-/-) cells also become protected from apoptosis when IκBα is specifically reconstituted at the OMM. Using various IκBα mutants, we demonstrate that apoptosis inhibition and NF-κB inhibition can be functionally and structurally separated. At mitochondria, IκBα stabilises the complex of VDAC1 and hexokinase II (HKII), thereby preventing Bax recruitment to VDAC1 and the release of cytochrome c for apoptosis induction. When IκBα is reduced in tumour cells with constitutively active NF-κB, they show an enhanced response to anticancer treatment in an in vivo xenograft tumour model. Our results reveal the unexpected activity of IκBα in guarding the integrity of the OMM against apoptosis induction and open possibilities for more specific interference in tumours with deregulated NF-κB.
H2.0-like homeobox regulates early hematopoiesis and promotes acute myeloid leukemia.
Kawahara Masahiro,Pandolfi Ashley,Bartholdy Boris,Barreyro Laura,Will Britta,Roth Michael,Okoye-Okafor Ujunwa C,Todorova Tihomira I,Figueroa Maria E,Melnick Ari,Mitsiades Constantine S,Steidl Ulrich
Homeobox domain-containing transcription factors are important regulators of hematopoiesis. Here, we report that increased levels of nonclustered H2.0-like homeobox (HLX) lead to loss of functional hematopoietic stem cells and formation of aberrant progenitors with unlimited serial clonogenicity and blocked differentiation. Inhibition of HLX reduces proliferation and clonogenicity of leukemia cells, overcomes the differentiation block, and leads to prolonged survival. HLX regulates a transcriptional program, including PAK1 and BTG1, that controls cellular differentiation and proliferation. HLX is overexpressed in 87% of patients with acute myeloid leukemia (AML) and independently correlates with inferior overall survival (n = 601, p = 2.3 × 10(-6)). Our study identifies HLX as a key regulator in immature hematopoietic and leukemia cells and as a prognostic marker and therapeutic target in AML.
Clusters of Tolerogenic B Cells Feature in the Dynamic Immunological Landscape of the Pregnant Uterus.
Benner Marilen,Feyaerts Dorien,García Celia Cartagena,Inci Nurcan,López Sergi Cedó,Fasse Esther,Shadmanfar Wijs,van der Heijden Olivier W H,Gorris Mark A J,Joosten Irma,Ferwerda Gerben,van der Molen Renate G
Well-timed interaction of correctly functioning maternal immune cells is essential to facilitate healthy placenta formation, because the uterine immune environment has to tolerate the semi-allogeneic fetus and allow adequate trophoblast invasion. Here, we assess the uterine immune signature before and during pregnancy. Extensive supervised and unsupervised flow cytometry clustering strategies not only show a general increase in immune memory throughout pregnancy but also reveal the continuous presence of B cells. Contrary to the belief that B cells are merely a consequence of uterine pathology, decidual B cells produce IL-10 and are found to be localized in clusters, together with Foxp3 T cells. Our findings therefore suggest a role for B cells in healthy pregnancy.
Fas ligand-mediated immune surveillance by T cells is essential for the control of spontaneous B cell lymphomas.
Afshar-Sterle Shoukat,Zotos Dimitra,Bernard Nicholas J,Scherger Anna K,Rödling Lisa,Alsop Amber E,Walker Jennifer,Masson Frederick,Belz Gabrielle T,Corcoran Lynn M,O'Reilly Lorraine A,Strasser Andreas,Smyth Mark J,Johnstone Ricky,Tarlinton David M,Nutt Stephen L,Kallies Axel
Loss of function of the tumor suppressor gene PRDM1 (also known as BLIMP1) or deregulated expression of the oncogene BCL6 occurs in a large proportion of diffuse large B cell lymphoma (DLBCL) cases. However, targeted mutation of either gene in mice leads to only slow and infrequent development of malignant lymphoma, and despite frequent mutation of BCL6 in activated B cells of healthy individuals, lymphoma development is rare. Here we show that T cells prevent the development of overt lymphoma in mice caused by Blimp1 deficiency or overexpression of Bcl6 in the B cell lineage. Impairment of T cell control results in rapid development of DLBCL-like disease, which can be eradicated by polyclonal CD8(+) T cells in a T cell receptor-, CD28- and Fas ligand-dependent manner. Thus, malignant transformation of mature B cells requires mutations that impair intrinsic differentiation processes and permit escape from T cell-mediated tumor surveillance.
Hypoxia and oxygenation induce a metabolic switch between pentose phosphate pathway and glycolysis in glioma stem-like cells.
Kathagen Annegret,Schulte Alexander,Balcke Gerd,Phillips Heidi S,Martens Tobias,Matschke Jakob,Günther Hauke S,Soriano Robert,Modrusan Zora,Sandmann Thomas,Kuhl Carsten,Tissier Alain,Holz Mareike,Krawinkel Lutz A,Glatzel Markus,Westphal Manfred,Lamszus Katrin
Fluctuations in oxygen tension during tissue remodeling impose a major metabolic challenge in human tumors. Stem-like tumor cells in glioblastoma, the most common malignant brain tumor, possess extraordinary metabolic flexibility, enabling them to initiate growth even under non-permissive conditions. We identified a reciprocal metabolic switch between the pentose phosphate pathway (PPP) and glycolysis in glioblastoma stem-like (GS) cells. Expression of PPP enzymes is upregulated by acute oxygenation but downregulated by hypoxia, whereas glycolysis enzymes, particularly those of the preparatory phase, are regulated inversely. Glucose flux through the PPP is reduced under hypoxia in favor of flux through glycolysis. PPP enzyme expression is elevated in human glioblastomas compared to normal brain, especially in highly proliferative tumor regions, whereas expression of parallel preparatory phase glycolysis enzymes is reduced in glioblastomas, except for strong upregulation in severely hypoxic regions. Hypoxia stimulates GS cell migration but reduces proliferation, whereas oxygenation has opposite effects, linking the metabolic switch to the "go or grow" potential of the cells. Our findings extend Warburg's observation that tumor cells predominantly utilize glycolysis for energy production, by suggesting that PPP activity is elevated in rapidly proliferating tumor cells but suppressed by acute severe hypoxic stress, favoring glycolysis and migration to protect cells against hypoxic cell damage.
Multi-parametric analysis of 57 SYNGAP1 variants reveal impacts on GTPase signaling, localization, and protein stability.
Meili Fabian,Wei William J,Sin Wun-Chey,Meyers Warren M,Dascalu Iulia,Callaghan Daniel B,Rogic Sanja,Pavlidis Paul,Haas Kurt
American journal of human genetics
SYNGAP1 is a neuronal Ras and Rap GTPase-activating protein with important roles in regulating excitatory synaptic plasticity. While many SYNGAP1 missense and nonsense mutations have been associated with intellectual disability, epilepsy, schizophrenia, and autism spectrum disorder (ASD), whether and how they contribute to individual disease phenotypes is often unknown. Here, we characterize 57 variants in seven assays that examine multiple aspects of SYNGAP1 function. Specifically, we used multiplex phospho-flow cytometry to measure variant impact on protein stability, pERK, pGSK3β, pp38, pCREB, and high-content imaging to examine subcellular localization. We find variants ranging from complete loss-of-function (LoF) to wild-type (WT)-like in their regulation of pERK and pGSK3β, while all variants retain at least partial ability to dephosphorylate pCREB. Interestingly, our assays reveal that a larger proportion of variants located within the disordered domain of unknown function (DUF) comprising the C-terminal half of SYNGAP1 exhibited higher LoF, compared to variants within the better studied catalytic domain. Moreover, we find protein instability to be a major contributor to dysfunction for only two missense variants, both located within the catalytic domain. Using high-content imaging, we find variants located within the C2 domain known to mediate membrane lipid interactions exhibit significantly larger cytoplasmic speckles than WT SYNGAP1. Moreover, this subcellular phenotype shows both correlation with altered catalytic activity and unique deviation from signaling assay results, highlighting multiple independent molecular mechanisms underlying variant dysfunction. Our multidimensional dataset allows clustering of variants based on functional phenotypes and provides high-confidence, multi-functional measures for making pathogenicity predictions.
PTEN loss in the Myf5 lineage redistributes body fat and reveals subsets of white adipocytes that arise from Myf5 precursors.
Sanchez-Gurmaches Joan,Hung Chien-Min,Sparks Cynthia A,Tang Yuefeng,Li Huawei,Guertin David A
The developmental origin of adipose tissue and what controls its distribution is poorly understood. By lineage tracing and gene expression analysis in mice, we provide evidence that mesenchymal precursors expressing Myf5--which are thought to give rise only to brown adipocytes and skeletal muscle--also give rise to a subset of white adipocytes. Furthermore, individual brown and white fats contain a mixture of adipocyte progenitor cells derived from Myf5(+) and Myf5(neg) lineages, the number of which varies with depot location. Subsets of white adipocytes originating from both Myf5(+) and Myf5(neg) precursors respond to β(3)-adrenoreceptor stimulation, suggesting "brite" adipocytes may also have multiple origins. We additionally find that deleting PTEN with myf5-cre causes lipomatosis and partial lipodystrophy by selectively expanding the Myf5(+) adipocyte lineages. Thus, the spectrum of adipocytes arising from Myf5(+) precursors is broader than previously thought, and differences in PI3K activity between adipocyte lineages alter body fat distribution.
Photoclickable MicroRNA for the Intracellular Target Identification of MicroRNAs.
Li Jinbo,Huang Lei,Xiao Xiao,Chen Yingjie,Wang Xingxing,Zhou Zhengquan,Zhang Chenyu,Zhang Yan
Journal of the American Chemical Society
MicroRNAs (miRNAs) are important gene regulators that bind with target genes and repress target gene expression at the post-transcriptional level. The identification of target genes associated with miRNAs inside different cells is a major challenge in miRNA chemical biology due to the lack of functional miRNAs bearing appropriate tags. Here we report photoclickable miRNAs as appropriately pretagged miRNAs that keep the intracellular function of miRNAs and allow the addition of molecular handles through photoclick reaction. The photoclickable miRNAs upon transfection inside cells were able to form functional complexes with target genes and repress target gene expression. Target genes associated with the photoclickable miRNAs in the complexes were then tagged with the molecular handle through photoclick reaction for pull-down and identification. Using photoclickable miR-106a, miR-27, and miR-122, we first verified that their intracellular function was comparable to that of intact miRNAs, which showed obvious advantage over corresponding biotinylated miRNAs. After attaching the biotin handle to the associated complexes containing the photoclickable miRNAs through the tetrazole-ene photoclick reaction, target genes previously bound with these miRNAs inside cells were successfully pulled town and analyzed. The application of this strategy was demonstrated by the identification of several new target genes of miR-122, followed by revealing a novel regulatory pathway in HepG2 cells with regard to the role of PEG10 in miR-122-promoted cell apoptosis.
DVC1 (C1orf124) is a DNA damage-targeting p97 adaptor that promotes ubiquitin-dependent responses to replication blocks.
Mosbech Anna,Gibbs-Seymour Ian,Kagias Konstantinos,Thorslund Tina,Beli Petra,Povlsen Lou,Nielsen Sofie Vincents,Smedegaard Stine,Sedgwick Garry,Lukas Claudia,Hartmann-Petersen Rasmus,Lukas Jiri,Choudhary Chunaram,Pocock Roger,Bekker-Jensen Simon,Mailand Niels
Nature structural & molecular biology
Ubiquitin-mediated processes orchestrate critical DNA-damage signaling and repair pathways. We identify human DVC1 (C1orf124; Spartan) as a cell cycle-regulated anaphase-promoting complex (APC) substrate that accumulates at stalled replication forks. DVC1 recruitment to sites of replication stress requires its ubiquitin-binding UBZ domain and PCNA-binding PIP box motif but is independent of RAD18-mediated PCNA monoubiquitylation. Via a conserved SHP box, DVC1 recruits the ubiquitin-selective chaperone p97 to blocked replication forks, which may facilitate p97-dependent removal of translesion synthesis (TLS) DNA polymerase η (Pol η) from monoubiquitylated PCNA. DVC1 knockdown enhances UV light-induced mutagenesis, and depletion of human DVC1 or the Caenorhabditis elegans ortholog DVC-1 causes hypersensitivity to replication stress-inducing agents. Our findings establish DVC1 as a DNA damage-targeting p97 adaptor that protects cells from deleterious consequences of replication blocks and suggest an important role of p97 in ubiquitin-dependent regulation of TLS.
Histone H2A.Z inheritance during the cell cycle and its impact on promoter organization and dynamics.
Nekrasov Maxim,Amrichova Jana,Parker Brian J,Soboleva Tatiana A,Jack Cameron,Williams Rohan,Huttley Gavin A,Tremethick David J
Nature structural & molecular biology
Although it has been clearly established that well-positioned histone H2A.Z-containing nucleosomes flank the nucleosome-depleted region (NDR) at the transcriptional start site (TSS) of active mammalian genes, how this chromatin-based information is transmitted through the cell cycle is unknown. We show here that in mouse trophoblast stem cells, the amount of histone H2A.Z at promoters decreased during S phase, coinciding with homotypic (H2A.Z-H2A.Z) nucleosomes flanking the TSS becoming heterotypic (H2A.Z-H2A). To our surprise these nucleosomes remained heterotypic at M phase. At the TSS, we identified an unstable heterotypic histone H2A.Z-containing nucleosome in G1 phase that was lost after DNA replication. These dynamic changes at the TSS mirror a global expansion of the NDR at S and M phases, which, unexpectedly, is unrelated to transcriptional activity. Coincident with the loss of histone H2A.Z at promoters, histone H2A.Z is targeted to the centromere when mitosis begins.
Oncogenic K-Ras decouples glucose and glutamine metabolism to support cancer cell growth.
Gaglio Daniela,Metallo Christian M,Gameiro Paulo A,Hiller Karsten,Danna Lara Sala,Balestrieri Chiara,Alberghina Lilia,Stephanopoulos Gregory,Chiaradonna Ferdinando
Molecular systems biology
Oncogenes such as K-ras mediate cellular and metabolic transformation during tumorigenesis. To analyze K-Ras-dependent metabolic alterations, we employed ¹³C metabolic flux analysis (MFA), non-targeted tracer fate detection (NTFD) of ¹⁵N-labeled glutamine, and transcriptomic profiling in mouse fibroblast and human carcinoma cell lines. Stable isotope-labeled glucose and glutamine tracers and computational determination of intracellular fluxes indicated that cells expressing oncogenic K-Ras exhibited enhanced glycolytic activity, decreased oxidative flux through the tricarboxylic acid (TCA) cycle, and increased utilization of glutamine for anabolic synthesis. Surprisingly, a non-canonical labeling of TCA cycle-associated metabolites was detected in both transformed cell lines. Transcriptional profiling detected elevated expression of several genes associated with glycolysis, glutamine metabolism, and nucleotide biosynthesis upon transformation with oncogenic K-Ras. Chemical perturbation of enzymes along these pathways further supports the decoupling of glycolysis and TCA metabolism, with glutamine supplying increased carbon to drive the TCA cycle. These results provide evidence for a role of oncogenic K-Ras in the metabolic reprogramming of cancer cells.
Rab11 regulates cell-cell communication during collective cell movements.
Ramel Damien,Wang Xiaobo,Laflamme Carl,Montell Denise J,Emery Gregory
Nature cell biology
Collective cell movements contribute to development and metastasis. The small GTPase Rac is a key regulator of actin dynamics and cell migration but the mechanisms that restrict Rac activation and localization in a group of collectively migrating cells are unknown. Here, we demonstrate that the small GTPases Rab5 and Rab11 regulate Rac activity and polarization during collective cell migration. We use photoactivatable forms of Rac to demonstrate that Rab11 acts on the entire group to ensure that Rac activity is properly restricted to the leading cell through regulation of cell-cell communication. In addition, we show that Rab11 binds to the actin cytoskeleton regulator Moesin and regulates its activation in vivo during migration. Accordingly, reducing the level of Moesin activity also affects cell-cell communication, whereas expressing active Moesin rescues loss of Rab11 function. Our model suggests that Rab11 controls the sensing of the relative levels of Rac activity in a group of cells, leading to the organization of individual cells in a coherent multicellular motile structure.
Lineage analysis of basal epithelial cells reveals their unexpected plasticity and supports a cell-of-origin model for prostate cancer heterogeneity.
Wang Zhu A,Mitrofanova Antonina,Bergren Sarah K,Abate-Shen Cory,Cardiff Robert D,Califano Andrea,Shen Michael M
Nature cell biology
A key issue in cancer biology is whether oncogenic transformation of different cell types of origin within an adult tissue gives rise to distinct tumour subtypes that differ in their prognosis and/or treatment response. We now show that initiation of prostate tumours in basal or luminal epithelial cells in mouse models results in tumours with distinct molecular signatures that are predictive of human patient outcomes. Furthermore, our analysis of untransformed basal cells reveals an unexpected assay dependence of their stem cell properties in sphere formation and transplantation assays versus genetic lineage tracing during prostate regeneration and adult tissue homeostasis. Although oncogenic transformation of basal cells gives rise to tumours with luminal phenotypes, cross-species bioinformatic analyses indicate that tumours of luminal origin are more aggressive than tumours of basal origin, and identify a molecular signature associated with patient outcome. Our results reveal the inherent plasticity of basal cells, and support a model in which different cells of origin generate distinct molecular subtypes of prostate cancer.
Characterization of the adipocyte cellular lineage in vivo.
Berry Ryan,Rodeheffer Matthew S
Nature cell biology
Mature adipocytes are generated through the proliferation and differentiation of precursor cells. Our previous studies identified adipocyte progenitors in white adipose tissue (WAT) as Lin(-):CD29(+):CD34(+):Sca-1(+):CD24(+) (CD24(+)) cells that are capable of generating functional WAT (ref. ). Here, we employ several Cre recombinase mouse models to identify the adipocyte cellular lineage in vivo. Although it has been proposed that white adipocytes are derived from endothelial and haematopoietic lineages, we find that neither of these lineages label white adipocytes. However, platelet-derived growth factor receptor α (PdgfRα)-Cre trace labels all white adipocytes. Analysis of WAT from PdgfRα-Cre reporter mice identifies CD24(+) and Lin(-):CD29(+):CD34(+):Sca-1(+): CD24(-) (CD24(-)) cells as adipocyte precursors. We show that CD24(+) cells generate the CD24(-) population in vivo and the CD24(-) cells express late markers of adipogenesis. From these data we propose a model where the CD24(+) adipocyte progenitors become further committed to the adipocyte lineage as CD24 expression is lost, generating CD24(-) preadipocytes. This characterization of the adipocyte cellular lineage will facilitate the study of the mechanisms that regulate WAT formation in vivo and WAT mass expansion in obesity.
Neutrophils prevent rectal bleeding in ulcerative colitis by peptidyl-arginine deiminase-4-dependent immunothrombosis.
Leppkes Moritz,Lindemann Aylin,Gößwein Stefanie,Paulus Susanne,Roth Dominik,Hartung Anne,Liebing Eva,Zundler Sebastian,Gonzalez-Acera Miguel,Patankar Jay V,Mascia Fabrizio,Scheibe Kristina,Hoffmann Markus,Uderhardt Stefan,Schauer Christine,Foersch Sebastian,Neufert Clemens,Vieth Michael,Schett Georg,Atreya Raja,Kühl Anja A,Bleich Andre,Becker Christoph,Herrmann Martin,Neurath Markus F
OBJECTIVE:Bleeding ulcers and erosions are hallmarks of active ulcerative colitis (UC). However, the mechanisms controlling bleeding and mucosal haemostasis remain elusive. DESIGN:We used high-resolution endoscopy and colon tissue samples of active UC (n = 36) as well as experimental models of physical and chemical mucosal damage in mice deficient for peptidyl-arginine deiminase-4 (PAD4), gnotobiotic mice and controls. We employed endoscopy, histochemistry, live-cell microscopy and flow cytometry to study eroded mucosal surfaces during mucosal haemostasis. RESULTS:Erosions and ulcerations in UC were covered by fresh blood, haematin or fibrin visible by endoscopy. Fibrin layers rather than fresh blood or haematin on erosions were inversely correlated with rectal bleeding in UC. Fibrin layers contained ample amounts of neutrophils coaggregated with neutrophil extracellular traps (NETs) with detectable activity of PAD. Transcriptome analyses showed significantly elevated expression in active UC. In experimentally inflicted wounds, we found that neutrophils underwent NET formation in a PAD4-dependent manner hours after formation of primary blood clots, and remodelled clots to immunothrombi containing citrullinated histones, even in the absence of microbiota. PAD4-deficient mice experienced an exacerbated course of dextrane sodium sulfate-induced colitis with markedly increased rectal bleeding (96 % vs 10 %) as compared with controls. PAD4-deficient mice failed to remodel blood clots on mucosal wounds eliciting impaired healing. Thus, NET-associated immunothrombi are protective in acute colitis, while insufficient immunothrombosis is associated with rectal bleeding. CONCLUSION:Our findings uncover that neutrophils induce secondary immunothrombosis by PAD4-dependent mechanisms. Insufficient immunothrombosis may favour rectal bleeding in UC.
Safety and immunogenicity of the novel tuberculosis vaccine ID93 + GLA-SE in BCG-vaccinated healthy adults in South Africa: a randomised, double-blind, placebo-controlled phase 1 trial.
Penn-Nicholson Adam,Tameris Michele,Smit Erica,Day Tracey A,Musvosvi Munyaradzi,Jayashankar Lakshmi,Vergara Julie,Mabwe Simbarashe,Bilek Nicole,Geldenhuys Hendrik,Luabeya Angelique Kany-Kany,Ellis Ruth,Ginsberg Ann M,Hanekom Willem A,Reed Steven G,Coler Rhea N,Scriba Thomas J,Hatherill Mark,
The Lancet. Respiratory medicine
BACKGROUND:A vaccine that prevents pulmonary tuberculosis in adults is needed to halt transmission in endemic regions. This trial aimed to assess the safety and immunogenicity of three administrations at varying doses of antigen and adjuvant of an investigational vaccine (ID93 + GLA-SE) compared with placebo in previously BCG-vaccinated healthy adults in a tuberculosis endemic country. METHODS:In this randomised, double-blind, placebo-controlled phase 1 trial, we enrolled HIV-negative, previously BCG-vaccinated adults (aged 18-50 years), with no evidence of previous or current tuberculosis disease, from among community volunteers in the Worcester region of Western Cape, South Africa. Participants were randomly assigned to receive varying doses of ID93 + GLA-SE or saline placebo at day 0, day 28, and day 112. Enrolment into each cohort was sequential. Cohort 1 participants were Mycobacterium tuberculosis uninfected (as defined by negative QuantiFERON [QFT] status), and received 10 μg ID93 plus 2 μg GLA-SE, or placebo; in cohorts 2-4, QFT-negative or positive participants received escalating doses of vaccine or placebo. Cohort 2 received 2 μg ID93 plus 2 μg GLA-SE; cohort 3 received 10 μg ID93 plus 2 μg GLA-SE; and cohort 4 received 10 μg ID93 plus 5 μg GLA-SE. Dose cohort allocation was sequential; randomisation within a cohort was according to a randomly-generated sequence (3 to 1 in cohort 1, 5 to 1 in cohorts 2-4). The primary endpoint was safety of ID93 + GLA-SE as defined by solicited and unsolicited adverse events up to 28 days after each study injection and serious adverse events for the duration of the study. Specific immune responses were measured by intracellular cytokine staining, flow cytometry, and ELISA. All analyses were done according to intention to treat, with additional per-protocol analyses for immunogenicity outcomes. This trial is registered with ClinicalTrials.gov, number NCT01927159. FINDINGS:Between Aug 30, 2013, and Sept 4, 2014, 227 individuals consented to participate; 213 were screened (three participants were not included as study number was already met and 11 withdrew consent before screening occurred, mostly due to relocation or demands of employment). 66 healthy, HIV-negative adults were randomly allocated to receive the vaccine (n=54) or placebo (n=12). All study participants received day 0 and day 28 study injections; five participants did not receive an injection on day 112. ID93 + GLA-SE was well tolerated; no severe or serious vaccine-related adverse events were recorded. Vaccine dose did not affect frequency or severity of adverse events, but mild injection site adverse events and flu-like symptoms were common in M tuberculosis-infected participants compared with uninfected participants. Vaccination induced durable antigen-specific IgG and Th1 cellular responses, which peaked after two administrations. Vaccine dose did not affect magnitude, kinetics, or profile of antibody and cellular responses. Earlier boosting and greater T-cell differentiation and effector-like profiles were seen in M tuberculosis-infected than in uninfected vaccinees. INTERPRETATION:Escalating doses of ID93 + GLA-SE induced similar antigen-specific CD4-positive T cell and humoral responses, with an acceptable safety profile in BCG-immunised, M tuberculosis-infected individuals. The T-cell differentiation profiles in M tuberculosis-infected vaccinees suggest priming through natural infection. While cohort sample sizes in this phase 1 trial were small and results should be interpreted in context, these data support efficacy testing of two administrations of the lowest (2 μg) ID93 vaccine dose in tuberculosis endemic populations. FUNDING:Aeras and the Paul G Allen Family Foundation.
Nrf2 regulates haematopoietic stem cell function.
Tsai Jennifer J,Dudakov Jarrod A,Takahashi Koichi,Shieh Jae-Hung,Velardi Enrico,Holland Amanda M,Singer Natalie V,West Mallory L,Smith Odette M,Young Lauren F,Shono Yusuke,Ghosh Arnab,Hanash Alan M,Tran Hien T,Moore Malcolm A S,van den Brink Marcel R M
Nature cell biology
Coordinating the balance between haematopoietic stem cell (HSC) quiescence and self-renewal is crucial for maintaining haematopoiesis lifelong. Equally important for haematopoietic function is modulating HSC localization within the bone marrow niches, as maintenance of HSC function is tightly controlled by a complex network of intrinsic molecular mechanisms and extrinsic signalling interactions with their surrounding microenvironment. In this study we demonstrate that nuclear factor erythroid 2-related factor 2 (Nfe2l2, or Nrf2), well established as a global regulator of the oxidative stress response, plays a regulatory role in several aspects of HSC homeostasis. Nrf2 deficiency results in an expansion of the haematopoietic stem and progenitor cell compartment due to cell-intrinsic hyperproliferation, which was accomplished at the expense of HSC quiescence and self-renewal. We further show that Nrf2 modulates both migration and retention of HSCs in their niche. Moreover, we identify a previously unrecognized link between Nrf2 and CXCR4, contributing, at least partially, to the maintenance of HSC function.
An official American Thoracic Society clinical practice guideline: the clinical utility of bronchoalveolar lavage cellular analysis in interstitial lung disease.
Meyer Keith C,Raghu Ganesh,Baughman Robert P,Brown Kevin K,Costabel Ulrich,du Bois Roland M,Drent Marjolein,Haslam Patricia L,Kim Dong Soon,Nagai Sonoko,Rottoli Paola,Saltini Cesare,Selman Moisés,Strange Charlie,Wood Brent,
American journal of respiratory and critical care medicine
BACKGROUND:The clinical utility of bronchoalveolar lavage fluid (BAL) cell analysis for the diagnosis and management of patients with interstitial lung disease (ILD) has been a subject of debate and controversy. The American Thoracic Society (ATS) sponsored a committee of international experts to examine all relevant literature on BAL in ILD and provide recommendations concerning the use of BAL in the diagnosis and management of patients with suspected ILD. PURPOSE:To provide recommendations for (1) the performance and processing of BAL and (2) the interpretation of BAL nucleated immune cell patterns and other BAL characteristics in patients with suspected ILD. METHODS:A pragmatic systematic review was performed to identify unique citations related to BAL in patients with ILD that were published between 1970 and 2006. The search was updated during the guideline development process to include published literature through March 2011. This is the evidence upon which the committee's conclusions and recommendations are based. RESULTS:Recommendations for the performance and processing of BAL, as well as the interpretation of BAL findings, were formulated by the committee. CONCLUSIONS:When used in conjunction with comprehensive clinical information and adequate thoracic imaging such as high-resolution computed tomography of the thorax, BAL cell patterns and other characteristics frequently provide useful information for the diagnostic evaluation of patients with suspected ILD.
Methotrexate hampers immunogenicity to BNT162b2 mRNA COVID-19 vaccine in immune-mediated inflammatory disease.
Haberman Rebecca H,Herati Ramin,Simon David,Samanovic Marie,Blank Rebecca B,Tuen Michael,Koralov Sergei B,Atreya Raja,Tascilar Koray,Allen Joseph R,Castillo Rochelle,Cornelius Amber R,Rackoff Paula,Solomon Gary,Adhikari Samrachana,Azar Natalie,Rosenthal Pamela,Izmirly Peter,Samuels Jonathan,Golden Brian,Reddy Soumya M,Neurath Markus F,Abramson Steven B,Schett Georg,Mulligan Mark J,Scher Jose U
Annals of the rheumatic diseases
OBJECTIVE:To investigate the humoral and cellular immune response to messenger RNA (mRNA) COVID-19 vaccines in patients with immune-mediated inflammatory diseases (IMIDs) on immunomodulatory treatment. METHODS:Established patients at New York University Langone Health with IMID (n=51) receiving the BNT162b2 mRNA vaccination were assessed at baseline and after second immunisation. Healthy subjects served as controls (n=26). IgG antibody responses to the spike protein were analysed for humoral response. Cellular immune response to SARS-CoV-2 was further analysed using high-parameter spectral flow cytometry. A second independent, validation cohort of controls (n=182) and patients with IMID (n=31) from Erlangen, Germany, were also analysed for humoral immune response. RESULTS:Although healthy subjects (n=208) and patients with IMID on biologic treatments (mostly on tumour necrosis factor blockers, n=37) demonstrate robust antibody responses (over 90%), those patients with IMID on background methotrexate (n=45) achieve an adequate response in only 62.2% of cases. Similarly, patients with IMID on methotrexate do not demonstrate an increase in CD8+ T-cell activation after vaccination. CONCLUSIONS:In two independent cohorts of patients with IMID, methotrexate, a widely used immunomodulator for the treatment of several IMIDs, adversely affected humoral and cellular immune response to COVID-19 mRNA vaccines. Although precise cut-offs for immunogenicity that correlate with vaccine efficacy are yet to be established, our findings suggest that different strategies may need to be explored in patients with IMID taking methotrexate to increase the chances of immunisation efficacy against SARS-CoV-2 as has been demonstrated for augmenting immunogenicity to other viral vaccines.
Convergent transcription induces transcriptional gene silencing in fission yeast and mammalian cells.
Gullerova Monika,Proudfoot Nick J
Nature structural & molecular biology
We show that convergent transcription induces transcriptional gene silencing (TGS) in trans for both fission yeast and mammalian cells. This method has advantages over existing strategies to induce gene silencing. Previous studies in fission yeast have characterized TGS as a cis-specific process involving RNA interference that maintains heterochromatic regions such as centromeres. In contrast, in mammalian cells, gene silencing is known to occur through a post-transcriptional mechanism that uses exogenous short interfering RNAs or endogenous microRNAs to inactivate mRNA. We now show that the introduction of convergent transcription plasmids into either Schizosaccharomyces pombe or mammalian cells allows the production of double-stranded RNA from inserted gene fragments, resulting in TGS of endogenous genes. We predict that using convergent transcription to induce gene silencing will be a generally useful strategy and allow for a fuller molecular understanding of the biology of TGS.
IL-6 receptor α defines effector memory CD8+ T cells producing Th2 cytokines and expanding in asthma.
Lee Naeun,You Sungyong,Shin Min Sun,Lee Won-Woo,Kang Ki Soo,Kim Sang Hyun,Kim Wan-Uk,Homer Robert J,Kang Min-Jong,Montgomery Ruth R,Dela Cruz Charles S,Shaw Albert C,Lee Patty J,Chupp Geoffrey L,Hwang Daehee,Kang Insoo
American journal of respiratory and critical care medicine
RATIONALE:Cytokine receptors can be markers defining different T-cell subsets and considered as therapeutic targets. The association of IL-6 and IL-6 receptor α (IL-6Rα) with asthma was reported, suggesting their involvement in asthma. OBJECTIVES:To determine whether and how IL-6Rα defines a distinct effector memory (EM) CD8+ T-cell population in health and disease. METHODS:EM CD8+ T cells expressing IL-6Rα (IL-6Rα(high)) were identified in human peripheral blood and analyzed for function, gene, and transcription factor expression. The relationship of these cells with asthma was determined using blood and sputum. MEASUREMENTS AND MAIN RESULTS:A unique population of IL-6Rα(high) EM CD8+ T cells was found in peripheral blood. These cells that potently proliferated, survived, and produced high levels of the Th2-type cytokines IL-5 and IL-13 had increased levels of GATA3 and decreased levels of T-bet and Blimp-1 in comparison with other EM CD8+ T cells. In fact, GATA3 was required for IL-6Rα expression. Patients with asthma had an increased frequency of IL-6Rα(high) EM CD8+ T cells in peripheral blood compared with healthy control subjects. Also, IL-6Rα(high) EM CD8+ T cells exclusively produced IL-5 and IL-13 in response to asthma-associated respiratory syncytial virus and bacterial superantigens. CONCLUSIONS:Human IL-6Rα(high) EM CD8+ T cells is a unique cell subset that may serve as a reservoir for effector CD8+ T cells, particularly the ones producing Th2-type cytokines, and expand in asthma.
Comparison of immunofixation, serum free light chain, and immunophenotyping for response evaluation and prognostication in multiple myeloma.
Paiva Bruno,Martinez-Lopez Joaquin,Vidriales Maria-Belen,Mateos Maria-Victoria,Montalban Maria-Angeles,Fernandez-Redondo Elena,Alonso Lourdes,Oriol Albert,Teruel Ana-Isabel,de Paz Raquel,Laraña José-Garcia,Bengoechea Enrique,Martin Alejandro,Mediavilla Joaquin Diaz,Palomera Luis,de Arriba Felipe,Bladé Joan,Orfao Alberto,Lahuerta Juan-Jose,San Miguel Jesus F
Journal of clinical oncology : official journal of the American Society of Clinical Oncology
PURPOSE:To investigate the impact of immunophenotypic response (IR) versus complete response (CR) and CR plus normal serum free light chain (sFLC) ratio (stringent CR) in elderly patients with multiple myeloma (MM) treated with novel agents. PATIENTS AND METHODS:From a total of 260 elderly patients newly diagnosed with MM included in the GEM05>65y trial, 102 patients achieving at least a partial response with ≥ 70% reduction in M-component after the six planned induction cycles were simultaneously analyzed by immunofixation, sFLC, and multiparameter flow cytometry (MFC) immunophenotyping; this population is the focus of this study. RESULTS:Forty-three percent of patients achieved CR, 30% achieved stringent CR, and 30% achieved IR. Patients in stringent CR showed no significant survival advantage compared with those in CR, whereas patients in IR showed significantly increased progression-free survival (PFS) and time to progression (TTP) compared with those in stringent CR or CR; this was confirmed by multivariate analysis (hazard ratio, 4.1; P = .01 for PFS). Discrepancies between the three techniques were relatively common. Notably, in all seven patients achieving IR but remaining immunofixation positive, the M-component disappeared in follow-up analysis. In contrast, MFC-positive patients who were immunofixation negative (n = 20) showed a tendency toward early reappearance of the M-component (median, 3 months). Similarly, in five of 11 stringent CR but MFC-positive patients, symptomatic disease progression was recorded at a median of 13 months after induction. CONCLUSION:Achieving an IR translates into superior PFS and TTP compared with conventional CR or stringent CR. These techniques provide complementary information and thus, an effort should be made to refine response criteria in MM.
Cigarette smoking impairs human pulmonary immunity to Mycobacterium tuberculosis.
O'Leary Seónadh M,Coleman Michelle M,Chew Wui Mei,Morrow Colette,McLaughlin Anne Marie,Gleeson Laura E,O'Sullivan Mary P,Keane Joseph
American journal of respiratory and critical care medicine
RATIONALE:Cigarette smoking is linked to important aspects of tuberculosis, such as susceptibility to infection, disease reactivation, mortality, transmission, and persistent infectiousness. The mechanistic basis for this remains poorly understood. OBJECTIVES:To compare the functional impairment seen in human alveolar macrophages (AM) from nonsmokers, smokers, and ex-smokers after infection with Mycobacterium tuberculosis (Mtb). METHODS:AM were acquired at bronchoscopy, and number and viability from smoking donors were compared with nonsmoking donors. AM were challenged in vitro with Mtb and intracellular bacterial viability was measured. Cytokine secretion was measured 24 hours postinfection by ELISA. Previously we determined the frequency of CD4(+)FoxP3(+) T cells in the presence or absence of allogeneic AM, and data were reanalyzed to separate the patient subjects according to smoking status. MEASUREMENTS AND MAIN RESULTS:There were significantly more AM from smokers compared with nonsmokers or ex-smokers (P < 0.01). AM from smokers could not control intracellular Mtb growth. Nonsmokers' AM generated significantly more tumor necrosis factor (TNF)-α, IFN-γ, and IL-1β after Mtb infection compared with uninfected AM (P < 0.05). However, Mtb-infected AM from smokers did not secrete significantly more TNF-α, IFN-γ, and IL-1β compared with uninfected smokers' AM. AM taken from ex-smokers also failed to secrete significantly increased TNF-α, IFN-γ, and IL-1β after Mtb infection. Both smokers' and nonsmokers' AM induced FoxP3(+) T regulatory cell phenotype responses in allogeneic admixed T cells (>4.8 fold; P < 0.05). Even after Mtb infection, AM continued to drive this regulatory phenotype. CONCLUSIONS:In smokers, the pulmonary compartment has a number of macrophage-specific immune impairments that provide some mechanistic explanations whereby cigarette smoking renders a patient susceptible to tuberculosis infection and disease.
Genetically Encoded Tools for Optical Dissection of the Mammalian Cell Cycle.
Sakaue-Sawano Asako,Yo Masahiro,Komatsu Naoki,Hiratsuka Toru,Kogure Takako,Hoshida Tetsushi,Goshima Naoki,Matsuda Michiyuki,Miyoshi Hiroyuki,Miyawaki Atsushi
Eukaryotic cells spend most of their life in interphase of the cell cycle. Understanding the rich diversity of metabolic and genomic regulation that occurs in interphase requires the demarcation of precise phase boundaries in situ. Here, we report the properties of two genetically encoded fluorescence sensors, Fucci(CA) and Fucci(SCA), which enable real-time monitoring of interphase and cell-cycle biology. We re-engineered the Cdt1-based sensor from the original Fucci system to respond to S phase-specific CUL4-mediated ubiquitylation alone or in combination with SCF-mediated ubiquitylation. In cultured cells, Fucci(CA) produced a sharp triple color-distinct separation of G1, S, and G2, while Fucci(SCA) permitted a two-color readout of G1 and S/G2. Fucci(CA) applications included tracking the transient G1 phase of rapidly dividing mouse embryonic stem cells and identifying a window for UV-irradiation damage in S phase. These results show that Fucci(CA) is an essential tool for quantitative studies of interphase cell-cycle regulation.
An immunological biomarker to predict MTX response in early RA.
Ponchel Frederique,Goëb Vincent,Parmar Rekha,El-Sherbiny Yasser,Boissinot Marjorie,El Jawhari Jehan,Burska Agata,Vital Edward M,Harrison Stephanie,Conaghan Philip G,Hensor Elizabeth,Emery Paul
Annals of the rheumatic diseases
OBJECTIVES:The therapeutic goal for patients with rheumatoid arthritis (RA) is clinical remission. This is best achieved by early diagnosis and appropriate therapeutic intervention. RA is associated with dysregulation of T-cell subsets (naïve, regulatory (Treg) and inflammation-related cells (IRC)) early in the disease. Our aim was to test the hypothesis that T-cell subset quantification can predict the achievement of clinical remission with early treatment in RA. METHODS:T-cell subsets were quantified in 108 drug-naïve, early RA patients commencing methotrexate (MTX) or MTX+antitumor necrosis factor (anti-TNF) and in 105 healthy controls (HC). The primary outcome assessed was remission (DAS28<2.6). A pilot study used frozen cells (38 patients and 35 HCs, see online supplementary material) and was validated with fresh blood (70 patients and 70 HCs). RESULTS:Immune dysregulation in early RA was confirmed with an association between age and reduced naïve cells compared with HCs (p=0.006), a lower age-adjusted Treg and higher IRC frequency (p=0.001). Anticitrullinated peptide antibody (ACPA) positivity was associated with lower naïve (p=0.031) and Treg frequencies (p=0.039). In 50 patients treated with MTX, ACPA/age-adjusted analysis demonstrated that higher naïve cell frequency (relative to HC) was associated with remission (OR 5.90 (1.66 to 20.98), p=0.006, sensitivity/specificity 62%/79%, Positive Predictive Value (PPV)/Negative Predictive Value (NPV) 66%/76%). Remission with MTX+anti-TNF (n=20) was not found to be associated with naïve cell frequency, and for patients with reduced naïve cells the remission rate increased from 24% (MTX) to 42% (MTX+anti-TNF). CONCLUSIONS:Baseline T-cell subset analysis has a value in predicting early RA remission with first therapy with MTX. Immunological analysis could be used in conjunction with clinical/serological features to predict response to MTX and help select the most appropriate therapy at disease presentation.
Single-Cell RNA Sequencing of the T Helper Cell Response to House Dust Mites Defines a Distinct Gene Expression Signature in Airway Th2 Cells.
Tibbitt Christopher Andrew,Stark Julian Mario,Martens Liesbet,Ma Junjie,Mold Jeff Eron,Deswarte Kim,Oliynyk Ganna,Feng Xiaogang,Lambrecht Bart Norbert,De Bleser Pieter,Nylén Susanne,Hammad Hamida,Arsenian Henriksson Marie,Saeys Yvan,Coquet Jonathan Marie
Naive CD4 T cells differentiate into functionally diverse T helper (Th) cell subsets. Th2 cells play a pathogenic role in asthma, yet a clear picture of their transcriptional profile is lacking. We performed single-cell RNA sequencing (scRNA-seq) of T helper cells from lymph node, lung, and airways in the house dust mite (HDM) model of allergic airway disease. scRNA-seq resolved transcriptional profiles of naive CD4 T, Th1, Th2, regulatory T (Treg) cells, and a CD4 T cell population responsive to type I interferons. Th2 cells in the airways were enriched for transcription of many genes, including Cd200r1, Il6, Plac8, and Igfbp7, and their mRNA profile was supported by analysis of chromatin accessibility and flow cytometry. Pathways associated with lipid metabolism were enriched in Th2 cells, and experiments with inhibitors of key metabolic pathways supported roles for glucose and lipid metabolism. These findings provide insight into the differentiation of pathogenic Th2 cells in the context of allergy.
Activated intestinal muscle cells promote preadipocyte migration: a novel mechanism for creeping fat formation in Crohn's disease.
Mao Ren,Doyon Genevieve,Gordon Ilyssa O,Li Jiannan,Lin Sinan,Wang Jie,Le Thi Hong Nga,Elias Michael,Kurada Satya,Southern Brian,Olman Mitchell,Chen Minhu,Zhao Shuai,Dejanovic Dina,Chandra Jyotsna,Mukherjee Pranab K,West Gail,Van Wagoner David R,Fiocchi Claudio,Rieder Florian
OBJECTIVE:Creeping fat, the wrapping of mesenteric fat around the bowel wall, is a typical feature of Crohn's disease, and is associated with stricture formation and bowel obstruction. How creeping fat forms is unknown, and we interrogated potential mechanisms using novel intestinal tissue and cell interaction systems. DESIGN:Tissues from normal, UC, non-strictured and strictured Crohn's disease intestinal specimens were obtained. The muscularis propria matrisome was determined via proteomics. Mesenteric fat explants, primary human preadipocytes and adipocytes were used in multiple ex vivo and in vitro cell migration systems on muscularis propria muscle cell derived or native extracellular matrix. Functional experiments included integrin characterisation via flow cytometry and their inhibition with specific blocking antibodies and chemicals. RESULTS:Crohn's disease muscularis propria cells produced an extracellular matrix scaffold which is in direct spatial and functional contact with the immediately overlaid creeping fat. The scaffold contained multiple proteins, but only fibronectin production was singularly upregulated by transforming growth factor-β1. The muscle cell-derived matrix triggered migration of preadipocytes out of mesenteric fat, fibronectin being the dominant factor responsible for their migration. Blockade of α5β1 on the preadipocyte surface inhibited their migration out of mesenteric fat and on 3D decellularised intestinal tissue extracellular matrix. CONCLUSION:Crohn's disease creeping fat appears to result from the migration of preadipocytes out of mesenteric fat and differentiation into adipocytes in response to an increased production of fibronectin by activated muscularis propria cells. These new mechanistic insights may lead to novel approaches for prevention of creeping fat-associated stricture formation.
Sensitive detection of RNAs in single cells by flow cytometry.
Yu H,Ernst L,Wagner M,Waggoner A
Nucleic acids research
A rapid and sensitive fluorescent in situ hybridization method has been developed to probe RNA contents of individual cells by flow cytometry. Fixed cells in suspension were hybridized with 5' end-fluorophore-labeled oligodeoxynucleotides complementary to defined regions of the RNA of interest and analyzed by flow cytometry. With this method, we monitored combinations of histone H4 mRNA, 18S rRNA and 28S rRNA levels in synchronized HeLa S3 cells by multicolor analysis. A fluorescence signal equivalent to 1800 copies of histone H4 mRNA per cell was detected with signal-to-background ratio of 5.4. If non-specific binding of the fluorophore-labeled probe can be reduced, as few as 100 copies of mRNA of the size of H4 could be detected in individual cells by flow cytometry.
Primary CNS CCND1/MYC-Positive Double-Hit B-Cell Lymphoma: A Case Report and Review of the Literature.
Sakurai Hiroko,Sugimoto Kei-Ji,Shimada Asami,Imai Hidenori,Wakabayashi Mutsumi,Sekiguchi Yasunobu,Ota Yasunori,Izutsu Koji,Takeuchi Kengo,Komatsu Norio,Noguchi Masaaki
Journal of clinical oncology : official journal of the American Society of Clinical Oncology
Association of Ipilimumab With Safety and Antitumor Activity in Women With Metastatic or Recurrent Human Papillomavirus-Related Cervical Carcinoma.
Lheureux Stephanie,Butler Marcus O,Clarke Blaise,Cristea Mihaela C,Martin Lainie P,Tonkin Katia,Fleming Gini F,Tinker Anna V,Hirte Hal W,Tsoref Daliah,Mackay Helen,Dhani Neesha C,Ghatage Prafull,Weberpals Johanne,Welch Stephen,Pham Nhu-An,Motta Vinicius,Sotov Valentin,Wang Lisa,Karakasis Katherine,Udagani Smitha,Kamel-Reid Suzanne,Streicher Howard Z,Shaw Patricia,Oza Amit M
Importance:Based on evidence of human papillomavirus (HPV)-induced immune evasion, immunotherapy may be an attractive strategy in cervical cancer. Ipilimumab is a fully humanized monoclonal antibody that blocks cytotoxic T-lymphocyte antigen-4 (CTLA-4), which acts to downregulate the T-cell immune response. Objective:To assess the safety and antitumor activity of ipilimumab in recurrent cervical cancer. Design, Setting, and Participants:A multicenter trial was designed for patients with metastatic cervical cancer (squamous cell carcinoma or adenocarcinoma) with measurable disease and progression after at least 1 line of platinum chemotherapy. A run-in safety cohort using ipilimumab, 3 mg/kg, every 21 days for 4 cycles in 6 patients was followed by a phase II cohort of ipilimumab, 10 mg/kg, every 21 days for 4 cycles and then 4 cycles of maintenance therapy every 12 weeks for patients demonstrating radiologic response or stabilization. Immune correlative studies were performed on peripheral blood before and after therapy on archival tissue and fresh tumor obtained prior to registration and 7 days after cycle 2. The study was conducted from December 3, 2012, to September 15, 2014. The data were analyzed from April 2016 to June 2016 and in July 2017. Main Outcomes and Measures:The primary end points were safety and objective response rate. Immune analyses were performed on blood and tumor tissue. Results:A total of 42 women (median age, 49 years; range, 23-78 years) were enrolled (29 [69%] squamous cell cervical cancer and 13 [31%] adenocarcinoma; 37 [93%] of 40 patients with tissue available for analysis had HPV-positive confirmation; there was no archival tissue for 2 women). Grade 3 toxic effects included diarrhea in 4 patients, 3 of whom had colitis. Of 34 patients evaluated for best response (Response Evaluation Criteria in Solid Tumors, version 1.1), 1 patient had partial response and 10 had stable disease. The median progression-free survival and overall survival were 2.5 months (95% CI, 2.1-3.2 months) and 8.5 months (95% CI, 3.6-not reached; 1 patient was still alive), respectively. Intratumoral pretreatment CD3, CD4, CD8, FoxP3, indoleamine 2,3-dioxygenase, and programmed cell death ligand 1 (PD-L1) expression was not predictive of benefit and did not significantly change with treatment. Multicolor flow cytometry on peripheral lymphocytes revealed a treatment-dependent increase of inducible T-cell costimulator, human leukocyte antigen-antigen D related, and PD-1 during initial treatment, which returned to baseline during maintenance. Conclusions and Relevance:Ipilimumab was tolerable in this population but did not show significant single-agent activity. Immune changes were induced by anti-CTLA-4 therapy but did not correlate with clinical activity. Changes in these markers may guide further treatment strategies.
IFN-λ resolves inflammation via suppression of neutrophil infiltration and IL-1β production.
Blazek Katrina,Eames Hayley L,Weiss Miriam,Byrne Adam J,Perocheau Dany,Pease James E,Doyle Sean,McCann Fiona,Williams Richard O,Udalova Irina A
The Journal of experimental medicine
The most studied biological role of type III interferons (IFNs) has so far been their antiviral activity, but their role in autoimmune and inflammatory diseases remains largely unexplored. Here, we show that treatment with IFN-λ2/IL-28A completely halts and reverses the development of collagen-induced arthritis (CIA) and discover cellular and molecular mechanisms of IL-28A antiinflammatory function. We demonstrate that treatment with IL-28A dramatically reduces numbers of proinflammatory IL-17-producing Th17 and γδ T cells in the joints and inguinal lymph nodes, without affecting T cell proliferative responses or levels of anticollagen antibodies. IL-28A exerts its antiinflammatory effect by restricting recruitment of IL-1b-expressing neutrophils, which are important for amplification of inflammation. We identify neutrophils as cells expressing high levels of IFN-λ receptor 1 (IFNLR1)-IL-28 receptor α (IL28RA) and targeted by IL-28A. Our data highlight neutrophils as contributors to the pathogenesis of autoimmune arthritis and present IFN-λs or agonists of IFNLR1-IL28RA as putative new therapeutics for neutrophil-driven inflammation.
Bcl11b is essential for group 2 innate lymphoid cell development.
Walker Jennifer A,Oliphant Christopher J,Englezakis Alexandros,Yu Yong,Clare Simon,Rodewald Hans-Reimer,Belz Gabrielle,Liu Pentao,Fallon Padraic G,McKenzie Andrew N J
The Journal of experimental medicine
Group 2 innate lymphoid cells (ILC2s) are often found associated with mucosal surfaces where they contribute to protective immunity, inappropriate allergic responses, and tissue repair. Although we know they develop from a common lymphoid progenitor in the bone marrow (BM), the specific lineage path and transcriptional regulators that are involved are only starting to emerge. After ILC2 gene expression analysis we investigated the role of Bcl11b, a factor previously linked to T cell commitment, in ILC2 development. Using combined Bcl11b-tom and Id2-gfp reporter mice, we show that Bcl11b is expressed in ILC2 precursors in the BM and maintained in mature ILC2s. In vivo deletion of Bcl11b, by conditional tamoxifen-induced depletion or by Bcl11b(-/-) fetal liver chimera reconstitution, demonstrates that ILC2s are wholly dependent on Bcl11b for their development. Notably, in the absence of Bcl11b there is a concomitant expansion of the RORγt(+) ILC3 population, suggesting that Bcl11b may negatively regulate this lineage. Using Nippostrongylus brasiliensis infection, we reveal that the absence of Bcl11b leads to impaired worm expulsion, caused by a deficit in ILC2s, whereas Citrobacter rodentium infection is cleared efficiently. These data clearly establish Bcl11b as a new factor in the differentiation of ILC2s.
DYRK1A controls the transition from proliferation to quiescence during lymphoid development by destabilizing Cyclin D3.
Thompson Benjamin J,Bhansali Rahul,Diebold Lauren,Cook Daniel E,Stolzenburg Lindsay,Casagrande Anne-Sophie,Besson Thierry,Leblond Bertrand,Désiré Laurent,Malinge Sébastien,Crispino John D
The Journal of experimental medicine
Pre-B and pre-T lymphocytes must orchestrate a transition from a highly proliferative state to a quiescent one during development. Cyclin D3 is essential for these cells' proliferation, but little is known about its posttranslational regulation at this stage. Here, we show that the dual specificity tyrosine-regulated kinase 1A (DYRK1A) restrains Cyclin D3 protein levels by phosphorylating T283 to induce its degradation. Loss of DYRK1A activity, via genetic inactivation or pharmacologic inhibition in mice, caused accumulation of Cyclin D3 protein, incomplete repression of E2F-mediated gene transcription, and failure to properly couple cell cycle exit with differentiation. Expression of a nonphosphorylatable Cyclin D3 T283A mutant recapitulated these defects, whereas inhibition of Cyclin D:CDK4/6 mitigated the effects of DYRK1A inhibition or loss. These data uncover a previously unknown role for DYRK1A in lymphopoiesis, and demonstrate how Cyclin D3 protein stability is negatively regulated during exit from the proliferative phases of B and T cell development.
CCL2-induced chemokine cascade promotes breast cancer metastasis by enhancing retention of metastasis-associated macrophages.
Kitamura Takanori,Qian Bin-Zhi,Soong Daniel,Cassetta Luca,Noy Roy,Sugano Gaël,Kato Yu,Li Jiufeng,Pollard Jeffrey W
The Journal of experimental medicine
Pulmonary metastasis of breast cancer cells is promoted by a distinct population of macrophages, metastasis-associated macrophages (MAMs), which originate from inflammatory monocytes (IMs) recruited by the CC-chemokine ligand 2 (CCL2). We demonstrate here that, through activation of the CCL2 receptor CCR2, the recruited MAMs secrete another chemokine ligand CCL3. Genetic deletion of CCL3 or its receptor CCR1 in macrophages reduces the number of lung metastasis foci, as well as the number of MAMs accumulated in tumor-challenged lung in mice. Adoptive transfer of WT IMs increases the reduced number of lung metastasis foci in Ccl3 deficient mice. Mechanistically, Ccr1 deficiency prevents MAM retention in the lung by reducing MAM-cancer cell interactions. These findings collectively indicate that the CCL2-triggered chemokine cascade in macrophages promotes metastatic seeding of breast cancer cells thereby amplifying the pathology already extant in the system. These data suggest that inhibition of CCR1, the distal part of this signaling relay, may have a therapeutic impact in metastatic disease with lower toxicity than blocking upstream targets.
The lysophosphatidylserine receptor GPR174 constrains regulatory T cell development and function.
Barnes Michael J,Li Chien-Ming,Xu Ying,An Jinping,Huang Yong,Cyster Jason G
The Journal of experimental medicine
Regulatory T cell (T reg cell) numbers and activities are tightly calibrated to maintain immune homeostasis, but the mechanisms involved are incompletely defined. Here, we report that the lysophosphatidylserine (LysoPS) receptor GPR174 is abundantly expressed in developing and mature T reg cells. In mice that lacked this X-linked gene, T reg cell generation in the thymus was intrinsically favored, and a higher fraction of peripheral T reg cells expressed CD103. LysoPS could act in vitro via GPR174 to suppress T cell proliferation and T reg cell generation. In vivo, LysoPS was detected in lymphoid organ and spinal cord tissues and was abundant in the colon. Gpr174(-/Y) mice were less susceptible to experimental autoimmune encephalomyelitis than wild-type mice, and GPR174 deficiency in T reg cells contributed to this phenotype. This study provides evidence that a bioactive lipid, LysoPS, negatively influences T reg cell accumulation and activity through GPR174. As such, GPR174 antagonists might have therapeutic potential for promoting immune regulation in the context of autoimmune disease.
High Graft CD8 Cell Dose Predicts Improved Survival and Enables Better Donor Selection in Allogeneic Stem-Cell Transplantation With Reduced-Intensity Conditioning.
Reshef Ran,Huffman Austin P,Gao Amy,Luskin Marlise R,Frey Noelle V,Gill Saar I,Hexner Elizabeth O,Kambayashi Taku,Loren Alison W,Luger Selina M,Mangan James K,Nasta Sunita D,Richman Lee P,Sell Mary,Stadtmauer Edward A,Vonderheide Robert H,Mick Rosemarie,Porter David L
Journal of clinical oncology : official journal of the American Society of Clinical Oncology
PURPOSE:To characterize the impact of graft T-cell composition on outcomes of reduced-intensity conditioned (RIC) allogeneic hematopoietic stem-cell transplantation (alloHSCT) in adults with hematologic malignancies. PATIENTS AND METHODS:We evaluated associations between graft T-cell doses and outcomes in 200 patients who underwent RIC alloHSCT with a peripheral blood stem-cell graft. We then studied 21 alloHSCT donors to identify predictors of optimal graft T-cell content. RESULTS:Higher CD8 cell doses were associated with a lower risk for relapse (adjusted hazard ratio [aHR], 0.43; P = .009) and improved relapse-free survival (aHR, 0.50; P = .006) and overall survival (aHR, 0.57; P = .04) without a significant increase in graft-versus-host disease or nonrelapse mortality. A cutoff level of 0.72 × 10(8) CD8 cells per kilogram optimally segregated patients receiving CD8(hi) and CD8(lo) grafts with differing overall survival (P = .007). Donor age inversely correlated with graft CD8 dose. Consequently, older donors were unlikely to provide a CD8(hi) graft, whereas approximately half of younger donors provided CD8(hi) grafts. Compared with recipients of older sibling donor grafts (consistently containing CD8(lo) doses), survival was significantly better for recipients of younger unrelated donor grafts with CD8(hi) doses (P = .03), but not for recipients of younger unrelated donor CD8(lo) grafts (P = .28). In addition, graft CD8 content could be predicted by measuring the proportion of CD8 cells in a screening blood sample from stem-cell donors. CONCLUSION:Higher graft CD8 dose, which was restricted to young donors, predicted better survival in patients undergoing RIC alloHSCT.
In vivo tracking of 'color-coded' effector, natural and induced regulatory T cells in the allograft response.
Fan Zhigang,Spencer Joel A,Lu Yan,Pitsillides Costas M,Singh Gurbakhshish,Kim Pilhan,Yun Seok H,Toxavidis Vasilis,Strom Terry B,Lin Charles P,Koulmanda Maria
Here we present methods to longitudinally track islet allograft-infiltrating T cells in live mice by endoscopic confocal microscopy and to analyze circulating T cells by in vivo flow cytometry. We developed a new reporter mouse whose T cell subsets express distinct, 'color-coded' proteins enabling in vivo detection and identification of effector T cells (T(eff) cells) and discrimination between natural and induced regulatory T cells (nT(reg) and iT(reg) cells). Using these tools, we observed marked differences in the T cell response in recipients receiving tolerance-inducing therapy (CD154-specific monoclonal antibody plus rapamycin) compared to untreated controls. These results establish real-time cell tracking as a powerful means to probe the dynamic cellular interplay mediating immunologic rejection or transplant tolerance.
STAT3 is a critical cell-intrinsic regulator of human unconventional T cell numbers and function.
Wilson Robert P,Ives Megan L,Rao Geetha,Lau Anthony,Payne Kathryn,Kobayashi Masao,Arkwright Peter D,Peake Jane,Wong Melanie,Adelstein Stephen,Smart Joanne M,French Martyn A,Fulcher David A,Picard Capucine,Bustamante Jacinta,Boisson-Dupuis Stephanie,Gray Paul,Stepensky Polina,Warnatz Klaus,Freeman Alexandra F,Rossjohn Jamie,McCluskey James,Holland Steven M,Casanova Jean-Laurent,Uzel Gulbu,Ma Cindy S,Tangye Stuart G,Deenick Elissa K
The Journal of experimental medicine
Unconventional T cells such as γδ T cells, natural killer T cells (NKT cells) and mucosal-associated invariant T cells (MAIT cells) are a major component of the immune system; however, the cytokine signaling pathways that control their development and function in humans are unknown. Primary immunodeficiencies caused by single gene mutations provide a unique opportunity to investigate the role of specific molecules in regulating human lymphocyte development and function. We found that individuals with loss-of-function mutations in STAT3 had reduced numbers of peripheral blood MAIT and NKT but not γδ T cells. Analysis of STAT3 mosaic individuals revealed that this effect was cell intrinsic. Surprisingly, the residual STAT3-deficient MAIT cells expressed normal levels of the transcription factor RORγt. Despite this, they displayed a deficiency in secretion of IL-17A and IL-17F, but were able to secrete normal levels of cytokines such as IFNγ and TNF. The deficiency in MAIT and NKT cells in STAT3-deficient patients was mirrored by loss-of-function mutations in IL12RB1 and IL21R, respectively. Thus, these results reveal for the first time the essential role of STAT3 signaling downstream of IL-23R and IL-21R in controlling human MAIT and NKT cell numbers.
The transcription factor Bcl11b is specifically expressed in group 2 innate lymphoid cells and is essential for their development.
Yu Yong,Wang Cui,Clare Simon,Wang Juexuan,Lee Song-Choon,Brandt Cordelia,Burke Shannon,Lu Liming,He Daqian,Jenkins Nancy A,Copeland Neal G,Dougan Gordon,Liu Pentao
The Journal of experimental medicine
Group 2 innate lymphoid cells (ILCs), or ILC2s, are a subset of recently identified ILCs, which play important roles in innate immunity by producing type 2 effector cytokines. Several transcription factors have been found to have critical functions in the development of both ILC2s and T cells. We report here that Bcl11b, a transcription factor essential in T cell lineage commitment and maintenance, is specifically expressed in progenitors committed to the ILC2 lineage and is required for ILC2 development. The Bcl11b gene is expressed in ∼28% of ILC progenitors (ILCPs; common helper innate lymphoid progenitors or ILCPs expressing either ID2 or promyelocytic leukemia zinc finger, respectively). Both in vitro and in vivo, these Bcl11b-expressing early ILCPs generate only ILC2s. Inactivation of Bcl11b causes a complete loss of ILC2 development from hematopoietic progenitors, which is confirmed upon immune challenge with either papain administration or influenza virus infection.
BAFF activation of the ERK5 MAP kinase pathway regulates B cell survival.
Jacque Emilie,Schweighoffer Edina,Tybulewicz Victor L J,Ley Steven C
The Journal of experimental medicine
B cell activating factor (BAFF) stimulation of the BAFF receptor (BAFF-R) is essential for the homeostatic survival of mature B cells. Earlier in vitro experiments with inhibitors that block MEK 1 and 2 suggested that activation of ERK 1 and 2 MAP kinases is required for BAFF-R to promote B cell survival. However, these inhibitors are now known to also inhibit MEK5, which activates the related MAP kinase ERK5. In the present study, we demonstrated that BAFF-induced B cell survival was actually independent of ERK1/2 activation but required ERK5 activation. Consistent with this, we showed that conditional deletion of ERK5 in B cells led to a pronounced global reduction in mature B2 B cell numbers, which correlated with impaired survival of ERK5-deficient B cells after BAFF stimulation. ERK5 was required for optimal BAFF up-regulation of Mcl1 and Bcl2a1, which are prosurvival members of the Bcl-2 family. However, ERK5 deficiency did not alter BAFF activation of the PI3-kinase-Akt or NF-κB signaling pathways, which are also important for BAFF to promote mature B cell survival. Our study reveals a critical role for the MEK5-ERK5 MAP kinase signaling pathway in BAFF-induced mature B cell survival and homeostatic maintenance of B2 cell numbers.
Genetic absence of PD-1 promotes accumulation of terminally differentiated exhausted CD8+ T cells.
Odorizzi Pamela M,Pauken Kristen E,Paley Michael A,Sharpe Arlene,Wherry E John
The Journal of experimental medicine
Programmed Death-1 (PD-1) has received considerable attention as a key regulator of CD8(+) T cell exhaustion during chronic infection and cancer because blockade of this pathway partially reverses T cell dysfunction. Although the PD-1 pathway is critical in regulating established "exhausted" CD8(+) T cells (TEX cells), it is unclear whether PD-1 directly causes T cell exhaustion. We show that PD-1 is not required for the induction of exhaustion in mice with chronic lymphocytic choriomeningitis virus (LCMV) infection. In fact, some aspects of exhaustion are more severe with genetic deletion of PD-1 from the onset of infection. Increased proliferation between days 8 and 14 postinfection is associated with subsequent decreased CD8(+) T cell survival and disruption of a critical proliferative hierarchy necessary to maintain exhausted populations long term. Ultimately, the absence of PD-1 leads to the accumulation of more cytotoxic, but terminally differentiated, CD8(+) TEX cells. These results demonstrate that CD8(+) T cell exhaustion can occur in the absence of PD-1. They also highlight a novel role for PD-1 in preserving TEX cell populations from overstimulation, excessive proliferation, and terminal differentiation.
Combined heterozygous loss of Ebf1 and Pax5 allows for T-lineage conversion of B cell progenitors.
Ungerbäck Jonas,Åhsberg Josefine,Strid Tobias,Somasundaram Rajesh,Sigvardsson Mikael
The Journal of experimental medicine
To investigate how transcription factor levels impact B-lymphocyte development, we generated mice carrying transheterozygous mutations in the Pax5 and Ebf1 genes. Whereas combined reduction of Pax5 and Ebf1 had minimal impact on the development of the earliest CD19(+) progenitors, these cells displayed an increased T cell potential in vivo and in vitro. The alteration in lineage fate depended on a Notch1-mediated conversion process, whereas no signs of de-differentiation could be detected. The differences in functional response to Notch signaling in Wt and Pax5(+/-)Ebf1(+/-) pro-B cells were reflected in the transcriptional response. Both genotypes responded by the generation of intracellular Notch1 and activation of a set of target genes, but only the Pax5(+/-)Ebf1(+/-) pro-B cells down-regulated genes central for the preservation of stable B cell identity. This report stresses the importance of the levels of transcription factor expression during lymphocyte development, and suggests that Pax5 and Ebf1 collaborate to modulate the transcriptional response to Notch signaling. This provides an insight on how transcription factors like Ebf1 and Pax5 preserve cellular identity during differentiation.
c-Myb is required for plasma cell migration to bone marrow after immunization or infection.
Good-Jacobson Kim L,O'Donnell Kristy,Belz Gabrielle T,Nutt Stephen L,Tarlinton David M
The Journal of experimental medicine
Plasma cell migration is crucial to immunity, but little is known about the molecular regulators of their migratory programs. Here, we detail the critical role of the transcription factor c-Myb in determining plasma cell location. In the absence of c-Myb, no IgG(+) antigen-specific plasma cells were detected in the bone marrow after immunization or virus infection. This was correlated with a dramatic reduction of plasma cells in peripheral blood, mislocalization in spleen, and an inability of c-Myb-deficient plasma cells to migrate along a CXCL12 gradient. Therefore, c-Myb plays an essential, novel role in establishing the long-lived plasma cell population in the BM via responsiveness to chemokine migration cues.
Autoimmunity-associated protein tyrosine phosphatase PEP negatively regulates IFN-α receptor signaling.
Holmes Derek A,Suto Eric,Lee Wyne P,Ou Qinglin,Gong Qian,Smith Hamish R C,Caplazi Patrick,Chan Andrew C
The Journal of experimental medicine
The protein tyrosine phosphatase PTPN22(C1858T) allelic polymorphism is associated with increased susceptibility for development of systemic lupus erythematosus (SLE) and other autoimmune diseases. PTPN22 (also known as LYP) and its mouse orthologue PEP play important roles in antigen and Toll-like receptor signaling in immune cell functions. We demonstrate here that PEP also plays an important inhibitory role in interferon-α receptor (IFNAR) signaling in mice. PEP co-immunoprecipitates with components of the IFNAR signaling complex. Pep(-/-) hematopoietic progenitors demonstrate increased IFNAR signaling, increased IFN-inducible gene expression, and enhanced proliferation and activation compared to Pep(+/+) progenitors in response to IFN-α. In addition, Pep(-/-) mice treated with IFN-α display a profound defect in hematopoiesis, resulting in anemia, thrombocytopenia, and neutropenia when compared to IFN-α-treated Pep(+/+) mice. As SLE patients carrying the PTPN22(C1858T) risk variant have higher serum IFN-α activity, these data provide a molecular basis for how type I IFNs and PTPN22 may cooperate to contribute to lupus-associated cytopenias.
Endothelial CD99 signals through soluble adenylyl cyclase and PKA to regulate leukocyte transendothelial migration.
Watson Richard L,Buck Jochen,Levin Lonny R,Winger Ryan C,Wang Jing,Arase Hisashi,Muller William A
The Journal of experimental medicine
CD99 is a critical regulator of leukocyte transendothelial migration (TEM). How CD99 signals during this process remains unknown. We show that during TEM, endothelial cell (EC) CD99 activates protein kinase A (PKA) via a signaling complex formed with the lysine-rich juxtamembrane cytoplasmic tail of CD99, the A-kinase anchoring protein ezrin, and soluble adenylyl cyclase (sAC). PKA then stimulates membrane trafficking from the lateral border recycling compartment to sites of TEM, facilitating the passage of leukocytes across the endothelium. Pharmacologic or genetic inhibition of EC sAC or PKA, like CD99 blockade, arrests neutrophils and monocytes partway through EC junctions, in vitro and in vivo, without affecting leukocyte adhesion or the expression of relevant cellular adhesion molecules. This is the first description of the CD99 signaling pathway in TEM as well as the first demonstration of a role for sAC in leukocyte TEM.
Glucose Metabolism Drives Histone Acetylation Landscape Transitions that Dictate Muscle Stem Cell Function.
Yucel Nora,Wang Yu Xin,Mai Thach,Porpiglia Ermelinda,Lund Peder J,Markov Glenn,Garcia Benjamin A,Bendall Sean C,Angelo Michael,Blau Helen M
The impact of glucose metabolism on muscle regeneration remains unresolved. We identify glucose metabolism as a crucial driver of histone acetylation and myogenic cell fate. We use single-cell mass cytometry (CyTOF) and flow cytometry to characterize the histone acetylation and metabolic states of quiescent, activated, and differentiating muscle stem cells (MuSCs). We find glucose is dispensable for mitochondrial respiration in proliferating MuSCs, so that glucose becomes available for maintaining high histone acetylation via acetyl-CoA. Conversely, quiescent and differentiating MuSCs increase glucose utilization for respiration and have consequently reduced acetylation. Pyruvate dehydrogenase (PDH) activity serves as a rheostat for histone acetylation and must be controlled for muscle regeneration. Increased PDH activity in proliferation increases histone acetylation and chromatin accessibility at genes that must be silenced for differentiation to proceed, and thus promotes self-renewal. These results highlight metabolism as a determinant of MuSC histone acetylation, fate, and function during muscle regeneration.
A 1-week intradermal dose-sparing regimen for rabies post-exposure prophylaxis (RESIST-2): an observational cohort study.
Cantaert Tineke,Borand Laurence,Kergoat Lauriane,Leng Chanthy,Ung Sivlin,In Sotheary,Peng Yiksing,Phoeun Chandara,Hing Chanthy,Taing Chun Navy,Saman Manil,Ong Sivuth,Mey Channa,Choeung Rithy,Ly Sowath,Dussart Philippe,Bourhy Hervé,Tarantola Arnaud
The Lancet. Infectious diseases
BACKGROUND:The international health authorities are backing an effort to eliminate canine-mediated rabies in humans by 2030. This effort will require improving access to adequate and timely rabies post-exposure prophylaxis as compliance is low with WHO-recommended regimens (given in four to five visits over 1 month). Access could be substantially improved by an abridged regimen to reduce doses, direct and indirect costs, and improve vaccine equity by better sharing of available vaccine. We aimed to compare rabies virus neutralising antibody titres before and after the fourth visit to determine whether that session was needed or the current regimen could be abridged. METHODS:In this observational cohort study, we measured rabies virus neutralising antibody titres using rapid fluorescent focus inhibition tests in 116 people bitten by dogs with laboratory-confirmed rabies and 20 control individuals. Percentages of circulating plasmablasts were determined by flow cytometry. All individuals had been referred to the rabies prevention clinic at Institut Pasteur in Cambodia and received two intradermal injections of post-exposure prophylaxis on days 0, 3, 7, and 28 (Thai Red Cross regimen) with or without equine rabies immunoglobulin, as per 2010 WHO recommendations. FINDINGS:All individuals had rabies virus neutralising antibody titres considered protective (≥0·5 IU/mL) and plasmablast activation on day 28 before the last injection. The median rabies virus neutralising antibody concentration in the group of individuals bitten by rabies virus-positive dogs was 1·08 IU/mL (IQR 0·37-3·09) on day 7, 26·86 (22·68-49·50) on day 28, and 26·74 (11·78-49·06) on day 42. No significant differences were observed in titres between days 28 and 42, after titres reached a plateau. These titres were reached notwithstanding equine rabies immunoglobulin use, age, sex, nutrition status as indicated by upper-arm circumference in children or BMI in adults, or dog infection status. Titres or plasmablast percentages did not increase between the day of the last injection and 2 weeks later. All patients were alive 1 year after post-exposure prophylaxis. INTERPRETATION:The fourth vaccine session on day 28 provides no additional benefit. Rabies post-exposure prophylaxis can be abridged to a two-dose, three-session, 1 week regimen to improve post-exposure prophylaxis coverage and equity at no risk to patients. FUNDING:Institut Pasteur.
Biomimetic Hydroxyapatite Nanorods Promote Bone Regeneration Accelerating Osteogenesis of BMSCs through T Cell-Derived IL-22.
Yu Fei,Lian Ruixian,Liu Lu,Liu Ting,Bi Chao,Hong Kan,Zhang Shuiquan,Ren Jiazi,Wang Haikun,Ouyang Ningjuan,Du Lin-Juan,Liu Yuan,Zhou Lujun,Liu Yan,Fang Bing,Li Yulin,Duan Sheng-Zhong,Xia Lunguo
Manipulations of morphological properties of nanobiomaterials have been demonstrated to modulate the outcome of osteoimmunomodulation and eventually osteogenesis through innate immune response. However, the functions and mechanisms of adaptive immune cells in the process of nanobiomaterials-mediated bone regeneration have remained unknown. Herein, we developed bone-mimicking hydroxyapatite (HAp) nanorods with different aspect ratios as model materials to investigate the impacts of the nanoshape features on osteogenesis and to explore the underlying mechanisms focusing on the functions of T cells and T cell-derived cytokines. HAp nanorods with different aspect ratios (HAp-0, HAp-30, and HAp-100) were implanted into mouse mandibular defect models. Micro-CT and hematoxylin and eosin staining demonstrated that HAp-100 had the best osteogenic effects. Flow cytometry analysis revealed that HAp-100 increased the percentage of T cells in injured mandibles. The osteogenic effects of HAp-100 were significantly blunted in injured mandibles of TCRβ mice. The Luminex xMAP assay and ELISA showed that HAp-100 induced a marked increase of interleukin (IL)-22 in injured mandibles. In cultured T cells, HAp-100 manifested the best capacity to induce the production of IL-22. Conditioned media from HAp-100-primed T cells promoted osteogenesis and JAK1/STAT3 activation in bone marrow stromal cells, all of which were abolished by neutralizing antibodies against IL-22. In summary, bone-mimicking HAp nanorods with different aspect ratios could regulate osteogenesis through modulation of T cells and IL-22 in the bone regeneration process. These findings provided insights for mediation of the immune response of T cells by nanomaterials on osteogenesis and strategies for designing biomaterials with osteoimmunomodulative functions.
C/EBPa controls acquisition and maintenance of adult haematopoietic stem cell quiescence.
Ye Min,Zhang Hong,Amabile Giovanni,Yang Henry,Staber Philipp B,Zhang Pu,Levantini Elena,Alberich-Jordà Meritxell,Zhang Junyan,Kawasaki Akira,Tenen Daniel G
Nature cell biology
In blood, the transcription factor C/EBPa is essential for myeloid differentiation and has been implicated in regulating self-renewal of fetal liver haematopoietic stem cells (HSCs). However, its function in adult HSCs has remained unknown. Here, using an inducible knockout model we found that C/EBPa-deficient adult HSCs underwent a pronounced increase in number with enhanced proliferation, characteristics resembling fetal liver HSCs. Consistently, transcription profiling of C/EBPa-deficient HSCs revealed a gene expression program similar to fetal liver HSCs. Moreover, we observed that age-specific Cebpa expression correlated with its inhibitory effect on the HSC cell cycle. Mechanistically we identified N-Myc as a downstream target of C/EBPa, and loss of C/EBPa resulted in de-repression of N-Myc. Our data establish C/EBPa as a central determinant in the switch from fetal to adult HSCs.
Role of CC chemokine receptor 9 in the progression of murine and human non-alcoholic steatohepatitis.
Morikawa Rei,Nakamoto Nobuhiro,Amiya Takeru,Chu Po-Sung,Koda Yuzo,Teratani Toshiaki,Suzuki Takahiro,Kurebayashi Yutaka,Ueno Akihisa,Taniki Nobuhito,Miyamoto Kentaro,Yamaguchi Akihiro,Shiba Shunsuke,Katayama Tadashi,Yoshida Kosuke,Takada Yoshiaki,Ishihara Rino,Ebinuma Hirotoshi,Sakamoto Michiie,Kanai Takanori
Journal of hepatology
BACKGROUND & AIMS:The number of patients with non-alcoholic steatohepatitis (NASH) is increasing globally. Recently, specific chemokine receptors have garnered interest as therapeutic targets in NASH. This is the first report to examine the role of the C-C chemokine receptor 9 (CCR9)/C-C chemokine receptor ligand 25 (CCL25) axis, and to reveal its therapeutic potential in NASH. METHODS:Patients with biopsy-proven non-alcoholic liver disease (NAFLD) were recruited and their serum and hepatic chemokine expression was examined. Furthermore, wild-type (WT) and Ccr9 mice were fed a high-fat high-cholesterol (HFHC) diet for 24 weeks to establish NASH. RESULTS:Serum CCL25, and hepatic CCR9 and CCL25 expression levels were increased in patients with NASH compared to healthy volunteers. Furthermore, Ccr9 mice were protected from HFHC diet-induced NASH progression both serologically and histologically. Flow cytometry and immunohistochemistry analysis showed that CCR9CD11b inflammatory macrophages accumulated in the inflamed livers of HFHC diet-fed mice, while the number was reduced in Ccr9 mice. Consistent with human NASH livers, CCR9 was also expressed on hepatic stellate cells (HSCs) in mice with NASH, while CCR9-deficient HSCs showed less fibrogenic potential in vitro. Administration of a CCR9 antagonist hampered further fibrosis progression in mice with NASH, supporting its potential clinical application. Finally, we showed that CCR9 blockade attenuated the development of NAFLD-related hepatocellular carcinoma in HF diet-fed mice injected with diethylnitrosamine. CONCLUSIONS:These results highlight the role of the CCR9/CCL25 axis on macrophage recruitment and fibrosis formation in a murine NASH model, providing new insights into therapeutic strategies for NASH. LAY SUMMARY:Herein, we show that a specific chemokine axis involving a receptor (CCR9) and its ligand (CCL25) contributes to the progression of non-alcoholic steatohepatitis and carcinogenesis in humans and mice. Furthermore, treatment with a CCR9 antagonist ameliorates the development of steatohepatitis and holds promise for the treatment of patients with non-alcoholic steatohepatitis.
Commensal microbiota influence systemic autoimmune responses.
Van Praet Jens T,Donovan Erin,Vanassche Inge,Drennan Michael B,Windels Fien,Dendooven Amélie,Allais Liesbeth,Cuvelier Claude A,van de Loo Fons,Norris Paula S,Kruglov Andrey A,Nedospasov Sergei A,Rabot Sylvie,Tito Raul,Raes Jeroen,Gaboriau-Routhiau Valerie,Cerf-Bensussan Nadine,Van de Wiele Tom,Eberl Gérard,Ware Carl F,Elewaut Dirk
The EMBO journal
Antinuclear antibodies are a hallmark feature of generalized autoimmune diseases, including systemic lupus erythematosus and systemic sclerosis. However, the processes underlying the loss of tolerance against nuclear self-constituents remain largely unresolved. Using mice deficient in lymphotoxin and Hox11, we report that approximately 25% of mice lacking secondary lymphoid organs spontaneously develop specific antinuclear antibodies. Interestingly, we find this phenotype is not caused by a defect in central tolerance. Rather, cell-specific deletion and in vivo lymphotoxin blockade link these systemic autoimmune responses to the formation of gut-associated lymphoid tissue in the neonatal period of life. We further demonstrate antinuclear antibody production is influenced by the presence of commensal gut flora, in particular increased colonization with segmented filamentous bacteria, and IL-17 receptor signaling. Together, these data indicate that neonatal colonization of gut microbiota influences generalized autoimmunity in adult life.
Immune response to intravenous immunoglobulin in patients with Kawasaki disease and MIS-C.
Zhu Yanfang P,Shamie Isaac,Lee Jamie C,Nowell Cameron J,Peng Weiqi,Angulo Shiela,Le Linh Nn,Liu Yushan,Miao Huilai,Xiong Hainan,Pena Cathleen J,Moreno Elizabeth,Griffis Eric,Labou Stephanie G,Franco Alessandra,Broderick Lori,Hoffman Hal M,Shimizu Chisato,Lewis Nathan E,Kanegaye John T,Tremoulet Adriana H,Burns Jane C,Croker Ben A,
The Journal of clinical investigation
BACKGROUNDMultisystem inflammatory syndrome in children (MIS-C) is a rare but potentially severe illness that follows exposure to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Kawasaki disease (KD) shares several clinical features with MIS-C, which prompted the use of intravenous immunoglobulin (IVIG), a mainstay therapy for KD. Both diseases share a robust activation of the innate immune system, including the IL-1 signaling pathway, and IL-1 blockade has been used for the treatment of both MIS-C and KD. The mechanism of action of IVIG in these 2 diseases and the cellular source of IL-1β have not been defined.METHODSThe effects of IVIG on peripheral blood leukocyte populations from patients with MIS-C and KD were examined using flow cytometry and mass cytometry (CyTOF) and live-cell imaging.RESULTSCirculating neutrophils were highly activated in patients with KD and MIS-C and were a major source of IL-1β. Following IVIG treatment, activated IL-1β+ neutrophils were reduced in the circulation. In vitro, IVIG was a potent activator of neutrophil cell death via PI3K and NADPH oxidase, but independently of caspase activation.CONCLUSIONSActivated neutrophils expressing IL-1β can be targeted by IVIG, supporting its use in both KD and MIS-C to ameliorate inflammation.FUNDINGPatient Centered Outcomes Research Institute; NIH; American Asthma Foundation; American Heart Association; Novo Nordisk Foundation; NIGMS; American Academy of Allergy, Asthma and Immunology Foundation.
Rapamycin antagonizes TNF induction of VCAM-1 on endothelial cells by inhibiting mTORC2.
Wang Chen,Qin Lingfeng,Manes Thomas D,Kirkiles-Smith Nancy C,Tellides George,Pober Jordan S
The Journal of experimental medicine
Recruitment of circulating leukocytes into inflamed tissues depends on adhesion molecules expressed by endothelial cells (ECs). Here we report that rapamycin pretreatment reduced the ability of TNF-treated ECs to capture T cells under conditions of venular flow. This functional change was caused by inhibition of TNF-induced expression of vascular cell adhesion molecule-1 (VCAM-1) and could be mimicked by knockdown of mammalian target of rapamycin (mTOR) or rictor, but not raptor, implicating mTORC2 as the target of rapamycin for this effect. Mechanistically, mTORC2 acts through Akt to repress Raf1-MEK1/2-ERK1/2 signaling, and inhibition of mTORC2 consequently results in hyperactivation of ERK1/2. Increased ERK1/2 activity antagonizes VCAM-1 expression by repressing TNF induction of the transcription factor IRF-1. Preventing activation of ERK1/2 reduced the ability of rapamycin to inhibit TNF-induced VCAM-1 expression. In vivo, rapamycin inhibited mTORC2 activity and potentiated activation of ERK1/2. These changes correlated with reduced endothelial expression of TNF-induced VCAM-1, which was restored via pharmacological inhibition of ERK1/2. Functionally, rapamycin reduced infiltration of leukocytes into renal glomeruli, an effect which was partially reversed by inhibition of ERK1/2. These data demonstrate a novel mechanism by which rapamycin modulates the ability of vascular endothelium to mediate inflammation and identifies endothelial mTORC2 as a potential therapeutic target.
Tumor-derived adenosine promotes macrophage proliferation in human hepatocellular carcinoma.
Wang Junfeng,Wang Yongchun,Chu Yifan,Li Zhixiong,Yu Xingjuan,Huang Zhijie,Xu Jing,Zheng Limin
Journal of hepatology
BACKGROUND & AIMS:Macrophages (Mϕ) represent a major component of tumor tissues and play an important role in both tumor progression and therapeutic response. Although tumor Mϕ are generally considered to be derived from circulating monocytes, emerging evidence indicates that tissue Mϕ pools can be maintained by self-renewal. We aimed to elucidate the contribution, phenotype, and regulatory mechanisms of proliferating Mϕ in human hepatocellular carcinoma (HCC). METHODS:Flow cytometry analyses were performed to examine the presence and phenotype of proliferating Mϕ in fresh HCC tissues. Dual immunofluorescence staining was applied to analyze the prognostic value of proliferating Mϕ. The underlying regulatory mechanisms were examined using human monocyte-derived Mϕ. RESULTS:Tumor-infiltrating Mϕ exhibited a significantly higher proliferative capacity than Mϕ in non-tumor tissues. A higher level of Mϕ proliferation was positively correlated with Mϕ density in the tumor and a poor prognosis in patients with HCC. Proliferating Mϕ were less differentiated (with increased CD206 expression) and were induced by the tumor cell-derived soluble small molecule, adenosine, but not proteins, lipids, or large peptides. Mechanistic studies demonstrated that autocrine granulocyte-macrophage colony-stimulating factor (GM-CSF) released by tumor-stimulated Mϕ could enhance A2A receptor expression on Mϕ and function synergistically with adenosine to elicit Mϕ proliferation in HCC. CONCLUSIONS:Local Mϕ proliferation is an important mechanism for Mϕ accumulation in HCC tissues. Tumor-derived adenosine functions synergistically with autocrine GM-CSF released from activated Mϕ, which promotes Mϕ proliferation. Thus, selective modulation of Mϕ accumulation at the source may provide a novel strategy for cancer therapy. LAY SUMMARY:Tumor-associated macrophages (TAMs) have been reported to play an essential role in both tumor progression and therapeutic response. A fundamental understanding of the mechanisms that regulate macrophage accumulation in tumors will undoubtedly lead to the development of strategies to target macrophages with high specificity and efficiency. The current study unveils a novel mechanism by which local proliferation is linked to macrophage accumulation in the tumor milieu, identifying potential targets for future immune-based anticancer therapies.
Polycomb Cbx family members mediate the balance between haematopoietic stem cell self-renewal and differentiation.
Klauke Karin,Radulović Višnja,Broekhuis Mathilde,Weersing Ellen,Zwart Erik,Olthof Sandra,Ritsema Martha,Bruggeman Sophia,Wu Xudong,Helin Kristian,Bystrykh Leonid,de Haan Gerald
Nature cell biology
The balance between self-renewal and differentiation of adult stem cells is essential for tissue homeostasis. Here we show that in the haematopoietic system this process is governed by polycomb chromobox (Cbx) proteins. Cbx7 is specifically expressed in haematopoietic stem cells (HSCs), and its overexpression enhances self-renewal and induces leukaemia. This effect is dependent on integration into polycomb repressive complex-1 (PRC1) and requires H3K27me3 binding. In contrast, overexpression of Cbx2, Cbx4 or Cbx8 results in differentiation and exhaustion of HSCs. ChIP-sequencing analysis shows that Cbx7 and Cbx8 share most of their targets; we identified approximately 200 differential targets. Whereas genes targeted by Cbx8 are highly expressed in HSCs and become repressed in progenitors, Cbx7 targets show the opposite expression pattern. Thus, Cbx7 preserves HSC self-renewal by repressing progenitor-specific genes. Taken together, the presence of distinct Cbx proteins confers target selectivity to PRC1 and provides a molecular balance between self-renewal and differentiation of HSCs.
Sulphydryl reactivity of the HLA-B27 epitope: accessibility of the free cysteine studied by flow cytometry.
MacLean L,Macey M,Lowdell M,Badakere S,Whelan M,Perrett D,Archer J
Annals of the rheumatic diseases
HLA-B27 has an unpaired cysteine on or near its serologically defined spondylitis associated epitope, and it has been argued that its sulphydryl side chain may be chemically reactive. In a previous study it was shown that chemical treatment of HLA-B27 cells with the sulphydryl binding agent p-chloromercuriphenylsulphonic acid (pCMPSA) specifically reduced binding of antibodies to HLA-B27 by up to 80%, as measured in a cellular enzyme linked immunosorbent assay (CELISA). The effect of sulphydryl blockade on intact B27 cells was investigated using flow cytometry. Compared with the CELISA, inhibition required higher concentrations of pCMPSA, and the degree of inhibition produced by a greater than or equal to 30 microM solution of pCMPSA as measured by flow cytometry (median 28.9%) was significantly lower than that measured by CELISA (median 73.6%; p = 1.6 x 10(-6)). Analysis of unfixed, cell surface HLA-B27 by flow cytometry suggests that on most B27 molecules the unpaired sulphydryl site is not available. On the basis of this evidence for modification after translation, a new 'altered self' hypothesis is proposed for the part which HLA-B27 plays in inflammatory disease.
Proteomic Analysis of Human Lung Development.
American journal of respiratory and critical care medicine
The current understanding of human lung development derives mostly from animal studies. Although transcript-level studies have analyzed human donor tissue to identify genes expressed during normal human lung development, protein-level analysis that would enable the generation of new hypotheses on the processes involved in pulmonary development are lacking. To define the temporal dynamic of protein expression during human lung development. We performed proteomics analysis of human lungs at 10 distinct times from birth to 8 years to identify the molecular networks mediating postnatal lung maturation. We identified 8,938 proteins providing a comprehensive view of the developing human lung proteome. The analysis of the data supports the existence of distinct molecular substages of alveolar development and predicted the age of independent human lung samples, and extensive remodeling of the lung proteome occurred during postnatal development. Evidence of post-transcriptional control was identified in early postnatal development. An extensive extracellular matrix remodeling was supported by changes in the proteome during alveologenesis. The concept of maturation of the immune system as an inherent part of normal lung development was substantiated by flow cytometry and transcriptomics. This study provides the first in-depth characterization of the human lung proteome during development, providing a unique proteomic resource freely accessible at Lungmap.net. The data support the extensive remodeling of the lung proteome during development, the existence of molecular substages of alveologenesis, and evidence of post-transcriptional control in early postnatal development.
Minimal PD-1 expression in mouse and human NK cells under diverse conditions.
Judge Sean J,Dunai Cordelia,Aguilar Ethan G,Vick Sarah C,Sturgill Ian R,Khuat Lam T,Stoffel Kevin M,Van Dyke Jonathan,Longo Dan L,Darrow Morgan A,Anderson Stephen K,Blazar Bruce R,Monjazeb Arta M,Serody Jonathan S,Canter Robert J,Murphy William J
The Journal of clinical investigation
PD-1 expression is a hallmark of both early antigen-specific T cell activation and later chronic stimulation, suggesting key roles in both naive T cell priming and memory T cell responses. Although significant similarities exist between T cells and NK cells, there are critical differences in their biology and functions reflecting their respective adaptive and innate immune effector functions. Expression of PD-1 on NK cells is controversial despite rapid incorporation into clinical cancer trials. Our objective was to stringently and comprehensively assess expression of PD-1 on both mouse and human NK cells under multiple conditions and using a variety of readouts. We evaluated NK cells from primary human tumor samples, after ex vivo culturing, and from multiple mouse tumor and viral models using flow cytometry, quantitative reverse-transcriptase PCR (qRT-PCR), and RNA-Seq for PD-1 expression. We demonstrate that, under multiple conditions, human and mouse NK cells consistently lack PD-1 expression despite the marked upregulation of other activation/regulatory markers, such as TIGIT. This was in marked contrast to T cells, which were far more prominent within all tumors and expressed PD-1. These data have important implications when attempting to discern NK from T cell effects and to determine whether PD-1 targeting can be expected to have direct effects on NK cell functions.
CD34+CD140b+ cells and circulating CXCL12 correlate with the angiographically assessed severity of cardiac allograft vasculopathy.
Schober Andreas,Hristov Mihail,Kofler Sieglinde,Forbrig Robert,Löhr Barbara,Heussen Nicole,Zhe Zhou,Akhtar Shamima,Schumann Uwe,Krötz Florian,Leibig Marcus,König Andreas,Kaczmarek Ingo,Reichart Bruno,Klauss Volker,Weber Christian,Sohn Hae-Young
European heart journal
AIMS:We sought to determine whether circulating vascular progenitor cells, such as endothelial progenitor cells (EPCs) or smooth muscle progenitor cells (SPCs), were associated with the severity of cardiac allograft vasculopathy (CAV). METHODS AND RESULTS:CD34(+)CD140b(+) SPCs and CD34(+)KDR(+) EPCs were measured in the peripheral circulation of 187 adult heart transplant recipients by flow cytometry. Cardiac allograft vasculopathy was quantified by angiography using a CAV-specific scoring system. Cardiac allograft vasculopathy was present in 84 patients (44.7%) and was classified as mild in 59 and severe in 25 cases. Circulating SPCs were more frequently detectable in CAV patients than in patients without CAV. The number of CD34(+)CD140b(+) cells showed a stepwise increase in patients with moderate and severe CAV. Smooth muscle progenitor cell counts were higher in patients with coronary stent implant compared with unstented patients with CAV. In contrast, peripheral CD34(+)KDR(+) EPC counts were not changed in CAV patients. Plasma CXCL12 levels correlated with the degree of CAV and SPC counts. None of the different immunosuppressive drug regimes was related to the SPC count or the CXCL12 levels. A multivariate regression analysis revealed that the SPC count was independently associated with the presence of CAV. CONCLUSION:Circulating SPCs, but not EPCs, and plasma CXCL12 concentrations are elevated in CAV patients, indicating that they play prominent roles in transplant arteriosclerosis.
Overexpression of methyl-CpG binding protein 2 impairs T(H)1 responses.
Yang Tianshu,Ramocki Melissa B,Neul Jeffrey L,Lu Wen,Roberts Luz,Knight John,Ward Christopher S,Zoghbi Huda Y,Kheradmand Farrah,Corry David B
Science translational medicine
The DNA binding protein methyl-CpG binding protein 2 (MeCP2) critically influences neuronal and brain function by modulating gene expression, and children with overexpression of the MECP2 gene exhibit postnatal neurological syndromes. We demonstrate that some children with MECP2 duplication also display variable immunological abnormalities that include reductions in memory T and B cells and natural killer cells and immunoglobulin assay responses. Moreover, whereas mice with MeCP2 overexpression were unable to control infection with the intra-macrophage parasite Leishmania major and secrete interferon-γ (IFN-γ) from involved lymph nodes, they were able to control airway fungal infection by Aspergillus niger and mount protective T helper cell type 2 (T(H)2)-dependent allergic responses. Relative to normal T cells, T(H) cells from children and mice with MECP2 duplication displayed similar impairments in IFN-γ secretion and T(H)1 responses that were due to both MeCP2-dependent suppression of IFN-γ transcription and sequestration of the IFN-γ locus as assessed by chromatin immunoprecipitation assay. Thus, overexpressed MeCP2 aberrantly suppresses IFN-γ secretion from T(H) cells, potentially leading to a partially immunodeficient state. Our findings establish a rational basis for identifying, treating, and preventing infectious complications potentially affecting children with MECP2 duplication.
PD-L1 blockade liberates intrinsic antitumourigenic properties of glycolytic macrophages in hepatocellular carcinoma.
Lu Li-Gong,Zhou Zhi-Ling,Wang Xu-Yan,Liu Bo-Yuan,Lu Jin-Ying,Liu Shuai,Zhang Guang-Bo,Zhan Mei-Xiao,Chen Yun
OBJECTIVE:Patients with increased PD-L1 host cells in tumours are more potent to benefit from antiprogrammed death-1/programmed death ligand-1 (PD-L1) treatment, but the underlying mechanism is still unclear. We aim to elucidate the nature, regulation and functional relevance of PD-L1 host cells in hepatocellular carcinoma (HCC). DESIGN:A total of untreated 184 HCC patients was enrolled randomly. C57BL/6 mice are given injection of Hepa1-6 cells to form autologous hepatoma. ELISpot, flow cytometry and real-time PCR are applied to analyse the phenotypic characteristics of PD-L1 cells isolated directly from HCC specimens paired with blood samples or generated from ex vivo and in vitro culture systems. Immunofluorescence and immunohistochemistry are performed to detect the presence of immune cells on paraffin-embedded and formalin-fixed samples. The underlying regulatory mechanisms of metabolic switching are assessed by both in vitro and in vivo studies. RESULTS:We demonstrate that PD-L1 host macrophages, which constructively represent the major cellular source of PD-L1 in HCC tumours, display an HLA-DRCD86 glycolytic phenotype, significantly produce antitumourigenic IL-12p70 and are polarised by intrinsic glycolytic metabolism. Mechanistically, a key glycolytic enzyme PKM2 triggered by hepatoma cell derived fibronectin 1, via a HIF-1α-dependent manner, concurrently controls the antitumourigenic properties and inflammation-mediated PD-L1 expression in glycolytic macrophages. Importantly, although increased PKM2 glycolytic macrophages predict poor prognosis of patients, blocking PD-L1 on these cells eliminates PD-L1-dominant immunosuppression and liberates intrinsic antitumourigenic properties. CONCLUSIONS:Selectively modulating the 'context' of glycolytic macrophages in HCC tumours might restore their antitumourigenic properties and provide a precise strategy for anticancer therapy.
ESE3/EHF, a promising target of rosiglitazone, suppresses pancreatic cancer stemness by downregulating CXCR4.
Zhou Tianxing,Liu Jing,Xie Yongjie,Yuan Shuai,Guo Yu,Bai Weiwei,Zhao Kaili,Jiang Wenna,Wang Hongwei,Wang Haotian,Zhao Tiansuo,Huang Chongbiao,Gao Song,Wang Xiuchao,Yang Shengyu,Hao Jihui
BACKGROUND AND AIMS:The crosstalk between cancer stem cells (CSCs) and their niche is required for the maintenance of stem cell-like phenotypes of CSCs. Here, we identified E26 transformation-specific homologous factor (EHF) as a key molecule in decreasing the sensitivity of pancreatic cancer (PC) cells to CSCs' niche stimulus. We also explored a therapeutic strategy to restore the expression of EHF. DESIGN:We used a LSL-Krasmice, LSL-Trp53 and Pdx1-Cre (KPC) mouse model and samples from patients with PC. Immunostaining, flow cytometry, sphere formation assays, anchorage-independent growth assay, in vivo tumourigenicity, reverse transcription PCR, chromatin immunoprecipitation (ChIP) and luciferase analyses were conducted in this study. RESULTS:CXCL12 derived from pancreatic stellate cells (PSCs) mediates the crosstalk between PC cells and PSCs to promote PC stemness. Tumorous EHF suppressed CSC stemness by decreasing the sensitivity of PC to CXCL12 stimulus and inhibiting the crosstalk between PC and CSC-supportive niches. Mechanically, EHF suppressed the transcription of the CXCL12 receptor CXCR4. EHF had a cell autonomous role in suppressing cancer stemness by inhibiting the transcription of , , and . Rosiglitazone suppressed PC stemness and inhibited the crosstalk between PC and PSCs by upregulating EHF. Preclinical KPC mouse cohorts demonstrated that rosiglitazone sensitised PDAC to gemcitabine therapy. CONCLUSIONS:EHF decreased the sensitivity of PC to the stimulus from PSC-derived CSC-supportive niche by negatively regulating tumorous CXCR4. Rosiglitazone could be used to target PC stem cells and the crosstalk between CSCs and their niche by upregulating EHF.
Therapeutic antiviral T cells noncytopathically clear persistently infected microglia after conversion into antigen-presenting cells.
Herz Jasmin,Johnson Kory R,McGavern Dorian B
The Journal of experimental medicine
Several viruses can infect the mammalian nervous system and induce neurological dysfunction. Adoptive immunotherapy is an approach that involves administration of antiviral T cells and has shown promise in clinical studies for the treatment of peripheral virus infections in humans such as cytomegalovirus (CMV), Epstein-Barr virus (EBV), and adenovirus, among others. In contrast, clearance of neurotropic infections is particularly challenging because the central nervous system (CNS) is relatively intolerant of immunopathological reactions. Therefore, it is essential to develop and mechanistically understand therapies that noncytopathically eradicate pathogens from the CNS. Here, we used mice persistently infected from birth with lymphocytic choriomeningitis virus (LCMV) to demonstrate that therapeutic antiviral T cells can completely purge the persistently infected brain without causing blood-brain barrier breakdown or tissue damage. Mechanistically, this is accomplished through a tailored release of chemoattractants that recruit antiviral T cells, but few pathogenic innate immune cells such as neutrophils and inflammatory monocytes. Upon arrival, T cells enlisted the support of nearly all brain-resident myeloid cells (microglia) by inducing proliferation and converting them into CD11c(+) antigen-presenting cells (APCs). Two-photon imaging experiments revealed that antiviral CD8(+) and CD4(+) T cells interacted directly with CD11c(+) microglia and induced STAT1 signaling but did not initiate programmed cell death. We propose that noncytopathic CNS viral clearance can be achieved by therapeutic antiviral T cells reliant on restricted chemoattractant production and interactions with apoptosis-resistant microglia.
Targeting phospholipase D1 attenuates intestinal tumorigenesis by controlling β-catenin signaling in cancer-initiating cells.
Kang Dong Woo,Choi Chi Yeol,Cho Yong-Hee,Tian Huasong,Di Paolo Gilbert,Choi Kang-Yell,Min Do Sik
The Journal of experimental medicine
Expression of the Wnt target gene phospholipase D1 (PLD1) is up-regulated in various carcinomas, including colorectal cancer (CRC). However, the mechanistic significance of its elevated expression in intestinal tumorigenesis remains unknown. In this study, we show that genetic and pharmacological targeting of PLD1 disrupts spontaneous and colitis-associated intestinal tumorigenesis in Apc(Min/+) and azoxymethane/dextran sodium sulfate mice models. Intestinal epithelial cell-specific PLD1 overexpression in Apc(Min/+) mice accelerated tumorigenesis with increased proliferation and nuclear β-catenin levels compared with Apc(Min/+) mice. Moreover, PLD1 inactivation suppressed the self-renewal capacity of colon cancer-initiating cells (CC-ICs) by decreasing expression of β-catenin via E2F1-induced microRNA (miR)-4496 up-regulation. Ultimately, low expression of PLD1 coupled with a low level of CC-IC markers was predictive of a good prognosis in CRC patients, suggesting in vivo relevance. Collectively, our data reveal that PLD1 has a crucial role in intestinal tumorigenesis via its modulation of the E2F1-miR-4496-β-catenin signaling pathway. Modulation of PLD1 expression and activity represents a promising therapeutic strategy for the treatment of intestinal tumorigenesis.
Blocking neutrophil diapedesis prevents hemorrhage during thrombocytopenia.
Hillgruber Carina,Pöppelmann Birgit,Weishaupt Carsten,Steingräber Annika Kathrin,Wessel Florian,Berdel Wolfgang E,Gessner J Engelbert,Ho-Tin-Noé Benoît,Vestweber Dietmar,Goerge Tobias
The Journal of experimental medicine
Spontaneous organ hemorrhage is the major complication in thrombocytopenia with a potential fatal outcome. However, the exact mechanisms regulating vascular integrity are still unknown. Here, we demonstrate that neutrophils recruited to inflammatory sites are the cellular culprits inducing thrombocytopenic tissue hemorrhage. Exposure of thrombocytopenic mice to UVB light provokes cutaneous petechial bleeding. This phenomenon is also observed in immune-thrombocytopenic patients when tested for UVB tolerance. Mechanistically, we show, analyzing several inflammatory models, that it is neutrophil diapedesis through the endothelial barrier that is responsible for the bleeding defect. First, bleeding is triggered by neutrophil-mediated mechanisms, which act downstream of capturing, adhesion, and crawling on the blood vessel wall and require Gαi signaling in neutrophils. Second, mutating Y731 in the cytoplasmic tail of VE-cadherin, known to selectively affect leukocyte diapedesis, but not the induction of vascular permeability, attenuates bleeding. Third, and in line with this, simply destabilizing endothelial junctions by histamine did not trigger bleeding. We conclude that specifically targeting neutrophil diapedesis through the endothelial barrier may represent a new therapeutic avenue to prevent fatal bleeding in immune-thrombocytopenic patients.
T cell development involves TRAF3IP3-mediated ERK signaling in the Golgi.
Zou Qiang,Jin Jin,Xiao Yichuan,Hu Hongbo,Zhou Xiaofei,Jie Zuliang,Xie Xiaoping,Li James Y H,Cheng Xuhong,Sun Shao-Cong
The Journal of experimental medicine
Generation of T lymphocytes in the thymus is guided by signal transduction from the T cell receptor (TCR), but the underlying mechanism is incompletely understood. Here we have identified a Golgi-associated factor, TRAF3-interacting protein 3 (TRAF3IP3), as a crucial mediator of thymocyte development. TRAF3IP3 deficiency in mice attenuates the generation of mature thymocytes caused by impaired thymocyte-positive selection. TRAF3IP3 mediates TCR-stimulated activation of the mitogen-activated protein kinase (MAPK) extracellular signal-regulated kinase (ERK) and its upstream kinase mitogen/extracellular signal-regulated kinase (MEK). Interestingly, TRAF3IP3 exerts this signaling function through recruiting MEK to the Golgi and, thereby, facilitating the interaction of MEK with its activator BRAF. Transgenic expression of a constitutively active MEK rescues the T cell development block in Traf3ip3 knockout mice. These findings establish TRAF3IP3 as a novel regulator of T cell development and suggest a Golgi-specific ERK signaling mechanism that regulates thymocyte development.
Functional reconstitution of HBV-specific CD8 T cells by in vitro polyphenol treatment in chronic hepatitis B.
Acerbi Greta,Montali Ilaria,Ferrigno Gennaro Domenico,Barili Valeria,Schivazappa Simona,Alfieri Arianna,Laccabue Diletta,Loglio Alessandro,Borghi Marta,Massari Marco,Rossi Marzia,Vecchi Andrea,Penna Amalia,Boni Carolina,Missale Gabriele,Lampertico Pietro,Del Rio Daniele,Ferrari Carlo,Fisicaro Paola
Journal of hepatology
BACKGROUND & AIMS:In chronic HBV infection, mitochondrial functions and proteostasis are dysregulated in exhausted HBV-specific CD8 T cells. To better characterise the potential involvement of deregulated protein degradation mechanisms in T cell exhaustion, we analysed lysosome-mediated autophagy in HBV-specific CD8 T cells. Bioactive compounds able to simultaneously target both mitochondrial functions and proteostasis were tested to identify optimal combination strategies to reconstitute efficient antiviral CD8 T cell responses in patients with chronic HBV infection. METHODS:Lysosome-mediated degradation pathways were analysed by flow cytometry in virus-specific CD8 T cells from patients with chronic HBV infection. Mitochondrial function, intracellular proteostasis, and cytokine production were evaluated in HBV-peptide-stimulated T cell cultures, in the presence or absence of the polyphenols resveratrol (RSV) and oleuropein (OLE) and their metabolites, either alone or in combination with other bioactive compounds. RESULTS:HBV-specific CD8 T cells from patients with CHB showed impaired autophagic flux. RSV and OLE elicited a significant improvement in mitochondrial, proteostasis and antiviral functions in CD8 T cells. Cytokine production was also enhanced by synthetic metabolites, which correspond to those generated by RSV and OLE metabolism in vivo, suggesting that these polyphenols may also display an effect after transformation in vivo. Moreover, polyphenolic compounds improved the T cell revitalising effect of mitochondria-targeted antioxidants and of programmed cell death protein 1/programmed cell death ligand 1 blockade. CONCLUSIONS:Simultaneously targeting multiple altered intracellular pathways with the combination of mitochondria-targeted antioxidants and natural polyphenols may represent a promising immune reconstitution strategy for the treatment of chronic HBV infection. LAY SUMMARY:In chronic hepatitis B, antiviral T lymphocytes are deeply impaired, with many altered intracellular functions. In vitro exposure to polyphenols, such as resveratrol and oleuropein, can correct some of the deregulated intracellular pathways and improve antiviral T cell function. This effect can be further strengthened by the association of polyphenols with antioxidant compounds in a significant proportion of patients. Thus, the combination of antioxidants and natural polyphenols represents a promising strategy for chronic hepatitis B therapy.
TWEAK/Fn14 signalling promotes cholangiocarcinoma niche formation and progression.
Dwyer Benjamin J,Jarman Edward J,Gogoi-Tiwari Jully,Ferreira-Gonzalez Sofia,Boulter Luke,Guest Rachel V,Kendall Timothy J,Kurian Dominic,Kilpatrick Alastair M,Robson Andrew J,O'Duibhir Eoghan,Man Tak Yung,Campana Lara,Starkey Lewis Philip J,Wigmore Stephen J,Olynyk John K,Ramm Grant A,Tirnitz-Parker Janina E E,Forbes Stuart J
Journal of hepatology
BACKGROUND & AIMS:Cholangiocarcinoma (CCA) is a cancer of the hepatic bile ducts that is rarely resectable and is associated with poor prognosis. Tumour necrosis factor-like weak inducer of apoptosis (TWEAK) is known to signal via its receptor fibroblast growth factor-inducible 14 (Fn14) and induce cholangiocyte and myofibroblast proliferation in liver injury. We aimed to characterise its role in CCA. METHODS:The expression of the TWEAK ligand and Fn14 receptor was assessed immunohistochemically and by bulk RNA and single cell transcriptomics of human liver tissue. Spatiotemporal dynamics of pathway regulation were comprehensively analysed in rat and mouse models of thioacetamide (TAA)-mediated CCA. Flow cytometry, qPCR and proteomic analyses of CCA cell lines and conditioned medium experiments with primary macrophages were performed to evaluate the downstream functions of TWEAK/Fn14. In vivo pathway manipulation was assessed via TWEAK overexpression in NICD/AKT-induced CCA or genetic Fn14 knockout during TAA-mediated carcinogenesis. RESULTS:Our data reveal TWEAK and Fn14 overexpression in multiple human CCA cohorts, and Fn14 upregulation in early TAA-induced carcinogenesis. TWEAK regulated the secretion of factors from CC-SW-1 and SNU-1079 CCA cells, inducing polarisation of proinflammatory CD206 macrophages. Pharmacological blocking of the TWEAK downstream target chemokine monocyte chemoattractant protein 1 (MCP-1 or CCL2) significantly reduced CCA xenograft growth, while TWEAK overexpression drove cancer-associated fibroblast proliferation and collagen deposition in the tumour niche. Genetic Fn14 ablation significantly reduced inflammatory, fibrogenic and ductular responses during carcinogenic TAA-mediated injury. CONCLUSION:These novel data provide evidence for the action of TWEAK/Fn14 on macrophage recruitment and phenotype, and cancer-associated fibroblast proliferation in CCA. Targeting TWEAK/Fn14 and its downstream signals may provide a means to inhibit CCA niche development and tumour growth. LAY SUMMARY:Cholangiocarcinoma is an aggressive, chemotherapy-resistant liver cancer. Interactions between tumour cells and cells that form a supportive environment for the tumour to grow are a source of this aggressiveness and resistance to chemotherapy. Herein, we describe interactions between tumour cells and their supportive environment via a chemical messenger, TWEAK and its receptor Fn14. TWEAK/Fn14 alters the recruitment and type of immune cells in tumours, increases the growth of cancer-associated fibroblasts in the tumour environment, and is a potential target to reduce tumour formation.
CCND1-CDK4-mediated cell cycle progression provides a competitive advantage for human hematopoietic stem cells in vivo.
Mende Nicole,Kuchen Erika E,Lesche Mathias,Grinenko Tatyana,Kokkaliaris Konstantinos D,Hanenberg Helmut,Lindemann Dirk,Dahl Andreas,Platz Alexander,Höfer Thomas,Calegari Federico,Waskow Claudia
The Journal of experimental medicine
Maintenance of stem cell properties is associated with reduced proliferation. However, in mouse hematopoietic stem cells (HSCs), loss of quiescence results in a wide range of phenotypes, ranging from functional failure to extensive self-renewal. It remains unknown whether the function of human HSCs is controlled by the kinetics of cell cycle progression. Using human HSCs and human progenitor cells (HSPCs), we report here that elevated levels of CCND1-CDK4 complexes promoted the transit from G0 to G1 and shortened the G1 cell cycle phase, resulting in protection from differentiation-inducing signals in vitro and increasing human leukocyte engraftment in vivo. Further, CCND1-CDK4 overexpression conferred a competitive advantage without impacting HSPC numbers. In contrast, accelerated cell cycle progression mediated by elevated levels of CCNE1-CDK2 led to the loss of functional HSPCs in vivo. Collectively, these data suggest that the transition kinetics through the early cell cycle phases are key regulators of human HSPC function and important for lifelong hematopoiesis.
Donor colonic CD103+ dendritic cells determine the severity of acute graft-versus-host disease.
Koyama Motoko,Cheong Melody,Markey Kate A,Gartlan Kate H,Kuns Rachel D,Locke Kelly R,Lineburg Katie E,Teal Bianca E,Leveque-El Mouttie Lucie,Bunting Mark D,Vuckovic Slavica,Zhang Ping,Teng Michele W L,Varelias Antiopi,Tey Siok-Keen,Wockner Leesa F,Engwerda Christian R,Smyth Mark J,Belz Gabrielle T,McColl Shaun R,MacDonald Kelli P A,Hill Geoffrey R
The Journal of experimental medicine
The primacy of the gastrointestinal (GI) tract in dictating the outcome of graft-versus-host disease (GVHD) is broadly accepted; however, the mechanisms controlling this effect are poorly understood. Here, we demonstrate that GVHD markedly enhances alloantigen presentation within the mesenteric lymph nodes (mLNs), mediated by donor CD103(+)CD11b(-) dendritic cells (DCs) that migrate from the colon under the influence of CCR7. Expansion and differentiation of donor T cells specifically within the mLNs is driven by profound levels of alloantigen, IL-12, and IL-6 promoted by Toll-like receptor (TLR) and receptor for advanced glycation end products (RAGE) signals. Critically, alloantigen presentation in the mLNs imprints gut-homing integrin signatures on donor T cells, leading to their emigration into the GI tract where they mediate fulminant disease. These data identify a critical, anatomically distinct, donor DC subset that amplifies GVHD. We thus highlight multiple therapeutic targets and the ability of GVHD, once initiated by recipient antigen-presenting cells, to generate a profound, localized, and lethal feed-forward cascade of donor DC-mediated indirect alloantigen presentation and cytokine secretion within the GI tract.
The Sm protein methyltransferase PRMT5 is not required for primordial germ cell specification in mice.
Li Ziwei,Yu Juehua,Hosohama Linzi,Nee Kevin,Gkountela Sofia,Chaudhari Sonal,Cass Ashley A,Xiao Xinshu,Clark Amander T
The EMBO journal
PRMT5 is a type II protein arginine methyltransferase with roles in stem cell biology, reprograming, cancer and neurogenesis. During embryogenesis in the mouse, it was hypothesized that PRMT5 functions with the master germline determinant BLIMP1 to promote primordial germ cell (PGC) specification. Using a Blimp1-Cre germline conditional knockout, we discovered that Prmt5 has no major role in murine germline specification, or the first global epigenetic reprograming event involving depletion of cytosine methylation from DNA and histone H3 lysine 9 dimethylation from chromatin. Instead, we discovered that PRMT5 functions at the conclusion of PGC reprograming I to promote proliferation, survival and expression of the gonadal germline program as marked by MVH. We show that PRMT5 regulates gene expression by promoting methylation of the Sm spliceosomal proteins and significantly altering the spliced repertoire of RNAs in mammalian embryonic cells and primordial cells.
Wnt activity and basal niche position sensitize intestinal stem and progenitor cells to DNA damage.
Tao Si,Tang Duozhuang,Morita Yohei,Sperka Tobias,Omrani Omid,Lechel André,Sakk Vadim,Kraus Johann,Kestler Hans A,Kühl Michael,Rudolph Karl Lenhard
The EMBO journal
Aging and carcinogenesis coincide with the accumulation of DNA damage and mutations in stem and progenitor cells. Molecular mechanisms that influence responses of stem and progenitor cells to DNA damage remain to be delineated. Here, we show that niche positioning and Wnt signaling activity modulate the sensitivity of intestinal stem and progenitor cells (ISPCs) to DNA damage. ISPCs at the crypt bottom with high Wnt/β-catenin activity are more sensitive to DNA damage compared to ISPCs in position 4 with low Wnt activity. These differences are not induced by differences in cell cycle activity but relate to DNA damage-dependent activation of Wnt signaling, which in turn amplifies DNA damage checkpoint activation. The study shows that instructed enhancement of Wnt signaling increases radio-sensitivity of ISPCs, while inhibition of Wnt signaling decreases it. These results provide a proof of concept that cell intrinsic levels of Wnt signaling modulate the sensitivity of ISPCs to DNA damage and heterogeneity in Wnt activation in the stem cell niche contributes to the selection of ISPCs in the context of DNA damage.
Space exploration by dendritic cells requires maintenance of myosin II activity by IP3 receptor 1.
Solanes Paola,Heuzé Mélina L,Maurin Mathieu,Bretou Marine,Lautenschlaeger Franziska,Maiuri Paolo,Terriac Emmanuel,Thoulouze Maria-Isabel,Launay Pierre,Piel Matthieu,Vargas Pablo,Lennon-Duménil Ana-Maria
The EMBO journal
Dendritic cells (DCs) patrol the interstitial space of peripheral tissues. The mechanisms that regulate their migration in such constrained environment remain unknown. We here investigated the role of calcium in immature DCs migrating in confinement. We found that they displayed calcium oscillations that were independent of extracellular calcium and more frequently observed in DCs undergoing strong speed fluctuations. In these cells, calcium spikes were associated with fast motility phases. IP₃ receptors (IP₃Rs) channels, which allow calcium release from the endoplasmic reticulum, were identified as required for immature DCs to migrate at fast speed. The IP₃R1 isoform was further shown to specifically regulate the locomotion persistence of immature DCs, that is, their capacity to maintain directional migration. This function of IP₃R1 results from its ability to control the phosphorylation levels of myosin II regulatory light chain (MLC) and the back/front polarization of the motor protein. We propose that by upholding myosin II activity, constitutive calcium release from the ER through IP₃R1 maintains DC polarity during migration in confinement, facilitating the exploration of their environment.
Lung microbiota promotes tolerance to allergens in neonates via PD-L1.
Gollwitzer Eva S,Saglani Sejal,Trompette Aurélien,Yadava Koshika,Sherburn Rebekah,McCoy Kathy D,Nicod Laurent P,Lloyd Clare M,Marsland Benjamin J
Epidemiological data point toward a critical period in early life during which environmental cues can set an individual on a trajectory toward respiratory health or disease. The neonatal immune system matures during this period, although little is known about the signals that lead to its maturation. Here we report that the formation of the lung microbiota is a key parameter in this process. Immediately following birth, neonatal mice were prone to develop exaggerated airway eosinophilia, release type 2 helper T cell cytokines and exhibit airway hyper-responsiveness following exposure to house dust mite allergens, even though their lungs harbored high numbers of natural CD4(+)Foxp3(+)CD25(+)Helios(+) regulatory T (Treg) cells. During the first 2 weeks after birth, the bacterial load in the lungs increased, and representation of the bacterial phyla shifts from a predominance of Gammaproteobacteria and Firmicutes towards Bacteroidetes. The changes in the microbiota were associated with decreased aeroallergen responsiveness and the emergence of a Helios(-) Treg cell subset that required interaction with programmed death ligand 1 (PD-L1) for development. Absence of microbial colonization(10) or blockade of PD-L1 during the first 2 weeks postpartum maintained exaggerated responsiveness to allergens through to adulthood. Adoptive transfer of Treg cells from adult mice to neonates before aeroallergen exposure ameliorated disease. Thus, formation of the airway microbiota induces regulatory cells early in life, which, when dysregulated, can lead to sustained susceptibility to allergic airway inflammation in adulthood.
Neutrophil granulocytes recruited upon translocation of intestinal bacteria enhance graft-versus-host disease via tissue damage.
Schwab Lukas,Goroncy Luise,Palaniyandi Senthilnathan,Gautam Sanjivan,Triantafyllopoulou Antigoni,Mocsai Attila,Reichardt Wilfried,Karlsson Fridrik J,Radhakrishnan Sabarinath V,Hanke Kathrin,Schmitt-Graeff Annette,Freudenberg Marina,von Loewenich Friederike D,Wolf Philipp,Leonhardt Franziska,Baxan Nicoleta,Pfeifer Dietmar,Schmah Oliver,Schönle Anne,Martin Stefan F,Mertelsmann Roland,Duyster Justus,Finke Jürgen,Prinz Marco,Henneke Philipp,Häcker Hans,Hildebrandt Gerhard C,Häcker Georg,Zeiser Robert
Acute graft-versus-host disease (GVHD) considerably limits wider usage of allogeneic hematopoietic cell transplantation (allo-HCT). Antigen-presenting cells and T cells are populations customarily associated with GVHD pathogenesis. Of note, neutrophils are the largest human white blood cell population. The cells cleave chemokines and produce reactive oxygen species, thereby promoting T cell activation. Therefore, during an allogeneic immune response, neutrophils could amplify tissue damage caused by conditioning regimens. We analyzed neutrophil infiltration of the mouse ileum after allo-HCT by in vivo myeloperoxidase imaging and found that infiltration levels were dependent on the local microbial flora and were not detectable under germ-free conditions. Physical or genetic depletion of neutrophils reduced GVHD-related mortality. The contribution of neutrophils to GVHD severity required reactive oxygen species (ROS) because selective Cybb (encoding cytochrome b-245, beta polypeptide, also known as NOX2) deficiency in neutrophils impairing ROS production led to lower levels of tissue damage, GVHD-related mortality and effector phenotype T cells. Enhanced survival of Bcl-xL transgenic neutrophils increased GVHD severity. In contrast, when we transferred neutrophils lacking Toll-like receptor-2 (TLR2), TLR3, TLR4, TLR7 and TLR9, which are normally less strongly activated by translocating bacteria, into wild-type C57BL/6 mice, GVHD severity was reduced. In humans, severity of intestinal GVHD strongly correlated with levels of neutrophils present in GVHD lesions. This study describes a new potential role for neutrophils in the pathogenesis of GVHD in both mice and humans.
The expression of Sox17 identifies and regulates haemogenic endothelium.
Clarke Raedun L,Yzaguirre Amanda D,Yashiro-Ohtani Yumi,Bondue Antoine,Blanpain Cedric,Pear Warren S,Speck Nancy A,Keller Gordon
Nature cell biology
Although it is well recognized that haematopoietic stem cells (HSCs) develop from a specialized population of endothelial cells known as haemogenic endothelium, the regulatory pathways that control this transition are not well defined. Here we identify Sox17 as a key regulator of haemogenic endothelial development. Analysis of Sox17-GFP reporter mice revealed that Sox17 is expressed in haemogenic endothelium and emerging HSCs and that it is required for HSC development. Using the mouse embryonic stem cell differentiation model, we show that Sox17 is also expressed in haemogenic endothelium generated in vitro and that it plays a pivotal role in the development and/or expansion of haemogenic endothelium through the Notch signalling pathway. Taken together, these findings position Sox17 as a key regulator of haemogenic endothelial and haematopoietic development.
Tumor endothelium FasL establishes a selective immune barrier promoting tolerance in tumors.
Motz Gregory T,Santoro Stephen P,Wang Li-Ping,Garrabrant Tom,Lastra Ricardo R,Hagemann Ian S,Lal Priti,Feldman Michael D,Benencia Fabian,Coukos George
We describe a new mechanism regulating the tumor endothelial barrier and T cell infiltration into tumors. We detected selective expression of the death mediator Fas ligand (FasL, also called CD95L) in the vasculature of human and mouse solid tumors but not in normal vasculature. In these tumors, FasL expression was associated with scarce CD8(+) infiltration and a predominance of FoxP3(+) T regulatory (Treg) cells. Tumor-derived vascular endothelial growth factor A (VEGF-A), interleukin 10 (IL-10) and prostaglandin E2 (PGE2) cooperatively induced FasL expression in endothelial cells, which acquired the ability to kill effector CD8(+) T cells but not Treg cells because of higher levels of c-FLIP expression in Treg cells. In mice, genetic or pharmacologic suppression of FasL produced a substantial increase in the influx of tumor-rejecting CD8(+) over FoxP3(+) T cells. Pharmacologic inhibition of VEGF and PGE2 produced a marked increase in the influx of tumor-rejecting CD8(+) over FoxP3(+) T cells that was dependent on attenuation of FasL expression and led to CD8-dependent tumor growth suppression. Thus, tumor paracrine mechanisms establish a tumor endothelial death barrier, which has a critical role in establishing immune tolerance and determining the fate of tumors.
Single-cell RNA sequencing reveals intrahepatic and peripheral immune characteristics related to disease phases in HBV-infected patients.
Zhang Chao,Li Jiesheng,Cheng Yongqian,Meng Fanping,Song Jin-Wen,Fan Xing,Fan Hongtao,Li Jing,Fu Yu-Long,Zhou Ming-Ju,Hu Wei,Wang Si-Yu,Fu Yuan-Jie,Zhang Ji-Yuan,Xu Ruo-Nan,Shi Ming,Hu Xueda,Zhang Zemin,Ren Xianwen,Wang Fu-Sheng
OBJECTIVE:A comprehensive immune landscape for HBV infection is pivotal to achieve HBV cure. DESIGN:We performed single-cell RNA sequencing of 2 43 000 cells from 46 paired liver and blood samples of 23 individuals, including six immune tolerant, 5 immune active (IA), 3 acute recovery (AR), 3 chronic resolved and 6 HBV-free healthy controls (HCs). Flow cytometry and histological assays were applied in a second HBV cohort for validation. RESULTS:Both IA and AR were characterised by high levels of intrahepatic exhausted CD8+ T (Tex) cells. In IA, Tex cells were mainly derived from liver-resident GZMK+ effector memory T cells and self-expansion. By contrast, peripheral CX3CR1+ effector T cells and GZMK+ effector memory T cells were the main source of Tex cells in AR. In IA but not AR, significant cell-cell interactions were observed between Tex cells and regulatory CD4+ T cells, as well as between Tex and FCGR3A+ macrophages. Such interactions were potentially mediated through human leukocyte antigen class I molecules together with their receptors CANX and LILRBs, respectively, contributing to the dysfunction of antiviral immune responses. By contrast, CX3CR1+GNLY+ central memory CD8+ T cells were concurrently expanded in both liver and blood of AR, providing a potential surrogate marker for viral resolution. In clinic, intrahepatic Tex cells were positively correlated with serum alanine aminotransferase levels and histological grading scores. CONCLUSION:Our study dissects the coordinated immune responses for different HBV infection phases and provides a rich resource for fully understanding immunopathogenesis and developing effective therapeutic strategies.
A three-dimensional engineered heterogeneous tumor model for assessing cellular environment and response.
Rodenhizer Darren,Dean Teresa,Xu Bin,Cojocari Dan,McGuigan Alison P
This protocol describes how to build and implement a three-dimensional (3D) cell culture system, TRACER (tissue roll for analysis of cellular environment and response), that enables analysis of cellular behavior and phenotype in hypoxic gradients. TRACER consists of infiltrating cells encapsulated in a hydrogel extracellular matrix (ECM) within a thin strip of porous cellulose scaffolding that is then rolled around an oxygen-impermeable mandrel for assembly of thick and layered 3D tissue constructs that develop cell-defined oxygen gradients. TRACER differs from other stacked-paper cell culture models because it is assembled from a single-piece scaffold, which facilitates rapid disassembly for analysis of different cell populations and metabolites. The protocol describes how to fabricate TRACER components, cell seeding in the scaffold, and scaffold assembly and disassembly. Furthermore, it provides methods to quantify live, dead, or proliferating cells, as well as gradients of oxygen using the nitroimidazole derivative EF5, in a layer-by-layer analysis with confocal microscopy or by flow cytometry of cells isolated from the TRACER scaffold. Additional methods to isolate live cells from TRACER layers for dose-response analysis with a clonogenic assay, as well as steps to extract RNA or fast-changing metabolites from TRACER layers, are also presented. Finally, we provide alternative steps to establish TRACER co-cultures for assessment of tumor cell invasion and metastasis, in this case in the absence of a hypoxic gradient. Although analysis time varies according to the assay chosen, scaffold fabrication and seeding typically take 2 h, and TRACER assembly takes 20 min on the day following scaffold seeding. The TRACER platform is designed for use by researchers and students who have basic tissue culture experience.
Redirecting cell-type specific cytokine responses with engineered interleukin-4 superkines.
Junttila Ilkka S,Creusot Remi J,Moraga Ignacio,Bates Darren L,Wong Michael T,Alonso Michael N,Suhoski Megan M,Lupardus Patrick,Meier-Schellersheim Martin,Engleman Edgar G,Utz Paul J,Fathman C Garrison,Paul William E,Garcia K Christopher
Nature chemical biology
Cytokines dimerize their receptors, with the binding of the 'second chain' triggering signaling. In the interleukin (IL)-4 and IL-13 system, different cell types express varying numbers of alternative second receptor chains (γc or IL-13Rα1), forming functionally distinct type I or type II complexes. We manipulated the affinity and specificity of second chain recruitment by human IL-4. A type I receptor-selective IL-4 'superkine' with 3,700-fold higher affinity for γc was three- to ten-fold more potent than wild-type IL-4. Conversely, a variant with high affinity for IL-13Rα1 more potently activated cells expressing the type II receptor and induced differentiation of dendritic cells from monocytes, implicating the type II receptor in this process. Superkines showed signaling advantages on cells with lower second chain numbers. Comparative transcriptional analysis reveals that the superkines induce largely redundant gene expression profiles. Variable second chain numbers can be exploited to redirect cytokines toward distinct cell subsets and elicit new actions, potentially improving the selectivity of cytokine therapy.
Highly efficient differentiation of human ES cells and iPS cells into mature pancreatic insulin-producing cells.
Zhang Donghui,Jiang Wei,Liu Meng,Sui Xin,Yin Xiaolei,Chen Song,Shi Yan,Deng Hongkui
Human pluripotent stem cells represent a potentially unlimited source of functional pancreatic endocrine lineage cells. Here we report a highly efficient approach to induce human embryonic stem (ES) cells and induced pluripotent stem (iPS) cells to differentiate into mature insulin-producing cells in a chemical-defined culture system. The differentiated human ES cells obtained by this approach comprised nearly 25% insulin-positive cells as assayed by flow cytometry analysis, which released insulin/C-peptide in response to glucose stimuli in a manner comparable to that of adult human islets. Most of these insulin-producing cells co-expressed mature beta cell-specific markers such as NKX6-1 and PDX1, indicating a similar gene expression pattern to adult islet beta cells in vivo. In this study, we also demonstrated that EGF facilitates the expansion of PDX1-positive pancreatic progenitors. Moreover, our protocol also succeeded in efficiently inducing human iPS cells to differentiate into insulin-producing cells. Therefore, this work not only provides a new model to study the mechanism of human pancreatic specialization and maturation in vitro, but also enhances the possibility of utilizing patient-specific iPS cells for the treatment of diabetes.
Control of Cdc28 CDK1 by a stress-induced lncRNA.
Nadal-Ribelles Mariona,Solé Carme,Xu Zhenyu,Steinmetz Lars M,de Nadal Eulàlia,Posas Francesc
Genomic analysis has revealed the existence of a large number of long noncoding RNAs (lncRNAs) with different functions in a variety of organisms, including yeast. Cells display dramatic changes of gene expression upon environmental changes. Upon osmostress, hundreds of stress-responsive genes are induced by the stress-activated protein kinase (SAPK) p38/Hog1. Using whole-genome tiling arrays, we found that Hog1 induces a set of lncRNAs upon stress. One of the genes expressing a Hog1-dependent lncRNA in antisense orientation is CDC28, the cyclin-dependent kinase 1 (CDK1) that controls the cell cycle in yeast. Cdc28 lncRNA mediates the establishment of gene looping and the relocalization of Hog1 and RSC from the 3' UTR to the +1 nucleosome to induce CDC28 expression. The increase in the levels of Cdc28 results in cells able to reenter the cell cycle more efficiently after stress. This may represent a general mechanism to prime expression of genes needed after stresses are alleviated.
Repression of the transcription factor Bach2 contributes to predisposition of IgG1 memory B cells toward plasma cell differentiation.
Kometani Kohei,Nakagawa Rinako,Shinnakasu Ryo,Kaji Tomohiro,Rybouchkin Andrei,Moriyama Saya,Furukawa Koji,Koseki Haruhiko,Takemori Toshitada,Kurosaki Tomohiro
Memory B cells are essential for generating rapid and robust secondary antibody responses. It has been thought that the unique cytoplasmic domain of IgG causes the prompt activation of antigen-experienced IgG memory B cells. To assess this model, we have generated a mouse containing IgG1 B cells that have never encountered antigen. We found that, upon challenge, antigen-experienced IgG1 memory B cells rapidly differentiated into plasma cells, whereas nonexperienced IgG1 B cells did not, suggesting the importance of the stimulation history. In addition, our results suggest that repression of the Bach2 transcription factor, which results from antigen experience, contributes to predisposition of IgG1 memory B cells to differentiate into plasma cells.
Antagonistic regulation by the transcription factors C/EBPα and MITF specifies basophil and mast cell fates.
Qi Xiaopeng,Hong Jessie,Chaves Lee,Zhuang Yonghua,Chen Yuhong,Wang Demin,Chabon Jacob,Graham Brian,Ohmori Keitaro,Li Yapeng,Huang Hua
It remains unclear whether basophils and mast cells are derived from a common progenitor. Furthermore, how basophil versus mast cell fate is specified has not been investigated. Here, we have identified a population of granulocyte-macrophage progenitors (GMPs) that were highly enriched in the capacity to differentiate into basophils and mast cells while retaining a limited capacity to differentiate into myeloid cells. We have designated these progenitor cells "pre-basophil and mast cell progenitors" (pre-BMPs). STAT5 signaling was required for the differentiation of pre-BMPs into both basophils and mast cells and was critical for inducing two downstream molecules: C/EBPα and MITF. We have identified C/EBPα as the critical basophil transcription factor for specifying basophil cell fate and MITF as the crucial transcription factor for specifying mast cell fate. C/EBPα and MITF silenced each other's transcription in a directly antagonistic fashion. Our study reveals how basophil and mast cell fate is specified.
Preexisting high frequencies of memory CD8+ T cells favor rapid memory differentiation and preservation of proliferative potential upon boosting.
Fraser Kathryn A,Schenkel Jason M,Jameson Stephen C,Vezys Vaiva,Masopust David
Memory CD8+ T cell quantity and quality determine protective efficacy against reinfection. Heterologous prime boost vaccination minimizes contraction of anamnestic effectors and maximizes memory CD8+ T cell quantity but reportedly erodes proliferative potential and protective efficacy. This study exploited heterologous prime boost vaccination to discover parameters regulating effector CD8+ T cell contraction and memory differentiation. When abundant memory T cells were established, boosting induced only 5-8 cell divisions, unusually rapid memory T cell differentiation as measured by phenotype and mitochondrial bioenergetic function, long-lived survival of 50% of effector T cells, and preservation of proliferative potential. Conversely, boosting in situations of low memory CD8+ T cell frequencies induced many cell divisions, increased contraction of effector cells, and caused senescence, low mitochondrial membrane potential, and poorly protective memory. Thus, anamnestic memory T cell differentiation is flexible, and abundant quantity can be achieved while maximizing protective efficacy and preserving proliferative potential.
Impaired antibacterial response of liver sinusoidal Vγ9Vδ2 T cells in patients with chronic liver disease.
Rha Min-Seok,Han Ji Won,Koh June-Young,Lee Ha Seok,Kim Jong Hoon,Cho Kyungjoo,Kim Soon Il,Kim Myoung Soo,Lee Jae Geun,Park Su-Hyung,Joo Dong Jin,Park Jun Yong,Shin Eui-Cheol
OBJECTIVE:The liver acts as a frontline barrier against diverse gut-derived pathogens, and the sinusoid is the primary site of liver immune surveillance. However, little is known about liver sinusoidal immune cells in the context of chronic liver disease (CLD). Here, we investigated the antibacterial capacity of liver sinusoidal γδ T cells in patients with various CLDs. DESIGN:We analysed the frequency, phenotype and functions of human liver sinusoidal γδ T cells from healthy donors and recipients with CLD, including HBV-related CLD (liver cirrhosis (LC) and/or hepatocellular carcinoma (HCC)), alcoholic LC and LC or HCC of other aetiologies, by flow cytometry and RNA-sequencing using liver perfusates obtained during living donor liver transplantation. We also measured the plasma levels of D-lactate and bacterial endotoxin to evaluate bacterial translocation. RESULTS:The frequency of liver sinusoidal Vγ9Vδ2 T cells was reduced in patients with CLD. Immunophenotypic and transcriptomic analyses revealed that liver sinusoidal Vγ9Vδ2 T cells from patients with CLD were persistently activated and pro-apoptotic. In addition, liver sinusoidal Vγ9Vδ2 T cells from patients with CLD showed significantly decreased interferon (IFN)-γ production following stimulation with bacterial metabolites and . The antibacterial IFN-γ response of liver sinusoidal Vγ9Vδ2 T cells significantly correlated with liver function, and inversely correlated with the plasma level of D-lactate in patients with CLD. Repetitive in vitro stimulation with induced activation, apoptosis and functional impairment of liver sinusoidal Vγ9Vδ2 T cells. CONCLUSION:Liver sinusoidal Vγ9Vδ2 T cells are functionally impaired in patients with CLD. Bacterial translocation and decreasing liver functions are associated with functional impairment of liver sinusoidal Vγ9Vδ2 T cells.
Human intestinal tissue-resident memory T cells comprise transcriptionally and functionally distinct subsets.
FitzPatrick Michael E B,Provine Nicholas M,Garner Lucy C,Powell Kate,Amini Ali,Irwin Sophie L,Ferry Helen,Ambrose Tim,Friend Peter,Vrakas Georgios,Reddy Srikanth,Soilleux Elizabeth,Klenerman Paul,Allan Philip J
Tissue-resident memory T (T) cells provide key adaptive immune responses in infection, cancer, and autoimmunity. However, transcriptional heterogeneity of human intestinal T cells remains undefined. Here, we investigate transcriptional and functional heterogeneity of human T cells through study of donor-derived T cells from intestinal transplant recipients. Single-cell transcriptional profiling identifies two transcriptional states of CD8 T cells, delineated by ITGAE and ITGB2 expression. We define a transcriptional signature discriminating these populations, including differential expression of cytotoxicity- and residency-associated genes. Flow cytometry of recipient-derived cells infiltrating the graft, and lymphocytes from healthy gut, confirm these CD8 T phenotypes. CD8 CD69CD103 T cells produce interleukin-2 (IL-2) and demonstrate greater polyfunctional cytokine production, whereas β2-integrinCD69CD103 T cells have higher granzyme expression. Analysis of intestinal CD4 T cells identifies several parallels, including a β2-integrin population. Together, these results describe the transcriptional, phenotypic, and functional heterogeneity of human intestinal CD4 and CD8 T cells.
Lymphocytic alveolitis is associated with the accumulation of functionally impaired HIV-specific T cells in the lung of antiretroviral therapy-naive subjects.
Neff C Preston,Chain Jennifer L,MaWhinney Samantha,Martin Allison K,Linderman Derek J,Flores Sonia C,Campbell Thomas B,Palmer Brent E,Fontenot Andrew P
American journal of respiratory and critical care medicine
RATIONALE:Lymphocytic alveolitis in HIV-1-infected individuals is associated with multiple pulmonary complications and a poor prognosis. Although lymphocytic alveolitis has been associated with viremia and an increased number of CD8(+) T cells in the lung, its exact cause is unknown. OBJECTIVES:To determine if HIV-1-specific T cells are associated with lymphocytic alveolitis in HIV-1-infected individuals. METHODS:Using blood and bronchoalveolar lavage (BAL) cells from normal control subjects and untreated HIV-1-infected individuals, we examined the frequency and functional capacity of HIV-1-specific T cells. MEASUREMENTS AND MAIN RESULTS:We found that HIV-1-specific T cells were significantly elevated in the BAL compared with blood of HIV-1-infected individuals and strongly correlated with T-cell alveolitis. Expression of Ki67, a marker of in vivo proliferation, was significantly reduced on HIV-1-specific T cells in BAL compared with blood, suggesting a diminished proliferative capacity. In addition, HIV-1-specific CD4(+) and CD8(+) T cells in BAL had higher expression of programmed death 1 (PD-1) and lower cytotoxic T-lymphocyte antigen 4 (CTLA-4) expression than those in the blood. A strong correlation between PD-1, but not CTLA-4, and HIV-1-specific T-cell proliferation was seen, and blockade of the PD-1/PD-L1 pathway augmented HIV-1-specific T-cell proliferation, suggesting that the PD-1 pathway was the main cause of reduced proliferation in the lung. CONCLUSIONS:These findings suggest that alveolitis associated with HIV-1 infection is caused by the recruitment of HIV-1-specific CD4(+) and CD8(+) T cells to the lung. These antigen-specific T cells display an impaired proliferative capacity that is caused by increased expression of PD-1.
T cell-derived protein S engages TAM receptor signaling in dendritic cells to control the magnitude of the immune response.
Carrera Silva Eugenio A,Chan Pamela Y,Joannas Leonel,Errasti Andrea E,Gagliani Nicola,Bosurgi Lidia,Jabbour Maurice,Perry Anthony,Smith-Chakmakova Faye,Mucida Daniel,Cheroutre Hilde,Burstyn-Cohen Tal,Leighton Jonathan A,Lemke Greg,Ghosh Sourav,Rothlin Carla V
Dendritic cell (DC) activation is essential for the induction of immune defense against pathogens, yet needs to be tightly controlled to avoid chronic inflammation and exaggerated immune responses. Here, we identify a mechanism of immune homeostasis by which adaptive immunity, once triggered, tempers DC activation and prevents overreactive immune responses. T cells, once activated, produced Protein S (Pros1) that signaled through TAM receptor tyrosine kinases in DCs to limit the magnitude of DC activation. Genetic ablation of Pros1 in mouse T cells led to increased expression of costimulatory molecules and cytokines in DCs and enhanced immune responses to T cell-dependent antigens, as well as increased colitis. Additionally, PROS1 was expressed in activated human T cells, and its ability to regulate DC activation was conserved. Our results identify a heretofore unrecognized, homeostatic negative feedback mechanism at the interface of adaptive and innate immunity that maintains the physiological magnitude of the immune response.
Notch simultaneously orchestrates multiple helper T cell programs independently of cytokine signals.
Bailis Will,Yashiro-Ohtani Yumi,Fang Terry C,Hatton Robin D,Weaver Casey T,Artis David,Pear Warren S
Two models are proposed to explain Notch function during helper T (Th) cell differentiation. One argues that Notch instructs one Th cell fate over the other, whereas the other posits that Notch function is dictated by cytokines. Here we provide a detailed mechanistic study investigating the role of Notch in orchestrating Th cell differentiation. Notch neither instructed Th cell differentiation nor did cytokines direct Notch activity, but instead, Notch simultaneously regulated the Th1, Th2, and Th17 cell genetic programs independently of cytokine signals. In addition to regulating these programs in both polarized and nonpolarized Th cells, we identified Ifng as a direct Notch target. Notch bound the Ifng CNS-22 enhancer, where it synergized with Tbet at the promoter. Thus, Notch acts as an unbiased amplifier of Th cell differentiation. Our data provide a paradigm for Notch in hematopoiesis, with Notch simultaneously orchestrating multiple lineage programs, rather than restricting alternate outcomes.
γδ T cells exhibit multifunctional and protective memory in intestinal tissues.
Sheridan Brian S,Romagnoli Pablo A,Pham Quynh-Mai,Fu Han-Hsuan,Alonzo Francis,Schubert Wolf-Dieter,Freitag Nancy E,Lefrançois Leo
The study of T cell memory and the target of vaccine design have focused on memory subsumed by T cells bearing the αβ T cell receptor. Alternatively, γδ T cells are thought to provide rapid immunity, particularly at mucosal borders. Here, we have shown that a distinct subset of mucosal γδ T cells mounts an immune response to oral Listeria monocytogenes (Lm) infection and leads to the development of multifunctional memory T cells capable of simultaneously producing interferon-γ and interleukin-17A in the murine intestinal mucosa. Challenge infection with oral Lm, but not oral Salmonella or intravenous Lm, induced rapid expansion of memory γδ T cells, suggesting contextual specificity to the priming pathogen. Importantly, memory γδ T cells were able to provide enhanced protection against infection. These findings illustrate that γδ T cells play a role with hallmarks of adaptive immunity in the intestinal mucosa.
Staining of cell surface human CD4 with 2'-F-pyrimidine-containing RNA aptamers for flow cytometry.
Davis K A,Lin Y,Abrams B,Jayasena S D
Nucleic acids research
We have used recombinant human CD4 presented on beads as an affinity matrix to screen a 2'-F-pyrimidine-containing RNA library with a complexity of approximately 10(14) molecules. Affinity-selected aptamers bind recombinant CD4 with low nanomolar equilibrium dissociation constants. These high-affinity aptamers conjugated to different fluorophores such as fluorescein and phycoerythrin were used to stain cells, expressing human CD4 on cell surface, for analysis by flow cytometry. Aptamers, conjugated to fluorophores, stained mouse T cells that express human CD4 on the surface, but not the control mouse T cells lacking human CD4. The control cells, however, do express mouse CD4 whose extracellular domain has 55% sequence identity to the human form. These human CD4-specific aptamers selectively stained CD4(+) T cells in a preparation of human peripheral blood mononuclear cells. These results and others suggest that aptamers are emerging as a versatile class of molecules that can be used for various diagnostic applications performed under different formats or platforms.
CXCR6 Inhibits Hepatocarcinogenesis by Promoting Natural Killer T- and CD4 T-Cell-Dependent Control of Senescence.
Mossanen Jana C,Kohlhepp Marlene,Wehr Alexander,Krenkel Oliver,Liepelt Anke,Roeth Anjali A,Möckel Diana,Heymann Felix,Lammers Twan,Gassler Nikolaus,Hermann Juliane,Jankowski Joachim,Neumann Ulf P,Luedde Tom,Trautwein Christian,Tacke Frank
BACKGROUND & AIMS:Inflammation in the liver provokes fibrosis, but inflammation is also important for tumor surveillance. Inhibitors of chemokine pathways, such as CXCL16 and CXCR6 regulation of lymphocyte trafficking, are being tested as antifibrotic agents, but their effects on the development of hepatocellular carcinoma (HCC) are unclear. We assessed the roles of CXCR6-dependent immune mechanisms in hepatocarcinogenesis. METHODS:C57BL/6J wild-type (WT) mice and CXCR6-deficient mice (Cxcr6) were given injections of diethylnitrosamine (DEN) to induce liver cancer and α-galactosylceramide to activate natural killer T (NKT) cells. We also performed studies in mice with conditional, hepatocyte-specific deletion of NEMO, which develop inflammation-associated liver tumors (Nemo and NemoCxcr6 mice). We collected liver tissues from patients with cirrhosis (n = 43), HCC (n = 35), and neither of these diseases (control individuals, n = 25). Human and mouse liver tissues were analyzed by histology, immunohistochemistry, flow cytometry, RNA expression arrays (from sorted hepatic lymphocytes), and matrix-assisted laser desorption/ionization imaging. Bone marrow was transferred from Cxcr6 or WT mice to irradiated C57BL/6J mice, and spleen and liver cells were analyzed by flow cytometry. CD4 T cells or NKT cells were isolated from the spleen and liver of CD45.1 WT mice and transferred into CXCR6-deficient mice after DEN injection. RESULTS:After DEN injection, CXCR6-deficient mice had a significantly higher tumor burden than WT mice and increased tumor progression, characterized by reduced intrahepatic numbers of invariant NKT and CD4 T cells that express tumor necrosis factor and interferon gamma. Livers of NemoCxcr6 mice had significantly more senescent hepatocytes than livers of Nemo mice. In studies of bone-marrow chimeras, adoptive cell transfer experiments, and analyses of Nemo mice, we found that NKT and CD4 T cells promote the removal of senescent hepatocytes to prevent hepatocarcinogenesis, and that this process required CXCR6. Injection of WT with α-galactosylceramide increased removal of senescent hepatocytes by NKT cells. We observed peritumoral accumulation of CXCR6-associated lymphocytes in human HCC, which appeared reduced compared with cirrhosis tissues. CONCLUSIONS:In studies of mice with liver tumors, we found that CXCR6 mediated NKT-cell and CD4 T-cell removal of senescent hepatocytes. Antifibrotic strategies to reduce CXCR6 activity in liver, or to reduce inflammation or modulate the immune response, should be tested for their effects on hepatocarcinogenesis.
Near-complete elimination of mutant mtDNA by iterative or dynamic dose-controlled treatment with mtZFNs.
Gammage Payam A,Gaude Edoardo,Van Haute Lindsey,Rebelo-Guiomar Pedro,Jackson Christopher B,Rorbach Joanna,Pekalski Marcin L,Robinson Alan J,Charpentier Marine,Concordet Jean-Paul,Frezza Christian,Minczuk Michal
Nucleic acids research
Mitochondrial diseases are frequently associated with mutations in mitochondrial DNA (mtDNA). In most cases, mutant and wild-type mtDNAs coexist, resulting in heteroplasmy. The selective elimination of mutant mtDNA, and consequent enrichment of wild-type mtDNA, can rescue pathological phenotypes in heteroplasmic cells. Use of the mitochondrially targeted zinc finger-nuclease (mtZFN) results in degradation of mutant mtDNA through site-specific DNA cleavage. Here, we describe a substantial enhancement of our previous mtZFN-based approaches to targeting mtDNA, allowing near-complete directional shifts of mtDNA heteroplasmy, either by iterative treatment or through finely controlled expression of mtZFN, which limits off-target catalysis and undesired mtDNA copy number depletion. To demonstrate the utility of this improved approach, we generated an isogenic distribution of heteroplasmic cells with variable mtDNA mutant level from the same parental source without clonal selection. Analysis of these populations demonstrated an altered metabolic signature in cells harbouring decreased levels of mutant m.8993T>G mtDNA, associated with neuropathy, ataxia, and retinitis pigmentosa (NARP). We conclude that mtZFN-based approaches offer means for mtDNA heteroplasmy manipulation in basic research, and may provide a strategy for therapeutic intervention in selected mitochondrial diseases.
The microRNA miR-155 controls CD8(+) T cell responses by regulating interferon signaling.
Gracias Donald T,Stelekati Erietta,Hope Jennifer L,Boesteanu Alina C,Doering Travis A,Norton Jillian,Mueller Yvonne M,Fraietta Joseph A,Wherry E John,Turner Martin,Katsikis Peter D
We found upregulation of expression of the microRNA miR-155 in primary effector and effector memory CD8(+) T cells, but low miR-155 expression in naive and central memory cells. Antiviral CD8(+) T cell responses and viral clearance were impaired in miR-155-deficient mice, and this defect was intrinsic to CD8(+) T cells, as miR-155-deficient CD8(+) T cells mounted greatly diminished primary and memory responses. Conversely, miR-155 overexpression augmented antiviral CD8(+) T cell responses in vivo. Gene-expression profiling showed that miR-155-deficient CD8(+) T cells had enhanced type I interferon signaling and were more susceptible to interferon's antiproliferative effect. Inhibition of the type I interferon-associated transcription factors STAT1 or IRF7 resulted in enhanced responses of miR-155-deficient CD8(+) T cells in vivo. We have thus identified a previously unknown role for miR-155 in regulating responsiveness to interferon and CD8(+) T cell responses to pathogens in vivo.
Deficiency in IL-17-committed Vγ4(+) γδ T cells in a spontaneous Sox13-mutant CD45.1(+) congenic mouse substrain provides protection from dermatitis.
Gray Elizabeth E,Ramírez-Valle Francisco,Xu Ying,Wu Shuang,Wu Zhihao,Karjalainen Klaus E,Cyster Jason G
Interleukin 17 (IL-17)-committed γδ T cells (γδT17 cells) participate in many immune responses, but their developmental requirements and subset specific functions remain poorly understood. Here we report that a commonly used CD45.1(+) congenic C57BL/6 mouse substrain is characterized by selective deficiency in Vγ4(+) γδT17 cells. This trait was due to a spontaneous mutation in the gene encoding the transcription factor Sox13 that caused an intrinsic defect in development of those cells in the neonatal thymus. The γδT17 cells migrated from skin to lymph nodes at low rates. In a model of psoriasis-like dermatitis, the Vγ4(+) γδT17 cell subset expanded considerably in lymph nodes and homed to inflamed skin. Sox13-mutant mice were protected from psoriasis-like skin changes, which identified a role for Sox13-dependent γδT17 cells in this inflammatory condition.
Activation of caspase-1 by the NLRP3 inflammasome regulates the NADPH oxidase NOX2 to control phagosome function.
Sokolovska Anna,Becker Christine E,Ip W K Eddie,Rathinam Vijay A K,Brudner Matthew,Paquette Nicholas,Tanne Antoine,Vanaja Sivapriya K,Moore Kathryn J,Fitzgerald Katherine A,Lacy-Hulbert Adam,Stuart Lynda M
Phagocytosis is a fundamental cellular process that is pivotal for immunity as it coordinates microbial killing, innate immune activation and antigen presentation. An essential step in this process is phagosome acidification, which regulates many functions of these organelles that allow phagosomes to participate in processes that are essential to both innate and adaptive immunity. Here we report that acidification of phagosomes containing Gram-positive bacteria is regulated by the NLRP3 inflammasome and caspase-1. Active caspase-1 accumulates on phagosomes and acts locally to control the pH by modulating buffering by the NADPH oxidase NOX2. These data provide insight into a mechanism by which innate immune signals can modify cellular defenses and establish a new function for the NLRP3 inflammasome and caspase-1 in host defense.
Next-generation RNA-based fluorescent biosensors enable anaerobic detection of cyclic di-GMP.
Wang Xin C,Wilson Stephen C,Hammond Ming C
Nucleic acids research
Bacteria occupy a diverse set of environmental niches with differing oxygen availability. Anaerobic environments such as mammalian digestive tracts and industrial reactors harbor an abundance of both obligate and facultative anaerobes, many of which play significant roles in human health and biomanufacturing. Studying bacterial function under partial or fully anaerobic conditions, however, is challenging given the paucity of suitable live-cell imaging tools. Here, we introduce a series of RNA-based fluorescent biosensors that respond selectively to cyclic di-GMP, an intracellular bacterial second messenger that controls cellular motility and biofilm formation. We demonstrate the utility of these biosensors in vivo under both aerobic and anaerobic conditions, and we show that biosensor expression does not interfere with the native motility phenotype. Together, our results attest to the effectiveness and versatility of RNA-based fluorescent biosensors, priming further development and application of these and other analogous sensors to study host-microbial and microbial-microbial interactions through small molecule signals.
The interplay between chromosome stability and cell cycle control explored through gene-gene interaction and computational simulation.
Frumkin Jesse P,Patra Biranchi N,Sevold Anthony,Ganguly Kumkum,Patel Chaya,Yoon Stephanie,Schmid Molly B,Ray Animesh
Nucleic acids research
Chromosome stability models are usually qualitative models derived from molecular-genetic mechanisms for DNA repair, DNA synthesis, and cell division. While qualitative models are informative, they are also challenging to reformulate as precise quantitative models. In this report we explore how (A) laboratory experiments, (B) quantitative simulation, and (C) seriation algorithms can inform models of chromosome stability. Laboratory experiments were used to identify 19 genes that when over-expressed cause chromosome instability in the yeast Saccharomyces cerevisiae To better understand the molecular mechanisms by which these genes act, we explored their genetic interactions with 18 deletion mutations known to cause chromosome instability. Quantitative simulations based on a mathematical model of the cell cycle were used to predict the consequences of several genetic interactions. These simulations lead us to suspect that the chromosome instability genes cause cell-cycle perturbations. Cell-cycle involvement was confirmed using a seriation algorithm, which was used to analyze the genetic interaction matrix to reveal an underlying cyclical pattern. The seriation algorithm searched over 10(14) possible arrangements of rows and columns to find one optimal arrangement, which correctly reflects events during cell cycle phases. To conclude, we illustrate how the molecular mechanisms behind these cell cycle events are consistent with established molecular interaction maps.
A microbial sensor for organophosphate hydrolysis exploiting an engineered specificity switch in a transcription factor.
Jha Ramesh K,Kern Theresa L,Kim Youngchang,Tesar Christine,Jedrzejczak Robert,Joachimiak Andrzej,Strauss Charlie E M
Nucleic acids research
A whole-cell biosensor utilizing a transcription factor (TF) is an effective tool for sensitive and selective detection of specialty chemicals or anthropogenic molecules, but requires access to an expanded repertoire of TFs. Using homology modeling and ligand docking for binding pocket identification, assisted by conservative mutations in the pocket, we engineered a novel specificity in an Acinetobacter TF, PobR, to 'sense' a chemical p-nitrophenol (pNP) and measured the response via a fluorescent protein reporter expressed from a PobR promoter. Out of 10(7) variants of PobR, four were active when dosed with pNP, with two mutants showing a specificity switch from the native effector 4-hydroxybenzoate (4HB). One of the mutants, pNPmut1 was then used to create a smart microbial cell responding to pNP production from hydrolysis of an insecticide, paraoxon, in a coupled assay involving phosphotriesterase (PTE) enzyme expressed from a separate promoter. We show the fluorescence of the cells correlated with the catalytic efficiency of the PTE variant expressed in each cell. High selectivity between similar molecules (4HB versus pNP), high sensitivity for pNP detection (∼2 μM) and agreement of apo- and holo-structures of PobR scaffold with predetermined computational models are other significant results presented in this work.
Effects of HDV infection and pegylated interferon α treatment on the natural killer cell compartment in chronically infected individuals.
Lunemann Sebastian,Malone David F G,Grabowski Jan,Port Kerstin,Béziat Vivien,Bremer Birgit,Malmberg Karl-Johan,Manns Michael P,Sandberg Johan K,Cornberg Markus,Ljunggren Hans-Gustaf,Wedemeyer Heiner,Björkström Niklas K
OBJECTIVE:Although hepatitis delta is considered an immune-mediated disease, adaptive immune responses to hepatitis delta virus (HDV) are hardly detectable. Thus, the role of other immune responses, including those mediated by natural killer (NK) cells, must be considered in HDV pathogenesis and in treatments with immune-stimulating agents such as interferon (IFN)α. However, the phenotype and function of NK cells in chronic HDV infection, or in HDV-infected individuals undergoing IFNα treatment, have not been extensively studied. DESIGN:We performed an extensive analysis of NK cells in chronically HDV-infected patients before and during treatment with IFNα, and compared the results with those for patients with HBV mono-infection as well as healthy controls. RESULTS:In untreated HDV-infected patients, a higher than normal frequency of NK cells was observed in peripheral blood with unaltered phenotypic NK cell differentiation status. In contrast, long-term IFNα treatment of HDV-infected patients caused a significant change in NK cell differentiation status, with selective loss of terminally differentiated NK cells and, in parallel, a relative enrichment in immature NK cell subsets. Treatment was associated with marked functional impairment of the NK cells, which was independent of the changes in NK cell differentiation status. Furthermore, treatment polarised NK cell IFN signalling from STAT4 towards STAT1 dependency. Strikingly, a high frequency of CD56(dim) NK cells at baseline was positively associated with IFNα treatment outcome in the patients. CONCLUSIONS:We describe in detail how HDV infection, and IFNα treatment of this infection, affects the NK cell compartment and what consequences this has for the functional capacity of NK cells.
Intrahepatic myeloid-cell aggregates enable local proliferation of CD8(+) T cells and successful immunotherapy against chronic viral liver infection.
Huang Li-Rung,Wohlleber Dirk,Reisinger Florian,Jenne Craig N,Cheng Ru-Lin,Abdullah Zeinab,Schildberg Frank A,Odenthal Margarete,Dienes Hans-Peter,van Rooijen Nico,Schmitt Edgar,Garbi Natalio,Croft Michael,Kurts Christian,Kubes Paul,Protzer Ulrike,Heikenwalder Mathias,Knolle Percy A
Chronic infection is difficult to overcome because of exhaustion or depletion of cytotoxic effector CD8(+) T cells (cytotoxic T lymphoytes (CTLs)). Here we report that signaling via Toll-like receptors (TLRs) induced intrahepatic aggregates of myeloid cells that enabled the population expansion of CTLs (iMATEs: 'intrahepatic myeloid-cell aggregates for T cell population expansion') without causing immunopathology. In the liver, CTL proliferation was restricted to iMATEs that were composed of inflammatory monocyte-derived CD11b(+) cells. Signaling via tumor-necrosis factor (TNF) caused iMATE formation that facilitated costimulation dependent on the receptor OX40 for expansion of the CTL population. The iMATEs arose during acute viral infection but were absent during chronic viral infection, yet they were still induced by TLR signaling. Such hepatic expansion of the CTL population controlled chronic viral infection of the liver after vaccination with DNA. Thus, iMATEs are dynamic structures that overcome regulatory cues that limit the population expansion of CTLs during chronic infection and can be used in new therapeutic vaccination strategies.
Cutaneous immunosurveillance and regulation of inflammation by group 2 innate lymphoid cells.
Roediger Ben,Kyle Ryan,Yip Kwok Ho,Sumaria Nital,Guy Thomas V,Kim Brian S,Mitchell Andrew J,Tay Szun S,Jain Rohit,Forbes-Blom Elizabeth,Chen Xi,Tong Philip L,Bolton Holly A,Artis David,Paul William E,Fazekas de St Groth Barbara,Grimbaldeston Michele A,Le Gros Graham,Weninger Wolfgang
Type 2 immunity is critical for defense against cutaneous infections but also underlies the development of allergic skin diseases. We report the identification in normal mouse dermis of an abundant, phenotypically unique group 2 innate lymphoid cell (ILC2) subset that depended on interleukin 7 (IL-7) and constitutively produced IL-13. Intravital multiphoton microscopy showed that dermal ILC2 cells specifically interacted with mast cells, whose function was suppressed by IL-13. Treatment of mice deficient in recombination-activating gene 1 (Rag1(-/-)) with IL-2 resulted in the population expansion of activated, IL-5-producing dermal ILC2 cells, which led to spontaneous dermatitis characterized by eosinophil infiltrates and activated mast cells. Our data show that ILC2 cells have both pro- and anti-inflammatory properties and identify a previously unknown interactive pathway between two innate populations of cells of the immune system linked to type 2 immunity and allergic diseases.
Natural and inducible TH17 cells are regulated differently by Akt and mTOR pathways.
Kim Jiyeon S,Sklarz Tammarah,Banks Lauren B,Gohil Mercy,Waickman Adam T,Skuli Nicolas,Krock Bryan L,Luo Chong T,Hu Weihong,Pollizzi Kristin N,Li Ming O,Rathmell Jeffrey C,Birnbaum Morris J,Powell Jonathan D,Jordan Martha S,Koretzky Gary A
Natural T helper 17 (nTH17) cells are a population of interleukin 17 (IL-17)-producing cells that acquire effector function in the thymus during development. Here we demonstrate that the serine/threonine kinase Akt has a critical role in regulating nTH17 cell development. Although Akt and the downstream mTORC1-ARNT-HIFα axis were required for generation of inducible TH17 (iTH17) cells, nTH17 cells developed independently of mTORC1. In contrast, mTORC2 and inhibition of Foxo proteins were critical for development of nTH17 cells. Moreover, distinct isoforms of Akt controlled the generation of TH17 cell subsets, as deletion of Akt2, but not of Akt1, led to defective generation of iTH17 cells. These findings define mechanisms regulating nTH17 cell development and reveal previously unknown roles of Akt and mTOR in shaping subsets of T cells.
T cells maintain an exhausted phenotype after antigen withdrawal and population reexpansion.
Utzschneider Daniel T,Legat Amandine,Fuertes Marraco Silvia A,Carrié Lucie,Luescher Immanuel,Speiser Daniel E,Zehn Dietmar
During chronic infection, pathogen-specific CD8(+) T cells upregulate expression of molecules such as the inhibitory surface receptor PD-1, have diminished cytokine production and are thought to undergo terminal differentiation into exhausted cells. Here we found that T cells with memory-like properties were generated during chronic infection. After transfer into naive mice, these cells robustly proliferated and controlled a viral infection. The reexpanded T cell populations continued to have the exhausted phenotype they acquired during the chronic infection. Thus, the cells underwent a form of differentiation that was stably transmitted to daughter cells. We therefore propose that during persistent infection, effector T cells stably differentiate into a state that is optimized to limit viral replication without causing overwhelming immunological pathology.
Enhancing the cellular uptake of Py-Im polyamides through next-generation aryl turns.
Meier Jordan L,Montgomery David C,Dervan Peter B
Nucleic acids research
Pyrrole-imidazole (Py-Im) hairpin polyamides are a class of programmable, sequence-specific DNA binding oligomers capable of disrupting protein-DNA interactions and modulating gene expression in living cells. Methods to control the cellular uptake and nuclear localization of these compounds are essential to their application as molecular probes or therapeutic agents. Here, we explore modifications of the hairpin γ-aminobutyric acid turn unit as a means to enhance cellular uptake and biological activity. Remarkably, introduction of a simple aryl group at the turn potentiates the biological effects of a polyamide targeting the sequence 5'-WGWWCW-3' (W =A/T) by up to two orders of magnitude. Confocal microscopy and quantitative flow cytometry analysis suggest this enhanced potency is due to increased nuclear uptake. Finally, we explore the generality of this approach and find that aryl-turn modifications enhance the uptake of all polyamides tested, while having a variable effect on the upper limit of polyamide nuclear accumulation. Overall this provides a step forward for controlling the intracellular concentration of Py-Im polyamides that will prove valuable for future applications in which biological potency is essential.
Bi-allelic Variants in the GPI Transamidase Subunit PIGK Cause a Neurodevelopmental Syndrome with Hypotonia, Cerebellar Atrophy, and Epilepsy.
Nguyen Thi Tuyet Mai,Murakami Yoshiko,Mobilio Sabrina,Niceta Marcello,Zampino Giuseppe,Philippe Christophe,Moutton Sébastien,Zaki Maha S,James Kiely N,Musaev Damir,Mu Weiyi,Baranano Kristin,Nance Jessica R,Rosenfeld Jill A,Braverman Nancy,Ciolfi Andrea,Millan Francisca,Person Richard E,Bruel Ange-Line,Thauvin-Robinet Christel,Ververi Athina,DeVile Catherine,Male Alison,Efthymiou Stephanie,Maroofian Reza,Houlden Henry,Maqbool Shazia,Rahman Fatima,Baratang Nissan V,Rousseau Justine,St-Denis Anik,Elrick Matthew J,Anselm Irina,Rodan Lance H,Tartaglia Marco,Gleeson Joseph,Kinoshita Taroh,Campeau Philippe M
American journal of human genetics
Glycosylphosphatidylinositol (GPI)-anchored proteins are critical for embryogenesis, neurogenesis, and cell signaling. Variants in several genes participating in GPI biosynthesis and processing lead to decreased cell surface presence of GPI-anchored proteins (GPI-APs) and cause inherited GPI deficiency disorders (IGDs). In this report, we describe 12 individuals from nine unrelated families with 10 different bi-allelic PIGK variants. PIGK encodes a component of the GPI transamidase complex, which attaches the GPI anchor to proteins. Clinical features found in most individuals include global developmental delay and/or intellectual disability, hypotonia, cerebellar ataxia, cerebellar atrophy, and facial dysmorphisms. The majority of the individuals have epilepsy. Two individuals have slightly decreased levels of serum alkaline phosphatase, while eight do not. Flow cytometric analysis of blood and fibroblasts from affected individuals showed decreased cell surface presence of GPI-APs. The overexpression of wild-type (WT) PIGK in fibroblasts rescued the levels of cell surface GPI-APs. In a knockout cell line, transfection with WT PIGK also rescued the GPI-AP levels, but transfection with the two tested mutant variants did not. Our study not only expands the clinical and known genetic spectrum of IGDs, but it also expands the genetic differential diagnosis for cerebellar atrophy. Given the fact that cerebellar atrophy is seen in other IGDs, flow cytometry for GPI-APs should be considered in the work-ups of individuals presenting this feature.
An engineered multicellular stem cell niche for the 3D derivation of human myogenic progenitors from iPSCs.
The EMBO journal
Fate decisions in the embryo are controlled by a plethora of microenvironmental interactions in a three-dimensional niche. To investigate whether aspects of this microenvironmental complexity can be engineered to direct myogenic human-induced pluripotent stem cell (hiPSC) differentiation, we here screened murine cell types present in the developmental or adult stem cell niche in heterotypic suspension embryoids. We identified embryonic endothelial cells and fibroblasts as highly permissive for myogenic specification of hiPSCs. After two weeks of sequential Wnt and FGF pathway induction, these three-component embryoids are enriched in Pax7-positive embryonic-like myogenic progenitors that can be isolated by flow cytometry. Myogenic differentiation of hiPSCs in heterotypic embryoids relies on a specialized structural microenvironment and depends on MAPK, PI3K/AKT, and Notch signaling. After transplantation in a mouse model of Duchenne muscular dystrophy, embryonic-like myogenic progenitors repopulate the stem cell niche, reactivate after repeated injury, and, compared to adult human myoblasts, display enhanced fusion and lead to increased muscle function. Altogether, we provide a two-week protocol for efficient and scalable suspension-based 3D derivation of Pax7-positive myogenic progenitors from hiPSCs.
Harnessing lipid signaling pathways to target specialized pro-angiogenic neutrophil subsets for regenerative immunotherapy.
To gain insights into neutrophil heterogeneity dynamics in the context of sterile inflammation and wound healing, we performed a pseudotime analysis of single-cell flow cytometry data using the spanning-tree progression analysis of density-normalized events algorithm. This enables us to view neutrophil transitional subsets along a pseudotime trajectory and identify distinct VEGFR1, VEGFR2, and CXCR4 high-expressing pro-angiogenic neutrophils. While the proresolving lipid mediator aspirin-triggered resolvin D1 (AT-RvD1) has a known ability to limit neutrophil infiltration, our analysis uncovers a mode of action in which AT-RvD1 leads to inflammation resolution through the selective reprogramming toward a therapeutic neutrophil subset. This accumulation leads to enhanced vascular remodeling in the skinfold window chamber and a proregenerative shift in macrophage and dendritic cell phenotype, resulting in improved wound closure after skin transplantation. As the targeting of functional immune subsets becomes the key to regenerative immunotherapies, single-cell pseudotime analysis tools will be vital in this field.
Targeting the IL-6-Yap-Snail signalling axis in synovial fibroblasts ameliorates inflammatory arthritis.
Symons Rebecca A,Colella Fabio,Collins Fraser L,Rafipay Alexandra J,Kania Karolina,McClure Jessica J,White Nathan,Cunningham Iain,Ashraf Sadaf,Hay Elizabeth,Mackenzie Kevin S,Howard Kenneth A,Riemen Anna H K,Manzo Antonio,Clark Susan M,Roelofs Anke J,De Bari Cosimo
Annals of the rheumatic diseases
OBJECTIVE:We aimed to understand the role of the transcriptional co-factor Yes-associated protein (Yap) in the molecular pathway underpinning the pathogenic transformation of synovial fibroblasts (SF) in rheumatoid arthritis (RA) to become invasive and cause joint destruction. METHODS:Synovium from patients with RA and mice with antigen-induced arthritis (AIA) was analysed by immunostaining and qRT-PCR. SF were targeted using and mice, crossed with fluorescent reporters for cell tracing and mice for conditional ablation. Fibroblast phenotypes were analysed by flow cytometry, and arthritis severity was assessed by histology. Yap activation was detected using Yap-Tead reporter cells and Yap-Snail interaction by proximity ligation assay. SF invasiveness was analysed using matrigel-coated transwells. RESULTS:Yap, its binding partner Snail and downstream target connective tissue growth factor were upregulated in hyperplastic human RA and in mouse AIA synovium, with Yap detected in SF but not macrophages. Lineage tracing showed polyclonal expansion of -expressing SF during AIA, with predominant expansion of the -lineage SF subpopulation descending from the embryonic joint interzone. -lineage SF showed increased expression of and adopted an erosive phenotype (podoplanin+Thy-1 cell surface antigen-), invading cartilage and bone. Conditional ablation of in -lineage cells or -expressing fibroblasts ameliorated AIA. Interleukin (IL)-6, but not tumour necrosis factor alpha (TNF-α) or IL-1β, Jak-dependently activated Yap and induced Yap-Snail interaction. SF invasiveness induced by IL-6 stimulation or Snail overexpression was prevented by Yap knockdown, showing a critical role for Yap in SF transformation in RA. CONCLUSIONS:Our findings uncover the IL-6-Yap-Snail signalling axis in pathogenic SF in inflammatory arthritis.
Cathepsin S inhibition suppresses systemic lupus erythematosus and lupus nephritis because cathepsin S is essential for MHC class II-mediated CD4 T cell and B cell priming.
Rupanagudi Khader Valli,Kulkarni Onkar P,Lichtnekert Julia,Darisipudi Murthy Narayana,Mulay Shrikant R,Schott Brigitte,Gruner Sabine,Haap Wolfgang,Hartmann Guido,Anders Hans-Joachim
Annals of the rheumatic diseases
OBJECTIVES:Major histocompatibility complex (MHC) class II-mediated priming of T and B lymphocytes is a central element of autoimmunity in systemic lupus erythematosus (SLE) and lupus nephritis. The cysteine protease cathepsin S degrades the invariant peptide chain during MHC II assembly with antigenic peptide in antigen-presenting cells; therefore, we hypothesised that cathepsin S inhibition would be therapeutic in SLE. METHODS:We developed a highly specific small molecule, orally available, cathepsin S antagonist, RO5461111, with suitable pharmacodynamic and pharmacokinetic properties that efficiently suppressed antigen-specific T cell and B cell priming in vitro and in vivo. RESULTS:When given to MRL-Fas(lpr) mice with SLE and lupus nephritis, RO5461111 significantly reduced the activation of spleen dendritic cells and the subsequent expansion and activation of CD4 T cells and CD4/CD8 double-negative T cells. Cathepsin S inhibition impaired the spatial organisation of germinal centres, suppressed follicular B cell maturation to plasma cells and Ig class switch. This reversed hypergammaglobulinemia and significantly suppressed the plasma levels of numerous IgG (but not IgM) autoantibodies below baseline, including anti-dsDNA. This effect was associated with less glomerular IgG deposits, which protected kidneys from lupus nephritis. CONCLUSIONS:Together, cathepsin S promotes SLE by driving MHC class II-mediated T and B cell priming, germinal centre formation and B cell maturation towards plasma cells. These afferent immune pathways can be specifically reversed with the cathepsin S antagonist RO5461111, which prevents lupus nephritis progression even when given after disease onset. This novel therapeutic strategy could correct a common pathomechanism of SLE and other immune complex-related autoimmune diseases.
Large-scale production of SAGE libraries from microdissected tissues, flow-sorted cells, and cell lines.
Khattra Jaswinder,Delaney Allen D,Zhao Yongjun,Siddiqui Asim,Asano Jennifer,McDonald Helen,Pandoh Pawan,Dhalla Noreen,Prabhu Anna-Liisa,Ma Kevin,Lee Stephanie,Ally Adrian,Tam Angela,Sa Danne,Rogers Sean,Charest David,Stott Jeff,Zuyderduyn Scott,Varhol Richard,Eaves Connie,Jones Steven,Holt Robert,Hirst Martin,Hoodless Pamela A,Marra Marco A
We describe the details of a serial analysis of gene expression (SAGE) library construction and analysis platform that has enabled the generation of >298 high-quality SAGE libraries and >30 million SAGE tags primarily from sub-microgram amounts of total RNA purified from samples acquired by microdissection. Several RNA isolation methods were used to handle the diversity of samples processed, and various measures were applied to minimize ditag PCR carryover contamination. Modifications in the SAGE protocol resulted in improved cloning and DNA sequencing efficiencies. Bioinformatic measures to automatically assess DNA sequencing results were implemented to analyze the integrity of ditag structure, linker or cross-species ditag contamination, and yield of high-quality tags per sequence read. Our analysis of singleton tag errors resulted in a method for correcting such errors to statistically determine tag accuracy. From the libraries generated, we produced an essentially complete mapping of reliable 21-base-pair tags to the mouse reference genome sequence for a meta-library of approximately 5 million tags. Our analyses led us to reject the commonly held notion that duplicate ditags are artifacts. Rather than the usual practice of discarding such tags, we conclude that they should be retained to avoid introducing bias into the results and thereby maintain the quantitative nature of the data, which is a major theoretical advantage of SAGE as a tool for global transcriptional profiling.
VAMP7 controls T cell activation by regulating the recruitment and phosphorylation of vesicular Lat at TCR-activation sites.
Larghi Paola,Williamson David J,Carpier Jean-Marie,Dogniaux Stéphanie,Chemin Karine,Bohineust Armelle,Danglot Lydia,Gaus Katharina,Galli Thierry,Hivroz Claire
The mechanisms by which Lat (a key adaptor in the T cell antigen receptor (TCR) signaling pathway) and the TCR come together after TCR triggering are not well understood. We investigate here the role of SNARE proteins, which are part of protein complexes involved in the docking, priming and fusion of vesicles with opposing membranes, in this process. Here we found, by silencing approaches and genetically modified mice, that the vesicular SNARE VAMP7 was required for the recruitment of Lat-containing vesicles to TCR-activation sites. Our results indicated that this did not involve fusion of Lat-containing vesicles with the plasma membrane. VAMP7, which localized together with Lat on the subsynaptic vesicles, controlled the phosphorylation of Lat, formation of the TCR-Lat-signaling complex and, ultimately, activation of T cells. Our findings suggest that the transport and docking of Lat-containing vesicles with target membranes containing TCRs regulates TCR-induced signaling.
T cell regulation mediated by interaction of soluble CD52 with the inhibitory receptor Siglec-10.
Bandala-Sanchez Esther,Zhang Yuxia,Reinwald Simone,Dromey James A,Lee Bo-Han,Qian Junyan,Böhmer Ralph M,Harrison Leonard C
Functionally diverse T cell populations interact to maintain homeostasis of the immune system. We found that human and mouse antigen-activated T cells with high expression of the lymphocyte surface marker CD52 suppressed other T cells. CD52(hi)CD4(+) T cells were distinct from CD4(+)CD25(+)Foxp3(+) regulatory T cells. Their suppression was mediated by soluble CD52 released by phospholipase C. Soluble CD52 bound to the inhibitory receptor Siglec-10 and impaired phosphorylation of the T cell receptor-associated kinases Lck and Zap70 and T cell activation. Humans with type 1 diabetes had a lower frequency and diminished function of CD52(hi)CD4(+) T cells responsive to the autoantigen GAD65. In diabetes-prone mice of the nonobese diabetic (NOD) strain, transfer of lymphocyte populations depleted of CD52(hi) cells resulted in a substantially accelerated onset of diabetes. Our studies identify a ligand-receptor mechanism of T cell regulation that may protect humans and mice from autoimmune disease.
GATA-3 controls the maintenance and proliferation of T cells downstream of TCR and cytokine signaling.
Wang Yunqi,Misumi Ichiro,Gu Ai-Di,Curtis T Anthony,Su Lishan,Whitmire Jason K,Wan Yisong Y
GATA-3 controls T helper type 2 (TH2) differentiation. However, whether GATA-3 regulates the function of mature T cells beyond TH2 determination remains poorly understood. We found that signaling via the T cell antigen receptor (TCR) and cytokine stimulation promoted GATA-3 expression in CD8(+) T cells, which controlled cell proliferation. Although GATA-3-deficient CD8(+) T cells were generated, their peripheral maintenance was impaired, with lower expression of the receptor for interleukin 7 (IL-7R). GATA-3-deficient T cells had defective responses to viral infection and alloantigen. The proto-oncoprotein c-Myc was a critical target of GATA-3 in promoting T cell proliferation. Our study thus demonstrates an essential role for GATA-3 in controlling the maintenance and proliferation of T cells and provides insight into immunoregulation.
A conserved human T cell population targets mycobacterial antigens presented by CD1b.
Van Rhijn Ildiko,Kasmar Anne,de Jong Annemieke,Gras Stephanie,Bhati Mugdha,Doorenspleet Marieke E,de Vries Niek,Godfrey Dale I,Altman John D,de Jager Wilco,Rossjohn Jamie,Moody D Branch
Human T cell antigen receptors (TCRs) pair in millions of combinations to create complex and unique T cell repertoires for each person. Through the use of tetramers to analyze TCRs reactive to the antigen-presenting molecule CD1b, we detected T cells with highly stereotyped TCR α-chains present among genetically unrelated patients with tuberculosis. The germline-encoded, mycolyl lipid-reactive (GEM) TCRs had an α-chain bearing the variable (V) region TRAV1-2 rearranged to the joining (J) region TRAJ9 with few nontemplated (N)-region additions. Analysis of TCRs by high-throughput sequencing, binding and crystallography showed linkage of TCRα sequence motifs to high-affinity recognition of antigen. Thus, the CD1-reactive TCR repertoire is composed of at least two compartments: high-affinity GEM TCRs, and more-diverse TCRs with low affinity for CD1b-lipid complexes. We found high interdonor conservation of TCRs that probably resulted from selection by a nonpolymorphic antigen-presenting molecule and an immunodominant antigen.
TRF2 inhibits a cell-extrinsic pathway through which natural killer cells eliminate cancer cells.
Biroccio Annamaria,Cherfils-Vicini Julien,Augereau Adeline,Pinte Sébastien,Bauwens Serge,Ye Jing,Simonet Thomas,Horard Béatrice,Jamet Karine,Cervera Ludovic,Mendez-Bermudez Aaron,Poncet Delphine,Grataroli Renée,de Rodenbeeke Claire T'kint,Salvati Erica,Rizzo Angela,Zizza Pasquale,Ricoul Michelle,Cognet Céline,Kuilman Thomas,Duret Helene,Lépinasse Florian,Marvel Jacqueline,Verhoeyen Els,Cosset François-Loïc,Peeper Daniel,Smyth Mark J,Londoño-Vallejo Arturo,Sabatier Laure,Picco Vincent,Pages Gilles,Scoazec Jean-Yves,Stoppacciaro Antonella,Leonetti Carlo,Vivier Eric,Gilson Eric
Nature cell biology
Dysfunctional telomeres suppress tumour progression by activating cell-intrinsic programs that lead to growth arrest. Increased levels of TRF2, a key factor in telomere protection, are observed in various human malignancies and contribute to oncogenesis. We demonstrate here that a high level of TRF2 in tumour cells decreased their ability to recruit and activate natural killer (NK) cells. Conversely, a reduced dose of TRF2 enabled tumour cells to be more easily eliminated by NK cells. Consistent with these results, a progressive upregulation of TRF2 correlated with decreased NK cell density during the early development of human colon cancer. By screening for TRF2-bound genes, we found that HS3ST4--a gene encoding for the heparan sulphate (glucosamine) 3-O-sulphotransferase 4--was regulated by TRF2 and inhibited the recruitment of NK cells in an epistatic relationship with TRF2. Overall, these results reveal a TRF2-dependent pathway that is tumour-cell extrinsic and regulates NK cell immunity.
Sensitivity of infectious SARS-CoV-2 B.1.1.7 and B.1.351 variants to neutralizing antibodies.
Planas Delphine,Bruel Timothée,Grzelak Ludivine,Guivel-Benhassine Florence,Staropoli Isabelle,Porrot Françoise,Planchais Cyril,Buchrieser Julian,Rajah Maaran Michael,Bishop Elodie,Albert Mélanie,Donati Flora,Prot Matthieu,Behillil Sylvie,Enouf Vincent,Maquart Marianne,Smati-Lafarge Mounira,Varon Emmanuelle,Schortgen Frédérique,Yahyaoui Layla,Gonzalez Maria,De Sèze Jérôme,Péré Hélène,Veyer David,Sève Aymeric,Simon-Lorière Etienne,Fafi-Kremer Samira,Stefic Karl,Mouquet Hugo,Hocqueloux Laurent,van der Werf Sylvie,Prazuck Thierry,Schwartz Olivier
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) B.1.1.7 and B.1.351 variants were first identified in the United Kingdom and South Africa, respectively, and have since spread to many countries. These variants harboring diverse mutations in the gene encoding the spike protein raise important concerns about their immune evasion potential. Here, we isolated infectious B.1.1.7 and B.1.351 strains from acutely infected individuals. We examined sensitivity of the two variants to SARS-CoV-2 antibodies present in sera and nasal swabs from individuals infected with previously circulating strains or who were recently vaccinated, in comparison with a D614G reference virus. We utilized a new rapid neutralization assay, based on reporter cells that become positive for GFP after overnight infection. Sera from 58 convalescent individuals collected up to 9 months after symptoms, similarly neutralized B.1.1.7 and D614G. In contrast, after 9 months, convalescent sera had a mean sixfold reduction in neutralizing titers, and 40% of the samples lacked any activity against B.1.351. Sera from 19 individuals vaccinated twice with Pfizer Cominarty, longitudinally tested up to 6 weeks after vaccination, were similarly potent against B.1.1.7 but less efficacious against B.1.351, when compared to D614G. Neutralizing titers increased after the second vaccine dose, but remained 14-fold lower against B.1.351. In contrast, sera from convalescent or vaccinated individuals similarly bound the three spike proteins in a flow cytometry-based serological assay. Neutralizing antibodies were rarely detected in nasal swabs from vaccinees. Thus, faster-spreading SARS-CoV-2 variants acquired a partial resistance to neutralizing antibodies generated by natural infection or vaccination, which was most frequently detected in individuals with low antibody levels. Our results indicate that B1.351, but not B.1.1.7, may increase the risk of infection in immunized individuals.
Differential responses of immune cells to type I interferon contribute to host resistance to viral infection.
Baranek Thomas,Manh Thien-Phong Vu,Alexandre Yannick,Maqbool Muhammad Ahmad,Cabeza Joaquin Zacarias,Tomasello Elena,Crozat Karine,Bessou Gilles,Zucchini Nicolas,Robbins Scott H,Vivier Eric,Kalinke Ulrich,Ferrier Pierre,Dalod Marc
Cell host & microbe
Type I interferons (IFNs) are central to antiviral defense, but how they orchestrate immune cell function is incompletely understood. We determined that IFNs produced during murine cytomegalovirus (MCMV) infection differentially affect dendritic cells (DCs) and natural killer (NK) cells. IFNs induce cell-intrinsic responses in DCs, activating antiproliferative, antiviral, and lymphocyte-activating gene networks, consistent with high activity of the transcription factor STAT1 in these cells. By comparison, NK cells exhibit lower STAT1 expression and reduced IFN responsiveness. Rather, IFNs indirectly affect NK cells by inducing IL-15, which activates the transcription factor E2F and stimulates genes promoting cell expansion. IFN cell-intrinsic responses are necessary in DCs, but not NK cells, for MCMV resistance. Thus, sensitivity to IFN-induced cytokines and differences in IFN receptor signaling program immune cells to mount distinct responses that promote viral control.
Transferrin-functionalized nanoparticles lose their targeting capabilities when a biomolecule corona adsorbs on the surface.
Salvati Anna,Pitek Andrzej S,Monopoli Marco P,Prapainop Kanlaya,Bombelli Francesca Baldelli,Hristov Delyan R,Kelly Philip M,Åberg Christoffer,Mahon Eugene,Dawson Kenneth A
Nanoparticles have been proposed as carriers for drugs, genes and therapies to treat various diseases. Many strategies have been developed to target nanomaterials to specific or over-expressed receptors in diseased cells, and these typically involve functionalizing the surface of nanoparticles with proteins, antibodies or other biomolecules. Here, we show that the targeting ability of such functionalized nanoparticles may disappear when they are placed in a biological environment. Using transferrin-conjugated nanoparticles, we found that proteins in the media can shield transferrin from binding to both its targeted receptors on cells and soluble transferrin receptors. Although nanoparticles continue to enter cells, the targeting specificity of transferrin is lost. Our results suggest that when nanoparticles are placed in a complex biological environment, interaction with other proteins in the medium and the formation of a protein corona can 'screen' the targeting molecules on the surface of nanoparticles and cause loss of specificity in targeting.
An epigenetic mechanism of resistance to targeted therapy in T cell acute lymphoblastic leukemia.
Knoechel Birgit,Roderick Justine E,Williamson Kaylyn E,Zhu Jiang,Lohr Jens G,Cotton Matthew J,Gillespie Shawn M,Fernandez Daniel,Ku Manching,Wang Hongfang,Piccioni Federica,Silver Serena J,Jain Mohit,Pearson Daniel,Kluk Michael J,Ott Christopher J,Shultz Leonard D,Brehm Michael A,Greiner Dale L,Gutierrez Alejandro,Stegmaier Kimberly,Kung Andrew L,Root David E,Bradner James E,Aster Jon C,Kelliher Michelle A,Bernstein Bradley E
The identification of activating NOTCH1 mutations in T cell acute lymphoblastic leukemia (T-ALL) led to clinical testing of γ-secretase inhibitors (GSIs) that prevent NOTCH1 activation. However, responses to these inhibitors have been transient, suggesting that resistance limits their clinical efficacy. Here we modeled T-ALL resistance, identifying GSI-tolerant 'persister' cells that expand in the absence of NOTCH1 signaling. Rare persisters are already present in naive T-ALL populations, and the reversibility of their phenotype suggests an epigenetic mechanism. Relative to GSI-sensitive cells, persister cells activate distinct signaling and transcriptional programs and exhibit chromatin compaction. A knockdown screen identified chromatin regulators essential for persister viability, including BRD4. BRD4 binds enhancers near critical T-ALL genes, including MYC and BCL2. The BRD4 inhibitor JQ1 downregulates expression of these targets and induces growth arrest and apoptosis in persister cells, at doses well tolerated by GSI-sensitive cells. Consistently, the GSI-JQ1 combination was found to be effective against primary human leukemias in vivo. Our findings establish a role for epigenetic heterogeneity in leukemia resistance that may be addressed by incorporating epigenetic modulators in combination therapy.
Prospective isolation of adult neural stem cells from the mouse subependymal zone.
Fischer Judith,Beckervordersandforth Ruth,Tripathi Pratibha,Steiner-Mezzadri Andrea,Ninkovic Jovica,Götz Magdalena
Neural stem cells (NSCs) have the remarkable capacity to self-renew and the lifelong ability to generate neurons in the adult mammalian brain. However, the molecular and cellular mechanisms contributing to these behaviors are still not understood. Now that prospective isolation of the NSCs has become feasible, these mechanisms can be studied. Here we describe a protocol for the efficient isolation of adult NSCs, by the application of a dual-labeling strategy on the basis of their glial identity and ciliated nature. The cells are isolated from the lateral ventricular subependymal zone (SEZ) of adult hGFAP-eGFP (human glial fibrillary acidic protein-enhanced green fluorescent protein) transgenic mice by fluorescence-activated cell sorting. Staining against prominin1 (CD133) allows the isolation of the NSCs (hGFAP-eGFP(+)/prominin1(+)), which can be further subdivided by labeling with the fluorescent epidermal growth factor. This protocol, which can be completed in 7 h, allows the assessment of quantitative changes in SEZ NSCs and the examination of their molecular and functional characteristics.
Proteomic screening of glutamatergic mouse brain synaptosomes isolated by fluorescence activated sorting.
Biesemann Christoph,Grønborg Mads,Luquet Elisa,Wichert Sven P,Bernard Véronique,Bungers Simon R,Cooper Ben,Varoqueaux Frédérique,Li Liyi,Byrne Jennifer A,Urlaub Henning,Jahn Olaf,Brose Nils,Herzog Etienne
The EMBO journal
For decades, neuroscientists have used enriched preparations of synaptic particles called synaptosomes to study synapse function. However, the interpretation of corresponding data is problematic as synaptosome preparations contain multiple types of synapses and non-synaptic neuronal and glial contaminants. We established a novel Fluorescence Activated Synaptosome Sorting (FASS) method that substantially improves conventional synaptosome enrichment protocols and enables high-resolution biochemical analyses of specific synapse subpopulations. Employing knock-in mice with fluorescent glutamatergic synapses, we show that FASS isolates intact ultrapure synaptosomes composed of a resealed presynaptic terminal and a postsynaptic density as assessed by light and electron microscopy. FASS synaptosomes contain bona fide glutamatergic synapse proteins but are almost devoid of other synapse types and extrasynaptic or glial contaminants. We identified 163 enriched proteins in FASS samples, of which FXYD6 and Tpd52 were validated as new synaptic proteins. FASS purification thus enables high-resolution biochemical analyses of specific synapse subpopulations in health and disease.
Induction of ICOS+CXCR3+CXCR5+ TH cells correlates with antibody responses to influenza vaccination.
Bentebibel Salah-Eddine,Lopez Santiago,Obermoser Gerlinde,Schmitt Nathalie,Mueller Cynthia,Harrod Carson,Flano Emilio,Mejias Asuncion,Albrecht Randy A,Blankenship Derek,Xu Hui,Pascual Virginia,Banchereau Jacques,Garcia-Sastre Adolfo,Palucka Anna Karolina,Ramilo Octavio,Ueno Hideki
Science translational medicine
Seasonal influenza vaccine protects 60 to 90% of healthy young adults from influenza infection. The immunological events that lead to the induction of protective antibody responses remain poorly understood in humans. We identified the type of CD4+ T cells associated with protective antibody responses after seasonal influenza vaccinations. The administration of trivalent split-virus influenza vaccines induced a temporary increase of CD4+ T cells expressing ICOS, which peaked at day 7, as did plasmablasts. The induction of ICOS was largely restricted to CD4+ T cells coexpressing the chemokine receptors CXCR3 and CXCR5, a subpopulation of circulating memory T follicular helper cells. Up to 60% of these ICOS+CXCR3+CXCR5+CD4+ T cells were specific for influenza antigens and expressed interleukin-2 (IL-2), IL-10, IL-21, and interferon-γ upon antigen stimulation. The increase of ICOS+CXCR3+CXCR5+CD4+ T cells in blood correlated with the increase of preexisting antibody titers, but not with the induction of primary antibody responses. Consistently, purified ICOS+CXCR3+CXCR5+CD4+ T cells efficiently induced memory B cells, but not naïve B cells, to differentiate into plasma cells that produce influenza-specific antibodies ex vivo. Thus, the emergence of blood ICOS+CXCR3+CXCR5+CD4+ T cells correlates with the development of protective antibody responses generated by memory B cells upon seasonal influenza vaccination.
Isolation, culture and evaluation of multilineage-differentiating stress-enduring (Muse) cells.
Kuroda Yasumasa,Wakao Shohei,Kitada Masaaki,Murakami Toru,Nojima Makoto,Dezawa Mari
Multilineage-differentiating stress-enduring (Muse) cells are distinct stem cells in mesenchymal cell populations with the capacity to self-renew, to differentiate into cells representative of all three germ layers from a single cell, and to repair damaged tissues by spontaneous differentiation into tissue-specific cells without forming teratomas. We describe step-by-step procedures for isolating and evaluating these cells. Muse cells are also a practical cell source for human induced pluripotent stem (iPS) cells with markedly high generation efficiency. They can be collected as cells that are double positive for stage-specific embryonic antigen-3 (SSEA-3) and CD105 from commercially available mesenchymal cells, such as adult human bone marrow stromal cells and dermal fibroblasts, or from fresh adult human bone marrow samples. Under both spontaneous and induced differentiation conditions, they show triploblastic differentiation. It takes 4-6 h to collect and 2 weeks to confirm the differentiation and self-renewal capacity of Muse cells.
Engineering bacterial thiosulfate and tetrathionate sensors for detecting gut inflammation.
Daeffler Kristina N-M,Galley Jeffrey D,Sheth Ravi U,Ortiz-Velez Laura C,Bibb Christopher O,Shroyer Noah F,Britton Robert A,Tabor Jeffrey J
Molecular systems biology
There is a groundswell of interest in using genetically engineered sensor bacteria to study gut microbiota pathways, and diagnose or treat associated diseases. Here, we computationally identify the first biological thiosulfate sensor and an improved tetrathionate sensor, both two-component systems from marine species, and validate them in laboratory Then, we port these sensors into a gut-adapted probiotic strain, and develop a method based upon oral gavage and flow cytometry of colon and fecal samples to demonstrate that colon inflammation (colitis) activates the thiosulfate sensor in mice harboring native gut microbiota. Our thiosulfate sensor may have applications in bacterial diagnostics or therapeutics. Finally, our approach can be replicated for a wide range of bacterial sensors and should thus enable a new class of minimally invasive studies of gut microbiota pathways.
Adapting human pluripotent stem cells to high-throughput and high-content screening.
Desbordes Sabrina C,Studer Lorenz
The increasing use of human pluripotent stem cells (hPSCs) as a source of cells for drug discovery, cytotoxicity assessment and disease modeling requires their adaptation to large-scale culture conditions and screening formats. Here, we describe a simple and robust protocol for the adaptation of human embryonic stem cells (hESCs) to high-throughput screening (HTS). This protocol can also be adapted to human induced pluripotent stem cells (hiPSCs) and high-content screening (HCS). We also describe a 7-d assay to identify compounds with an effect on hESC self-renewal and differentiation. This assay can be adapted to a variety of applications. The procedure involves the culture expansion of hESCs, their adaptation to 384-well plates, the addition of small molecules or other factors, and finally data acquisition and processing. In this protocol, the optimal number of hESCs plated in 384-well plates has been adapted to HTS/HCS assays of 7 d.
Probing RNA recognition by human ADAR2 using a high-throughput mutagenesis method.
Wang Yuru,Beal Peter A
Nucleic acids research
Adenosine deamination is one of the most prevalent post-transcriptional modifications in mRNA. In humans, ADAR1 and ADAR2 catalyze this modification and their malfunction correlates with disease. Recently our laboratory reported crystal structures of the human ADAR2 deaminase domain bound to duplex RNA revealing a protein loop that binds the RNA on the 5' side of the modification site. This 5' binding loop appears to be one contributor to substrate specificity differences between ADAR family members. In this study, we endeavored to reveal detailed structure-activity relationships in this loop to advance our understanding of RNA recognition by ADAR2. To achieve this goal, we established a high-throughput mutagenesis approach which allows rapid screening of ADAR variants in single yeast cells and provides quantitative evaluation for enzymatic activity. Using this approach, we determined the importance of specific amino acids at 19 different positions in the ADAR2 5' binding loop and revealed six residues that provide essential structural elements supporting the fold of the loop and key RNA-binding functional groups. This work provided new insight into RNA recognition by ADAR2 and established a new tool for defining structure-function relationships in ADAR reactions.
Engineering of temperature- and light-switchable Cas9 variants.
Richter Florian,Fonfara Ines,Bouazza Boris,Schumacher Charlotte Helene,Bratovič Majda,Charpentier Emmanuelle,Möglich Andreas
Nucleic acids research
Sensory photoreceptors have enabled non-invasive and spatiotemporal control of numerous biological processes. Photoreceptor engineering has expanded the repertoire beyond natural receptors, but to date no generally applicable strategy exists towards constructing light-regulated protein actuators of arbitrary function. We hence explored whether the homodimeric Rhodobacter sphaeroides light-oxygen-voltage (LOV) domain (RsLOV) that dissociates upon blue-light exposure can confer light sensitivity onto effector proteins, via a mechanism of light-induced functional site release. We chose the RNA-guided programmable DNA endonuclease Cas9 as proof-of-principle effector, and constructed a comprehensive library of RsLOV inserted throughout the Cas9 protein. Screening with a high-throughput assay based on transcriptional repression in Escherichia coli yielded paRC9, a moderately light-activatable variant. As domain insertion can lead to protein destabilization, we also screened the library for temperature-sensitive variants and isolated tsRC9, a variant with robust activity at 29°C but negligible activity at 37°C. Biochemical assays confirmed temperature-dependent DNA cleavage and binding for tsRC9, but indicated that the light sensitivity of paRC9 is specific to the cellular setting. Using tsRC9, the first temperature-sensitive Cas9 variant, we demonstrate temperature-dependent transcriptional control over ectopic and endogenous genetic loci. Taken together, RsLOV can confer light sensitivity onto an unrelated effector; unexpectedly, the same LOV domain can also impart strong temperature sensitivity.