Cytometry by time of flight identifies distinct signatures in patients with systemic sclerosis, systemic lupus erythematosus and Sjögrens syndrome.
van der Kroef Maarten,van den Hoogen Lucas L,Mertens Jorre S,Blokland Sofie L M,Haskett Scott,Devaprasad Abhinandan,Carvalheiro Tiago,Chouri Eleni,Vazirpanah Nadia,Cossu Marta,Wichers Catherina G K,Silva-Cardoso Sandra C,Affandi Alsya J,Bekker Cornelis P J,Lopes Ana P,Hillen Maarten R,Bonte-Mineur Femke,Kok Marc R,Beretta Lorenzo,Rossato Marzia,Mingueneau Michaël,van Roon Joel A G,Radstake Timothy R D J
European journal of immunology
Systemic sclerosis (SSc), systemic lupus erythematosus (SLE) and primary Sjögrens syndrome (pSS) are clinically distinct systemic autoimmune diseases (SADs) that share molecular pathways. We quantified the frequency of circulating immune-cells in 169 patients with these SADs and 44 healty controls (HC) using mass-cytometry and assessed the diagnostic value of these results. Alterations in the frequency of immune-cell subsets were present in all SADs compared to HC. Most alterations, including a decrease of CD56 NK-cells in SSc and IgM Bcells in pSS, were disease specific; only a reduced frequency of plasmacytoid dendritic cells was common between all SADs Strikingly, hierarchical clustering of SSc patients identified 4 clusters associated with different clinical phenotypes, and 9 of the 12 cell subset-alterations in SSc were also present during the preclinical-phase of the disease. Additionally, we found a strong association between the use of prednisone and alterations in B-cell subsets. Although differences in immune-cell frequencies between these SADs are apparent, the discriminative value thereof is too low for diagnostic purposes. Within each disease, mass cytometry analyses revealed distinct patterns between endophenotypes. Given the lack of tools enabling early diagnosis of SSc, our results justify further research into the value of cellular phenotyping as a diagnostic aid.
Single-cell mass cytometry on peripheral blood identifies immune cell subsets associated with primary biliary cholangitis.
Jang Jin Sung,Juran Brian D,Cunningham Kevin Y,Gupta Vinod K,Son Young Min,Yang Ju Dong,Ali Ahmad H,Enninga Elizabeth Ann L,Sung Jaeyun,Lazaridis Konstantinos N
The relationship between primary biliary cholangitis (PBC), a chronic cholestatic autoimmune liver disease, and the peripheral immune system remains to be fully understood. Herein, we performed the first mass cytometry (CyTOF)-based, immunophenotyping analysis of the peripheral immune system in PBC at single-cell resolution. CyTOF was performed on peripheral blood mononuclear cells (PBMCs) from PBC patients (n = 33) and age-/sex-matched healthy controls (n = 33) to obtain immune cell abundance and marker expression profiles. Hierarchical clustering methods were applied to identify immune cell types and subsets significantly associated with PBC. Subsets of gamma-delta T cells (CD3TCRgd), CD8 T cells (CD3CD8CD161PD1), and memory B cells (CD3CD19CD20CD24CD27) were found to have lower abundance in PBC than in control. In contrast, higher abundance of subsets of monocytes and naïve B cells were observed in PBC compared to control. Furthermore, several naïve B cell (CD3CD19CD20CD24CD27) subsets were significantly higher in PBC patients with cirrhosis (indicative of late-stage disease) than in those without cirrhosis. Alternatively, subsets of memory B cells were lower in abundance in cirrhotic relative to non-cirrhotic PBC patients. Future immunophenotyping investigations could lead to better understanding of PBC pathogenesis and progression, and also to the discovery of novel biomarkers and treatment strategies.
In depth comparative phenotyping of blood innate myeloid leukocytes from healthy humans and macaques using mass cytometry.
Elhmouzi-Younes Jamila,Palgen Jean-Louis,Tchitchek Nicolas,Delandre Simon,Namet Inana,Bodinham Caroline L,Pizzoferro Kathleen,Lewis David J M,Le Grand Roger,Cosma Antonio,Beignon Anne-Sophie
Cytometry. Part A : the journal of the International Society for Analytical Cytology
Comparative immune-profiling of innate responses in humans and non-human primates is important to understand the pathogenesis of infectious and chronic inflammatory diseases as well as for the preclinical development of vaccines and immune therapies. However, direct comparisons of the two species are rare and were never performed using mass cytometry. Here, whole-blood-derived leukocytes from healthy humans and cynomolgus macaques were analyzed with mass cytometry. Two similar panels of around 30 monoclonal antibodies targeting human markers associated with innate myeloid cells to stain fixed human and macaque leukocytes were constructed. To compare the circulating innate cells from the two primate species, an analysis pipeline combining a clustering analysis by the Spanning-tree Progression Analysis of Density-normalized Events (SPADE) algorithm with a two-step hierarchical clustering of cells nodes and markers was used. Identical SPADE settings were applied to both datasets, except for the 20 clustering markers which slightly differed. A correlation analysis designed to compare the phenotypes of human and macaque cell nodes and based on 16 markers, including 15 shared clustering markers and CD19 for humans or CD20 for macaques, revealed similarities and differences between staining patterns. This study unique by the number of individuals (26 humans and 5 macaques) and the use of mass cytometry certainly contributes to better assess the advantages and limits of the use of non-human primates in preclinical research. © 2017 International Society for Advancement of Cytometry.
Mass Cytometry Analysis Reveals the Landscape and Dynamics of CD32a CD4 T Cells From Early HIV Infection to Effective cART.
Coindre Sixtine,Tchitchek Nicolas,Alaoui Lamine,Vaslin Bruno,Bourgeois Christine,Goujard Cecile,Avettand-Fenoel Veronique,Lecuroux Camille,Bruhns Pierre,Le Grand Roger,Beignon Anne-Sophie,Lambotte Olivier,Favier Benoit,
Frontiers in immunology
CD32a has been proposed as a specific marker of latently HIV-infected CD4 T cells. However, CD32a was recently found to be expressed on CD4 T cells of healthy donors, leading to controversy on the relevance of this marker in HIV persistence. Here, we used mass cytometry to characterize the landscape and variation in the abundance of CD32a CD4 T cells during HIV infection. To this end, we analyzed CD32a CD4 T cells in primary HIV infection before and after effective combination antiretroviral therapy (cART) and in healthy donors. We found that CD32a CD4 T cells include heterogeneous subsets that are differentially affected by HIV infection. Our analysis revealed that naive (), central memory (), and effector/memory () CD32a CD4 T-cell clusters that co-express LILRA2- and CD64-activating receptors were more abundant in primary HIV infection and cART stages. Conversely, LILRA2 CD32a CD4 T-cell clusters of either the T, T, or T phenotype were more abundant in healthy individuals. Finally, an activated CD32a CD4 T cell cluster co-expressing LILRA2, CD57, and NKG2C was more abundant in all HIV stages, particularly during primary HIV infection. Overall, our data show that multiple abundance modifications of CD32a CD4 T-cell subsets occur in the early phase of HIV infection, and some of which are conserved after effective cART. Our study brings a better comprehension of the relationship between CD32a expression and CD4 T cells during HIV infection.
Candidate Markers for Stratification and Classification in Rheumatoid Arthritis.
Bader Lucius,Gullaksen Stein-Erik,Blaser Nello,Brun Morten,Bringeland Gerd Haga,Sulen André,Gjesdal Clara Gram,Vedeler Christian,Gavasso Sonia
Frontiers in immunology
Rheumatoid arthritis (RA) is a chronic autoimmune, inflammatory disease, characterized by synovitis in small- and medium-sized joints and, if not treated early and efficiently, joint damage, and destruction. RA is a heterogeneous disease with a plethora of treatment options. The pro-inflammatory cytokine tumor necrosis factor (TNF) plays a central role in the pathogenesis of RA, and TNF inhibitors effectively repress inflammatory activity in RA. Currently, treatment decisions are primarily based on empirics and economic considerations. However, the considerable interpatient variability in response to treatment is a challenge. Markers for a more exact patient classification and stratification are lacking. The objective of this study was to identify markers in immune cell populations that distinguish RA patients from healthy donors with an emphasis on TNF signaling. We employed mass cytometry (CyTOF) with a panel of 13 phenotyping and 10 functional markers to explore signaling in unstimulated and TNF-stimulated peripheral blood mononuclear cells from 20 newly diagnosed, untreated RA patients and 20 healthy donors. The resulting high-dimensional data were analyzed in three independent analysis pipelines, characterized by differences in both data clean-up, identification of cell subsets/clustering and statistical approaches. All three analysis pipelines identified p-p38, IkBa, p-cJun, p-NFkB, and CD86 in cells of both the innate arm (myeloid dendritic cells and classical monocytes) and the adaptive arm (memory CD4 T cells) of the immune system as markers for differentiation between RA patients and healthy donors. Inclusion of the markers p-Akt and CD120b resulted in the correct classification of 18 of 20 RA patients and 17 of 20 healthy donors in regression modeling based on a combined model of basal and TNF-induced signal. Expression patterns in a set of functional markers and specific immune cell subsets were distinct in RA patients compared to healthy individuals. These signatures may support studies of disease pathogenesis, provide candidate markers for response, and non-response to TNF inhibitor treatment, and aid the identification of future therapeutic targets.
Immune monitoring using mass cytometry and related high-dimensional imaging approaches.
Hartmann Felix J,Bendall Sean C
Nature reviews. Rheumatology
The cellular complexity and functional diversity of the human immune system necessitate the use of high-dimensional single-cell tools to uncover its role in multifaceted diseases such as rheumatic diseases, as well as other autoimmune and inflammatory disorders. Proteomic technologies that use elemental (heavy metal) reporter ions, such as mass cytometry (also known as CyTOF) and analogous high-dimensional imaging approaches (including multiplexed ion beam imaging (MIBI) and imaging mass cytometry (IMC)), have been developed from their low-dimensional counterparts, flow cytometry and immunohistochemistry, to meet this need. A growing number of studies have been published that use these technologies to identify functional biomarkers and therapeutic targets in rheumatic diseases, but the full potential of their application to rheumatic disease research has yet to be fulfilled. This Review introduces the underlying technologies for high-dimensional immune monitoring and discusses aspects necessary for their successful implementation, including study design principles, analytical tools and future developments for the field of rheumatology.
Mass cytometry as a platform for the discovery of cellular biomarkers to guide effective rheumatic disease therapy.
Nair Nitya,Mei Henrik E,Chen Shih-Yu,Hale Matthew,Nolan Garry P,Maecker Holden T,Genovese Mark,Fathman C Garrison,Whiting Chan C
Arthritis research & therapy
The development of biomarkers for autoimmune diseases has been hampered by a lack of understanding of disease etiopathogenesis and of the mechanisms underlying the induction and maintenance of inflammation, which involves complex activation dynamics of diverse cell types. The heterogeneous nature and suboptimal clinical response to treatment observed in many autoimmune syndromes highlight the need to develop improved strategies to predict patient outcome to therapy and personalize patient care. Mass cytometry, using CyTOF®, is an advanced technology that facilitates multiparametric, phenotypic analysis of immune cells at single-cell resolution. In this review, we outline the capabilities of mass cytometry and illustrate the potential of this technology to enhance the discovery of cellular biomarkers for rheumatoid arthritis, a prototypical autoimmune disease.
Single-cell mass cytometry reveals distinct populations of brain myeloid cells in mouse neuroinflammation and neurodegeneration models.
Ajami Bahareh,Samusik Nikolay,Wieghofer Peter,Ho Peggy P,Crotti Andrea,Bjornson Zach,Prinz Marco,Fantl Wendy J,Nolan Garry P,Steinman Lawrence
Neuroinflammation and neurodegeneration may represent two poles of brain pathology. Brain myeloid cells, particularly microglia, play key roles in these conditions. We employed single-cell mass cytometry (CyTOF) to compare myeloid cell populations in the experimental autoimmune encephalomyelitis (EAE) model of multiple sclerosis, the R6/2 model of Huntington's disease (HD) and the mutant superoxide dismutase 1 (mSOD1) model of amyotrophic lateral sclerosis (ALS). We identified three myeloid cell populations exclusive to the CNS and present in each disease model. Blood-derived monocytes comprised five populations and migrated to the brain in EAE, but not in HD and ALS models. Single-cell analysis resolved differences in signaling and cytokine production within similar myeloid populations in EAE compared to HD and ALS models. Moreover, these analyses highlighted α5 integrin on myeloid cells as a potential therapeutic target for neuroinflammation. Together, these findings illustrate how neuropathology may differ between inflammatory and degenerative brain disease.
Maturing Human CD127+ CCR7+ PDL1+ Dendritic Cells Express AIRE in the Absence of Tissue Restricted Antigens.
Fergusson Joannah R,Morgan Michael D,Bruchard Melanie,Huitema Leonie,Heesters Balthasar A,van Unen Vincent,van Hamburg Jan Piet,van der Wel Nicole N,Picavet Daisy,Koning Frits,Tas Sander W,Anderson Mark S,Marioni John C,Holländer Georg A,Spits Hergen
Frontiers in immunology
Expression of the Autoimmune regulator (AIRE) outside of the thymus has long been suggested in both humans and mice, but the cellular source in humans has remained undefined. Here we identify AIRE expression in human tonsils and extensively analyzed these "extra-thymic AIRE expressing cells" (eTACs) using combinations of flow cytometry, CyTOF and single cell RNA-sequencing. We identified AIRE+ cells as dendritic cells (DCs) with a mature and migratory phenotype including high levels of antigen presenting molecules and costimulatory molecules, and specific expression of CD127, CCR7, and PDL1. These cells also possessed the ability to stimulate and re-stimulate T cells and displayed reduced responses to toll-like receptor (TLR) agonists compared to conventional DCs. While expression of was enriched within CCR7+CD127+ DCs, single-cell RNA sequencing revealed expression of to be transient, rather than stable, and associated with the differentiation to a mature phenotype. The role of AIRE in central tolerance induction within the thymus is well-established, however our study shows that expression within the periphery is not associated with an enriched expression of tissue-restricted antigens (TRAs). This unexpected finding, suggestive of wider functions of AIRE, may provide an explanation for the non-autoimmune symptoms of APECED patients who lack functional AIRE.
Neuroinflammation at single cell level: What is new?
Brandão W N,De Oliveira M G,Andreoni R T,Nakaya H,Farias A S,Peron Jps
Journal of leukocyte biology
Multiple sclerosis is a chronic and demyelinating disease of the central nervous system (CNS), most prevalent in women, and with an important social and economic cost worldwide. It is triggered by self-reacting lymphocytes that infiltrate the CNS and initiate neuroinflammation. Further, axonal loss and neuronal death takes place, leading to neurodegeneration and brain atrophy. The murine model for studying MS, experimental autoimmune encephalomyelitis (EAE), consists in immunizing mice with myelin-derived epitopes. APCs activate encephalitogenic T CD4 and CD8 lymphocytes that migrate mainly to the spinal cord resulting in neuroinflammation. Most of the knowledge on the pathophysiology and treatment of MS was obtained from EAE experiments, as Th17 cells, anti-alpha4 blocking Abs and the role of microbiota. Conversely, recent technology breakthroughs, such as CyTOF and single-cell RNA-seq, promise to revolutionize our understanding on the mechanisms involved both in MS and EAE. In fact, the importance of specific cellular populations and key molecules in MS/EAE is a constant matter of debate. It is well accepted that both Th1 and Th17 T CD4 lymphocytes play a relevant role in disease initiation after re-activation in situ. What is still under constant investigation, however, is the plasticity of the lymphocyte population, and the individual contribution of both resident and inflammatory cells for the progression or recovery of the disease. Thus, in this review, new findings obtained after single-cell analysis of blood and central nervous system infiltrating cells from MS/EAE and how they have contributed to a better knowledge on the cellular and molecular mechanisms of neuroinflammation are discussed.
B cell alterations during BAFF inhibition with belimumab in SLE.
Ramsköld Daniel,Parodis Ioannis,Lakshmikanth Tadepally,Sippl Natalie,Khademi Mohsen,Chen Yang,Zickert Agneta,Mikeš Jaromír,Achour Adnane,Amara Khaled,Piehl Fredrik,Brodin Petter,Gunnarsson Iva,Malmström Vivianne
BACKGROUND:Systemic lupus erythematosus (SLE) is a systemic autoimmune disease, which exhibits multiple B cell abnormalities including expanded populations of memory B cells and elevated levels of autoantibodies. Belimumab is a monoclonal antibody targeting the B cell cytokine BAFF (a.k.a. BLyS), approved for the treatment of SLE. METHODS:In this prospective cohort study, B cells from peripheral blood of 23 SLE patients initiating belimumab treatment and followed longitudinally for up to three years, were assessed using mass cytometry. FINDINGS:B cells decreased during the study period, with a rapid decrease of both naïve and CD11cCD21 B cells at the first follow-up visit, followed by a continuous reduction at subsequent follow-ups. In contrast, plasma cells and switched memory B cells remained stable throughout the study. The observed immunological changes correlated with early, but not late, clinical improvements. Moreover, high baseline B cell counts were predictive of failure to attain low disease activity. In summary, our data unveiled both rapid and gradual later therapy-associated alterations of both known and unforeseen B cell phenotypes. INTERPRETATION:Our results suggest that evaluation of B cell counts might prove useful prior to initiation of belimumab treatment and that early treatment evaluation and discontinuation might underestimate delayed clinical improvements resultant of late B cell changes.
High-dimensional single-cell proteomics analysis identifies immune checkpoint signatures and therapeutic targets in ulcerative colitis.
Fuchs Sebastian,Sawas Nadia,Staedler Nicolas,Schubert David A,D'Andrea Annalisa,Zeiser Robert,Piali Luca,Hruz Petr,Frei Andreas P
European journal of immunology
Immune checkpoints are regulators of immune cells and play key roles in the modulation of immune responses. The role of checkpoints in autoimmune disease is poorly understood but likely to be central since checkpoint inhibition during cancer treatment can cause autoimmunity. We generated a high-dimensional single-cell proteomics data set from PBMCs of healthy individuals and patients with ulcerative colitis (UC) by mass cytometry, enabling systems-wide analyses of immune cell frequencies and cell type-specific expression patterns of 12 immune checkpoints. Subtle but significant changes in immune cell frequencies and checkpoint expression were observed between UC patients on different treatment regimens and between patients and healthy controls. Most strikingly, UC patients showed a reduced number of peripheral NK-cells and those cells showed an altered phenotype including increased TIGIT expression. Based on these results, we modulated NK-cell function ex vivo through targeting of TIGIT pathway members. In summary, we describe a pattern of changes in immune cell abundance and checkpoint expression as a basis for UC patient stratification and we show modulation of a corresponding immune cell subset through checkpoint targeting. Our approach can be used for the identification of pathogenic immune cell subsets and guide target selection in autoimmunity and chronic inflammation.
Mass Cytometry Identifies Distinct Subsets of Regulatory T Cells and Natural Killer Cells Associated With High Risk for Type 1 Diabetes.
Barcenilla Hugo,Åkerman Linda,Pihl Mikael,Ludvigsson Johnny,Casas Rosaura
Frontiers in immunology
Type 1 diabetes (T1D) is characterized by autoimmune destruction of insulin producing β-cells. The time from onset of islet autoimmunity to manifest clinical disease can vary widely in length, and it is fairly uncharacterized both clinically and immunologically. In the current study, peripheral blood mononuclear cells from autoantibody-positive children with high risk for T1D, and from age-matched healthy individuals, were analyzed by mass cytometry using a panel of 32 antibodies. Surface markers were chosen to identify multiple cell types including T, B, NK, monocytes, and DC, and antibodies specific for identification of differentiation, activation and functional markers were also included in the panel. By applying dimensional reduction and computational unsupervised clustering approaches, we delineated in an unbiased fashion 132 phenotypically distinct subsets within the major immune cell populations. We were able to identify an effector memory Treg subset expressing HLA-DR, CCR4, CCR6, CXCR3, and GATA3 that was increased in the high-risk group. In addition, two subsets of NK cells defined by CD16 CD8 CXCR3 and CD16 CD8 CXCR3 CD11c were also higher in the same subjects. High-risk individuals did not show impaired glucose tolerance at the time of sampling, suggesting that the changes observed were not the result of metabolic imbalance, and might be potential biomarkers predictive of T1D.