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International audience; The interleukin-12 (IL-12) family comprises the only heterodimeric cytokines mediating diverse functional effects. We previously reported a striking bimodal IL-12p70 response to lipopolysaccharide (LPS) stimulation in healthy donors. Herein, we demonstrate that interferon β (IFNβ) is a major upstream determinant of IL-12p70 production, which is also associated with numbers and activation of circulating monocytes. Integrative modeling of proteomic, genetic, epigenomic, and cellular data confirms IFNβ as key for LPS-induced IL-12p70 and allowed us to compare the relative effects of each of these parameters on variable cytokine responses. Clinical relevance of our findings is supported by reduced IFNβ-IL-12p70 responses in patients hospitalized with acute severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection or chronically infected with hepatitis C (HCV). Importantly, these responses are resolved after viral clearance. Our systems immunology approach defines a better understanding of IL-12p70 and IFNβ in healthy and infected persons, providing insights into how common genetic and epigenetic variation may impact immune responses to bacterial infection.

Background Tuberculosis (TB) is caused by Mycobacterium tuberculosis (Mtb) infection and is a major public health problem. Clinical challenges include the lack of a blood-based test for active disease. Current blood-based tests, such as QuantiFERON (QFT) do not distinguish active TB disease from asymptomatic Mtb infection. Methods We hypothesized that TruCulture, an immunomonitoring method for whole-blood stimulation, could discriminate active disease from latent Mtb infection (LTBI). We stimulated whole blood from patients with active TB and compared with LTBI donors. Mtb-specific antigens and live bacillus Calmette-Guérin (BCG) were used as stimuli, with direct comparison to QFT. Protein analyses were performed using conventional and digital enzyme-linked immunosorbent assay (ELISA), as well as Luminex. Results TruCulture showed discrimination of active TB cases from LTBI (P < .0001, AUC = .81) compared with QFT (P = .45, AUC = .56), based on an interferon γ (IFNγ) readout after Mtb antigen (Ag) stimulation. This result was replicated in an independent cohort (AUC = .89). In exploratory analyses, TB stratification could be further improved by the Mtb antigen to BCG IFNγ ratio (P < .0001, AUC = .91). Finally, the combination of digital ELISA and transcriptional analysis showed that LTBI donors with high IFNγ clustered with patients with TB, suggesting the possibility to identify subclinical disease. Conclusions TruCulture offers a next-generation solution for whole-blood stimulation and immunomonitoring with the possibility to discriminate active and latent infection., We tested TruCulture, an immunomonitoring tool, to identify active disease from latent Mtb infection. TruCulture showed improved discrimination of tuberculosis cases from LTBI as compared with QuantiFERON. Tuberculosis stratification could be further improved by the Mtb Ag:BCG IFNγ ratio.

Despite improvement in clinical management, allogeneic hematopoietic stem cell transplantation (HSCT) is still hampered by high morbidity and mortality rates, mainly due to graft versus host disease (GvHD). Recently, it has been demonstrated that the allogeneic immune response might be influenced by external factors such as tissues microenvironment or host microbiota. Here we used high throughput metabolomics to analyze two cohorts of genotypically HLA-identical related recipient and donor pairs. Metabolomic profiles markedly differ between recipients and donors. At the onset of acute GvHD, in addition to host-derived metabolites, we identify significant variation in microbiota-derived metabolites, especially in aryl hydrocarbon receptor (AhR) ligands, bile acids and plasmalogens. Altogether, our findings support that the allogeneic immune response during acute GvHD might be influenced by bile acids and by the decreased production of AhR ligands by microbiota that could limit indoleamine 2,3-dioxygenase induction and influence allogeneic T cell reactivity., Graft versus host disease (GvHD) still hinders allogeneic hematopoietic stem cell transplantation. Here, the authors use metabolomics to analyze two cohorts of paired transplant recipients and donors, identifying significant differences in both host- and microbiota-derived metabolites.

The importance of interleukin-17A (IL-17A) in the pathogenesis of axial spondyloarthritis (SpA) has been demonstrated by the success of IL-17A blockade. However, the nature of the cell populations that produce this important proinflammatory cytokine remains poorly defined. We undertook this study to characterize the major IL-17A-producing blood cell populations in the peripheral blood of patients with axial SpA, with a focus on mucosal-associated invariant T (MAIT) cells, a population known to be capable of producing IL-17.We evaluated IL-17A production from 5 sorted peripheral blood cell populations, namely, MAIT cells, γδ T cells, CD4+ T cells, CD8+ T cells, and neutrophils, before and after stimulation with phorbol myristate acetate, the calcium ionophore A23187, and β-1,3-glucan. Expression of IL-17A transcripts and protein were determined using nCounter and ultra-sensitive Simoa technology, respectively. MAIT cells from the axial entheses of non-axial SpA control patients (n = 5) were further characterized using flow cytometric immunophenotyping and quantitative polymerase chain reaction, and the production of IL-17 was assessed following stimulation.On a per-cell basis, MAIT cells from peripheral blood produced the most IL-17A compared to CD4+ T cells (P 0.01), CD8+ T cells (P 0.0001), and γδ T cells (P 0.0001). IL-17A was not produced by neutrophils. Gene expression analysis also revealed significantly higher expression of IL17A and IL23R in MAIT cells. Stimulation of peripheral blood MAIT cells with anti-CD3/CD28 and IL-7 and/or IL-18 induced strong expression of IL17F. MAIT cells were present in the normal, unaffected entheses of control patients who did not have axial SpA and showed elevated AHR, JAK1, STAT4, and TGFB1 transcript expression with inducible IL-17A protein. IL-18 protein expression was evident in spinal enthesis digests.Both peripheral blood MAIT cells and resident MAIT cells in normal axial entheses contribute to the production of IL-17 and may play important roles in the pathogenesis of axial SpA.

International audience; Objective: Clinical trials of the anti-interleukin-17A (anti-IL-17A) antibody secukinumab have demonstrated a crucial role of the cytokine IL-17A in the pathogenesis of spondyloarthritis (SpA); however, its cellular source in this condition remains a matter of controversy. Group 3 innate lymphoid cells (ILC3s) have been recently identified as potent producers of proinflammatory cytokines, including IL-17A and IL-22, in a number of different tissues. This study was undertaken to characterize the presence and composition of ILCs, and investigate whether these cells are an important source of IL-17A, in the synovial tissue (ST) of patients with SpA.Methods: Matched ST, synovial fluid, and peripheral blood (PB) samples were obtained from SpA patients with actively inflamed knee joints. ILC subsets were characterized by flow cytometry. Gene expression analysis at the single-cell level was performed directly ex vivo and after in vitro activation. An IL-17A enzyme-linked immunospot assay was used to detect IL-17A-secreting cells.Results: ILCs, and particularly NKp44+ ILC3s, were expanded in inflamed arthritic joints. Single-cell expression analysis demonstrated that ST ILCs were clearly distinguishable from ST T cells and from their PB counterparts. Expression of the Th17 signature transcripts RORC, AHR, and IL23R was detected in a large proportion of ST ILC3s. These cells were capable of inducing expression of IL22 and CSF2, but not IL17A, in response to in vitro restimulation.Conclusion: Our findings demonstrate that absolute and relative numbers of ILC3s are enriched in the synovial joints of patients with SpA. However, these cells are not a significant source of IL-17A in this disease.

International audience; Chronic inflammatory diseases (CIDs) cause significant morbidity and are a considerable burden for the patients in terms of pain, impaired function, and diminished quality of life. Important progress in CID treatment has been obtained with biological therapies, such as tumor-necrosis-factor blockers. However, more than a third of the patients fail to respond to these inhibitors and are exposed to the side effects of treatment, without the benefits. Therefore, there is a strong interest in developing tools to predict response of patients to biologics. Areas covered: The authors searched PubMed for recent studies on biomarkers for disease assessment and prediction of therapeutic responses, focusing on the effect of TNF blockers on immune responses in spondyloarthritis (SpA), and other CID, in particular rheumatoid arthritis and inflammatory bowel disease. Conclusions will be drawn about the possible development of predictive biomarkers for response to treatment. Expert opinion: No validated biomarker is currently available to predict treatment response in CID. New insight could be generated through the development of new bioinformatic modeling approaches to combine multidimensional biomarkers that explain the different genetic, immunological and environmental determinants of therapeutic responses.

The cellular and molecular processes involved in acute graft-versus-host disease (aGVHD) development early after allogeneic hematopoietic cell transplantation (HCT) in humans remain largely unknown. We have performed multiparameter immunophenotyping and molecular profiling of CD4+ and CD8+ T cells in 2 independent cohorts of patients undergoing HCT, as well as in their HLA-identical sibling donors. Cellular profiling using spectral flow cytometry showed an incomplete reconstitution of the T-cell compartment in recipients without aGVHD early after transplantation, as well as a shift toward an effector memory phenotype, paralleled by depletion of the naive T-cell pool. Molecular profiling of T-cell populations in donors vs recipients without aGVHD revealed increased pathway activity of >40 gene modules in recipients. These pathways were associated in particular with T-cell activation, adhesion, migration, and effector functions. Cellular profiles from recipients developing aGVHD displayed an enrichment of cells with a T memory stem cell–like phenotype compared with recipients without aGVHD. Comparison of gene profiles from these recipients revealed that transforming growth factor-β (TGF-β) signaling was most significantly downregulated, whereas the pathway activity of NF-κB–associated transcription factors and signaling pathways were increased, at aGVHD onset. This study suggests that the integration of cellular and molecular profiles provides new insights into the development of aGVHD in humans.

Introduction La place centrale de l’IL-17A dans la physiopathologie de la spondyloarthrite axiale (AxSpA) a ete confirmee par le succes des anti-IL-17A. Cependant, la nature des populations cellulaires produisant cette importante cytokine pro-inflammatoire reste mal definie. Nous avons donc caracterise les principales populations cellulaires productrices d’IL-17A dans la AxSpA, et plus particulierement une population de lymphocytes T non conventionnels, les MAIT (Mucosal Associated Invariant T cells). Patients et methodes La production d’IL-17A de 5 populations de cellules sanguines peripheriques triees, a savoir les MAIT, les lymphocytes Tγ δ, les T CD4+, les T CD8+ et les neutrophiles a ete evaluee avant et apres stimulation par l’association de PMA, d’un ionophore calcique A23187 et de la B1,3 glucane. L’expression genique et proteique d’IL-17A etait respectivement determinee par la technologie nCounter et la technologie ultra-sensible SimoA. Les MAIT provenant d’entheses axiales de temoins sains (n = 5) etaient caracterises par cytometrie en flux, qPCR et leur production d’IL-17 evaluee apres stimulation. Resultats Dans le sang, a l’echelle cellulaire, les MAIT produisaient la plus grande quantite d’IL-17A par rapport aux T CD4+ (p Conclusion Les MAIT circulants et les MAIT residents dans les entheses axiales contribuent a la production d’IL-17 et pourraient jouer un role important dans la physiopathologie de l’AxSpA.

A Publisher Correction to this article was published on 03 May 2018; International audience; The quantification and characterization of circulating immune cells provide key indicators of human health and disease. To identify the relative effects of environmental and genetic factors on variation in the parameters of innate and adaptive immune cells in homeostatic conditions, we combined standardized flow cytometry of blood leukocytes and genome-wide DNA genotyping of 1,000 healthy, unrelated people of Western European ancestry. We found that smoking, together with age, sex and latent infection with cytomegalovirus, were the main non-genetic factors that affected variation in parameters of human immune cells. Genome-wide association studies of 166 immunophenotypes identified 15 loci that showed enrichment for disease-associated variants. Finally, we demonstrated that the parameters of innate cells were more strongly controlled by genetic variation than were those of adaptive cells, which were driven by mainly environmental exposure. Our data establish a resource that will generate new hypotheses in immunology and highlight the role of innate immunity in susceptibility to common autoimmune diseases.

T helper 17 (Th17) cells have crucial functions in mucosal immunity and the pathogenesis of several chronic inflammatory diseases. The lineage-specific transcription factor, RORγt, encoded by the RORC gene modulates Th17 polarization and function, as well as thymocyte development. Here we define several regulatory elements at the human RORC locus in thymocytes and peripheral CD4

T helper 17 (Th17) cells have crucial functions in mucosal immunity and the pathogenesis of several chronic inflammatory diseases. The lineage-specific transcription factor, RORγt, encoded by the RORC gene modulates Th17 polarization and function, as well as thymocyte development. Here we define several regulatory elements at the human RORC locus in thymocytes and peripheral CD4+ T lymphocytes, with CRISPR/Cas9-guided deletion of these genomic segments supporting their role in RORγt expression. Mechanistically, T cell receptor stimulation induces cyclosporine A-sensitive histone modifications and P300/CBP acetylase recruitment at these elements in activated CD4+ T cells. Meanwhile, NFAT proteins bind to these regulatory elements and activate RORγt transcription in cooperation with NF-kB. Our data thus demonstrate that NFAT specifically regulate RORγt expression by binding to the RORC locus and promoting its permissive conformation., The master transcription factor RORγt, encoded by the RORC gene, controls the polarization of CD4+ T cells expressing interleukin-17 (Th17). Here the authors describe several regulatory elements at the RORC locus that are recognized by NFAT and NFkB to induce a permissive epigenetic configuration of the RORC gene for RORγt expression and Th17 differentiation.

Background IL-17A-inhibition has been very successful in AxSpA but the cell populations targeted by this new therapeutic remain unknown. This question is relevant because the recent failure of anti-IL-23 in AxSpA demonstrated that IL-23-independent IL-17-producing cells may be of particular relevance for SpA pathogenesis. Some data from literature suggests the involvement of MAIT, γδ T, and neutrophils as IL-17A producing cells in AxSpA (1–3). However, even though they may be responsible for IL-17A-mediated inflammation, it is still unclear which is the major IL-17A-producing cell population in this disease. Objectives To assess and compare gene expression profiles of neutrophils, MAIT, γδ, CD4+ and CD8+ T cells from AxSpA patients. Methods We recruited 5 healthy donors and 10 patients with a diagnosis of AxSpA according to the ASAS criteria. We compared the gene expression profiles of 5 sorted cell populations: 3 innate cell populations (neutrophils, MAIT and γδ T cells) and 2 adaptive cell populations (CD4+T and CD8+T) after cell stimulation by PMA + A23187 (calcium ionophore) + β1,3 glucan (extracted from Aspergillus fumigatus hyphae). Published data suggested that neutrophils stimulation by Aspergillus fumigatus induces IL-17A production by these cells(4). For each of these cell populations, cytokine production and the expression of a panel of 755 genes (Autoimmune discovery panel from Nanostring including 43 genes for which a polymorphism was associated with AS) were assessed. Patient and control groups were compared with a Mann-Whitney test and comparison of cell populations was performed by a multigroup comparison. Results There was no significant difference between patients and controls regarding gene expression profile of neutrophils, γδ T, CD4+T and CD8+T. We observed that 34 genes were differentially expressed between patients and controls in MAIT cells (p = 0.03, q = 0.1). In particular, T cell activation genes (TBX21, AHR, ZAP70) and cell interaction genes (ITAG6, CTNNB1, ICAM2, ITGB2, SELL) were decreased in patients. Among AxSpA patients, MAIT cells were those significantly showing the highest level of IL-17A expression. IL23R and RORC were also more expressed by MAIT compared to others cell populations. IL-17A expression was very low in neutrophils but we observed that 18 out of the 43 AS associated genes were mainly expressed by neutrophils (p Conclusion These preliminary data confirm that the innate immune cells could play an important role in AxSpA. MAIT cells are at the forefront of the expression of IL-17A before γδ T, CD4+T and CD8+T. Neutrophils do not appear to participate in the production of IL-17A, but the high expression of AS linked genes in these cells suggests their involvement in AxSpA. References [1] Appel H, et al. Analysis of IL-17(+) cells in facet joints of patients with spondyloarthritis suggests that the innate immune pathway might be of greater relevance than the Th17-mediated adaptive immune response. Arthritis Res Ther. 2011 [2] Kenna TJ, et al. Enrichment of circulating interleukin-17-secreting interleukin-23 receptor-positive γ/δ T cells in patients with active ankylosing spondylitis. Arthritis Rheum. 2012 [3] Gracey E, et al. IL-7 primes IL-17 in mucosal-associated invariant T (MAIT) cells, which contribute to the Th17-axis in ankylosing spondylitis. Ann Rheum Dis. 2016 [4] Taylor PR, et al. Activation of neutrophils by autocrine IL-17A-IL-17RC interactions during fungal infection is regulated by IL-6, IL-23, RORγt and dectin-2. Nat Immunol. 2014 Disclosure of Interests nicolas rosine: None declared, Surya Koturan: None declared, Hanane Yahia: None declared, Claire Leloup: None declared, Elisabetta Bianchi: None declared, Corinne Miceli Richard Grant/research support from: MSD, Pfizer, AbbVie, Biogen, UCB, Novartis, Consultant for: Abbvie, Novartis, BMS, Lars Rogge: None declared

Background: Anti-TNF therapy has revolutionized treatment of many chronic inflammatory diseases, including rheumatoid arthritis, Crohn”s disease and spondyloarthritis (SpA). However, clinical efficacy of TNF-inhibitors (TNFi) is limited by a high rate of non-responsiveness (30-40%) both in SpA and other diseases, which exposes a substantial fraction of patients to important side-effects without clinical benefit. Despite TNFi having been used extensively for many years, it is still not possible to determine which patients will respond to TNFi before treatment initiation. Objectives: In this study, we have tested the hypothesis that the functional analysis of immune responses of patients before and after anti-TNF therapy may not only improve our understanding of the molecular mechanisms of TNF-blocker activity, but also identify correlates of therapeutic responses in SpA patients. Methods: To facilitate the potential translation of our findings into a clinical setting, we have used standardized whole-blood stimulation assays (“TruCulture” assays; Duffy et al., 2014), and have minimized sources of pre-analytical variability, implementing a highly sensitive and robust pipeline to assess immune functions in patients. Results: We show that anti-TNF therapy induces profound changes in patients’ immune responses, affecting predominantly several innate immune pathways. Of note, all these effects could be measured in stimulated, but not in resting immune cells, and were already detectable early, after a single TNFi injection. Modular transcriptional repertoire analysis revealed that TNFi affect immune responses via multiple mechanisms, such as directing monocyte/macrophage polarization and the inhibition of a TNF- and IL-1-dependent feed-forward loop of NF-kB activation. On the other hand, the action of anti-TNF treatment was surprisingly selective, in that it did not affect the IL-6/Th17 or Th1 arm of the patients’ immune response. These findings have important implications for therapy, given the recent approval of anti-IL-17 antibodies as an alternative agent for the treatment of several chronic inflammatory diseases. To investigate the concept that the immune status of a patient will define their response to TNFi treatment, we have used machine-learning algorithms to identify, in whole-blood stimulation assays, immunological transcripts that correlate with clinical efficacy of TNFi. We found that high expression, before treatment initiation, of molecules associated with leukocyte invasion/migration and inflammatory processes predisposes to favorable outcome of anti-TNF therapy, while high-level expression of cytotoxic molecules was associated with poor therapeutic responses to TNF-blockers. Conclusion: These findings suggest that SpA patients whose immune response is characterized by strong, NF-kB-mediated inflammation are more likely to benefit from TNFi treatment than patients with an active T/NK-cell component. This proof-of-concept study establishes a new framework to explore patient stratification in chronic inflammatory diseases according to the patients’ immune phenotype. Reference: [1] Duffy, D., et al. Functional Analysis via Standardized Whole-Blood Stimulation Systems Defines the Boundaries of a Healthy Immune Response to Complex Stimuli. Immunity 40, 436-450 (2014). Disclosure of Interests: None declared

International audience; Objective: Secukinumab (anti-interleukin-17A [anti-IL-17A]) is an effective therapy for ankylosing spondylitis and psoriatic arthritis, the prototypical forms of spondyloarthritis (SpA). We undertook this study to determine whether secukinumab modulates the immunopathology of target lesions without blunting systemic immune responses, using peripheral SpA as a model.Methods: Twenty patients with active peripheral SpA were included in a 12-week open-label trial with secukinumab (300 mg once weekly from baseline to week 4 and then every 4 weeks thereafter). Outcomes included clinical response, cytokine production by peripheral blood cells using TruCulture technology, and histologic and real-time quantitative polymerase chain reaction analysis of synovial biopsy samples before and after treatment.Results: All patients completed the 12-week study without severe adverse events (AEs) or severe treatment-related AEs. The efficacy end point, the number of patients meeting the American College of Rheumatology 20% improvement criteria (achieving an ACR20 response) at 12 weeks, was achieved by 13 of the 20 patients, of whom 8 achieved an ACR50 response and 5 achieved an ACR70 response, with rapid and significant improvements in all clinical disease activity measures. Clinical improvement in joint counts was associated with a histologic decrease in synovial sublining macrophages (P = 0.028) and neutrophils (P = 0.004), both of which are sensitive synovial biomarkers of inflammatory response in peripheral SpA, as well as with decreased synovial expression of IL-17A messenger RNA (mRNA) (P = 0.010) but not of tumor necrosis factor mRNA. Systemically, secukinumab treatment decreased the C-reactive protein level and the erythrocyte sedimentation rate (both P < 0.01), and also decreased matrix metalloproteinase 3 production in the TruCulture system (P < 0.05). However, with the exception of IL-17A itself, the capacity of peripheral blood cells to produce a broad panel of cytokines and chemokines upon stimulation with microbial antigens was not affected.Conclusion: This mechanism-of-action study in peripheral SpA indicates that clinical improvement with secukinumab treatment is paralleled by immunomodulation of inflamed target tissues without compromising systemic immune responses.

International audience; Multi-parametric flow cytometry is a key technology for characterization of immune cell phenotypes. However, robust high-dimensional post-analytic strategies for automated data analysis in large numbers of donors are still lacking. Here, we report a computational pipeline, called FlowGM, which minimizes operator input, is insensitive to compensation settings, and can be adapted to different analytic panels. A Gaussian Mixture Model (GMM)-based approach was utilized for initial clustering, with the number of clusters determined using Bayesian Information Criterion. Meta-clustering in a reference donor permitted automated identification of 24 cell types across four panels. Cluster labels were integrated into FCS files, thus permitting comparisons to manual gating. Cell numbers and coefficient of variation (CV) were similar between FlowGM and conventional gating for lymphocyte populations, but notably FlowGM provided improved discrimination of "hard-to-gate" monocyte and dendritic cell (DC) subsets. FlowGM thus provides rapid high-dimensional analysis of cell phenotypes and is amenable to cohort studies.

Lim et al. show that IL-12 can drive IFN-γ production by human group 2 innate lymphoid cells., Group 2 innate lymphoid cells (ILC2) include IL-5– and IL-13–producing CRTh2+CD127+ cells that are implicated in early protective immunity at mucosal surfaces. Whereas functional plasticity has been demonstrated for both human and mouse ILC3 subsets that can reversibly give rise to IFN-γ–producing ILC1, plasticity of human or mouse ILC2 has not been shown. Here, we analyze the phenotypic and functional heterogeneity of human peripheral blood ILC2. Although subsets of human CRTh2+ ILC2 differentially express CD117 (c-kit receptor), some ILC2 surface phenotypes are unstable and can be modulated in vitro. Surprisingly, human IL-13+ ILC2 can acquire the capacity to produce IFN-γ, thereby generating plastic ILC2. ILC2 cultures demonstrated that IFN-γ+ ILC2 clones could be derived and were stably associated with increased T-BET expression. The inductive mechanism for ILC2 plasticity was mapped to the IL-12–IL-12R signaling pathway and was confirmed through analysis of patients with Mendelian susceptibility to mycobacterial disease due to IL-12Rβ1 deficiencies that failed to generate plastic ILC2. We also detected IL-13+IFN-γ+ ILC2 ex vivo in intestinal samples from Crohn’s disease patients. These results demonstrate cytokine production plasticity for human ILC2 and further suggest that environmental cues can dictate ILC phenotype and function for these tissue-resident innate effector cells.

Background:It is currently not known if TNF or IL-17A inhibitors have an impact on immune cell frequencies in axial Spondyloarthritis (AxSpA). This question is important to understand the impact of biologics on the immune system. Data from clinical trials didn’t show significant modification on immune cells and especially on lymphocytes. But regarding the risk of infections linked to these treatments lymphocyte cell subsets are certainly disturbed. Moreover, biologics could affect subsets of cells with an unusual phenotype.Objectives:To identify the phenotype of cell subsets affected by biologics.Methods:We used an “unsupervised approach” to analyze CD4+ T cells and CD8+T cells subsets. Contrary to a “supervised approach”, this strategy takes advantages of the fluorescence emitted by all of the surface markers used to characterize the cells at the same time. The objective was on the one hand to overcome statistical problems related to the number of patients and the repetition of the tests and on the other hand to increase the sensitivity of the analysis by identifying and analyzing new cell populations. The first step was to cluster the cells based on a selection of 12 T cells markers characteristic of the classical cell subsets and the stage of maturation to obtain cell clusters with a phenotype based on the combination of these 12 markers. Then, we were able to describe “a posteriori” the change of frequency of the clusters identified. The second step was to create a visualization of the cells affected to confirm their existence in a classical flow cytometry gate. With this pipeline, we analyzed CD4 and CD8 T cells isolated from a group of AxSpA patients (n=7) before and after 3 months of TNF therapy and a group of patients (n=6) before and after 4 months of IL-17A therapy.Results:We observed that after biologics CD4 and CD8 T cells frequencies did not change but there was a redistribution of the different clusters analyzed. Specifically, we identified for CD4+T cells after anti TNF treatment an increase of 2 clusters (CD4+CD27+CD45RA+Va7.2intCD161int and CD4+CD27-CD45RA-CCR6+CD161int) and a decrease of 3 clusters (CD4+CD27+CD45RA+CRTH2intCD161int, CD4+ CD27+CD45RA+CXCR3+, CD4+CD27+CD45RA+gdint CD161int) and for CD8+T cells a decrease of 1 cluster after treatment (CD8+ CD27+CD45RA+CD161+CXCR3+) and an increase of 1 cluster (CD8+ CD27+CD45RA+). The clusters affected by anti-IL-17A therapy were different. For CD4+T cells, we identified a decrease of 2 clusters (CD4+CD27+CD45RA+CXCR5+ CD161+ and CD4+CD27+CD45RA- CXCR3+CCR6+CD161+) and an increase of 2 clusters (CD4+CD27+CD45RA+gdintCD161+, CD4+CD27+ CRTH2intCCR6+) and for CD8+T cells a decrease of 1 cluster (CD8+CD27+CD45RA+ CXCR3+CRTH2intCD161int) and an increase of 1 cluster (CD8+CD27+CD45RA+CXCR3intCD161-).Conclusion:We identified 5 different clusters in CD4+T cells affected by anti TNF and 4 by anti-IL-17A. We identified 2 clusters in CD8+T cells affected by anti TNF and 2 by anti-IL-17A. The phenotypes of these clusters were unexpected and raised new questions about the effect of biologics in AxSpA. We were also able to create a visualization of these cells affected by biologics in a “classic gating view” which will help us to perform scRNAseq. With this unique approach, we show an impact of biologics on the frequency of very specific subset of CD4+ and CD8+ T cells in AxSpAFigure 1.Disclosure of Interests:None declared

Background The response to TNF-blockers in axial spondyloarthritis (AxSpA) is at least partially influenced by HLA-B27 through a still poorly understood mechanism.Objectives Given that HLA-B27 regulates the gut microbiota composition in rats1,2, we seek to evaluate the predictive value of the gut microbiota composition in AxSpA patients on their responsiveness to TNF-blockers.Methods A total of 58 patients was monocentrically recruited between October 2014 and May 2015. At baseline, these patients had an active disease despite NSAIDs intake and were eligible for treatment with a TNF-blocker, while having no history of inflammatory bowel disease (IBD). The mean BASDAI (̑extpm}SD) was 45.6{̑extpm}21.4; ASDAS 2.8{̑extpm}0.9 and CRP 9.7{̑extpm11.4 mg/L. Among these patients, 56 fulfilled the ASAS classification criteria (imaging arm) with sacro-iliitis on X-rays (n=37) or objective signs of inflammation on MRI (n=48). Two patients fulfilled the clinical arm. These patients were not subjected to antibiotics within 3 months before stool sample collection. Bacterial 16S rRNA gene sequencing of the V3-V4 region was performed on stools samples before and 3 months after TNF-blocker treatment. Beta diversity metrics were calculated on the abundance of operational taxonomic units (OTU) after their taxonomic assignment on quality-filtered sequences.Results Principal component analysis (PCA) ordination of Bray-Curtis similarity revealed that current smoking (compared with never or ever smokers) and HLA-B27 genotype were significantly associated with the overall composition of the microbiota at baseline. Meanwhile, the abundance of eleven bacterial OTUs was influenced by HLA-B27 genotype at baseline but not after 3 month of treatment. In contrast, we identified a bacterial signature that was linked to the smoking behaviour independently of TNF-blocker treatment, whereas the BASDAI and ASDAS indices were significantly associated to the general composition of the gut microbiota after the 3 month treatment. In line with a previous report3, the abundance of Ruminococcus gnavus was not associated with disease activity in the absence of IBD. Interestingly, the abundance of 5 and 7 bacterial OTUs at baseline was associated with the response to TNF-blockers assessed by BASDAI and ASDAS, respectively. Among these candidates, the abundance of one bacterial OTUs belonging to the Clostridiales order was associated with a better response to the treatment and with the HLA-B27 genotype.Conclusions Anti-TNF treatment was found to modulate the HLA-B27-induced variations of the intestinal microbiota of AxSpA patients. Moreover, the abundance of a subset of OTUs at baseline was found to predict the responsiveness to TNF-blockers. Further functional studies will be conducted to assess how these taxa can be use as predictors of the treatment outcome.References [1] Gill, et al. Effects of HLA-B27 on Gut Microbiota in Experimental Spondyloarthritis Implicate an Ecological Model of Dysbiosis. Arthritis Rheumatol2017.[2] Lin, et al. HLA-B27 and human β2-microglobulin affect the gut microbiota of transgenic rats. PLoS One2014.[3] Breban, et al. Faecal microbiota study reveals specific dysbiosis in spondyloarthritis.Annal Rheum Dis2017.Disclosure of Interest None declared

Introduction Secukinumab (anti-IL-17A) is an effective therapy for ankylosing spondylitis(AS) and psoriatic arthritis (PsA), the prototypical forms of spondyloarthritis (SpA). Objectives This study assessed if secukinumab modulates the immunopathology of target lesions without blunting systemic immune responses, using peripheral SpA (pSpA) as model. Methods 20 active peripheral SpA patients were included in a 12 week open-label trial with secukinumab (300 mg weekly for 4 weeks followed by every 4 weeks). Outcomes included clinical response, cytokine production by peripheral blood cells using TruCulture technology, and histological and qPCR analysis of synovial biopsies before and after treatment. Results All patients completed the 12 week study, without SAEs or severe treatment-related AEs. The primary efficacy endpoint, EULAR DAS 28 response at wk 12, was achieved by 18/20 patients (10 good and 8 moderate responders), with rapid and significant improvements in all clinical disease activity measurements. Clinical improvement in joint counts was associated with histological decrease in synovial sublining macrophages (p=0.028) and neutrophils (p=0.004), sensitive synovial biomarkers of response in pSpA, as well as with decreased synovial expression of IL-17A (p=0.010) but not TNF. Systemically, secukinumab treatment decreased CRP (p Conclusions This mechanism-of-action study in pSpA indicates that clinical improvement upon secukinumab treatment is paralleled by immunomodulation of the inflamed target tissues without compromising systemic immune responses. Disclosure of interest M. van de Sande Grant/research support from: Novartis, Eli Lilly, Boehringer Ingelheim, Consultant for: Abbvie, Novartis, Speakers bureau: x, L. van Mens: None declared, S. Menegatti: None declared, I. Blijdorp: None declared, J. de Jong: None declared, I. Fluri: None declared, T. Latuhihin: None declared, A. van Kuijk Grant/research support from: x, Consultant for: Novartis, Celgene, L. Rogge: None declared, N. Yeremenko: None declared, D. Baeten Grant/research support from: Pfizer, MSD, Abbvie, UCB, Novartis, Janssen, Boehringer Ingelheim, Consultant for: Pfizer, MSD, Abbvie, UCB, Novartis, Janssen, Boehringer Ingelheim, Eli Lily, Roche, BMS, Glenmark, Employee of: UCB

Innate lymphoid cells (ILCs) represent innate versions of T helper and cytotoxic T cells that differentiate from committed ILC precursors (ILCPs). How ILCPs give rise to mature tissue-resident ILCs remains unclear. Here, we identify circulating and tissue ILCPs in humans that fail to express the transcription factors and cytokine outputs of mature ILCs but have these signature loci in an epigenetically poised configuration. Human ILCPs robustly generate all ILC subsets in vitro and in vivo. While human ILCPs express low levels of retinoic acid receptor (RAR)-related orphan receptor C (RORC) transcripts, these cells are found in RORC-deficient patients and retain potential for EOMES+ natural killer (NK) cells, interferon gamma-positive (IFN-γ+) ILC1s, interleukin (IL)-13+ ILC2s, and for IL-22+, but not for IL-17A+ ILC3s. Our results support a model of tissue ILC differentiation ("ILC-poiesis"), whereby diverse ILC subsets are generated in situ from systemically distributed ILCPs in response to local environmental signals. A.I.L. is a scholar in Pasteur-Paris University (PPU) International PhD program and supported by FP7 under grant agreement 317057 (HOMIN) and the Fondation ARC. R.S. was supported by EMBO (ALTF 1201-2014) and Marie Curie (H2020-MSCA-IF-2014). Other support included grants from the Institut Pasteur, Inserm, Laboratoire d'Excellence REVIVE (ANR-10-LBX-73), and FP7 under grant agreement HEALTH-F3-2012-305578 (PathCO) and 317057 (HOMIN). J.P.D. is a founder of AXENIS and a member of its advisory board.

Objective Recent genome-wide association studies have revealed numerous genetic associations between specific single-nucleotide polymorphisms (SNPs) and immune-mediated inflammatory diseases. The current challenge is to identify associations of the genetic variants with effector mechanisms implicated in pathogenesis. This study was undertaken to investigate the link between genetic variation at loci associated with spondyloarthritis (SpA) and the effector function of CD4+ T lymphocyte subsets involved in chronic inflammatory disease. Methods Expression of Th17 and Th1 cytokines and transcription factors was measured in CD4+ T cells isolated from patients with SpA. Correlation analyses were performed to assess potential associations of these expression levels with the patient's genotype at loci genetically linked to SpA. Results The effector functions of Th17 and Th1 cells in patients with SpA were found to be under combinatorial control by multiple SNPs at genes associated with the interleukin-23 (IL-23)/Th17 pathway. Patients with SpA carrying risk-associated alleles of genes in the IL-23/Th17 pathway expressed the highest levels of genes involved in the differentiation and function of Th17 and Th1 cells, whereas the presence of protective alleles was associated with low-level expression of these genes. In contrast, variation at loci that were genetically linked to SpA, but not associated with the IL-23 pathway, did not affect the expression of Th17- and Th1-specific genes, suggesting that these SNPs may contribute to the pathogenesis of SpA through distinct cellular mechanisms. Conclusion These results show that genetic variations at genes associated with the IL-23 signaling pathway may influence the effector functions of Th17 and Th1 cells in patients with SpA. These findings provide a framework to delineate the mechanisms by which genetic variants contribute to pathology.

Introduction Many efforts have been attempted to improve our understanding of GVHD pathophysiology but few focused on human GVHD. Most studies have focused on the central role played by immune cells. However, recent researches have highlighted the influence of tissue microenvironment or of host microbiota in regulating allo-immune response. In human, circulating metabolites are produced by both tissues and gut microbiota. Among them, many biochemical compounds regulate immune cells function and could be the bridge between microbiota, tissues and the immune system. The aim of this study was to characterize metabolomics alterations associated with allo-HSCT and to determine biochemical pathway specifically involved during acute GVHD. Methods We collected donors and recipients' frozen plasma from a monocentric (n=43) and a multicentric cohort (n=56) of patients who underwent transplantation from an HLA-identical sibling in France. Donors' samples were collected before stem cell collection. Recipient's samples were obtained at acute GVHD onset or at day + 90 in patients without GVHD. After protein removal, plasma extracts were analyzed by two separate reverse phase (RP)/UPLC-MS/MS methods with positive ion mode electrospray ionization (ESI), one for analysis by RP/UPLC-MS/MS with negative ion mode ESI and one for analysis by HILIC/UPLC-MS/MS with negative ion mode ESI. Compounds were identified by comparison to library entries of purified standards or recurrent unknown entities. Peaks were quantified using area-under-the-curve. All statistical analyses were performed with R 3.5.0 and the MetaboAnalystR package. Results More than 800 circulating metabolites were identified in the 2 cohorts, belonging to lipid (43.6%), amino acid (22.2%), xenobiotics (19.1%), nucleotide (4.2%), carbohydrate (3.6%), cofactors and vitamins (3.4%), peptide (2.8%) and energy (1.1%) pathways. After allo-HSCT, the comparison of metabolomics profiles of recipients without GVHD with those of healthy related donors revealed major changes in 222 metabolites (with a significance threshold at p 2 and p After comparison of recipients with or without GvHD, we were able to identify specific metabolomics changes associated with the onset of GvHD, irrespective of age, sex and feeding of patients. Acute GvHD was associated with a major decrease of plasmalogen and lysoplasmalogen (FC2, p Discussion Our results demonstrate that allo-HSCT is associated with major metabolomics changes in recipients, which might me due to drug intakes, metabolic stress or microbiota alteration. GvHD was characterized by specific changes in complex lipid metabolism and amino acid metabolites that are involved in immune cells regulation and inflammation. This study highlights the potential role of circulating metabolites in GvHD pathophysiology that could be targeted for prophylaxis or treatment. Figure Figure. Disclosures Peffault De Latour: Pfizer Inc.: Consultancy, Honoraria, Research Funding; Alexion Pharmaceuticals, Inc.: Consultancy, Honoraria, Research Funding; Novartis: Consultancy, Honoraria, Research Funding; Amgen Inc.: Research Funding.

HIV-1 infection is characterized by a progressive decline in CD4 + T cells leading to a state of profound immunodeficiency. IL-2 therapy has been shown to improve CD4 + counts beyond that observed with antiretroviral therapy. Recent phase III trials revealed that despite a sustained increase in CD4 + counts, IL-2-treated patients did not experience a better clinical outcome [Abrams D, et al. (2009) N Engl J Med 361(16):1548–1559]. To explain these disappointing results, we have studied phenotypic, functional, and molecular characteristics of CD4 + T cell populations in IL-2-treated patients. We found that the principal effect of long-term IL-2 therapy was the expansion of two distinct CD4 + CD25 + T cell populations (CD4 + CD25 lo CD127 lo FOXP3 + and CD4 + CD25 hi CD127 lo FOXP3 hi ) that shared phenotypic markers of Treg but could be distinguished by the levels of CD25 and FOXP3 expression. IL-2-expanded CD4 + CD25 + T cells suppressed proliferation of effector cells in vitro and had gene expression profiles similar to those of natural regulatory CD4 + CD25 hi FOXP3 + T cells (Treg) from healthy donors, an immunosuppressive T cell subset critically important for the maintenance of self-tolerance. We propose that the sustained increase of the peripheral Treg pool in IL-2-treated HIV patients may account for the unexpected clinical observation that patients with the greatest expansion of CD4 + T cells had a higher relative risk of clinical progression to AIDS.

Jakmip1 belongs to a family of three related genes encoding proteins rich in coiled-coils. Jakmip1 is expressed predominantly in neuronal and lymphoid cells and colocalizes with microtubules. We have studied the expression of Jakmip1 mRNA and protein in distinct subsets of human primary lymphocytes. Jakmip1 is absent in naive CD8+ and CD4+ T lymphocytes from peripheral blood but is highly expressed in Ag-experienced T cells. In cord blood T lymphocytes, induction of Jakmip1 occurs upon TCR/CD28 stimulation and parallels induction of effector proteins, such as granzyme B and perforin. Further analysis of CD8+ and CD4+ T cell subsets showed a higher expression of Jakmip1 in the effector CCR7− and CD27− T cell subpopulations. In a gene expression follow-up of the development of CMV-specific CD8+ response, Jakmip1 emerged as one of the most highly up-regulated genes from primary infection to latent stage. To investigate the relationship between Jakmip1 and effector function, we monitored cytotoxicity of primary CD8+ T cells silenced for Jakmip1 or transduced with the full-length protein or the N-terminal region. Our findings point to Jakmip1 being a novel effector memory gene restraining T cell-mediated cytotoxicity.

The COP9 signalosome (CSN) is a conserved, multisubunit complex first identified as a developmental regulator in plants. Gene inactivation of single CSN subunits results in early embryonic lethality in mice, indicating that the CSN is essential for mammalian development. The pleiotropic function of the CSN may be related to its ability to remove the ubiquitin-like peptide Nedd8 from cullin-RING ubiquitin ligases, such as the SCF complex, and therefore regulate their activity. However, the mechanism of CSN regulatory action on cullins has been debated, since, paradoxically, the CSN has an inhibitory role in vitro, while genetic evidence supports a positive regulatory role in vivo. We have targeted expression of CSN subunits 4 and 5 in human cells by lentivirus-mediated small hairpin RNA delivery. Down-regulation of either subunit resulted in disruption of the CSN complex and in Cullin1 hyperneddylation. Functional consequences of CSN down-regulation were decreased protein levels of Skp2, the substrate recognition subunit of SCF(Skp2), and stabilization of a Skp2 target, the cyclin-dependent kinase inhibitor p27(Kip1). CSN down-regulation caused an impairment in cell proliferation, which could be partially reversed by suppression of p27(Kip1). Moreover, restoring Skp2 levels in CSN-deficient cells recovered cell cycle progression, indicating that loss of Skp2 in these cells plays an important role in their proliferation defect. Our data indicate that the CSN is necessary to ensure the assembly of a functional SCF(Skp2) complex and therefore contributes to cell cycle regulation of human cells.

Integrin adhesion receptors can act as signaling receptors that transmit information from the extracellular environment to the interior of the cell, affecting many fundamental cellular processes, such as cell motility, proliferation, differentiation, and survival. Integrin signaling depends on the formation of organized sub-membrane complexes that comprise cytoskeletal, adapter, and signaling molecules. The identification of molecules that interact with the cytoplasmic domain of integrins has been the focus of research aimed to elucidating the mechanistic basis of integrin signal transduction. We have identified RanBPM as a novel interactor of the beta(2) integrin LFA-1 in a yeast-two-hybrid screen. In the same assay, RanBPM also interacted with the beta(1) integrin cytoplasmic domain. We demonstrate that RanBPM is a peripheral membrane protein and that integrins and RanBPM interact in vitro and in vivo and co-localize at the cell membrane. We find that RanBPM is phosphorylated on serine residues; phosphorylation of RanBPM is increased by stress stimuli and decreased by treatment with the p38 kinase inhibitor SB203580. Transfection of RanBPM synergizes with LFA-1-mediated adhesion in the transcriptional activation of an AP-1-dependent promoter, indicating that the two proteins interact functionally as well. We suggest that RanBPM may constitute a molecular scaffold that contributes to coupling LFA-1 and other integrins with intracellular signaling pathways.

RING finger proteins have been implicated in many fundamental cellular processes, including the control of gene expression. A key regulator of light-dependent development in Arabidopsis thaliana is the constitutive photomorphogenesis protein 1 (atCOP1), a RING finger protein that plays an essential role in translating light/dark signals into specific changes in gene transcription. atCOP1 binds the basic leucine zipper factor HY5 and suppresses its transcriptional activity through a yet undefined mechanism that results in HY5 degradation in response to darkness. Furthermore, the pleiotropic phenotype of atCOP1 mutants indicates that atCOP1 may be a central regulator of several transcriptional pathways. Here we report the cloning and characterization of the human orthologue of atCOP1. Human COP1 (huCOP1) distributes both to the cytoplasm and the nucleus of cells and shows a striking degree of sequence conservation with atCOP1, suggesting the possibility of a functional conservation as well. In co-immunoprecipitation assays huCOP1 specifically binds basic leucine zipper factors of the Jun family. As a functional consequence of this interaction, expression of huCOP1 in mammalian cells down-regulates c-Jun-dependent transcription and the expression of the AP-1 target genes, urokinase and matrix metalloproteinase 1. The RING domain of huCOP1 displays ubiquitin ligase activity in an autoubiquitination assay in vitro; however, suppression of AP-1-dependent transcription by huCOP1 occurs in the absence of changes in c-Jun protein levels, suggesting that this inhibitory effect is independent of c-Jun degradation. Our findings indicate that huCOP1 is a novel regulator of AP-1-dependent transcription sharing the important properties of Arabidopsis COP1 in the control of gene expression.

Genomic-scale gene expression profiling in combination with the availability of a draft sequence of the human genome is beginning to revolutionize the way immunology is done. The possibility of measuring levels of gene expression for tens of thousands of genes simultaneously and in a quantitative fashion aids in the definition of a comprehensive molecular phenotype of cells and cellular processes of the immune system in health and disease. T helper lymphocytes are an essential element of appropriate immune responses to pathogens. To achieve effective immunity, T helper cells differentiate into at least two specialized subsets that direct type 1 and type 2 immune responses. Here, I discuss recent progress that has been made in our understanding of the genetic program that controls the development and functional properties of helper T cell subsets.

International audience; Standardization of immunophenotyping procedures has become a high priority. We have developed a suite of whole-blood, syringe-based assay systems that can be used to reproducibly assess induced innate or adaptive immune responses. By eliminating preanalytical errors associated with immune monitoring, we have defined the protein signatures induced by (1) medically relevant bacteria, fungi, and viruses; (2) agonists specific for defined host sensors; (3) clinically employed cytokines; and (4) activators of T cell immunity. Our results provide an initial assessment of healthy donor reference values for induced cytokines and chemokines and we report the failure to release interleukin-1α as a common immunological phenotype. The observed naturally occurring variation of the immune response may help to explain differential susceptibility to disease or response to therapeutic intervention. The implementation of a general solution for assessment of functional immune responses will help support harmonization of clinical studies and data sharing.

Background The aim of the study was to define T lymphocyte reactivity to selected peptides of an islet antigen IA-2, associated with Type 1 diabetes. Methods We used 10 peptides selected from the IA-2 molecule due to their predicted ability to bind to HLA-DRB1*0401, a Type 1 diabetes-associated allele. We tested 21 identical twin pairs discordant for the disease and 15 control subjects and then followed them prospectively; seven non-diabetic twins developed diabetes. Results Twins of identical pairs tended to respond to different peptides suggesting that the T cell response is, to a degree, shaped by non-genetically determined factors (p

CD4+ T helper 1 (Th1) cells and Th2 cells are distinguished based on the pattern of cytokines they are able to produce. Selectin ligands and chemokine receptors are differentially expressed in Th1 and Th2 cells, providing a basis for tissue-specific recruitment of helper T-cell subsets. However, the modes and mechanisms regulating tissue-specific localization of Th1 and Th2 cells are still largely unknown. Here, we show the preferential expression on Th1 cells of the integrin 6/β1, which is distinctly regulated by the Th1-inducing cytokines interleukin-12 (IL-12) and interferon-alfa (IFN-). The pattern of integrin 6/β1 regulation closely mirrors that of the chemokine receptor CCR1. Analysis of signal transducer and activator of transcription 4 (Stat4) activation by IL-12 and IFN- shows distinct signaling kinetics by these cytokines, correlating with the pattern of CCR1 and integrin 6/β1 expression. Unlike IFN-, the ability of IL-12 to generate prolonged intracellular signals appears to be critical for inducing integrin 6/β1 upregulation in Th1 cells. The expression and upregulation of CCR1 and 6/β1 integrin promotes the migration of Th1 cells. These findings suggest that the exquisite regulation of integrin 6/β1 and CCR1 may play an important role in tissue-specific localization of Th1 cells.

During the past year significant advances have been made in our understanding of the factors contributing to the differentiation of CD4+ T helper cell subsets. These have been driven, in part, by the realization that cytokines from the innate immune response, such as interleukin-12 (IL-12) and interferons (IFNs), play a critical role in T cell subset differentiation. This review covers some of the most recent data concerning the divergent role that IFNs have in the differentiation of human versus mouse T helper cell subsets. In this review we discuss the molecular basis for the specie-specific effect of type I IFN on the selective induction of Th1 type immune responses. Furthermore, since IFN-beta is used in the treatment of multiple sclerosis (MS) we discuss the potential effects of such treatment and the value of the Th1/Th2 paradigm in MS.

IRF-1-deficient mice show a striking defect in the development of T helper 1 (Th1) cells. In the present report, we investigate the expression of IRF-1 during differentiation of human T helper cells. No significant differences of IRF-1 mRNA expression were found in established Th1 and Th2 cells; however, interleukin 12 (IL-12) induced a strong up-regulation of IRF-1 transcripts in Th1 but not in Th2 cells. We demonstrate that IL-12-induced up-regulation of IRF-1 is mediated by signal transducer and activator of transcription-4, which binds to the interferon (IFN)-gamma-activated sequence present in the promoter of the IRF-1 gene. Strong IL-12-dependent activation of a reporter gene construct containing the IRF-1 IFN-gamma-activated sequence element provides further evidence for the key role of signal transducer and activator of transcription-4 in the IL-12-induced up-regulation of IRF-1 transcripts in T cells. IRF-1 expression was strongly induced after stimulation of naive CD4(+) T cells via the T cell receptor, irrespective of the cytokines present at priming, indicating that this transcription factor does not play a major role in initiating a Th1-specific transcriptional cascade in differentiating helper T cells. However, our finding that IRF-1 is a target gene of IL-12 suggests that some of the IL-12-induced effector functions of Th1 cells may be mediated by IRF-1.

Continuous administration of soluble protein antigen to BALB/c mice inhibits the development of Th1 and induces selective differentiation of Th2 cells. Here we show that interleukin (IL)-12, administered together with soluble protein through a mini-osmotic pump implanted subcutaneously, not only prevents the inhibition of Th1 cell development, but stimulates higher interferon (IFN)-gamma production than in mice receiving IL-12 alone. In parallel to co-stimulation of Th1 cell development, co-administration of IL-12 blocks the Th2 response induced by soluble protein. IL-12 administered in adjuvant with antigen or intraperitoneally 2 days after the immunization does not break the inhibition of Th1 but can still decrease the Th2 response induced by pretreatment with soluble protein antigen. In contrast to IL-12, co-administration of IL-2 or IFN-gamma does not affect the diversion to Th2 induced by soluble antigen. Thus IL-12, but not IL-2 nor IFN-gamma, converts in vivo the inhibitory signal for Th1 cell development delivered by soluble antigen into an immunogenic one, while blocking a positive signal for Th2 cell differentiation. A molecular basis for the co-stimulation of Th1 priming and the prevention of Th2 differentiation by IL-12 in vivo is provided by the observation that transcripts encoding the IL-12 receptor beta2 chain, which is required for IL-12 signaling and Th1 cell development, are selectively inhibited by soluble antigen but are enhanced by IL-12 co-administration.

International audience; Understanding the heterogeneity of human CD4(+)FOXP3(+) regulatory T cells (Tregs) and their potential for lineage reprogramming is of critical importance for moving Treg therapy into the clinics. Using multiparameter single-cell analysis techniques, we explored the heterogeneity and functional diversity of human Tregs in healthy donors and in patients after allogeneic hematopoietic stem cell transplantation (alloHSCT). Human Tregs displayed a level of complexity similar to conventional CD4(+) effector T cells with respect to the expression of transcription factors, homing receptors and inflammatory cytokines. Single-cell profiling of the rare Treg producing interleukin-17A or interferon-gamma showed an overlap of gene expression signatures of Th17 or Th1 cells and of Tregs. To assess whether Treg homeostasis is affected by an inflammatory and lymphopenic environment, we characterized the Treg compartment in patients early after alloHSCT. This analysis suggested a marked depletion of Treg with a naive phenotype in patients developing acute graft-versus-host disease, compared with tolerant patients. However, single-cell profiling showed that CD4(+) FOXP3(+) T cells maintain the Treg gene expression signature and Treg-suppressive activity was preserved. Our study establishes that heterogeneity at the single-cell level, rather than lineage reprogramming of CD4(+)FOXP3(+) T cells, explains the remarkable complexity and functional diversity of human Tregs.