Adrien Six

Regulatory T-Cell Responses to Low-Dose Interleukin-2 in HCV-Induced Vasculitis

BACKGROUND Patients with vasculitis induced by the hepatitis C virus (HCV) have reduced levels of regulatory T cells (Tregs). Resolution of HCV infection correlates with cure of vasculitis and the recovery of Treg levels. We reasoned that interleukin-2, a cytokine that promotes Treg survival and function, could be beneficial for patients with vasculitis that is resistant to HCV therapy. METHODS We investigated the safety and immunologic effects of the administration of low-dose interleukin-2 in a prospective open-label, phase 1-phase 2a study. Ten patients with HCV-induced vasculitis that was refractory to conventional antiviral therapy, rituximab therapy, or both and who were not receiving glucocorticoid or immunosuppressant therapy, received one course of interleukin-2 (1.5 million IU per day) for 5 days, followed by three 5-day courses of 3 million IU per day at weeks 3, 6, and 9. Both the safety of the treatment and its effectiveness were evaluated, the latter by monitoring the Treg response and the clinical signs of HCV vasculitis. RESULTS No adverse events reached a level higher than grade 1. The treatment did not induce effector T-cell activation, vasculitis flare, or increased HCV viremia. We observed a reduction in cryoglobulinemia in 9 of 10 patients and improvement of vasculitis in 8 of 10. Administration of low-dose interleukin-2 was followed by an increase in the percentage of CD4+, CD25(high), forkhead box P3 (FOXP3+) Tregs [E(max) (maximum value)÷baseline value×100=420%] with potent suppressive activity in all subjects and by a concomitantly decreased proportion of marginal-zone B cells. Transcriptome studies of peripheral-blood mononuclear cells revealed that interleukin-2 induced a global attenuation of the signatures for inflammation and oxidative stress mediators. CONCLUSIONS The trial showed that low-dose interleukin-2 was not associated with adverse effects and led to Treg recovery and concomitant clinical improvement in patients with HCV-induced vasculitis, an autoimmune condition. (Funded by the French Agency for Research on AIDS and Viral Hepatitis [ANRS] and others; ClinicalTrials.gov number, NCT00574652.).

Restoration of regulatory and effector T cell balance and B cell homeostasis in systemic lupus erythematosus patients through vitamin D supplementation.

IntroductionSystemic lupus erythematosus (SLE) is a T and B cell-dependent autoimmune disease characterized by the appearance of autoantibodies, a global regulatory T cells (Tregs) depletion and an increase in Th17 cells. Recent studies have shown the multifaceted immunomodulatory effects of vitamin D, notably the expansion of Tregs and the decrease of Th1 and Th17 cells. A significant correlation between higher disease activity and lower serum 25-hydroxyvitamin D levels [25(OH)D] was also shown.MethodsIn this prospective study, we evaluated the safety and the immunological effects of vitamin D supplementation (100 000 IU of cholecalciferol per week for 4 weeks, followed by 100 000 IU of cholecalciferol per month for 6 months.) in 20 SLE patients with hypovitaminosis D.ResultsSerum 25(OH)D levels dramatically increased under vitamin D supplementation from 18.7±6.7 at day 0 to 51.4±14.1 (p<0.001) at 2 months and 41.5±10.1 ng/mL (p<0.001) at 6 months. Vitamin D was well tolerated and induced a preferential increase of naïve CD4+ T cells, an increase of regulatory T cells and a decrease of effector Th1 and Th17 cells. Vitamin D also induced a decrease of memory B cells and anti-DNA antibodies. No modification of the prednisone dosage or initiation of new immunosuppressant agents was needed in all patients. We did not observe SLE flare during the 6 months follow-up period.ConclusionsThis preliminary study suggests the beneficial role of vitamin D in SLE patients and needs to be confirmed in randomized controlled trials.

Phenotypic and Functional Similarity of Gut Intraepithelial and Systemic T Cells in a Teleost Fish

Gut-associated lymphocytes were described in fish, but their involvement in immune responses is still unknown. In rainbow trout, intraepithelial lymphocytes (IELs) are scattered between gut epithelial cells, but neither Peyer’s patches nor mesenteric lymph nodes were identified. Rainbow trout IELs contain mainly T cells, because they expressed transcripts of T cell marker homologs of CD8, CD4, CD28, CD3ε, TCRζ, TCRγ, and TCRβ and lacked IgM. However, trout IELs did not show specific homing to the gut mucosa, which in mammals defines IELs as a distinctive mucosal population. A detailed analysis of the TCRβ repertoire of rainbow trout IELs was performed in both naive and virus-infected animals. TCRβ transcripts of rainbow trout IELs were highly diverse and polyclonal in adult naive individuals, in sharp contrast with the restricted diversity of IEL oligoclonal repertoires described in birds and mammals. Significant modifications of the trout IEL TCRβ repertoire were observed after a systemic infection with a fish rhabdovirus and were especially marked for Vβ4-bearing receptors as previously reported for spleen cells. Thus, we could not find any specific properties of the trout IEL TCRβ repertoire compared with the spleen and pronephros TCRβ repertoire, which questions the reality of a distinct IEL compartment in teleosts. Our findings suggest that a highly diversified αβ ΤCR repertoire is maintained in fish IELs in the absence of Peyer’s patches and mesenteric lymph nodes, whereas the restricted diversity of mouse αβ IELs is attributed to multiple cycles of activation and recirculation, allowing a progressive narrowing of the repertoire.

Interleukin-21 modulates Th1 and Th17 responses in giant cell arteritis

OBJECTIVE Giant cell arteritis (GCA) is a large-vessel vasculitis of unknown origin. Recent findings indicate that at least 2 separate lineages of CD4+ T cells, Th1 and Th17 cells, participate in vascular inflammation. The pathways driving these T cell differentiations are incompletely understood, but may provide novel therapeutic targets. This study was undertaken to identify cytokines involved in the pathogenesis of GCA. METHODS Thirty GCA patients fulfilling the American College of Rheumatology criteria, with active disease or disease in remission, and 30 age-matched controls were included. Levels of 27 cytokines were determined in culture supernatants, and flow cytometric analysis of peripheral blood mononuclear cells (PBMCs) and immunohistochemical analysis of temporal artery samples were performed. RESULTS Multiparametric analysis of cytokines produced by PBMCs associated with GCA disease activity identified a signature involving interleukin-2 receptor (IL-2R), IL-12, interferon-γ (IFNγ), IL-17A, IL-21, and granulocyte-macrophage colony-stimulating factor (GM-CSF). An expansion of Th1 and Th17 cells and a decrease in Treg cells were observed in the peripheral blood of patients with active GCA. An expansion of IL-21-producing CD4+ T cells was also observed in patients with active GCA and correlated positively with Th17 and Th1 cell expansion. Immunohistochemical analysis revealed IFNγ, IL-17A, and IL-21 expression within inflammatory infiltrates. Stimulation of purified CD4+ T cells with IL-21 increased Th1 and Th17 cell frequencies and decreased FoxP3 expression. In contrast, blockade of IL-21 using IL-21R-Fc markedly decreased the production of IL-17A and IFNγ and increased FoxP3 expression. CONCLUSION Our findings indicate that IL-21 plays a critical role in modulating Th1 and Th17 responses and Treg cells in GCA, and might represent a potential target for novel therapy.

Teleost fish mount complex clonal IgM and IgT responses in spleen upon systemic viral infection

Upon infection, B-lymphocytes expressing antibodies specific for the intruding pathogen develop clonal responses triggered by pathogen recognition via the B-cell receptor. The constant region of antibodies produced by such responding clones dictates their functional properties. In teleost fish, the clonal structure of B-cell responses and the respective contribution of the three isotypes IgM, IgD and IgT remain unknown. The expression of IgM and IgT are mutually exclusive, leading to the existence of two B-cell subsets expressing either both IgM and IgD or only IgT. Here, we undertook a comprehensive analysis of the variable heavy chain (VH) domain repertoires of the IgM, IgD and IgT in spleen of homozygous isogenic rainbow trout (Onchorhynchus mykiss) before, and after challenge with a rhabdovirus, the Viral Hemorrhagic Septicemia Virus (VHSV), using CDR3-length spectratyping and pyrosequencing of immunoglobulin (Ig) transcripts. In healthy fish, we observed distinct repertoires for IgM, IgD and IgT, respectively, with a few amplified μ and τ junctions, suggesting the presence of IgM- and IgT-secreting cells in the spleen. In infected animals, we detected complex and highly diverse IgM responses involving all VH subgroups, and dominated by a few large public and private clones. A lower number of robust clonal responses involving only a few VH were detected for the mucosal IgT, indicating that both IgM(+) and IgT(+) spleen B cells responded to systemic infection but at different degrees. In contrast, the IgD response to the infection was faint. Although fish IgD and IgT present different structural features and evolutionary origin compared to mammalian IgD and IgA, respectively, their implication in the B-cell response evokes these mouse and human counterparts. Thus, it appears that the general properties of antibody responses were already in place in common ancestors of fish and mammals, and were globally conserved during evolution with possible functional convergences.

The Astonishing Diversity of Ig Classes and B Cell Repertoires in Teleost Fish

With lymphoid tissue anatomy different than mammals, and diverse adaptations to all aquatic environments, fish constitute a fascinating group of vertebrate to study the biology of B cell repertoires in a comparative perspective. Fish B lymphocytes express immunoglobulin (Ig) on their surface and secrete antigen-specific antibodies in response to immune challenges. Three antibody classes have been identified in fish, namely IgM, IgD, and IgT, while IgG, IgA, and IgE are absent. IgM and IgD have been found in all fish species analyzed, and thus seem to be primordial antibody classes. IgM and IgD are normally co-expressed from the same mRNA through alternative splicing, as in mammals. Tetrameric IgM is the main antibody class found in serum. Some species of fish also have IgT, which seems to exist only in fish and is specialized in mucosal immunity. IgM/IgD and IgT are expressed by two different sub-populations of B cells. The tools available to investigate B cell responses at the cellular level in fish are limited, but the progress of fish genomics has started to unravel a rich diversity of IgH and immunoglobulin light chain locus organization, which might be related to the succession of genome remodelings that occurred during fish evolution. Moreover, the development of deep sequencing techniques has allowed the investigation of the global features of the expressed fish B cell repertoires in zebrafish and rainbow trout, in steady state or after infection. This review provides a description of the organization of fish Ig loci, with a particular emphasis on their heterogeneity between species, and presents recent data on the structure of the expressed Ig repertoire in healthy and infected fish.

The past, present, and future of immune repertoire biology - the rise of next-generation repertoire analysis

T and B cell repertoires are collections of lymphocytes, each characterized by its antigen-specific receptor. We review here classical technologies and analysis strategies developed to assess immunoglobulin (IG) and T cell receptor (TR) repertoire diversity, and describe recent advances in the field. First, we describe the broad range of available methodological tools developed in the past decades, each of which answering different questions and showing complementarity for progressive identification of the level of repertoire alterations: global overview of the diversity by flow cytometry, IG repertoire descriptions at the protein level for the identification of IG reactivities, IG/TR CDR3 spectratyping strategies, and related molecular quantification or dynamics of T/B cell differentiation. Additionally, we introduce the recent technological advances in molecular biology tools allowing deeper analysis of IG/TR diversity by next-generation sequencing (NGS), offering systematic and comprehensive sequencing of IG/TR transcripts in a short amount of time. NGS provides several angles of analysis such as clonotype frequency, CDR3 diversity, CDR3 sequence analysis, V allele identification with a quantitative dimension, therefore requiring high-throughput analysis tools development. In this line, we discuss the recent efforts made for nomenclature standardization and ontology development. We then present the variety of available statistical analysis and modeling approaches developed with regards to the various levels of diversity analysis, and reveal the increasing sophistication of those modeling approaches. To conclude, we provide some examples of recent mathematical modeling strategies and perspectives that illustrate the active rise of a “next-generation” of repertoire analysis.

T cell diversity and TcR repertoires in teleost fish.

In vertebrates, the diverse and extended range of antigenic motifs is matched to large populations of lymphocytes. The concept of immune repertoire was proposed to describe this diversity of lymphocyte receptors--IG and TR--required for the recognition specificity. Immune repertoires have become useful tools to describe lymphocyte and receptor populations during the immune system development and in pathological situations. In teleosts, the presence of conventional T cells was first proposed to explain graft rejection and optimized specific antibody production. The discovery of TR genes definitely established the reality of conventional T cells in fish. The development of genomic and EST databases recently led to the description of several key T cell markers including CD4, CD8, CD3, CD28, CTLA4, as well as important cytokines, suggesting the existence of different T helper (Th) subtypes, similar to the mammalian Th1, Th2 and Th17. Over the last decade, repertoire studies have demonstrated that both public and private responses occur in fish as they do in mammals, and in vitro specific cytotoxicity assays have been established. While such typical features of T cells are similar in both fish and mammals, the structure of particular repertoires such as the one of gut intra-epithelial lymphocytes seems to be very different. Future studies will further reveal the particular characteristics of teleost T cell repertoires and adaptive responses.

Preserving the B-Cell Compartment Favors Operational Tolerance in Human Renal Transplantation

Transplanted individuals in operational tolerance (OT) maintain long-term stable graft function after completely stopping immunosuppression. Understanding the mechanisms involved in OT can provide valuable information about pathways to human transplantation tolerance. Here we report that operationally tolerant individuals display quantitative and functional preservation of the B-cell compartment in renal transplantation. OT exhibited normal numbers of circulating total B cells, naive, memory and regulatory B cells (Bregs) as well as preserved B-cell receptor repertoire, similar to healthy individuals. In addition, OT also displayed conserved capacity to activate the cluster of differentiation 40 (CD40)/signal transducer and activator of transcription 3 (STAT3) signaling pathway in Bregs, in contrast, with chronic rejection. Rather than expansion or higher activation, we show that the preservation of the B-cell compartment favors OT.

Behçet’s disease physiopathology: a contemporary review

Behçet’s disease, also known as the Silk Road Disease, is a rare systemic vasculitis disorder of unknown etiology. Recurrent attacks of acute inflammation characterize Behçet’s disease. Frequent oral aphthous ulcers, genital ulcers, skin lesions and ocular lesions are the most common manifestations. Inflammation is typically self-limiting in time and relapsing episodes of clinical manifestations represent a hallmark of Behçet’s disease. Other less frequent yet severe manifestations that have a major prognostic impact involve the eyes, the central nervous system, the main large vessels and the gastrointestinal tract. Behçet’s disease has a heterogeneous onset and is associated with significant morbidity and premature mortality. This study presents a current immunological review of the disease and provides a synopsis of clinical aspects and treatment options.