Quentin Simon

Regulatory B cells: an exciting target for future therapeutics in transplantation.

Transplantation is the preferred treatment for most end-stage solid organ diseases. Despite potent immunosuppressive agents, chronic rejection remains a real problem in transplantation. For many years, the predominant immunological focus of research into transplant rejection has been T cells. The pillar of immunotherapy in clinical practice is T cell-directed, which efficiently prevents acute T cell-mediated allograft rejection. However, the root of late allograft failure is chronic rejection and the humoral arm of the immune response now emerges as an important factor in transplantation. Thus, the potential effects of Abs and B cell infiltrate on transplants have cast B cells as major actors in late graft rejection. Consequently, a number of recent drugs target either B cells or plasma cells. However, immunotherapies, such as the anti-CD20 B cell-depleting antibody, can generate deleterious effects on the transplant, likely due to the deletion of beneficial population. The positive contribution of regulatory B (Breg) cells or B10 cells has been reported in the case of transplantation, mainly in mice models and highlights the primordial role that some populations of B cells can play in graft tolerance. Yet, this regulatory aspect remains poorly characterized in clinical transplantation. Thus, total B cell depletion treatments should be avoided and novel approaches should be considered that manipulate the different B cell subsets. This article provides an overview of the current knowledge on the link between Breg cells and grafts, and reports a number of data advising Breg cells as a new target for future therapeutic approaches.

Human regulatory B cells control the TFH cell response

Background Follicular helper T (TFH) cells support terminal B‐cell differentiation. Human regulatory B (Breg) cells modulate cellular responses, but their control of TFH cell–dependent humoral immune responses is unknown. Objective We sought to assess the role of Breg cells on TFH cell development and function. Methods Human T cells were polyclonally stimulated in the presence of IL‐12 and IL‐21 to generate TFH cells. They were cocultured with B cells to induce their terminal differentiation. Breg cells were included in these cultures, and their effects were evaluated by using flow cytometry and ELISA. Results B‐cell lymphoma 6, IL‐21, inducible costimulator, CXCR5, and programmed cell death protein 1 (PD‐1) expressions increased on stimulated human T cells, characterizing TFH cell maturation. In cocultures they differentiated B cells into CD138+ plasma and IgD−CD27+ memory cells and triggered immunoglobulin secretions. Breg cells obtained by Toll‐like receptor 9 and CD40 activation of B cells prevented TFH cell development. Added to TFH cell and B‐cell cocultures, they inhibited B‐cell differentiation, impeded immunoglobulin secretions, and expanded Foxp3+CXCR5+PD‐1+ follicular regulatory T cells. Breg cells modulated IL‐21 receptor expressions on TFH cells and B cells, and their suppressive activities involved CD40, CD80, CD86, and intercellular adhesion molecule interactions and required production of IL‐10 and TGF‐&bgr;. Conclusion Human Breg cells control TFH cell maturation, expand follicular regulatory T cells, and inhibit the TFH cell–mediated antibody secretion. These novel observations demonstrate a role for the Breg cell in germinal center reactions and suggest that deficient activities might impair the TFH cell–dependent control of humoral immunity and might lead to the development of aberrant autoimmune responses. Graphical abstract Figure. No Caption available.

B cell subset distribution is altered in patients with severe periodontitis

Several studies have recently highlighted the implication of B cells in physiopathogenesis of periodontal disease by showing that a B cell deficiency leads to improved periodontal parameters. However, the detailed profiles of circulating B cell subsets have not yet been investigated in patients with severe periodontitis (SP). We hypothesised that an abnormal distribution of B cell subsets could be detected in the blood of patients with severe periodontal lesions, as already reported for patients with chronic inflammatory diseases as systemic autoimmune diseases. Fifteen subjects with SP and 13 subjects without periodontitis, according to the definition proposed by the CDC periodontal disease surveillance work group, were enrolled in this pilot observational study. Two flow cytometry panels were designed to analyse the circulating B and B1 cell subset distribution in association with the RANKL expression. A significantly higher percentage of CD27+ memory B cells was observed in patients with SP. Among these CD27+ B cells, the proportion of the switched memory subset was significantly higher. At the same time, human B1 cells, which were previously associated with a regulatory function (CD20+CD69-CD43+CD27+CD11b+), decreased in SP patients. The RANKL expression increased in every B cell subset from the SP patients and was significantly greater in activated B cells than in the subjects without periodontitis. These preliminary results demonstrate the altered distribution of B cells in the context of severe periodontitis. Further investigations with a larger cohort of patients can elucidate if the analysis of the B cell compartment distribution can reflect the periodontal disease activity and be a reliable marker for its prognosis (clinical trial registration number: NCT02833285, B cell functions in periodontitis).

LAG-3 Inhibitory Receptor Expression Identifies Immunosuppressive Natural Regulatory Plasma Cells

Summary B lymphocytes can suppress immunity through interleukin (IL)‐10 production in infectious, autoimmune, and malignant diseases. Here, we have identified a natural plasma cell subset that distinctively expresses the inhibitory receptor LAG‐3 and mediates this function in vivo. These plasma cells also express the inhibitory receptors CD200, PD‐L1, and PD‐L2. They develop from various B cell subsets in a B cell receptor (BCR)‐dependent manner independently of microbiota in naive mice. After challenge they upregulate IL‐10 expression via a Toll‐like receptor‐driven mechanism within hours and without proliferating. This function is associated with a unique transcriptome and epigenome, including the lowest amount of DNA methylation at the Il10 locus compared to other B cell subsets. Their augmented accumulation in naive mutant mice with increased BCR signaling correlates with the inhibition of memory T cell formation and vaccine efficacy after challenge. These natural regulatory plasma cells may be of broad relevance for disease intervention. Graphical Abstract Figure. No caption available. HighlightsLAG‐3 expression identifies natural regulatory plasma cellsLAG‐3+CD138hi plasma cells express IL‐10 within hours of stimulationLAG‐3+CD138hi plasma cells have a unique epigenome poised to express IL‐10LAG‐3+CD138hi plasma cells develop via an antigen‐specific mechanism &NA; Plasma cells secrete antibodies and play a key role in host defense against infection. Lino et al. identify a novel subset of natural regulatory plasma cells characterized by the expression of LAG‐3 that develops at steady state independently of microbiota, and respond to innate stimulation by producing immunosuppressive IL‐10.

Defective regulation of autoreactive IL-6-producing transitional B lymphocytes is associated with disease in patients with systemic sclerosis

Systemic sclerosis (SSc) has the highest case‐specific mortality of any rheumatic disease, and no effective therapy is available. A clear manifestation of SSc is the presence of autoantibodies. However, the origin of autoantibody‐producing B lymphocytes, their mechanisms of activation and autoantibody production, and their role remain unclear. This study was undertaken to identify mechanisms that contribute to pathogenic B cell generation and involvement in SSc and to assess the altered distribution and function of B cells in SSc patients.

Regulatory B Cells

B cells are often defined as progenitors of antibody-producing cells, with a critical role in adaptive immunity. Over the years, we have come to appreciate that different subsets of B cells play diverse roles in humoral immune responses, including modulation of effector T cell response by antigen presentation, costimulation, and cytokine production, as well as in innate immunity; for example, through the production of natural antibodies directed to T cell independent antigens or antibody-mediated FcγR-mediated polarization of macrophages. More recently, a subpopulation of B cells with the ability to negatively regulate cellular immune responses and inflammation is increasingly being interrogated for their role in infectious diseases, autoimmunity, and cancer, while gaining traction as a viable target for therapeutic manipulation for immunomodulation.

Antigen-Specificity in the Thymic Development and Peripheral Activity of CD4+FOXP3+ T Regulatory Cells

CD4+Foxp3+ T regulatory cells (Treg) are essential for the life of the organism, in particular because they protect the host against its own autoaggressive CD4+Foxp3− T lymphocytes (Tconv). Treg distinctively suppress autoaggressive immunity while permitting efficient defense against infectious diseases. This split effect indicates that Treg activity is controlled in an antigen-specific manner. This specificity is achieved first by the formation of the Treg repertoire during their development, and second by their activation in the periphery. This review presents novel information on the antigen-specificity of Treg development in the thymus, and Treg function in the periphery. These aspects have so far remained imprecisely understood due to the lack of knowledge of the actual antigens recognized by Treg during the different steps of their life, so that most previous studies have been performed using artificial antigens. However, recent studies identified some antigens mediating the positive selection of autoreactive Treg in the thymus, and the function of Treg in the periphery in autoimmune and allergic disorders. These investigations emphasized the remarkable specificity of Treg development and function. Indeed, the development of autoreactive Treg in the thymus was found to be mediated by single autoantigens, so that the absence of one antigen led to a dramatic loss of Treg reacting toward that antigen. The specificity of Treg development is important because the constitution of the Treg repertoire, and especially the presence of holes in this repertoire, was found to crucially influence human immunopathology. Indeed, it was found that the development of human immunopathology was permitted by the lack of Treg against the antigens driving the autoimmune or allergic T cell responses rather than by the impairment of Treg activation or function. The specificity of Treg suppression in the periphery is therefore intimately associated with the mechanisms shaping the formation of the Treg repertoire during their development. This novel information refines significantly our understanding of the antigen-specificity of Treg protective function, which is required to envision how these cells distinctively regulate unwanted immune responses as well as for the development of appropriate approaches to optimally harness them therapeutically in autoimmune, malignant, and infectious diseases.

A1.21 In-depth characterisation of CD24highCD38high transitional human B cells reveals different regulatory profiles

CD24high CD38high transitional B cells represent a key stage in the developmental pathway of B cell peripheral tolerance and functional maturation. These B cells have been widely ascribed regulatory functions. However, the phenotypic and functional overlap between these cells and regulatory B cells remains controversial. In this study, we use multi-colour flow cytometry with bioinformatic analyses and functional studies to show that CD24high CD38high B cells can be differentiated into multiple subsets. The study also reveals for the first time that human transitional B cells encompass transitional type 1 (T1) and T2 B cells but also distinct anergic T3 B cells as well as IL-10-producing CD27+ transitional B cells. Interestingly, the latter two subsets differentially regulate CD4+ T cell proliferation and polarisation towards Th1 effector cells. Additional analyses show that the percentage of T3 B cells is reduced while the frequency of CD27+ transitional B cells is increased in patients with autoimmune diseases compared with matched healthy individuals. This study provides evidence for the existence of different transitional B cell subsets each displaying unique phenotypic and regulatory functional profiles. Furthermore, the study indicates that altered distribution of transitional B cells subsets highlights different regulatory defects in autoimmune diseases

AB0032 In-Depth Characterization of Cd24Highcd38High Transitional Human B Cells Reveals Different Regulatory Profiles and Are Abnormally Distributed in Autoimmune Diseases

Background CD24high CD38high transitional B cells represent a key stage in the developmental pathway of B cell peripheral tolerance and functional maturation. These B cells have been widely ascribed regulatory functions. However, the phenotypic and functional overlap between these cells and regulatory B cells remains controversial. Objectives In this study, we use multi-color flow cytometry with bioinformatic analyses and functional studies to show that CD24high CD38high B cells can be differentiated into multiple subsets. Results The study also reveals for the first time that human transitional B cells encompass transitional type 1 (T1) and T2 B cells but also distinct anergic T3 B cells as well as IL-10-producing CD27+ transitional B cells. Interestingly, the latter two subsets differentially regulate CD4+ T cell proliferation and polarization towards Th1 effector cells. Additional analyses show that the percentage of T3 B cells is reduced while the frequency of CD27+ transitional B cells is increased in patients with autoimmune diseases compared with matched healthy individuals. Conclusions This study provides evidence for the existence of different transitional B cell subsets each displaying unique phenotypic and regulatory functional profiles.Furthermore, the study indicates that altered distribution of transitional B cells subsets highlights different regulatory defects in autoimmune diseases. Disclosure of Interest None declared

A8.31 Characterisation of human transitional B-cells though their regulatory functions

Transitional B cells represent a cortical link between the initial B cell compartment in bone marrow and the mature peripheral B cell repertoire. The prior characterisation of human transitional B-cells has suggested that this population could be subdivised into two major subsets: the immature T1-Bcells CD19+ CD10high CD24high CD38high IgMhigh IgDlow CD21low and tne intermediate cells mentionned as T2-B cells CD19+ CD10 + CD24high CD38high IgMhigh IgDhigh CD21high. Recently B-cell regulation have been shed in light and a lot of evidences suggested that regulatory B cells (Bregs) are contained within the CD24high CD38high transitional B-cell subsets. In this study, we wanted to fully characterised the composition of the CD24high CD38high population using phenotypic and functionnal approaches. First, we used a non manual and non subjective gating technique with 10 color flow cytometry to identify the different subsets contained in the CD24high CD38high population through FLOCK (Flow cytometry without K) algorythm program. We showed that non less than 5 different populations are present in the parent gate. Three population are included in transitional B-cell subset confirmed by in vitro functional analysis. The two other populations belong to memory B-cells. In the second part of the work, the different populations were sorted and their supressive capacities in a coculture model of B cells and activated T-cell were analysed. We demonstrated that B-cell subets have different regulatory potency on the inhibition of T-cell proliferation and Th1 cytokine production. These data bring new clarifications on the precise nature of regulatory B cells in human and their supressive functions. Regulatory B-cell supress T-cell proliferation through a TGFβ/IDO axis and are deficient in chronic humoral rejection In kidney transplantation, B cell is increasingly identified as an important determinant for graft outcome. Chronic antibody mediated rejection (cABMR) represent today the major complication in clinic. Patients with cABMR display a unique B cell phenotype characterised by a disturbance in B cell distribution. Here, the aim of our study was to research if cABMR B cells present also impaired regulatories functions. Experiments are realised in 12 stable patients (ST) (delay from graft >12 months, no rejection, proteinuria <0.5g/24h, PRA <10%, no DSA and a one year biopsy without allograft glomerulopathy or rejection), 14 patients with cABMR (positive DSA, allograft glomerulopathy and/or C4d staining) and 17 controls (blood donors). B-cell functions were analysed using coculture with B and T cells. Proliferation and Th1 cytokine secretion of isolated CFSE labelled T cells in presence of autologous B cells was compared between the 3 groups. T cells were stimulated with anti CD3 and anti CD28, and co-cultured with CpG-activated B cells. Suppressive cytokines like TGFβ and IL-10 and the regulatory enzyme indoleamine 2,3-dioxygenase (IDO) were evaluated during the course of the culture. First, we have demonstrated that compared with situation of stable graft function and healthy volunteers, B cells from cABMR patients display an impaired regulatory function on T cell proliferation and Th1 production. Furthermore, B cells induce more T regulatory cells (Tregs) in coculture after 4 days in HV and ST groups compared to cABMR group (p<10-3). We also evidenced that TGFβ and IDO expressed by B cells are involved in inhibition of T cell proliferation and Treg cells generation in HV and ST patients. We additionally shown that B cells from cABMR patients present a failure of TGFβ and IL-10 secretion in co-culture associated with a significantly decrease of IDO production leading to a defect in the capacity of activated-B cells to induce Treg cells and control T cell responses. Finally, B cells from cABMR patients display functional abnormalities as evidenced by the loss of B cell suppressive activity. This work represents interesting perspectives with the discovered of new cellular targets who can be might guide therapy in transplantation in the future.