Persistent humoral immune memory against pathogens and vaccines is notably supported by two major long-lived players: plasma cells continuously secreting antibodies (Abs), and antigen (Ag)-watching memory B-cells. The latter are capable upon re-challenge by Ag to rapidly yield more abundant Abs, functionally diversified by class-switching, and with supposedly higher affinity than those from naive cells. Like T-cells, B-cells fall in various functional compartments notably regarding memory, the frontiers of which remains fuzzy. Whether B-cell receptor (BCR) class-switching significantly contributes to their functional split remains controversial. It is also clear that memory B-cells need support from T follicular helper (Tfh) cells and stromal cells of lymphoid organs, and there are indications that B-cells might also reciprocally modulate their supportive micro-environment. Most but not all memory B-cells are class-switched, and memory responses to Ag include (but not only consist into) high affinity class-switched Abs. Class-switching can also sometimes shorten B-cell lifespan. Altogethrer, we clearly still have a very poor understanding of the impact of the class of membrane BCR and secreted Abs on immune response polarity and immune memory, and on the whole network of cell interactions which support immune responses in lymphoid tissues. More in-depth study is needed for B-cell intrinsic features connected to class-switching, and also for B-cell extrinsic features dependent from Tfh and the lymphoid stroma. With the global objective of better understanding the link between class switching and the processes of immune response polarization and immune memory, we will thus simultaneously consider not only B-cells but also their main partners, lymphoid stromal cells and Tfh, and develop models where B cells specifically produce one specific Ig class but with diversified BCR and TCR repertoires, and where Ag-specific cells can be followed, studied and eventually sorted. While various tools are available and will be developed along the project, implementing them into an ultimate model for fine studies of the cell interactions that support immune memory will by itself constitute one of the aims of the project, paving the way for future still more ambitious studies. The project combines the expertise of Team 1 about B-cell intrinsic features, B-cell fate and class-switching, Team 2, with expertise in Tfh physiology and fate and Team 3 which masters the characterization of the stromal populations organizing lymphoid tissues and their functional cross-talk with B and Tfh lymphoid cells. Teams will jointly explore both BCR-class-dependent intrinsic B-cell phenotypes and B-cell impact on the other cell populations of lymphoid tissues. B-cell memory is a major issue not only for basic immunology, but also for the vaccinology and immunopathology fields. The issue of long-term protection against pathogens after primary infection or vaccination is of increasing importance since vaccination in infancy is now widely proposed for the prevention of multiple infections (and HPV or HCV-associated sarcomas). Besides these benefits, it remains unclear to what extent post-vaccinal memory can ensure lifelong protection. This is of especially crucial interest for those pathogens potentially yielding more severe infections in adults than children. Besides protection, humoral memory is also involved in long-term adverse reactions in auto-immune or allergic settings. Beyond basic immunology, the “immune memory issues” are thus both pertinent to prevention or treatment of infectious diseases on one hand, to immunotherapy of cancer of inflammatory diseases on the other hand, and finally to the long-term management of immunoallergic diseases, where in-depth understanding (and potentially control) of immune memory would be of clinical importance and might translate into effective immunotherapy protocols.

IgA responses, as the most ambivalent facets of immunity, are also strongly involved into immunopathology. At both systemic and mucosal sites, IgA responses mix inflammation with implication in tolerance. Their mutual dependence with the microbiota illustrates a crucial [gut – immunity axis] which, when deregulated, may end as a [gut – kidney axis] and lead to IgA nephropathy (IgAN), the most frequent chronic kidney disease. Our understanding of what differs in IgA molecules involved in immunity vs tolerance vs IgAN remains controversial. Many variables impact IgA properties: from synthesis in bone marrow vs gut associated lymphoid tissue (GALT), polymerization, glycosylation, affinity for a given antigen, V-region properties, IgA1/IgA2 balance, specific functions of the IgA BCR and/or of the B-cell subset of origin. Our aim is to demultiplex these variables, by exploring and manipulating the heterogeneity of IgA responses, based on both studies of human samples (from donors and IgAN patients), and mouse models adapted to in vivo mechanistic studies of IgA properties and nephritogenicity. We will explore (1) the diversity of IgA-producing cells (2) the complexity of the corresponding secreted IgA in terms of repertoire, glycosylation and polymerization (3) the cytokine and metabolic environment of IgA+ cells, (4) the IgA-microbiota network and (5) the dysregulated IgA production in IgAN. Human B subsets will be studied by cell cytometry, functional studies and RNAseq, in parallel to mouse models engineered to produce human IgA1 and develop IgAN, in which in vivo studies will manipulate the microbiota, the diet and the cytokine environment of GALT B-cells. While biotherapies based on IgA monoclonal Abs are explored, in-depth understanding of the protective vs nephritogenic effects of IgA is mandatory. Alternative therapies for modulating IgA production by targeting diet or microbiota parameters would also be of crucial pertinence for patients affected with IgAN.

The genome is divided into several compartments: the nucleosome, supranucleosome and nucleus. Gene position is precise and dynamic in the nucleus; genes can interact with each other via their enhancer and promoter regions. The immunoglobulin heavy chain locus harbors two enhancers: 3'RR and Eµ-MAR. We will study the role of these enhancers on nuclear territory dynamics and how they maintain the genomic integrity of B lymphocytes. Nuclear positioning of immunoglobulin loci, chromatin status, 3D folding, and B-cell genome integrity will be examined in murine models by partial or total deletion for these two enhancers. This project will elucidate the mechanisms that govern the dynamics of territories and this project will contribute to our understanding of the mechanisms that govern the balance between legitimate and illegitimate interactions.

Chronic inflammatory diseases (IDs) are the third cause of death in developed countries, after cancer and cardiovascular disorders, and their prevalence is growing in westernized countries. These diseases constitute a heterogeneous group of illnesses, including non-exhaustively, rheumatic diseases (rheumatoid arthritis (RA)), autoimmune systemic diseases (systemic lupus erythematosus (SLE)), and inflammatory bowel disorders (IBDs). All these diseases, which appear clinically different, share many similarities, such as common genetic background, common pathophysiological pathways and not surprisingly similar treatments. They are characterized by an autoimmune response with circulating autoantibodies secreted by B cells, which are activated by a specific subset of effector CD4+ T cells, follicular helper T cells (Tfh). Tissue lesions in these pathologies involve another subset of effector T cell, the IL17-secreting T cells (Th17). Interestingly, we recently highlighted the crucial role of CD95L in SLE pathogenesis. CD95L (FasL) belongs to the TNF family. While its receptor CD95 (Fas) is ubiquitously expressed, CD95L is mainly detected at the surface of lymphocytes and NK cells where it plays a pivotal role in the elimination of infected and transformed cells. CD95L is a transmembrane protein acting through cell-to-cell contact but it can be cleaved by metalloproteases, releasing a soluble ligand (cleaved CD95L or cl-CD95L) whose biological function remains to be defined. We observed that cl-CD95L is increased in lupus patients, and this soluble ligand aggravates inflammation in SLE by inducing non-apoptotic signaling pathways (NF-?B and PI3K). CD95 harbors an intracellular stretch designated death domain (DD). Binding of membrane-bound CD95L to CD95 leads to the recruitment of the adaptor protein FADD via the DD. FADD in turn aggregates the initiator caspase-8 and caspase-10. The CD95/FADD/caspase complex is called death-inducing signalling complex (DISC) and implements the apoptotic signaling pathway. In contrast, cl-CD95L fails to form DISC, but instead triggers the formation of a non-apoptotic complex termed motility-inducing signaling complex (MISC) inducing a Ca2+ response. Recent data from our group highlighted that this Ca2+ response occurred through the direct recruitment of PLC?1 by CD95. Indeed, in presence of cl-CD95L, the juxtamembrane region of CD95, called calcium-inducing domain (CID), binds PLC?1 to induce endothelial transmigration of Th17 cells in SLE (Immunity, 2017). Moreover, a chimeric molecule consisting of the CID conjugated to the cell-penetrating domain (designated TAT-CID) binds PLC?1 and prevents its recruitment to CD95. Strikingly, injections of TAT-CID in lupus-prone mice dampen the accumulation of Th17 cells in inflamed organs and alleviate clinical symptoms. Our preliminary data indicate that cl-CD95L also triggers endothelial transmigration of Tfh cells and this process is inhibited by TAT-CID. Furthermore, cl-CD95L favors the activation of Tfh cells and by doing so, their ability to promote the differentiation of B cells into Ig-producing plasma cells. These data urge us to investigate the molecular mechanisms by which cl-CD95L stimulates Tfh cells and decipher whether the therapeutic effect of TAT-CID in lupus-prone mice is related to a combined action on Th17 and Tfh cells. Our consortium intends to address whether i) cl-CD95L is increased not only in the sera of SLE patients but also in those of RA and IBD patients and ii) how this ligand promotes migration/differentiation in Th17 and Tfh cells. Also, using Protein-fragment complementation assay (PCA), high-throughput screening will be performed iii) to identify new inhibitors of CD95/PLC?1 interaction. In conclusion, this project will extend our observations on the role of cl-CD95L in several Th17 and/or Tfh-driven IDs and translate those results into innovative therapeutic molecules for ID patients.