B cells produce antibodies (Ab), play an essential role in the immune system and are important therapeutic targets. As hybridomas, B cells are also key for biotechnological production of recombinant monoclonal immunoglobulin (Ig) reagents. The possibility of engineering the B-cell genome for Ig production has potentially far-reaching interests in human health through vaccines, cancer, auto-immunity, infectious or genetic diseases and biomedical diagnosis applications. However, primary B-cells remain difficult to modify genetically. Gene editing technology is not commonly used in B cells to induce the production of a desired Ab instead of the cells’s own Ig. We have recently discovered a new system for efficient gene delivery to human and murine B-cells, which we propose to exploit for CRISPR/cas9 genome editing. Three laboratories will collaborate to develop tools and therapeutically-relevant applications in human and murine models. The efforts will initially be focused on editing the Ig heavy chain gene locus. One goal is to precisely redirect antibody specificity by induced antigenic-specificity replacement (iASR). Well-described Ab will be used to test iASR strategies in vitro and in vivo. Another goal is to modify IgH constant region to force a specific class switch recombination (iCSR). This strategy will be developed to generate IgM Abs for diagnostics and blood cell typing. Overall this project’s ambition is to overcome technological limitations to obtain an efficient platform for B-cell enginering and vectored antibody therapy.

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.

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.

The “TIE-Skip” project (36 months, 2 teams) will address scientific issues pertinent to the B-cell-lineage and their implications in immunopathology, with specific focus on antibody-secreting plasma cells. The error-prone V(D)J recombination process generates out-of-frame V(D)J junctions in ~2/3 of cases, leading to the appearance of premature stop codons on immunoglobulin (Ig) transcripts. Whereas the nonsense-mediated mRNA decay (NMD) pathway ensures efficient degradation of nonproductive Ig mRNAs, less is known about the impact of alternative splicing with regard to the production of truncated Ig with internal deletions. In this demand, we seek to determine the impact of truncated-Ig chains produced after exon skipping events eliminating the variable (V) exon. The rationale comes from our recent study showing that the production of aberrant Ig light chains, encoded by alternatively spliced mRNAs lacking V exons, induces ER stress-associated apoptosis in antibody-secreting cells (Srour et al, J Exp Med 2016). Exon skipping events eliminating the V exon can be induced by nonsense codons (physiological nonsense-associated exon skipping) or, by antisense oligonucleotides (AON) hybridizing the donor splice site of V exon on Ig pre-mRNAs (therapeutic AON-mediated Ig exon skipping). Experiments addressing the toxicity of V-domain less Ig chains will be performed in both normal and autoimmune (lupus) conditions. We already obtained a proof of concept for this antisense-mediated Ig exon skipping approach, by using AON hybridizing Ig lambda light chain RNAs in human PC lines. To extend this study, we wish to analyze the extent of exon skipping during splicing of Ig transcripts and, to address whether AON strategies eliminating the V exon could open new therapeutic perspectives for the treatment of PC diseases (Delpy et al., Patent n°PCT/EP2016/078475). Overall, this proposal should have major impact on basic research and public health, and should bring added value for a new therapeutic Ig exon skipping approach. These findings should open new avenues for the treatment of plasma cell disorders.

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.