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.

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.

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.

AL amyloidosis is a rare but devastating disease caused by the tissue/organ deposition of amyloid fibrils composed of a monoclonal immunoglobulin light chain (LC). Despite extensive biochemical studies and clinical investigations, the pathophysiology of this disease is still misunderstood due to the absence of a reliable animal model. We have recently uncovered for the first time the conditions to reproduce AL amyloidosis in a proprietary transgenic mouse model. In the present project, we will use this original and unique animal model to better understand the events leading to in vivo fibril formation, the intrinsic toxicity of amyloid LC and their effects on different biological processes, including impaired hemostasis. We will also take advantage on this long-awaited model to validate two complementary approaches aiming at removing AL amyloid deposits from organs.