Characteristics and role of B cells in SSc

In this project called BIMID (B cells in IMIDs), we hypothesize  that B cell characteristics and roles may vary depending on the recognized autoantigen within the same AID, and on their localization.

The objectives of the BIMID project are to better characterize and compare B cell subpopulations, functions, and intracellular pathways  between SSc patients and healthy controls, between SSc patients with different autoantibody specificities (anti-topoisomerase 1 and anti-centromere) and between circulating and skin-resident B cells, in order to highlight shared characteristics and differences between these populations. (i) Comparing phenotypes and functions from B cells collected from these 2 different SSc subpopulations with different serotypes will decipher whether the contribution of B cell to the pathophysiology in SSc is dependent (i) on the autoimmune condition itself (SSc), versus on the patient’s serotype or (ii) on the intrinsic B cells functions (differentiation stage, production of Ab, production of cytokines, main functional/activation pathways, metabolism pathway…).

The ultimate goals of the project are (i) to decipher B cells characteristics and function in these 2 SSc populations, (ii) to find diagnosis and/or follow-up markers, and (iii) new drug targets for B cells.

To achieve these goals, we will use combination of technologies based on in-depth phenotype, transcriptomics and proteomics both on blood and tissue B cells. First, we plan to use an innovative technique, consisting in a single- cell multiomics approach (10X Genomics - Immune profiling method). It permits to have access to multiple layers of information about B cells: (i) BCR sequencing, leading access to the isotype of B cells, and to the range of clonality and of somatic hypermutations (ii) expression of some cell surface protein , (iii) whole transcriptome analysis, to appreciate the functional program of these specific B cells, and then their pathological roles in terms of cytokine production, activation pathways. This approach will be completed by an extensive analysis of B cell surface markers. Using a spectral flow cytometry technique, we will study the expression of more than 40 proteins on the surface of B cells. This technology will allow to analyze (i) the subpopulations of B cells that are dysregulated during SSc, and then to access to clues to understand their ontogeny and (ii) their resting or activation phenotypes. This project is funded by CPER support (2 yrs 200 k€).

This in-depth characterization of B cells will provide a better understanding of the characteristics and, consequently, the pathophysiological role of B cells in SSc. Indeed, we propose to analyze B cells by innovative technologies that allow an almost exhaustive characterization of these cells. Such an integrative description of B cells features has never been conducted in SSc. It could help elucidate their ontogeny, shedding light on the events that led to their emergence and activation, and be useful to the comprehension of their pathogenic roles, deciphering their activation pathway, and their mechanisms of action (cytokine synthesis, for example).  We will also compare B cells between patients with different autoantibody specificities, who have different organ involvement and prognosis. This study could help to found common and/or different dysregulated pathways, and understand how B cells of different specificity could play different roles in SSc pathogenesis. It could occur through their autoantibody production (which is suggested by previous results of our team (Chepy et al. 2022; Chepy, Vivier, et al. 2025)) and/or cytokine production and interaction with other cells. Comparing B cells between the circulating blood and an affected tissue (i.e. the skin), could help understand the mechanisms by which B cells are attracted in the skin, and how they exert their pathogenic role in the tissue. This is of importance, because, at present, therapies targeting B cells in AIDs mainly focus on circulating B cells, while their effect on tissue-resident B cells remains less certain. The recent development of CAR-T cells could enable better targeting of these tissue-resident B cells.

To complete these approaches, we will use the mouse model of SSc based on repeated immunisation of C57Bl/6 mice against TOPO-1 in the presence of Freund's complete adjuvant, which was shown to induce cutaneous and pulmonary fibrosis in mice. We master this model with the development of autoreactive TOPO-1 specific B cells, and we propose here to analyze the modes of action of TOPO-1 autoreactive B cell : Secretion of anti-TOPO-1 Aab (ATA) & Aab independent modes of action: secretion of pro-inflammatory/fibrosing cytokines, interaction with other immune cells, interaction with FB/EC.

We will study the role of immunization against TOPO-1 in the exacerbation of pulmonary fibrosis in a well- characterized murine model of SSc (bleomycin model by intrapulmonary instillation). Mice will be repeatedly immunized against TOPO-1, then receive intratracheal instillation of Bleomycin. We will analyse the infiltration and localization of B cells in the lungs, and count and characterize the TOPO-1 autoreactive B cells in these lungs using flow cytometry approaches. Finally, from the serum of immunized mice, ATA IgG will be purified by affinity chromatography. In parallel, from the splenocytes, we will sort TOPO-1 autoreactive B cells by flow cytometry for V(D)J sequencing of their BCR. From these sequences, we will produce ATA monoclonal antibodies. These antibodies, whether purified or monoclonal, will then be passively transferred to healthy or SSc mice to study their pathogenic potential in the induction or aggravation of the disease. The presence of ATA autoreactive B cells will also be sought in target organs: we will study their phenotypes and functions: local secretion of antibodies, secretion of pro-inflammatory/fibrotic cytokines...This model will be used to assess therapeutic intervention targeting TOPO-1 autoreactive B cells with specific cell therapies (development of CAAR T cells in collaboration with IHU Immun4Cure).