Deciphering tolerance breakdown in a human B-cell mediated autoimmune disease – Autoimmuni-B

To determine the origin and repertoire of auto-immune B cell clones targeting GpIIbIIIa, the main platelet antigen target, in the spleen of ITP patients after B cell reconstitution, the consortium will use novel approaches. This will combine
1) a new in vitro single cell differentiation assay for human B cells allowing us to test their Ab specificity and to obtain anti-GpIIbIIIa VH sequences for memory and germinal center B cells,
2) innovative microfluidic systems allowing the identification of anti-GPIIbIIIa-specific single plasma cells (PC) and the affinity of the Ab they produce, their sorting and VH/VL sequencing. Sequences obtained for anti-GPIIbIIIa-specific single B cells will be searched in high-throughput sequencing data in order to identify clonally related cells in germinal center, memory B cells and PC and then provide for the first time a global view of an auto-immune response in the human spleen.

- Identification of a rituximab-resistant memory B cell subset with unique characteristics, allowing it to survive the B-cell depletion in a dormant state and to become reactivated after clearance of the drug. This rituximab-resistant subset is vulnerable to anti-CD19 therapy in vitro, suggesting new therapeutic interventions in this disease
- Identification of anti-GPIIbIIIa plasma cells within distinct lymphoid compartments and harboring a wide range of affinities by microfluidic assays

- characterization of plasma cell gene expression profiles in pathological contexts, based on paired blood, spleen and bone marrow samples
- isolation of GPIIbIIIa-specific plasma cells for Ig repertoire sequencing and correlation with antigen affinity

«Rituximab-resistant splenic memory B cells and newly-engaged naive B cells fuel relapses in patients with immune thrombocytopenia«
Crickx, E., Chappert, P., Sokal, A., Weller, S., Azzaoui, I, Vandenberghe, A., Bonnard, G., Rossi, G., Fadeev, T., Storck, S., Fadjallah, J., Meignin, V., Rivière, E., Audia, S., Godeau, B., Michel, M., Weill, J.-C., Reynaud, C.-A. and Mahévas, M.
Sci. Transl. Med., in press (2021)

Which deregulation occurs in human autoimmune diseases and what is the dynamic contribution of each subset of B cells in this process is the central question of this proposal. We will take advantage of a unique model in which different clinical scenarios in immune thrombocytopenia (ITP), an autoimmune disease caused by antibody-mediated platelet destruction, delineate multiple fundamental aspects.
At first, this study will focus on patients relapsing around 6 months after B cell depletion and undergoing splenectomy, which offers the opportunity to study the restart of the disease prior to any new treatment with access to the target organ and allows us to explore the different scenarios of tolerance breakage. To determine the origin and repertoire of auto-immune B cell clones targeting GpIIbIIIa, the main platelet antigen target, in the spleen of ITP patients after B cell reconstitution, the consortium will use novel approaches. This will combine, 1) a new in vitro single cell differentiation assay for human B cells allowing us to test their Ab specificity and to obtain anti-GpIIbIIIa VH sequences for memory and germinal center B cells, 2) innovative microfluidic systems allowing the identification of anti-GPIIbIIIa-specific single plasma cells (PC) and the affinity of the Ab they produce, their sorting and VH/VL sequencing. Sequences obtained for anti-GPIIbIIIa-specific single B cells will be searched in high-throughput sequencing data in order to identify clonally related cells in germinal center, memory B cells and PC and then provide for the first time a global view of an auto-immune response in the human spleen.
Relapses observed in ITP after B cell depletion during restart of B cell lymphopoiesis may suggest a defect in B cell negative selection of autoreactive clones. At what step of B cell development does the tolerance breakdown against platelet antigens occur and is this mechanism restricted to defined platelet glycoprotein antigens, is the second question asked by this proposal. As readout of defective tolerance checkpoints in B cell development, we will analyze, using our single cell in vitro assay, the polyreactivity (against dsDNA, insulin and LPS) and autoreactivity against nuclear and platelet antigens, of immature (central tolerance) and naïve B cells (peripheral tolerance) in the spleen of ITP patients and healthy donors. One hypothesis is that BAFF excess, which occurs in sera and spleen after B-cell depletion, could impact the threshold of negative selection of autoreactive newly formed B cells. This will also be tested in blood along a prospective trial in which ITP patients are treated with a B cell depleting mAb (Rituximab) in the presence or not of BAFF-neutralizing antibodies (Belimumab) until the B cell reconstitution.
Finally, overall changes in the environment driving pathogenic plasma cell development could be reflected at the level of the individual PC itself. Splenic PC from ITP patients displayed a specific genetic signature but it was previously not possible to link antigen specificity to plasma cell gene profiling. To properly compare pure PC populations, we will study bone marrow biopsies from patients, 6 months after rituximab and splenectomy. Using a double microfluidic bioassay for the detection of anti- GPIIbIIIa and/or anti-tetanus specificities, the consortium will perform single-cell RNAseq after sorting of antigen-specific PC. Analysis of such specific PC, in the absence of renewal by memory B cells due to rituximab depletion, will provide a unique signature of long-lived PC generated by a chronic immune response.
This project will help to understand fundamental mechanisms that lead to relapse and tolerance breakdown in a model of auto-immune disease and offers a unique opportunity to advance our knowledge of plasma cells to identify pathogenic molecular signatures. This could help to define novel markers or targets to develop more personalized therapies.