*Purpose: During the inflammatory process, the immune system capacity to regulate itself is mandatory to balance effector positive feedback, avoid allergies and autoimmunity. Different subsets of B cells have been described with regulatory properties. Granzyme B (GZMB)+Bcells have been shown to be increased in blood from transplanted patients who tolerate their graft without immunosuppressive treatment and to enhance graft tolerance in mice model, validating their protective role in transplantation. Althought the regulatory functions are dependent of GZMB, GZMB+Bcells does not induce any cytotoxic effect in target cells. To date, we are far from understanding the complexity of the genesis, phenotype and function of GZMB+ Bcells.

*Methods: We have previously shown that GZMB+Bcells can be expanded in vitro from sorted B cells stimulated with a cocktail of IL-2, IL-21, IgG IgA IgM F(ab)’2, CpG oligodeoxynucleotides and CD40L, while keeping their suppressive properties. We analyzed GZMB+Bregs transcriptome in two healthy donors by scRNAseq to identify a Breg signature. We also analyzed CD3/CD28 stimulated CD4+CD25- T cells alone or in cocultures with either GZMB+Bregs or resting B cells to characterize their effects on T cells beyond the decreased proliferation observed in vitro.

*Results: RNA differential expression in CD3/CD28 activated CD4+ CD25- T cell compartment does corroborate in vitro GZMB+ Bregs antiproliferative functions, but it does also highlight an inhibition T cell function through a decrease of different immune pathways. We also identified different potential GZMB+ Bregs ligands that may be responsible of T cell differential expression. We analyzed gene enrichment in the GZMB+Bregs compared to the unstimulated cells to identify a Breg signature endorsing the activated state and the regulatory functions of GZMB+ Bregs.

*Conclusions: These results are an important step in GZMB+Bregs characterization. However, although we highlighted the signature of in vitro induced regulatory cells, the signature is highly impacted by their proliferative status compared to the unstimulated B cells. To go through this bias, we will look for this GZMB+Bregs signature in the B cell compartment of tolerant patients.

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