*Purpose: We previously generated in vitro Toll-like receptor (TLR)-activated B cells became regulatory B cells (Bregs) that suppress T cell proliferation and promote allograft tolerance. We have now investigated the contribution of Breg antigen presentation function and BCR recognition in the suppressive property of Bregs cells.

*Methods: Direct and indirect allorecognitions were assessed in B6 recipient mice injected with BALB/c Bregs-TLR or B6 Bregs-TLR, respectively. B cells were activated with CpG ODN 1668 for 3 days with the addition of LPS, PMA, and ionomycin for the last 5 hours to generate Bregs-TLR. To generate various Bregs-TLR, B cells were isolated from allogenic BALB/c, syngeneic B6, and MHC class II (MHC-II)-deficient B6 mice. In vitro suppression assays and regulatory T cell induction were measured by flow cytometry after co-culture with autologous or allogeneic T cells and Bregs-TLR. In vivo studies were performed in an islet transplant model in which diabetic B cell-deficient uMT (B6) mice received BALB/c islets together with either BALB/c, B6 or MHC-II KO B6.Bregs-TLR. Controls were naïve B cells.

*Results: In vitro colcultures showed that B6.Bregs-TLR equally suppressed syngeneic B6 , allogeneic BALB/c and third party C3H CD4⁺ T cell proliferation mediated by CD3/CD28 stimulation. Likewise, B6.Bregs-TLR cells promoted Treg development in similar proportions in the 3 strains of CD4+ T cells. We next compared the respective efficacy of syngeneic (B6) or allogeneic (BALB/c) Bregs-TLR in preventing rejection of BALB/c islets transplanted in B cell deficient μMT (B6) recipient mice. Adoptive transfer of BALB/c.Bregs-TLR accelerated graft rejection as all recipients rejected BALB/c islets in less than 10 days after transplantation. In contrast, the transfer of syngeneic B6.Bregs-TLR cells led to 54% long-term engraftment (>100 days). Experiments on the role of Breg antigen presentation by MHC-II also revealed that the lack of MHC-II didn’t alter Breg suppression or graft survival times.

*Conclusions: Although Bal/c.Bregs-TLR and B6.Bregs-TLR have similar tolerogenic Breg phenotypes and in vitro suppression of T cell proliferation, in vivo graft survival outcomes are different. Direct pathway of donor B cells and recipient T cells could be a positive effect on graft reject although donor B cells have regulatory properties. It suggests that regulatory B cells rely on varied mechanisms to regulate T cell immunity.

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