*Purpose: We previously generated Toll-like receptor (TLR)-activated B cells in rodent settings. These B cells can suppress T cell proliferation via in vitro mixed lymphocytes reaction and promote allograft tolerance as Regulatory B cells (Bregs). A continuous effort toward Bregs drove us to generate TLR-activated B cells in Nonhuman primates using the same protocol in the murine model. In this study, we evaluated the efficacy of their phenotype, function, and mechanism to suppress T cell responses.

*Methods: Purified splenic NHP B cells were activated with CpG ODN 2006-5G for 3 days with the addition of LPS, PMA, and ionomycin for the last 5 hours. They were then harvested and co-cultured with CFSE-labeled autologous T cells stimulated with polyclonal CD3/CD28 beads or PHA. On day 4, proliferation was measured by flow cytometry. In some cultures, anti-PD1, or anti-GzmB was added to evaluate their role in suppressing T cell proliferation.

*Results: The majority of CpG/LPI-NHP B cells had immature phenotypes, CD20+CD21+CD27-IgM+ on surface. In in vitro experiment, they effectively suppressed CD4+ and CD8+ T cell proliferation and reduced their viability upon stimulation, while B cells with only CpG activation did not. The CpG/LPI-NHP B cells expressed higher levels of LAP, a surrogate marker for TGF-β, and programmed death (PD1), but not expressed a higher level of IL-10 compared to naïve NHP B cells. Instead, it seemed that Granzyme B (GzmB) was instrumental to the suppressive effect because the blockade of GzmB increased the proliferation in the coculture.

*Conclusions: We have successfully generated NHP Bregs with in vitro suppressive capacity toward T cell responses. Here we show for the first time that CpG-activated NHP B cells exert regulatory function. Our studies suggest that Bregs may provide a great option for regulatory cell-based therapy. We are pursuing the translational potential of Bregs in a large animal model by evaluating their regulatory function in vivo.

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