Background: Our previous studies document the potent capacity of regulatory B cells (Bregs) to mitigate allograft immunity. However, we have been largely unsuccessful in demonstrating their function in vitro. Recently, others have shown that human B cells activated with CpG and CD40L can suppress T cell proliferation by Granzyme B (GzmB). We thus generated suppressive murine B cells in vitro to delineate the mechanisms by which Bregs might exert suppressive function.

Methods: Purified splenic C57BL/6 B cells were activated with CpG ODN 1668 or 2395 for 3 days, adding LPS, PMA, and ionomycin for the last 5 hours (CpG/LPI-B cells). They were then harvested and co-cultured with CFSE-labeled syngeneic T cells stimulated with polyclonal CD3/CD28 beads or irradiated BALB/c splenocytes. At day 4, proliferation was measured by flow cytometry. In some cultures, anti-IL-10, anti-TGF-β, or anti-GzmB was added to evaluate their role in suppressing T cell proliferation. Contact dependence of suppression was also assessed by a transwell assay.

Results: Activated CpG/LPI-B cells effectively suppressed CD4+ T cell proliferation by >45%, while B cells with only CpG activation did not (<5%). The CpG/LPI-B cells expressed higher levels of LAP, TIM1, IL-10, CD25, and GzmB. However, assays with B cells from IL-10^-/- and TIM1^-/- mice or with blocking antibodies suggest that the suppression is not IL-10, TGF-β, or TIM1-dependent, but GzmB-dependent. Furthermore, CpG/LPI-B cells were not suppressive (<5%) in our transwell studies, implicating contact dependence. We observed similar T cell suppression with allogeneic stimulators.

Conclusion: We have successfully generated B cells with in vitro suppressive capacity. Here we show for the first time that CpG-activated murine B cells exert regulatory function via a mechanism involving GzmB, independent of IL-10. They also require direct contact with the target T cells. These results suggest that B cells rely on varied mechanisms to regulate T cell immunity. We are currently testing their antigen-specificity and ability to prolong allograft survival in an islet transplantation model.

CITATION INFORMATION: Kojima L, Kimura S, Lee K, Schuetz C, Washburn L, Deng K, Tector H, Amundsen B, Ndishabandi D, Markmann A, Markmann-Porch M, Oura T, Alessandrini A, Kim J, Yeh H, Markmann J. Suppression of T Cell Proliferation by CpG-Activated B Cells Depends on Granzyme-B and Cell-Cell Contact, but Not on IL-10. Am J Transplant. 2017;17 (suppl 3).

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