Background: We previously demonstrated the potent capacity of regulatory B cells (Bregs) to mitigate allograft immunity. Recently, we produced TLR-activated B cells in vitro that can suppress T cell proliferation in a Granzyme B-dependent manner. Here, we further elucidate how these Bregs exert their suppressive capacity and evaluate their regulatory function in vivo.

Methods: Purified splenic B cells from B6, IL-10^-/-, or TGF-β^-/- mice were activated with CpG ODN 1668 for 3 days. LPS, PMA, and ionomycin were also added for the last 5 hours (CpG/LPI Bregs). These Bregs were then cultured with syngeneic T cells and CD3/CD28 beads. Proliferation was measured by flow cytometry on day 4. Cytokine secretion from the activation and suppression assays was measured using a Luminex assay. For in vivo studies, B cell deficient [mu]MT mice received CpG/LPI Bregs and BALB/c islets.

Results: CpG/LPI Bregs have an IgM^hi IgD^lo CD24^hi immature B cell phenotype, with increased CD25, CD80/86, and LAP expression. IL-10 is often cited as a Breg marker, and our Luminex results confirmed increased IL-10 secretion. The upregulated CD25 raised concerns that the Bregs were depleting IL-2 essential for T cell proliferation. However, we saw significantly higher supernatant IL-2 levels in the presence of these Bregs. To confirm this result, we sorted on CD25^– or CD25⁺ Bregs and did not see a difference in proliferation. Despite the abundance of IL-2, we did not observe Treg induction when we cultured CD4⁺Foxp3^– naïve T cells with the Bregs. In our in vivo studies, we adoptively transferred B6 CpG/LPI Bregs, which induced islet allograft tolerance in 40% of the recipients. Transferred IL-10^-/- Bregs promoted tolerance to a similar degree, demonstrating that this is an IL-10-independent model. Since LAP expression was also upregulated on CpG/LPI Bregs, we sought to determine whether TGF-β is necessary. Our TGF-β^-/- Bregs were unable to prolong graft survival, implicating TGF-β dependence.

Conclusion: Our in vitro generated CpG/LPI Bregs promoted tolerance of allogeneic islets, which was IL-10-independent but TGF-β-dependent. Their in vitro suppressive capacity was due to neither IL-2 depletion nor Treg induction. These results raise the possibility of producing TLR-activated Bregs in vitro for therapeutic use in transplantation.

CITATION INFORMATION: Kojima L., Lee K., Deng K., Tector H., Dai C., Kimura S., Rickert C., Yeh H., Markmann J. TLR-Activated Bregs Promote Islet Allograft Tolerance in an IL-10-Independent and TGF-β-Dependent Manner Am J Transplant. 2017;17 (suppl 3).

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