Mechanisms of action, optimization and application of Bregs

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PROJECT SUMMARY / ABSTRACT It has been suggested that the future of medicine rests in cell and gene therapy. While this assertion may be premature, it seems clear that these innovative therapies have extraordinary, paradigm shifting potential. For cell-based therapies, this is best exemplified by the powerful impact of chimeric antigen receptor (CAR) T cells in the treatment of hematological malignancies refractory to current standard of care (5). In the transplant realm, MSC’s, stem cell derived islets, and facilitator cells to promote bone marrow tolerance to renal grafts have shown encouraging results as cell-based therapeutics (6-8). In addition, numerous early phase clinical trials are underway exploring the potential of regulatory T cells (Tregs) to mitigate rejection of liver and kidney allografts (9, 10). More recently, CAR technology has been employed to generate CAR Tregs as more potent, off-the-shelf, donor antigen-specific regulatory populations (11). In the current proposal, we investigate the regulatory properties in the other arm of adaptive immunity to focus on the regulatory activities of B cells. After finding that B cells were required for tolerance in varied experimental transplant models and that B cells (or Bregs) isolated from mice tolerant to islets could adoptively transfer tolerance to otherwise untreated B cell deficient hosts, we sought to expand the tolerogenic Breg population ex vivo. In so doing, we made the unexpected observation that even naïve B cells stimulated ex vivo by Toll-like receptors (TLRs) manifested potent suppressive activity in MLRs and prevented graft rejection in vivo (12). With further study, we demonstrate that a variety of B cell activating signals induce Breg suppression and that, depending on the activating trigger, the mechanism of suppression in vivo varies. Our overarching hypothesis is that Breg suppression is executed through antigen presentation in the context of local immunoregulatory cytokine elaboration, such as TGF-ß and IL-10. Supporting this notion, we found that B cell specificity for the donor was essential to suppressive function, perhaps indicating cognate Breg-T cell/Treg communication (13, 14). Also noteworthy is our finding that clonal Breg populations with specificity for donor antigens exhibited the greatest suppressive potency in vitro and in vivo (13). Based on these findings, in Aim 3, we will conduct innovative studies to determine whether Bregs, imbued with donor specificity, through either transient or permanent expression of a donor-specific CAR, prevent allograft rejection. Furthermore, recent studies indicate that CAR can be delivered effectively in vivo using lipid nanoparticles (LNP) decorated with antibodies to target select cell populations, such as host B cells (15). The LNP approach developed for vaccine delivery also lends itself to parallel transfer and expression of mRNA payloads encoding suppressive molecules (IL-10, TGF-ß, etc.) to augment potency (16). Collectively, our proposed work will advance understanding of Breg mechanisms of differentiation and suppression, optimize Breg function and evaluate the potential of Bregs and CAR Bregs as cellular therapeutics.