CD40L-stimulated B cells for ex vivo expansion of polyspecific nonhuman primate regulatory T cells for translational studies.

BACKGROUND The therapeutic applications of regulatory T cells (Tregs) include treating autoimmune diseases, graft-versus-host disease, and induction of transplantation tolerance. For ex vivo expanded Tregs to be used in deceased donor transplantation, they must be able to suppress T-cell responses to a broad range of HLA. Here, we present a novel approach for the expansion of polyspecific Tregs in Cynomolgus macaques that was adapted from a Good Manufacturing Practice-compliant protocol. RESULTS Tregs were isolated by fluorescence-activated cell sorting (FACS) and expanded in the presence of a panel of CD40L-stimulated B cells (CD40L-sBc). Prior to Treg culture, CD40L-sBc were expanded in vitro from multiple MHC-disparate macaques. Expanded Tregs expressed high levels of FoxP3 and Helios, a high percentage of TSDR demethylation, and strong suppression of naive T-cell responses in vitro. In addition, these Tregs produced low levels of inflammatory cytokines and were able to expand post-cryopreservation. Specificity assays confirmed that these Tregs were suppressive upon activation by any antigen-presenting cell (APCs) whose MHC was shared by CD40L-sBc used during expansion, proving they are polyspecific. CONCLUSIONS We developed an approach for the expansion of highly suppressive MCM polyspecific Tregs through the use of a combination of CD40L-engeniered B cells with the potential to be translated to clinical studies. To our knowledge, this is the first report that uses a pool of MHC-mismatched CD40L-sBc to create polyspecific Tregs suitable for use in deceased-donor transplants.