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

The therapeutic applications of regulatory T cells (T_regs ) include treating autoimmune diseases, graft-versus-host disease and induction of transplantation tolerance. For ex-vivo expanded T_regs to be used in deceased donor transplantation, they must be able to suppress T cell responses to a broad range of human leukocyte antigen (HLA). Here, we present a novel approach for the expansion of polyspecific T_regs in cynomolgus macaques that was adapted from a good manufacturing practice-compliant protocol. T_regs 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 T_reg culture, CD40L-sBc were expanded in vitro from multiple major histocompatibility complex (MHC)-disparate macaques. Expanded T_regs expressed high levels of forkhead box protein 3 (FoxP3) and Helios, a high percentage of T_reg -specific demethylated region (TSDR) demethylation and strong suppression of naïve T cell responses in vitro. In addition, these T_regs produced low levels of inflammatory cytokines and were able to expand post-cryopreservation. Specificity assays confirmed that these T_regs were suppressive upon activation by any antigen-presenting cells (APCs) whose MHC was shared by CD40L-sBc used during expansion, proving that they are polyspecific. We developed an approach for the expansion of highly suppressive cynomolgus macaque polyspecific T_regs through the use of a combination of CD40L-engineered 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 T_regs suitable for use in deceased-donor transplants.