Tregs integrate native and CAR-mediated costimulatory signals for control of allograft rejection.

Tregs expressing chimeric antigen receptors (CAR-Tregs) are a promising tool to promote transplant tolerance. The relationship between CAR structure and Treg function was studied in xenogeneic, immunodeficient mice, revealing advantages of CD28-encoding CARs. However, these models could underrepresent interactions between CAR-Tregs, antigen-presenting cells (APCs), and donor-specific Abs. We generated Tregs expressing HLA-A2-specific CARs with different costimulatory domains and compared their function in vitro and in vivo using an immunocompetent model of transplantation. In vitro, the CD28-encoding CAR had superior antigen-specific suppression, proliferation, and cytokine production. In contrast, in vivo, Tregs expressing CARs encoding CD28, ICOS, programmed cell death 1, and GITR, but not 4-1BB or OX40, all extended skin allograft survival. To reconcile in vitro and in vivo data, we analyzed effects of a CAR encoding CD3ζ but no costimulatory domain. These data revealed that exogenous costimulation from APCs can compensate for the lack of a CAR-encoded CD28 domain. Thus, Tregs expressing a CAR with or without CD28 are functionally equivalent in vivo, mediating similar extension of skin allograft survival and controlling the generation of anti-HLA-A2 alloantibodies. This study reveals a dimension of CAR-Treg biology and has important implications for the design of CARs for clinical use in Tregs.

Systematic testing and specificity mapping of alloantigen-specific chimeric antigen receptors in regulatory T cells.

Chimeric antigen receptor (CAR) technology can be used to engineer the antigen specificity of regulatory T cells (Tregs) and improve their potency as an adoptive cell therapy in multiple disease models. As synthetic receptors, CARs carry the risk of immunogenicity, particularly when derived from nonhuman antibodies. Using an HLA-A*02:01-specific CAR (A2-CAR) encoding a single-chain variable fragment (Fv) derived from a mouse antibody, we developed a panel of 20 humanized A2-CARs (hA2-CARs). Systematic testing demonstrated variations in expression, and ability to bind HLA-A*02:01 and stimulate human Treg suppression in vitro. In addition, we developed a new method to comprehensively map the alloantigen specificity of CARs, revealing that humanization reduced HLA-A cross-reactivity. In vivo bioluminescence imaging showed rapid trafficking and persistence of hA2-CAR Tregs in A2-expressing allografts, with eventual migration to draining lymph nodes. Adoptive transfer of hA2-CAR Tregs suppressed HLA-A2+ cell-mediated xenogeneic graft-versus-host disease and diminished rejection of human HLA-A2+ skin allografts. These data provide a platform for systematic development and specificity testing of humanized alloantigen-specific CARs that can be used to engineer specificity and homing of therapeutic Tregs.