Functional effects of chimeric antigen receptor co-receptor signaling domains in human Tregs

SummaryAntigen-specific regulatory T cells (Tregs) engineered with chimeric antigen receptor (CARs) are a potent immunosuppressive cellular therapy in multiple disease models. To date the majority of CAR Treg studies employed second generation CARs, encoding a CD28 or 4-1BB co-receptor signaling domain and CD3ζ, but it was not known if this CAR design was optimal for Tregs. Using an HLA-A2-specific CAR platform and human Tregs, we compared ten CARs with different co-receptor signaling domains and systematically tested their function. Tregs expressing a CAR encoding wild-type CD28 were markedly superior to all other CARs tested in anin vivomodel of graft-versus-host disease. In vitro assays revealed stable expression of Helios and ability to suppress CD80 expression on DCs as keyin vitropredictors ofin vivofunction. This comprehensive study of CAR signaling-domain variants in Tregs can be leveraged to optimize CAR design for use in antigen-specific Treg therapy.