Functional effects of chimeric antigen receptor co-receptor signaling domains in human regulatory T cells

Antigen-specific regulatory T cells (T-regs) engineered with chimeric antigen receptors (CARs) are a potent immuno-suppressive cellular therapy in multiple disease models and could overcome shortcomings of polyclonal T(reg )therapy. CAR therapy was initially developed with conventional T cells, which have different signaling requirements than do Tregs. To date, most of the CAR T-reg studies used second-generation CARs, encoding a CD28 or 4-1BB co-receptor signaling domain and CD3 zeta, but it was not known if this CAR design was optimal for T-regs. Using a human leuko-cyte antigen-A2-specific CAR platform and human Legs, we compared 10 CARs with different co-receptor signaling domains and systematically tested their function and CAR-stimulated gene expression profile. T-regs expressing a CAR encoding CD28wt were markedly superior to all other CARs tested in an in vivo model of graft-versus-host disease. In vitro assays revealed stable expression of Helios and an ability to suppress CD80 expression on dendritic cells as key in vitro predictors of in vivo function. This comprehensive study of CAR signaling domain variants in T-regs can be leveraged to optimize CAR design for use in antigen-specific T(reg )therapy.