Antigen-specific regulatory T cells (Tregs) engineered with chimeric antigen receptors (CARs) are a potent immunosuppressive cellular therapy in multiple disease models and could overcome shortcomings of polyclonal Treg therapy. CAR therapy was initially developed with conventional T cells, which have different signaling requirements than do Tregs. To date, most of the CAR Treg studies used 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 a human leukocyte antigen–A2–specific CAR platform and human Tregs, we compared 10 CARs with different co-receptor signaling domains and systematically tested their function and CAR-stimulated gene expression profile. Tregs 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 Tregs can be leveraged to optimize CAR design for use in antigen-specific Treg therapy.

CD4 + effector lymphocytes (Teff) are traditionally classified by the cytokines they produce. To determine the states that Teff actually adopt in frontline tissues in vivo , we applied single-cell transcriptome and chromatin analysis on colonic Teff cells, in germ-free or conventional mice, or after challenge with a range of phenotypically biasing microbes. Subsets were marked by expression of interferon-signature or myeloid-specific transcripts, but transcriptome or chromatin structure could not resolve discrete clusters fitting classic T H subsets. At baseline or at different times of infection, transcripts encoding cytokines or proteins commonly used as T H markers distributed in a polarized continuum, which was also functionally validated. Clones derived from single progenitors gave rise to both IFN-γ and IL17-producing cells. Most transcriptional variance was tied to the infecting agent, independent of the cytokines produced, and chromatin variance primarily reflected activity of AP1 and IRF transcription factor families, not the canonical subset master regulators T-bet, GATA3, RORγ.

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