Antigen-specific regulatory T cells (T_regs) engineered with chimeric antigen receptors (CARs) are a potent immunosuppressive 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 T_regs. To date, most of the CAR T_reg 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 T_regs. Using a human leukocyte antigen–A2–specific CAR platform and human T_regs, 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.

嵌合抗原受体（CARs）改造的抗原特异性调节性T细胞（T _regs）是一种有效的免疫抑制性细胞疗法，可用于多种疾病模型，并可克服多克隆T _reg疗法的缺点。CAR疗法最初是用传统的T细胞开发的，它们与_Tregs相比具有不同的信号传导要求。迄今为止，大多数CAR T _reg研究都使用第二代CAR，它们编码CD28或4-1BB共受体信号传导域和CD3ζ，但尚不清楚该CAR设计是否最适合T _reg。使用人类白细胞抗原–A2特异性CAR平台和人类T _regs，我们比较了10个具有不同共受体信号传导域的CAR，并系统地测试了它们的功能和CAR刺激的基因表达谱。在移植物抗宿主疾病的体内模型中，表达CAR编码CD28wt的T _regs明显优于所有其他CAR。体外测定显示Helios的稳定表达和抑制树突状细胞上CD80表达的能力是体内功能的关键体外预测指标。这种全面的CAR信号领域的研究在T变体_的REG可以利用可优化汽车设计中的抗原特异性T使用_REG治疗。