Infusion of regulatory T cells (Tregs) engineered with a chimeric antigen receptor (CAR) targeting donor-derived human leukocyte antigen (HLA) is a promising strategy to promote transplant tolerance. Here, we describe an anti-HLA-A2 CAR (A2-CAR) generated by grafting the complementarity-determining regions (CDRs) of a human monoclonal anti-HLA-A2 antibody into the framework regions of the Herceptin 4D5 single-chain variable fragment and fusing it with a CD28-ζ signaling domain. The CDR-grafted A2-CAR maintained the specificity of the original antibody. We then generated HLA-A2 mono-specific human CAR Tregs either by deleting the endogenous T-cell receptor (TCR) via CRISPR/Cas9 and introducing the A2-CAR using lentiviral transduction or by directly integrating the CAR construct into the TCR alpha constant locus using homology-directed repair. These A2-CAR⁺TCR^deficient human Tregs maintained both Treg phenotype and function in vitro. Moreover, they selectively accumulated in HLA-A2-expressing islets transplanted from either HLA-A2 transgenic mice or deceased human donors. A2-CAR⁺TCR^deficient Tregs did not impair the function of these HLA-A2⁺ islets, whereas similarly engineered A2-CAR⁺TCR^deficientCD4⁺ conventional T cells rejected the islets in less than 2 weeks. A2-CAR⁺TCR^deficient Tregs delayed graft-versus-host disease only in the presence of HLA-A2, expressed either by co-transferred peripheral blood mononuclear cells or by the recipient mice. Altogether, we demonstrate that genome-engineered mono-antigen-specific A2-CAR Tregs localize to HLA-A2-expressing grafts and exhibit antigen-dependent in vivo suppression, independent of TCR expression. These approaches may be applied towards developing precision Treg cell therapies for transplant tolerance.

注入具有嵌合抗原受体 (CAR) 靶向供体来源的人类白细胞抗原 (HLA) 的调节性 T 细胞 (Tregs) 是促进移植耐受性的一种有前景的策略。在这里，我们描述了通过将人单克隆抗 HLA-A2 抗体的互补决定区 (CDR) 移植到赫赛汀 4D5 单链可变片段的框架区而产生的抗 HLA-A2 CAR (A2-CAR)并将其与 CD28-ζ 信号域融合。CDR 移植的 A2-CAR 保持了原始抗体的特异性。然后我们通过删除内源性 T 细胞受体 (TCR) 来生成 HLA-A2 单特异性人 CAR Tregs通过CRISPR/Cas9 并使用慢病毒转导或通过使用同源定向修复将 CAR 构建体直接整合到 TCR α 恒定基因座中来引入 A2-CAR。这些 A2-CAR ⁺ TCR^缺陷的人类 Treg 保持了 Treg 表型和功能体外. 此外，它们选择性地积聚在从 HLA-A2 转基因小鼠或已故人类供体移植的 HLA-A2 表达胰岛中。A2-CAR ⁺ TCR^缺陷的Tregs 不会损害这些 HLA-A2 ⁺胰岛的功能，而类似设计的 A2-CAR ⁺ TCR^缺陷CD4 ⁺传统 T 细胞在不到 2 周的时间内排斥胰岛。A2-CAR ⁺ TCR^缺陷性 Tregs 延迟移植-相对- 仅在 HLA-A2 存在时宿主疾病，由共转移的外周血单核细胞或受体小鼠表达。总之，我们证明了基因组工程化的单抗原特异性 A2-CAR Treg 定位于表达 HLA-A2 的移植物并表现出抗原依赖性体内抑制，独立于 TCR 表达。这些方法可用于开发针对移植耐受的精确 Treg 细胞疗法。