Pluripotent stem cells (PSCs) are a promising source of allogeneic T cells for off-the-shelf immunotherapies. However, the process of differentiating genetically engineered PSCs to generate mature T cells requires that the same molecular elements that are crucial for the selection of these cells be removed to prevent alloreactivity. Here we show that antigen-restricted mature T cells can be generated in vitro from PSCs edited via CRISPR to lack endogenous T cell receptors (TCRs) and class I major histocompatibility complexes. Specifically, we used T cell precursors from RAG1^−/−RAG2^−/−B2M^−/− human PSCs expressing a single TCR, and a murine stromal cell line providing the cognate human major histocompatibility complex molecule and other critical signals for T cell maturation. Possibly owing to the absence of TCR mispairing, the generated T cells showed substantially better tumour control in mice than T cells with an intact endogenous TCR. Introducing the T cell selection components into the stromal microenvironment of the PSCs overcomes inherent biological challenges associated with the development of T cell immunotherapies from allogeneic PSCs.

多能干细胞 (PSC) 是用于现成免疫疗法的同种异体 T 细胞的有前途的来源。然而，分化基因工程 PSC 以生成成熟 T 细胞的过程需要去除对这些细胞的选择至关重要的相同分子元件，以防止同种异体反应。在这里，我们展示了抗原限制性成熟 T 细胞可以在体外从通过 CRISPR 编辑的 PSC 产生，以缺乏内源性 T 细胞受体 (TCR) 和 I 类主要组织相容性复合物。具体来说，我们使用来自表达单一 TCR 的RAG1 ^−/− RAG2 ^{− /−} B2M ^{− /−}人 PSC 的 T 细胞前体，以及提供同源人主要组织相容性复合物分子和 T 细胞成熟的其他关键信号的鼠基质细胞系。可能由于不存在 TCR 错配，所产生的 T 细胞在小鼠体内表现出比具有完整内源 TCR 的 T 细胞更好的肿瘤控制能力。将 T 细胞选择成分引入 PSC 的基质微环境，克服了与同种异体 PSC 开发 T 细胞免疫疗法相关的固有生物学挑战。