Generation of antigen-specific mature T cells from RAG1 -/- RAG2 -/- B2M -/- stem cells by engineering their microenvironment.

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.