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

Pluripotent stem cells (PSCs) present promising engineering opportunities as sources of allogeneic T cells for off-the-shelf immunotherapies. However, the complex in vitro process of differentiation required to generate mature T cells from genetically engineered PSCs introduces unique challenges, as positive selection is dependent on the same molecular machinery that must be removed to prevent alloreactivity. We report an organoid-based method in which engineering of the stromal microenvironment induced generation of antigen-restricted, mature, conventional T cells from PSCs which lacked all endogenous TCR and Class I MHC expression. Transgenic signals required to rescue positive selection were supplied by separate cellular sources; T cell precursors from edited PSC expressed a single TCR, and a murine stromal line provided cognate human MHC and other critical T cell development signals. Edited T cells exhibited superior tumour control in vivo likely due to the absence of TCR mispairing. This in vitro method overcomes key biological impediments inherent to developing T cell immunotherapies from allogeneic PSCs. Overall design: scRNA-sequencing of T cells, derived in vitro from human PSCs and sorted from PBMC