Strength of CAR signaling determines T cell versus ILC differentiation from pluripotent stem cells

Abstract

Generation of chimeric antigen receptor (CAR) Tcells from pluripotent stem cells (PSCs) will enable advances in cancer immunotherapy. Understanding how CARs affect Tcell differentiation from PSCs is important for this effort. The recently described artificial thymic organoid (ATO) system supports invitro differentiation of PSCs to Tcells. Unexpectedly, PSCs transduced with a CD19-targeted CAR resulted in diversion of Tcell differentiation to the innate lymphoid cell 2 (ILC2) lineage in ATOs. Tcells and ILC2s are closely related lymphoid lineages with shared developmental and transcriptional programs. Mechanistically, we show that antigen-independent CAR signaling during lymphoid development enriched for ILC2-primed precursors at the expense of Tcell precursors. We applied this understanding to modulate CAR signaling strength through expression level, structure, and presentation of cognate antigen to demonstrate that the Tcell-versus-ILC lineage decision can be rationally controlled in either direction, providing a framework for achieving CAR-T cell development from PSCs.