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

Generation of chimeric antigen receptor (CAR) T cells from pluripotent stem cells (PSCs) will enable advances in cancer immunotherapy. A detailed understanding of the effects of CARs on T cell differentiation from PSCs is important to this effort. The recently described artificial thymic organoid (ATO) system supports in vitro differentiation of PSCs to T cells. Unexpectedly, PSCs transduced with a CD19-targeted CAR resulted in diversion of T cell differentiation to the ILC2 lineage in ATOs. T cells and ILC2s are closely related lymphoid lineages which share certain developmental and transcriptional programs. Mechanistically, we show that antigen-independent CAR signaling during early lymphoid development was evident in ILC2-primed lymphoid precursors. We applied this understanding to rationally modulate CAR signaling strength through changes to CAR expression level, structure, or expression of cognate antigen and demonstrate that the T-versus-ILC lineage decision can be controlled in either direction, providing a framework for achieving normal CAR-T cell development from PSCs. The goal of this project was to analyze and compare transcriptome of lymphoid progenitors generated in early stage ATOs at single cell level between H1 and H1-CAR ATOs