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 by single cell RNA-seq of ILC2-primed precursors. We applied this understanding to rationally modulate CAR signaling strength through changes to CAR expression level, structure, or expression of cognate antigen which demonstrate that the T-versus-ILC decision can be controlled in either direction, including restoration of normal CAR-T cell development. To further characterize the CAR ILC2-like cells, we sorted H1-CAR DN cells, containing ILC2s, as DAPI-eGFP+CD3-TCRαβ-CD8α-CD4- from week 6 ATOs in biological triplicates and compared them with the previously published ATO derived CD8SP T cell gene expression (GSE116015)