- Li, Suwen;
- Wang, Chloe S;
- Montel-Hagen, Amélie;
- Chen, Ho-Chung;
- Lopez, Shawn;
- Zhou, Olivia;
- Dai, Kristy;
- Tsai, Steven;
- Satyadi, William;
- Botero, Carlos;
- Wong, Claudia;
- Casero, David;
- Crooks, Gay M;
- Seet, Christopher S
Generation of chimeric antigen receptor (CAR) T cells from pluripotent stem cells (PSCs) will enable advances in cancer immunotherapy. Understanding how CARs affect T cell differentiation from PSCs is important for 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 innate lymphoid cell 2 (ILC2) lineage in ATOs. T cells 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 T cell precursors. We applied this understanding to modulate CAR signaling strength through expression level, structure, and presentation of cognate antigen to demonstrate that the T cell-versus-ILC lineage decision can be rationally controlled in either direction, providing a framework for achieving CAR-T cell development from PSCs.