UCLA

T cells and Type 2 innate lymphoid cells (ILC2) are closely related lymphoid lineages which share certain developmental and transcriptional programs, including a requirement for Notch and IL-7 signaling during differentiation. The recently described artificial thymic organoid (ATO) system supports in vitro differentiation of human pluripotent stem cells (PSCs) to mature αβT cells. Unexpectedly, PSCs transduced with a CD19-targeted chimeric antigen receptor (CAR) resulted in ILC2-biased lymphopoiesis and a block in T cell differentiation in ATOs. PSC-derived CAR-ILC2s expressed classical ILC2 markers and gene expression, and surprisingly responded to both cytokine stimuli and antigen-induced CAR signaling. Mechanistically, single cell RNA-seq of early lymphoid precursors in ATOs revealed evidence of CAR activation in ILC2 precursors, and rational modulation of CAR signaling could restore generation of functional CAR-T cells from PSCs or, conversely, divert CAR-T cell differentiation to ILC2 and, to a lesser extent, NK/ILC1 lineages. Taken together, our findings shed light on human T and ILC2 lineage development and provide a framework for applying CAR technology to the generation of multiple lymphoid lineages from PSCs.