Strength of CAR signaling reveals bifurcation of T and ILC2 lineage differentiation from pluripotent stem cells

Abstract

Abstract T cells and type 2 innate lymphoid cells (ILC2) are two closely related lymphoid lineages that share certain developmental and transcriptional programs, including a requirement for Notch and IL-7 signaling during differentiation. We recently developed an artificial thymic organoid (ATO) system that supports in vitro differentiation of human pluripotent stem cells (PSCs) to mature αβT cells. We show here that lentiviral introduction of a CD19-targeted chimeric antigen receptor (CAR) into PSCs surprisingly resulted in ILC2-biased lymphopoiesis from PSCs at the expense of T cell differentiation. PSC-derived ILC2s expressed the classical ILC2 markers CD25, CD200R, and CRTH2 as well GATA3, ID2, and TCF7, and responded to both cytokine stimuli and antigen-dependent CAR signaling by producing IL-5 and IL-13. To understand CAR-mediated lineage diversion from T to ILC2, we performed single cell RNAseq of PSC-derived hematopoietic progenitor cells before the phenotypic onset of either T or ILC2 differentiation. This revealed a gene signature of T cell receptor signaling, suggesting aberrant CAR activation at the earliest stages of lymphocyte development. We established that CAR signaling in ATOs was antigen-independent and thus likely driven by tonic signaling. We applied this finding to rationally modulate the T/ILC2 lineage decision by fine-tuning CAR signaling strength during lymphocyte development from PSCs, permitting restoration of conventional CAR-T cell differentiation or, conversely, directed differentiation of isogenic antigen-specific CAR-ILC2s. Taken together, our findings shed light on human ILC2 development and inform the applied differentiation of both ILC2s and conventional CAR-T cells from PSCs. Supported by Tobacco-Related Disease Research Program Predoctoral Award (SL), Broad Stem Cell Research Center UCLA Fellowship (SL, CSS), CIRM Bridges Program (CB), NIH T32 (ST), NIH K08CA235525 (CSS).