Strength of CAR signaling determines T cell versus ILC differentiation from pluripotent stem cells. 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. Overall design: 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

从多能干细胞（pluripotent stem cells, PSCs）制备嵌合抗原受体（chimeric antigen receptor, CAR）T细胞，将推动癌症免疫疗法的发展。深入理解CAR对多能干细胞向T细胞分化的调控效应，对该研究领域的推进至关重要。近期报道的人工胸腺类器官（artificial thymic organoid, ATO）体系可支持多能干细胞体外定向分化为T细胞。令人意外的是，经靶向CD19的CAR转导的多能干细胞，在人工胸腺类器官中会使T细胞分化路径转向ILC2谱系。T细胞与ILC2均为亲缘关系紧密的淋巴谱系，二者共享部分发育与转录调控程序。机制层面研究表明，在ILC2定向的淋巴前体细胞中，早期淋巴发育阶段存在不依赖抗原的CAR信号激活。基于上述发现，我们通过调控CAR表达水平、CAR结构或同源抗原表达来合理调节CAR信号强度，证实可双向控制T细胞与ILC谱系的命运抉择，为实现多能干细胞定向生成正常CAR-T细胞提供了理论框架。整体实验设计：本项目旨在从单细胞水平，对比分析H1细胞系与H1-CAR细胞系在早期人工胸腺类器官中培养得到的淋巴祖细胞的转录组特征。