Joint single-cell transcriptomics and epigenomics analysis reveal key regulators of CAR T cell stemness and antitumor immunity

In this study, we revealed the molecular network governing the differentiation of CAR T cells into transcriptionally and epigenetically distinct subsets. Using two mouse cancer models with different sensitivities to CAR T-cell therapy, we showed that CD8+ CAR T cells transitioned from the stem-like to effector-like subset in B-cell ALL but developed into exhausted T cells in the solid tumor. By simultaneously profiling transcriptomic and epigenomic analyses in single cells, we demonstrated that lineage-defining TFs were often controlled by exceptionally high numbers of cis-regulatory elements and regulated distinct chromatin states foreshadowing transcriptional changes during T cell differentiation. Different CAR T-cell subsets were governed by distinct gene regulatory networks with TFs as hubs. We showed that FOXP1 was a hub TF in the stem-like network and promoted the antitumor response and stemness of CAR T cells while limiting their transition to the effector-like subset. In contrast, KLF2, a hub TF in the effector-like network, controlled the lineage choice between effector-like and exhausted subsets by driving the effector program and suppressing the exhaustion program. Overall design: CAR T cells isolated from spleen, bone marrow or tumor were sorted for scRNA-Seq or multiome anallysis.

本研究揭示了调控嵌合抗原受体T细胞（CAR T cell）向转录组与表观基因组特征迥异的亚群分化的分子网络。本研究采用两种对CAR T细胞疗法敏感性存在差异的小鼠癌症模型，证实CD8+ CAR T细胞在B细胞急性淋巴细胞白血病（B-cell ALL）模型中可由干细胞样亚群向效应样亚群转化，而在实体瘤模型中则分化为耗竭性T细胞。通过同时开展单细胞转录组与表观基因组联合分析，本研究证实，谱系特异性转录因子（Transcription Factor, TF）通常受数量极多的顺式调控元件（cis-regulatory elements）调控，并可调控独特的染色质状态，该状态可预示T细胞分化过程中的转录组变化。不同CAR T细胞亚群由以转录因子为核心的独特基因调控网络所支配。本研究证实，FOXP1是干细胞样调控网络中的核心转录因子，可增强CAR T细胞的抗肿瘤应答与干细胞干性，同时抑制其向效应样亚群的转化。与之相反，KLF2作为效应样调控网络中的核心转录因子，可通过激活效应程序、抑制耗竭程序，调控效应样亚群与耗竭亚群之间的谱系选择。实验整体设计：从脾脏、骨髓或肿瘤组织中分离的CAR T细胞经分选后，用于单细胞RNA测序（scRNA-Seq）或多组学（multiome）分析。