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. CAR T cells isolated from spleen, bone marrow or tumor were sorted for scRNA-Seq or multiome anallysis.