Dysfunction in T cells limits the efficacy of cancer immunotherapy. We profiled the epigenome, transcriptome, and enhancer connectome of exhaustion-prone GD2-targeting HA-28z chimeric antigen receptor (CAR) T cells and control CD19-targeting CAR T cells, which present less exhaustion-inducing tonic signaling, at multiple points during their ex vivo expansion. We found widespread, dynamic changes in chromatin accessibility and three-dimensional (3D) chromosome conformation preceding changes in gene expression, notably at loci proximal to exhaustion-associated genes such as PDCD1, CTLA4, and HAVCR2, and increased DNA motif access for AP-1 family transcription factors, which are known to promote exhaustion. Although T cell exhaustion has been studied in detail in mice, we find that the regulatory networks of T cell exhaustion differ between species and involve distinct loci of accessible chromatin and cis-regulated target genes in human CAR T cell exhaustion. Deletion of exhaustion-specific candidate enhancers of PDCD1 suppress the expression of PD-1 in an in vitro model of T cell dysfunction and in HA-28z CAR T cells, suggesting enhancer editing as a path forward in improving cancer immunotherapy.

T细胞功能障碍限制了癌症免疫疗法的功效。我们分析了易耗竭的 GD2 靶向 HA-28z 嵌合抗原受体 (CAR) T 细胞和对照 CD19 靶向 CAR T 细胞的表观基因组、转录组和增强子连接组，这些细胞在多个时间点呈现较少的耗竭诱导强直信号。他们的离体扩张。我们发现染色质可及性和三维 (3D) 染色体构象的广泛动态变化先于基因表达的变化，特别是在与衰竭相关基因（如PDCD1、CTLA4和HAVCR2 ）接近的基因座上，并且增加了对 AP-1 家族转录因子的 DNA 基序访问，这些转录因子已知会促进衰竭。尽管已经在小鼠中详细研究了 T 细胞耗竭，但我们发现 T 细胞耗竭的调节网络在物种之间存在差异，并且涉及人类 CAR T 细胞耗竭中可接近的染色质和顺式调节的靶基因的不同位点。在 T 细胞功能障碍的体外模型和 HA-28z CAR T 细胞中，删除PDCD1的衰竭特异性候选增强子会抑制 PD-1 的表达，这表明增强子编辑是改善癌症免疫治疗的一条途径。