Costimulatory domains direct distinct fates of CAR-driven T cell dysfunction [ATAC-seq]. Costimulatory domains direct distinct fates of CAR-driven T cell dysfunction [ATAC-seq]

Chimeric antigen receptor (CAR) engineered T cells often fail to enact effector functions after infusion into patients. Understanding the biological pathways that lead CAR T cells to failure is of critical importance in the design of more effective therapies. We developed and validated an in vitro model that drives T cell dysfunction by chronic CAR stimulation and interrogated how CAR costimulatory domains contribute to T cell failure. We found that dysfunctional CD28-based CARs targeting CD19 bear hallmarks of classical T cell exhaustion while dysfunctional 41BB-based CARs are phenotypically, transcriptionally and epigenetically distinct. We confirmed activation of this unique transcriptional program in CAR T cells that failed to control clinical disease. Further, we demonstrate that 41BB-dependent activation of the transcription factor FOXO3 is a significant contributor to this dysfunction and disruption of FOXO3 significantly improves 41BB-based CAR T cell function. These findings identify that chronic activation of 41BB leads to novel state of T cell dysfunction that can be alleviated by genetic modification of FOXO3. Overall design: Comparative profiling analysis of ATAC-seq data for CD19-directed human CAR T cells with CD28 and 41BB during chronic activation by CD19+ ALL cell line Nalm6. T cells were sequenced in technical triplicate.

嵌合抗原受体（Chimeric antigen receptor, CAR）修饰的T细胞在输注至患者体内后通常无法发挥效应功能。阐明介导CAR-T细胞功能衰竭的生物学通路，对于开发更有效的治疗方案至关重要。我们构建并验证了一种通过慢性CAR刺激诱导T细胞功能衰竭的体外模型，并以此探究CAR共刺激结构域如何导致T细胞功能衰竭。我们发现，靶向CD19的基于CD28的功能衰竭型CAR具有经典T细胞耗竭的特征，而基于41BB的功能衰竭型CAR在表型、转录组和表观基因组层面均存在显著差异。我们在无法控制临床疾病进程的CAR-T细胞中，证实了该独特转录程序的激活。此外，我们证实，转录因子FOXO3的41BB依赖性激活是该功能衰竭的重要诱因，而敲除FOXO3可显著改善基于41BB的CAR-T细胞的功能。本研究结果表明，41BB的慢性激活会引发一种新型的T细胞功能衰竭状态，而通过对FOXO3进行基因编辑可缓解该状态。
实验设计概述：对分别携带CD28与41BB共刺激结构域的靶向CD19的人源CAR-T细胞，在经CD19阳性急性淋巴细胞白血病细胞系Nalm6慢性激活过程中的转座酶可及性测序（Assay for Transposase-Accessible Chromatin using sequencing, ATAC-seq）数据进行比较谱式分析。所有T细胞样本均设置3次技术重复进行测序。