Costimulatory domains direct distinct fates of CAR-drivenT cell dysfunction [RNA-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. Comparative gene expression profiling analysis of RNA-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 collected from five different donors and each sample was sequenced in technical triplicate.

嵌合抗原受体（CAR）工程化T细胞在输注至患者体内后，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具有关键意义。本研究开发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，以此探究CAR共刺激结构域如何介导T细胞衰竭过程。我们发现，靶向CD19的功能衰竭型基于CD28的CAR-T细胞具有经典T细胞耗竭的特征，而功能衰竭型基于41BB的CAR-T细胞则在表型、转录组及表观遗传层面均存在显著差异。我们在无法控制临床疾病的CAR-T细胞中，验证了这一独特转录程序的激活状态。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的慢性激活会诱导一种全新的T细胞功能衰竭状态，通过对FOXO3进行基因修饰即可缓解该状态。本数据集包含针对CD19阳性急性淋巴细胞白血病（Acute Lymphoblastic Leukemia, ALL）细胞系Nalm6介导的慢性激活过程中，携带CD28或41BB的CD19靶向人CAR-T细胞的RNA测序（RNA-seq）数据的比较基因表达谱分析结果。研究中T细胞取自5名不同供者，每个样本均完成3次技术重复测序。