CD26 CAR-T cells have attenuated mitochondrial and glycolytic metabolic profiling

Multiple targets of chimeric antigen receptor T cells (CAR-T cells) are shared expressed by tumor cells and T cells, these self-antigens may stimulate CAR-T cells continuously during the expansion. Persistent exposure to antigens is considered to cause metabolic reprogramming of T cells and the metabolic profiling is critical in determining the cell fate and effector function of CAR-T cells. However, whether the stimulation of self-antigens during CAR-T cell generation could remodel the metabolic profiling is unclear. In this study, we aim to investigate the metabolic characteristics of CD26 CAR-T cells, which expressed CD26 antigens themselves. The mitochondrial biogenesis of CD26 and CD19 CAR-T cells during expansion was evaluated by the mitochondrial content, mitochondrial DNA copy numbers and genes involved in mitochondrial regulation. The metabolic profiling was investigated by the ATP production, mitochondrial quality and the expression of metabolism-related genes. Furthermore, we assessed the phenotypes of CAR-T cells through memory-related markers. We reported that CD26 CAR-T cells had elevated mitochondrial biogenesis, ATP production and oxidative phosphorylation at early expansion stage. However, the mitochondrial biogenesis, mitochondrial quality, oxidative phosphorylation and glycolytic activity were all weakened at later expansion stage. On the contrary, CD19 CAR-T cells did not exhibit such characteristics. CD26 CAR-T cells showed distinctive metabolic profiling during expansion that was extremely unfavorable to cell persistence and function. These findings may provide new insights for the optimization of CD26 CAR-T cells in terms of metabolism.嵌合抗原受体T细胞（chimeric antigen receptor T cells，CAR-T细胞）的多个靶标可被肿瘤细胞与T细胞共表达，这类自身抗原可在CAR-T细胞扩增过程中持续刺激CAR-T细胞。抗原持续暴露被认为可诱导T细胞发生代谢重编程，而代谢谱对于决定CAR-T细胞的细胞命运与效应功能至关重要。然而，在CAR-T细胞制备过程中，自身抗原刺激是否会重塑其代谢谱，目前尚不明确。本研究旨在探究自身表达CD26抗原的CD26 CAR-T细胞的代谢特征。本研究通过线粒体含量、线粒体DNA拷贝数以及线粒体调控相关基因的表达水平，对扩增阶段CD26与CD19 CAR-T细胞的线粒体生物发生情况进行了评估；通过ATP生成水平、线粒体质量以及代谢相关基因的表达，对其代谢谱展开分析。此外，本研究通过记忆相关标志物对CAR-T细胞的表型进行了评估。研究结果显示，在扩增早期，CD26 CAR-T细胞的线粒体生物发生、ATP生成与氧化磷酸化水平均显著升高，但在扩增后期，其线粒体生物发生、线粒体质量、氧化磷酸化以及糖酵解活性均出现明显减弱。与之相反，CD19 CAR-T细胞并未表现出此类特征。CD26 CAR-T细胞在扩增过程中呈现出的独特代谢谱，极不利于细胞存活与功能发挥。本研究结果可为CD26 CAR-T细胞的代谢优化策略提供新的理论参考。