Effects of fumarate on the metabolic reprogramming of aging T-cells associated with rescue of the T-cell anti-tumor functions

Changes in mitochondrial bioenergetics in aging T-cells play a key role in the decrease in T-cell function and tumor resistance with aging. The bioactive sphingolipid ceramide, induced by aging stress, is a major player in these changes, as its accumulation at the mitochondrial membranes of both mouse and human aging T-cells induces ceramide-dependent mitophagy, which decreases T-cell viability, cytokine secretion, and anti-tumor activity. Besides lipid signaling, our findings show a general depletion of tricarboxylic acid cycle (TCA) metabolites in aging T-cells, with pools of fumarate, malate, and argininosuccinate significantly decreasing in both mouse and human aging T-cells. In vivo supplementation of fumarate to mouse T-cells seemed to correct some of the functional defects observed in aging, with a protection of T-cell viability, cytokine production, and tumor killing capacity in co-cultures with tumor cells. The fumarate supplementation also led to a decrease in ceramide-dependent mitophagy in the aging T-cells, indicating an interplay between ceramide-dependent mitophagy and fumarate metabolism. Here, the RNAseq data shows a reversal of the genetic signature of aging in T-cells upon supplying fumarate, associated with a significant increase in different metabolic enzymes. Overall design: 24-month old C57BL/6 mice were treated with 10mg/kg fumarate for 2 months. After two months of treatment, T-cells were collected from mice treated with fumarate or vehicle, as well as young 2 month old mice. T-cells were then activated in vitro with plate-bound CD3 (2ug/ml) and CD28 (5ug/ml) in the presence of IL-2 (100IU/ml). T-cells were then collected and RNA was extracted for RNAseq.

衰老T细胞的线粒体生物能学（mitochondrial bioenergetics）改变，在衰老相关T细胞功能下降与抗肿瘤能力减弱过程中发挥关键作用。由衰老应激诱导的生物活性鞘脂神经酰胺（ceramide），是介导上述改变的核心因子：在小鼠与人类衰老T细胞的线粒体膜（mitochondrial membranes）中积累的神经酰胺，可引发神经酰胺依赖性线粒体自噬（ceramide-dependent mitophagy），进而降低T细胞存活率、细胞因子分泌能力与抗肿瘤活性。除脂质信号通路外，本研究结果还显示，衰老T细胞中存在三羧酸循环（tricarboxylic acid cycle, TCA）代谢物的普遍耗竭，其中延胡索酸盐（fumarate）、苹果酸盐（malate）及精氨琥珀酸（argininosuccinate）的代谢池水平在小鼠与人类衰老T细胞中均显著下降。向小鼠T细胞体内补充延胡索酸盐，可部分纠正衰老相关的功能缺陷，能够在与肿瘤细胞的共培养体系中保护T细胞存活率、细胞因子产生能力与肿瘤杀伤能力。此外，延胡索酸盐补充还可降低衰老T细胞中的神经酰胺依赖性线粒体自噬水平，提示神经酰胺依赖性线粒体自噬与延胡索酸代谢之间存在相互调控关系。本研究的RNA测序（RNAseq）数据显示，向衰老T细胞补充延胡索酸盐后，其衰老相关基因表达特征发生逆转，同时伴随多种代谢酶的表达水平显著升高。整体实验设计：将24月龄C57BL/6小鼠以10mg/kg剂量连续2个月给予延胡索酸盐处理。处理2个月后，分别从延胡索酸盐处理组、赋形剂（vehicle）对照组小鼠以及2月龄年轻小鼠体内分离T细胞。随后，在体外使用平板包被的CD3（2μg/ml）与CD28（5μg/ml），并添加白细胞介素2（IL-2，100IU/ml）活化T细胞。收集活化后的T细胞，提取RNA并进行RNA测序。