Carbon-monoxide Induced PERK Regulated Autophagy Reprograms Anti-Tumor T Cells with Robust Immunometabolic Phenotype

Mitochondria and Endoplasmic reticulum (ER) share structural and functional networks and activates well-orchestrated signaling processes to shape a cell's fate and function. While persistent ER stress (ERS) response leads to mitochondrial collapse, moderate ERS condition promotes mitochondrial function. Strategies to boost anti-tumor T cell function by targeting ERMitochondria crosstalk have not been exploited yet. Thus, we used carbon monoxide (CO), a short-lived gaseous molecule, to test if engaging moderate ERS conditions can improve T cells mitochondrial function and anti-tumor function. Using melanoma antigen specific T cells, we identify that CO induced transient activation of ERS sensor 'protein kinase R-like endoplasmic reticulum kinase (PERK)' dramatically increase anti-tumor T cell function. Furthermore, COinduced PERK activation temporarily halts protein translation and induces protective autophagy (that includes mitophagy). To get further insight, we used LC3-GFP to differentiate between the cells that prepare themselves to undergo active autophagy (LC3-GFPpos) and those that fail to enter into the process (LC3-GFPneg). We found that LC3-GFPpos T cells show robust anti-tumor potential whereas, LC3-GFPneg cells show T regulatory-like phenotype, harbor dysfunctional mitochondria, and accumulate abnormal metabolite content. These anomalous ratios of metabolites render the cells with a hypermethylated state and distinct epigenetic profile limiting their anti-tumor activity. Overall, the study highlights that ERS activated autophagy pathways modify mitochondrial function and epigenetically reprogram the T cells towards a superior antitumor phenotype to achieve robust tumor control. Overall design: Examination of mRNA profiles in T cells using RNA-seq. Examination of chromatin accessibility in T cells using ATAC-seq.

线粒体与内质网（Endoplasmic Reticulum, ER）共享结构与功能网络，并协同激活高度有序的信号传导过程，进而调控细胞的命运与功能。持续的内质网应激（ER Stress, ERS）应答会引发线粒体功能崩溃，而适度的内质网应激条件则可改善线粒体功能。目前，通过靶向内质网-线粒体串扰以增强抗肿瘤T细胞功能的策略尚未得到开发利用。为此，我们利用短寿命气态分子一氧化碳（Carbon Monoxide, CO），探究适度内质网应激能否改善T细胞的线粒体功能与抗肿瘤活性。我们以黑色素瘤抗原特异性T细胞为模型，发现一氧化碳诱导的内质网应激感受器蛋白激酶R样内质网激酶（Protein Kinase R-like Endoplasmic Reticulum Kinase, PERK）的瞬时激活，可显著增强T细胞的抗肿瘤功能。此外，一氧化碳诱导的PERK激活可暂时抑制蛋白质翻译，并诱导保护性自噬（包括线粒体自噬）。为进一步阐明其机制，我们利用LC3-GFP标记区分两类T细胞：一类是准备启动活跃自噬的细胞（LC3-GFP阳性，LC3-GFPpos），另一类是无法进入自噬过程的细胞（LC3-GFP阴性，LC3-GFPneg）。实验结果显示，LC3-GFP阳性T细胞具备极强的抗肿瘤潜能；而LC3-GFP阴性T细胞则呈现调节性T细胞样表型，伴随线粒体功能障碍与异常代谢物蓄积。代谢物的异常比例使此类细胞处于高甲基化状态，并具备独特的表观遗传特征，进而限制其抗肿瘤活性。综上，本研究证实，内质网应激激活的自噬通路可重塑线粒体功能，并通过表观遗传重编程将T细胞转化为更优异的抗肿瘤表型，从而实现高效的肿瘤控制。实验整体设计：通过RNA测序（RNA-seq）检测T细胞的mRNA表达谱；通过转座酶可及性测序（ATAC-seq）检测T细胞的染色质开放状态。