The transcription factor Bhlhe40 programs mitochondrial regulation of resident CD8+ T cell fitness and functionality

Tissue-resident memory CD8+ T cells (Trm) share characteristics and core residency gene programs with tumor-infiltrating lymphocytes (TILs). However, the transcriptional, metabolic and epigenetic regulation of Trm cell and TIL differentiation, maintenance and function are largely undefined. Furthermore, it is unknown whether metabolic and epigenetic manipulations can be employed to promote Trm/TIL functional programs for cancer immunotherapy. Here we report a critical mechanism programming the mitochondrial and epigenetic regulation of Trm/TIL functionality in situ. We found that mouse and human Trm/TIL expressed the transcription factor BHLHE40 and the BHLHE40-associated gene signature. Bhlhe40 was specifically required for Trm and TIL development and polyfunctionality. Local PD-1 signaling inhibited TIL Bhlhe40 expression, and Bhlhe40 was critical for TILs reinvigoration following anti-PD therapy. Mechanistically, Bhlhe40 sustained Trm/TIL mitochondrial fitness for the promotion of a functional epigenetic state. Building on these findings, we also identified an epigenetic and metabolic regimen that promoted Trm/TIL gene signatures associated with tissue residency and poly-functionality through in vitro T cell screening. Strikingly, this regimen empowered the anti-tumor activity of CD8+ T cells and possessed therapeutic potential even at an advanced tumor stage in mouse models. Our results provide mechanistic insights on the local regulation of Trm and TIL function, and offer a viable strategy for developing novel immunotherapeutic means for cancer. Examination of RNA expression in: in vitro activated CD8+ T cell from WT or Bhlhe40 deficient mice; TIL cells and Splenic CD8+ T cells from B16-OVA bearing WT mice; Trm cells from IAV infected WT or Bhlhe40 deficient mice and Splenic CD8+ T cells from IAV infected WT mice.

组织驻留记忆CD8+ T细胞（Tissue-resident memory CD8+ T cells, Trm）与肿瘤浸润淋巴细胞（tumor-infiltrating lymphocytes, TILs）共享特征及核心驻留基因程序。然而，目前针对Trm细胞与TILs的分化、维持及功能的转录、代谢与表观遗传调控机制，仍未得到充分阐明。此外，尚不清楚能否通过代谢与表观遗传操控手段，激活Trm/TIL的功能程序以应用于癌症免疫治疗。本研究揭示了一种可原位调控Trm/TIL功能的线粒体与表观遗传关键机制。我们发现，小鼠及人类的Trm/TIL均表达转录因子BHLHE40及其相关基因特征谱。Bhlhe40对Trm与TIL的发育及多功能活性至关重要。局部程序性死亡受体1（Programmed Cell Death Protein 1, PD-1）信号通路可抑制TIL的Bhlhe40表达，而Bhlhe40在抗PD治疗后TIL的功能重振过程中发挥关键作用。从机制层面而言，Bhlhe40可维持Trm/TIL的线粒体健康状态，进而促进功能性表观遗传状态的形成。基于上述发现，我们通过体外T细胞筛选，确定了一种表观遗传与代谢调控方案，该方案可促进与组织驻留及多功能性相关的Trm/TIL基因特征。值得注意的是，该方案可增强CD8+ T细胞的抗肿瘤活性，且在小鼠模型的晚期肿瘤阶段仍具备治疗潜力。本研究结果为Trm与TIL功能的局部调控机制提供了新的认知，并为开发新型癌症免疫治疗手段提供了可行策略。本研究的RNA表达检测涵盖以下样本：野生型（wild type, WT）或Bhlhe40缺陷型小鼠的体外活化CD8+ T细胞；携带B16-OVA肿瘤的野生型小鼠的肿瘤浸润淋巴细胞与脾脏CD8+ T细胞；甲型流感病毒（Influenza A virus, IAV）感染的野生型或Bhlhe40缺陷型小鼠的Trm细胞，以及甲型流感病毒感染的野生型小鼠的脾脏CD8+ T细胞。