Zhou Xianfei, Cai Miaoguo, Yang Fan, Huang Luoshun, Ling Yisheng, Zhang Yang, Nie Hanqiu, Xing Renwei
Department of Hepatobiliary Surgery, Municipal Hospital Affiliated to Taizhou University, Taizhou, China.
Department of Radiation Oncology, Taizhou Hospital of Zhejiang Province Affiliated to Wenzhou Medical University, Taizhou, China.
Genes Immun. 2025 Feb;26(1):45-53. doi: 10.1038/s41435-024-00315-1. Epub 2024 Dec 23.
The hypoxic microenvironment is an essential feature of solid tumors. Autophagy has been controversial in its role in immune regulation. This project aims to elucidate the impact of autophagy in pancreatic cancer (PC) under specific conditions (hypoxia) on CD8 T cells and the regulatory mechanisms behind it.The levels of HIF1α and autophagy were analyzed by western blot (WB) and immunofluorescence (IF). The effects of HIF1α on cell autophagy were assessed in normoxic or hypoxic treatments using KC7F2 (HIF-1 channel inhibitor) or chloroquine (autophagy inhibitor). CD8 T cells were co-cultured with PC cells to assess the cytotoxicity using lactate dehydrogenase (LDH) and Hoechst/PI staining. The content of cytokines and the activation level of CD8 T cells were measured by enzyme-linked immunosorbent assay (ELISA) and flow cytometry. MHC-I expression in PC cells (membranes) was analyzed using quantitative reverse transcription polymerase chain reaction (qRT-PCR), WB, IF, and flow cytometry. Humanized immune-reconstituted mice were applied to investigate the impact of HIF1α-induced autophagy on in vivo immunity.When cells were in hypoxia, the levels of HIF1α and autophagy were higher compared to normoxic conditions. Treatment with KC7F2 resulted in similar levels of HIF1α and autophagy as those in normoxic state. Chloroquine treatment reversed the autophagy level to the normoxic state. The autophagy level of PC cells transfected with oe-HIF1α was increased, with reduced MHC-I expression on cells (membranes), which impaired the cytotoxicity of CD8 T cells, and thus decreasing the probability of recognition and attack by CD8 T cells when co-cultured with them. In mice, overexpression of HIF1α hindered the immune suppressive function of CD8 T cells and facilitated the immune escape of PC by reducing antigen presentation of MHC-I.Under hypoxia, HIF1α-induced autophagy reduces the cytotoxicity of CD8 T cells by repressing MHC-I expression.
缺氧微环境是实体瘤的一个基本特征。自噬在免疫调节中的作用一直存在争议。本项目旨在阐明特定条件（缺氧）下胰腺癌（PC）中的自噬对CD8 T细胞的影响及其背后的调控机制。通过蛋白质免疫印迹法（WB）和免疫荧光法（IF）分析HIF1α和自噬水平。使用KC7F2（HIF-1通道抑制剂）或氯喹（自噬抑制剂）在常氧或缺氧处理中评估HIF1α对细胞自噬的影响。将CD8 T细胞与PC细胞共培养，使用乳酸脱氢酶（LDH）和Hoechst/PI染色评估细胞毒性。通过酶联免疫吸附测定（ELISA）和流式细胞术测量细胞因子含量和CD8 T细胞的活化水平。使用定量逆转录聚合酶链反应（qRT-PCR）、WB、IF和流式细胞术分析PC细胞（细胞膜）中MHC-I的表达。应用人源化免疫重建小鼠研究HIF1α诱导的自噬对体内免疫的影响。当细胞处于缺氧状态时，与常氧条件相比，HIF1α和自噬水平更高。用KC7F2处理导致HIF1α和自噬水平与常氧状态相似。氯喹处理将自噬水平逆转至常氧状态。用oe-HIF1α转染的PC细胞自噬水平升高，细胞（细胞膜）上MHC-I表达降低，这损害了CD8 T细胞的细胞毒性，因此在与它们共培养时降低了被CD8 T细胞识别和攻击的概率。在小鼠中，HIF1α的过表达通过减少MHC-I的抗原呈递阻碍了CD8 T细胞的免疫抑制功能并促进了PC的免疫逃逸。在缺氧条件下，HIF1α诱导的自噬通过抑制MHC-I表达降低CD8 T细胞的细胞毒性。
