Glutamine deprivation impairs function of infiltrating CD8(+ )T cells in hepatocellular carcinoma by inducing mitochondrial damage and apoptosis

BACKGROUND: The functions of infiltrating CD8(+ )T cells are often impaired due to tumor cells causing nutrient deprivation in the tumor microenvironment. Thus, the mechanisms of CD8(+ )T cell dysfunction have become a hot research topic, and there is increased interest on how changes in metabolomics correlate with CD8(+ )T cell dysfunction. AIM: To investigate whether and how glutamine metabolism affects the function of infiltrating CD8(+ )T cells in hepatocellular carcinoma. METHODS: Immunohistochemical staining and immunofluorescence were performed on surgically resected liver tissues from patients. Differentially expressed genes in infiltrating CD8(+ )T cells in hepatocellular carcinoma were detected using RNA sequencing. Activated CD8(+ )T cells were co-cultured with Huh-7 cells for 3 d. The function and mitochondrial status of CD8(+ )T cells were analyzed by flow cytometry, quantitative real-time polymerase chain reaction, and transmission electron microscopy. Next, CD8(+ )T cells were treated with the mitochondrial protective and damaging agents. Functional alterations in CD8(+ )T cells were detected by flow cytometry. Then, complete medium without glutamine was used to culture cells and their functional changes and mitochondrial status were detected. RESULTS: There were a large number of infiltrating PD-1(+)CD8(+ )T cells in liver cancer tissues. Next, we co-cultured CD8(+ )T cells and Huh-7 cells to explore the regulatory effect of hepatoma cells on CD8(+ )T cells. Flow cytometry results revealed increased PD-1 expression and decreased secretion of perforin (PRF1) and granzyme B (GZMB) by CD8(+ )T cells in the co-culture group. Meanwhile, JC-1 staining was decreased and the levels of reactive oxygen species and apoptosis were increased in CD8(+ )T cells of the co-culture group; additionally, the mitochondria of these cells were swollen. When CD8(+ )T cells were treated with the mitochondrial protective and damaging agents, their function was restored and inhibited, respectively, through the mitochondrial damage and apoptotic pathways. Subsequently, complete medium without glutamine was used to culture cells. As expected, CD8(+ )T cells showed functional downregulation, mitochondrial damage, and apoptosis. CONCLUSION: Glutamine deprivation impairs the function of infiltrating CD8(+ )T cells in hepatocellular carcinoma through the mitochondrial damage and apoptotic pathways.