| Objectives To investigate the regulatory effect of glutamine(Gln)on the function of cytotoxic T lymphocytes(CTL)and its mechanism in order to further broaden the ideas of clinical liver cancer diagnosis and treatment.Methods Immunohistochemical and immunofluorescence staining was performed on surgically resected liver tissues from patients;human CTL cells were sorted from venous blood and cultured in vitro,induced and activated by CD3,CD28 and IL-2 and co-cultured with hepatoma cell Huh-7;the differential expression of CTL cell genes was detected by RNA sequencing;murine CTL cells cultured in vitro were induced and activated by CD3,CD28 and IL-2 and co-cultured with hepatoma cell Hepa1-6;Proteins were extracted and Western blot was used to detect the expression of programmed cell death protein 1(PD-1),T cell immunoglobulin and mucin domain-containing protein 3(TIM-3),glucose-regulated protein 78(GRP78),protein kinase RNA-like endoplasmic reticulum kinase(PERK),glutaminase(GLS)and solute carrier 1 member 5(SLC1A5);the concentrations of Granzyme B,perforin and Gln in the cell culture supernatant were detected by ELISA and colorimetry;the morphology of endoplasmic reticulum was observed by transmission electron microscopy;endoplasmic reticulum stress inhibition assay,Gln deprivation assay and glutaminase inhibition assay were performed to detect ER stress and changes in cell function.Statistical data were analyzed using independent sample t-test and one-way ANOVA,and SPSS 21.0 was used to analyze the data,and P<0.05 was considered statistically significant.Results 1 Immunohistochemistry and immunofluorescence showed that a large number of PD-1~+CD8~+T cells were expressed in liver cancer tissues.2 RNA-seq results showed that the expression of genes involved in ER-related responses and regulation by external stimulus was up-regulated in CD8~+T cells co-cultured with hepatoma cells;the expression of genes involved in cell proliferation and cell secretion was down-regulated.3 The expression of PD-1 and TIM-3 in CTLL-2 cells co-cultured with Hepa1-6 was remarkably increased;the secretion of Granzyme B and perforin was significantly reduced;meanwhile,the expression of GRP78 and P-PERK was significantly increased(P less than 0.05),and the endoplasmic reticulum was obviously swollen.4 4-PBA inhibition experiments showed that the expression of PD-1 and TIM-3 was decreased after ER stress was inhibited;however,the secretion of Granzyme B and perforin was increased(P less than 0.05).5Colorimetric results showed that the co-culture group consumed more Gln;after Gln deprivation,the expression of PD-1 and TIM-3 was increased and the secretion of Granzyme B and perforin was decreased in CTLL-2 cells;meanwhile,the expression of GRP78 and P-PERK was increased and the endoplasmic reticulum was significantly swollen;while,under the costimulation of 4-PBA and Gln deprivation,the amount of GRP78 and P-PERK proteins was decreased(P are less than 0.05),and the significantly swollen endoplasmic reticulum was improved.6 The expression of GLS2 was significantly lower in both CTLL-2 cells co-cultured with Hepa1-6 and deprived of Gln.However,GLS1 of Hepa1-6 deprived of Gln was significantly decreased(P are less than 0.05).7There was no significant difference in the expression of PD-1,TIM-3,Granzyme B,perforin,GRP78 and P-PERK in CTLL-2 cells treated with CB 839(P are more than 0.05);while the expression of PD-1 and TIM-3 in CTLL-2 cells treated with Compound 968 was increased,and the secretion of Granzyme B and perforin was decreased;meanwhile,the expression of GRP78 and P-PERK proteins was increased(P values are less than 0.05),and the endoplasmic reticulum was significantly swollen.Conclusions Glutamine deprivation decreases the function of hepatocarcinoma-infiltrating CTL cells through the GLS2-ER stress pathway.Figure 21;Table 1;Reference 163... |
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