Exosomes derived from hepatocellular carcinoma inhibit CD4+ and CD8+ T cell function through the PD-1/PD-L1 pathway

T cell function is significantly inhibited in hepatocellular carcinoma (HCC), although T cells play an important role in killing cancers. To illustrate the mechanisms of T cell inhibition in liver cancer, CD4(+) and CD8(+) T cell quantity and function were investigated in our research. First, we counted the numbers of CD4(+) and CD8(+ )T cells in HCC peripheral blood by flow cytometry and found that CD4(+) and CD8(+) T cells were not obviously decreased compared to levels found in healthy volunteers. However, we found that the secretion of IFN-gamma from CD4(+) and CD8(+) T cells, an important factor for T cell activation, was significantly decreased compared to the control group. Furthermore, we wondered whether CD4(+) and CD8(+) T cell suppression was mediated by hepatocellular carcinoma exosomes. Exosomes were successfully isolated from Huh-7 cell supernatant and verified through NanoSight analysis and transmission electron microscopy (TEM). CD4(+)PD-1(-) and CD8(+)PD-1(- )T cells were sorted from the peripheral blood of healthy volunteers by flow cytometry and cultured with RPMI-1640 medium. Then, CD4(+)PD-1(-) and CD8(+)PD-1(-) T cells were treated with HCC exosomes, and Western blot assays were used to discover whether levels of programmed cell death protein 1 (PD-1), a negative modulator of T cell response, were upregulated on these T cells. To examine how HCC exosomes are involved in CD4(+) and CD8(+) T cell inhibition, we researched whether PD-L1, the ligand for the PD-1 protein, was upregulated in HCC exosomes using a Western blot assay. Our experiments indicate that exosomes containing PD-L1 derived from hepatocellular carcinoma might induce PD-1 upregulation, resulting in CD4(+) and CD8(+) T cell suppression.