[Interleukin-12 over-expression in malignant melanoma B16 cells reduces programmed death-1 expression on T cells in mice with immune reconstitution].

OBJECTIVE: To investigate whether interleukin-12 (IL-12) over-expression in malignant melanoma B16 cells affects the expression level of programmed death-1 (PD-1) on T cells in mice during immune microenvironment reconstruction. METHODS: B16 cells were transfected with an IL-12 expression lentiviral vector, and IL-12 over-expression in the cells was verified qPCR and ELISA. Plate cloning assay was used to compare the cell proliferation activity between B16 cells and B16/IL-12 cells. The expression of IL-12 protein in B16/IL-12 cells-derived tumor tissue were detected by ELISA. C57BL/6 mice were inoculated with B16 cells or B16/IL-12 cells, and 14 days later the proportion of T cells with high expression of PD-1 in the tumor-draining lymph nodes was detected by flow cytometry. Mouse models of immune reconstitution established by 650 cGy X-ray radiation were inoculated with B16 (B16+RT group) or B16/IL-12 (B16/IL-12+RT group) cells, with the mice without X-ray radiation prior to B16 cell inoculation as controls. Tumor growth in the mice was recorded at different time points, and on day 14, flow cytometry was performed to detect the proportion of T cells with high PD-1 expression in the tumor-draining lymph nodes and in the tumor tissue. RESULTS: B16 cells infected with the IL-12-overexpressing lentiviral vector showed significantly increased mRNA and protein levels of IL-12 (P < 0.001) without obvious changes in cell viability (P>0.05). B16/IL-12 cells expressed higher levels of IL-12 than B16 cells in vivo (P < 0.01). In the tumor-bearing mouse models, the proportion of CD4 (+) PD-1(+) T cells was significantly lower in B16/IL-12 group than in B16 group (P < 0.01). In the mice with X-ray radiation-induced immune reconstitution, PD-1 expressions on CD4(+) T cells (P < 0.05) and CD8+ T cells (P < 0.01) were significantly higher in B16+ RT group than in the control mice and in B16/IL-12+RT group (P < 0.01 or 0.001); the tumors grew more slowly in B16/IL-12+RT group than in B16 + RT group (P < 0.001). CONCLUSIONS: During immune microenvironment reconstruction, overexpression IL-12 in the tumor microenvironment can reduce the percentage of PD-1 (+) T cells and suppress the growth of malignant melanoma in mice.