OAZI-1 overexpression in mouse B16-F1 melanoma cells can effectively stimulate DC immune activity / 中国免疫学杂志

ABSTRACT

Objective:To analyze, at cellular level, whether the mouse B16-F1 melanoma cells with OAZI-1 overexpression could activate antigen-presenting cells and promote the phagocytotic and antigen-presenting efficiencies of mouse peritoneal macrophage and bone marrow derived DC on tumor cells. Methods:The plasmid pcDNA3. 1(+)/OAZI-1 was transfected into B16-F1 cells by Li-pofectamine2000 reagent. The positive clones with OAZI-1 overexpression ( B16/OAZI-1 ) were identified by Western blot assay and RT-PCR. Macrophages from abdominal cavity and DC from bone marrow were collected from BALB/c mouse. The B16-F1 cells transfected with the pcDNA3. 1(+) (B16/3. 1) were used as the control cells in this experiment. B16-F1 cells and macrophages were co-cultured for 4 h at a 1∶5 ratio and DC were co-cultured with B16-F1 cells at 1∶1 ratio for 4 h. And then the phagocytotic efficiencies were assayed by flow cytometry. DC were co-cultured with B16-F1 cells at 1∶1 ratio for 24 h and then the expression of mature DC surface marker molecules CD40,CD80,CD86 were determined by flow cytometry. The DC activated by the tumor cells were co-cultured with mouse spleen lymphocytes for 24 h, and then IFN-γ content in culture medium was analyzed by ELISA. Results: Phagocytotic assay showed that,compared to the control cells,the OAZI-1 overexpression in B16-F1 cells significantly enhanced the engulfment of B16-F1 cells by macrophages ( 24. 7% vs 53. 9% ) and DC ( 8. 2% vs 13. 8%) . When DC were co-incubated with OAZI-1 overexpressed B16-F1 for 24 h,the expression levels of CD40,CD80,CD86 on the DC surface,which were the molecular markers for matured DC,increased from 24. 2%,20. 8% and 16. 4% to 46. 8%,32. 5% and 36. 1% respectively. Co-culture of tumor-activated DC with the spleen lymphocytes resulted in an increased IFN-γcontent in the culture medium(32. 9 pg/ml vs 15. 1 pg/ml). Conclusion:The tumor cells with OAZI-1 overexpression can be engulfed more efficiently by macrophages and DC. And this process can induce the maturation and activation of DCs. Matured DC could induce T cell activation and then activate the anti-tumor immune response.