Conclusion
DCs transduced with LV-SMP30 can dramatically enhance specific CD3+CD8+ T cell immune responses against mouse hepatocarcinoma cells in vitro and in vivo. These findings lend significant support to the development of the DC-based SMP30 antigen vaccine for hepatocarcinoma immunotherapy.
Methods
A recombinant lentiviral vector (LV-SMP30) expressing SMP30 was constructed and transduced into DCs. The expression of SMP30 was detected by western blot. Mouse bone marrow-derived DCs were divided into four groups: LV-SMP30 group (transduced with LV-SMP30), Protein group (co-cultured with SMP30 protein), LV group (transduced with the empty vector) and Untreated group (the normal DCs). The effect of LV-SMP30 on DCs was detected through surface markers (CD123, CD11c, CD80 and CD86) and cytokine production. The activation and proliferation of CD3+CD8+ T cells were detected by CCK-8 kit. Flow cytometry was used to detect CD3+CD8+ T cell-mediated cytotoxicity. After construction of a mouse subcutaneous xenograft model, the volume and growth of tumors in different groups were observed. The changes in serum immune indexes in the treated groups were compared with those in the control group.
Results
The LV-SMP30 recombinant was constructed and transduced into DCs successfully, and LV-SMP30-transduced DCs stably expressed SMP30. The percentages of expression in the LV-SMP30 and Protein groups were significantly higher than those in the LV or Untreated groups (P<0.05). Meanwhile, after the DCs were cultured for 72 hours, the levels of IL-2, IL-6, IL-12, and IFN-γ were significantly higher in the LV-SMP30 and Protein groups than in the LV group or Untreated group (P<0.05). After the DCs were continuously cultured for one week, however, the cytokine levels in the LV-SMP30 group were significantly higher than those in the Protein group (P<0.05). In addition, CD3+CD8+ T cell proliferation and activation levels were substantially higher in the LV-SMP30 and Protein groups than in the LV or Untreated groups (P<0.05). Furthermore, as the ratio of effectors/target cells increasing in the LV-SMP30 group, CD3+CD8+ T cell-mediated cytotoxicity in H22 cells became higher (0:1, 10:1; 20:1; 40:1, respectively). In comparison to the control group, the cytotoxicity of the LV-SMP30 group was considerably increased at the ratios of 10:1, 20:1 and 40:1 (P<0.05). However, in the case of Hep1-6 cells, there was no significant difference in CD3+CD8+ T cell-mediated cytotoxicity among the groups. In addition, when compared with other groups, the mice in the LV-SMP30 group showed the most volume reduction, the slowest tumor growth, and the highest level of IL-2 and IFN-γ (P<0.05).