Objective: To design the suitable sequence siRNA of Foxp3 to interfere the function of regulatory T cells(Treg), and to evaluate whether the suppression of Treg could enhance the anti-tumor immune response in hepatocellular carcinoma patients or not.
Methods: Foxp3-specific siRNAs by chemical synthesis were delivered into regulatory T cells. The inhibition efficiencies of Foxp3-specific siRNAs were evaluated by real-time PCR and fluorescently stained for intracellular Foxp3 and analyzed using multiparameter FCM. The Foxp3(+) Treg subpopulation was selectively analyzed for surface expression levels of CD127, CTLA-4 and GITR. The suppression of Treg to CD4(+)CD25(-) T cells or anti-tumor specific CD8(+) T-cell responses induced by tumor specific antigen (NY-ESO-1b) was evaluated by CFSE, Elispot and Pentamer analysis.
Results: A subpopulation of Tregs with reduced levels of Foxp3 mRNA and protein mediated by siRNA was CD127 up-regulation and CTLA-4 or GITR down-regulation compared with those in Foxp3(high) Tregs. Knockdown of intracellular Foxp3 in Treg reduced suppression of Foxp3+ Treg to proliferative capacity of CD4(+)CD25(-) T cells. IFN-gamma released of NY-ESO-1-specific CD8(+) T cells from the group of Tregs with Foxp3 specific siRNA transfected was increased as compared with the group of Treg with non-specific control siRNA transfected(132+/-55 vs 27+/-11, P<0.05). Frequency of NY-ESO-1b-specific CD8(+) T cells by Pentamer analysis from the group of Tregs with Foxp3 specific siRNA transfected was increased as compared with the group of Treg with non-specific control siRNA transfected (0.21%+/-0.17% vs 0.57%+/-0.39%, P<0.05).
Conclusion: Knockdown of intracellular Foxp3 in Treg mediated by siRNAs can inhibit the suppression of Foxp3+ Treg and enhance the anti-tumor immune response in hepatocellular carcinoma patients in vitro.