Objective: To employ the technology of interfering RNA (RNAi) to identify the role of Foxp3 in the in vitro suppressive effect of human regulatory T cell (Treg) on effector T cells.
Methods: Expanded human Treg were transfected with siRNA targeting Foxp3 genes. The transfection efficiency, the level of corresponding gene and its protein expression were measured by fluorescent microscopy, fluorescence-activated cell sorting (FACS), real-time PCR and Western blot respectively. The phenotypes of Treg were analyzed by FACS. The siRNA transfected Treg was then co-cultured with porcine PBMC or human PBMC-stimulated autologous CD4+CD25- T cells. Their proliferations were examined by WST-1. Treg and autologous CD4+CD25- T cell-related suppressive cytokines were assessed by ELISA.
Results: A 68% transfection efficiency in expanded Treg was achieved for Foxp3 siRNA. Real-time PCR revealed a 61.4% mRNA knockdown induced by siRNA targeting Foxp3 genes in Treg versus the control (P < 0.01). Some Treg-associated surface markers were significantly altered versus the control. And the production of suppressive cytokines was lowered. These changes were correlated with the diminished Treg activity in suppressing the proliferation of effector CD4+CD25- T cells. There was 83% suppression by non-transfected Treg vs 48% suppression by Foxp3 siRNA transfected Treg in xeno-immune response (P < 0.05); and 65% suppression by non-transfected Treg vs 48% suppression by Foxp3 siRNA transfected Treg in allo-immune response (P < 0.01).
Conclusion: Foxp3 is a key intracellular marker for maintaining the phenotypes and functions of Treg.