CD4+CD25+Foxp3+ regulatory T cells suppress NKG2D-mediated NK cell cytotoxicity in peripheral blood

Background: Studies have shown that CD4(+)CD25(+)Foxp3(+)Treg cells suppress NKG2D expression on NK cells via a cell contactdependent mechanism and increased TGF-beta and IL-10 production in some cancer models. We herein aimed to explore whether CD4(+)CD25(+)Foxp3(+)Tregs suppress NKG2D-mediated NK cell cytotoxicity in peripheral blood and elucidate the exact mechanism underlying this phenomenon. Methods: To explore the function of NKG2D, NK cell cultures were treated with an NKG2D-blocking antibody to block these receptors. Additionally, TGF-beta- and IL-10-blocking antibodies were added to NK and CD4(+)CD25(+)Foxp3(+)Treg cell cocultures to evaluate whether the latter cells suppress NKG2D expression of NK cells via increasing the production of TGF-beta and IL-10. The expression of NKG2D on NK cells was detected by 3-color flow cytometry, and NK cell activity was assessed by 3 assays: a nonradioactive cytotoxicity assay, an ELISA measuring IFN-gamma production and a flow cytometry assay to evaluate CD107a expression. Results: Blocking NKG2D decreased NK cell cytotoxicity, IFN-gamma production and CD107a expression. Moreover, blocking TGF-beta and IL-10 substantially increased the NKG2D expression in NK and CD4(+)CD25(+)Foxp3(+)Treg cell cocultures. Similarly, blocking TGF-beta and IL-10 enhanced NK cell cytotoxicity, IFN-gamma production and CD107a expression; Transwell insert assays also revealed increased IFN-gamma production and CD107a and NKG2D expression. Conclusion: CD4(+)CD25(+)Foxp3(+)Tregs suppress NKG2D-mediated NK cell cytotoxicity in peripheral blood via a cell contact-dependent mechanism and increased TGF-beta and IL-10 production.