Geng Xu, Li Ming, Cui Bin, Lu Chao, Liu Xiaowen, Zhang Peng, Liu Bin, Ma Chunyan, Shen Yajuan, Lu Zhiming
Department of Clinical Laboratory.
Department of Central Laboratory.
Medicine (Baltimore). 2019 May;98(22):e15722. doi: 10.1097/MD.0000000000015722.
Studies have shown that CD4CD25Foxp3Treg cells suppress NKG2D expression on NK cells via a cell contact-dependent mechanism and increased TGF-β and IL-10 production in some cancer models. We herein aimed to explore whether CD4CD25Foxp3Tregs suppress NKG2D-mediated NK cell cytotoxicity in peripheral blood and elucidate the exact mechanism underlying this phenomenon.
To explore the function of NKG2D, NK cell cultures were treated with an NKG2D-blocking antibody to block these receptors. Additionally, TGF-β- and IL-10-blocking antibodies were added to NK and CD4CD25Foxp3Treg cell cocultures to evaluate whether the latter cells suppress NKG2D expression of NK cells via increasing the production of TGF-β 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-γ production and a flow cytometry assay to evaluate CD107a expression.
Blocking NKG2D decreased NK cell cytotoxicity, IFN-γ production and CD107a expression. Moreover, blocking TGF-β and IL-10 substantially increased the NKG2D expression in NK and CD4CD25Foxp3Treg cell cocultures. Similarly, blocking TGF-β and IL-10 enhanced NK cell cytotoxicity, IFN-γ production and CD107a expression; Transwell insert assays also revealed increased IFN-γ production and CD107a and NKG2D expression.
CD4CD25Foxp3Tregs suppress NKG2D-mediated NK cell cytotoxicity in peripheral blood via a cell contact-dependent mechanism and increased TGF-β and IL-10 production.
研究表明，CD4⁺CD25⁺Foxp3⁺调节性T细胞（Treg细胞）通过细胞接触依赖性机制抑制自然杀伤细胞（NK细胞）上NKG2D的表达，并在一些癌症模型中增加转化生长因子-β（TGF-β）和白细胞介素-10（IL-10）的产生。我们在此旨在探讨CD4⁺CD25⁺Foxp3⁺Treg细胞是否抑制外周血中NKG2D介导的NK细胞细胞毒性，并阐明这一现象背后的确切机制。
为了探究NKG2D的功能，用NKG2D阻断抗体处理NK细胞培养物以阻断这些受体。此外，将TGF-β和IL-10阻断抗体添加到NK细胞与CD4⁺CD25⁺Foxp3⁺Treg细胞的共培养物中，以评估后一种细胞是否通过增加TGF-β和IL-10的产生来抑制NK细胞的NKG2D表达。通过三色流式细胞术检测NK细胞上NKG2D的表达，并通过三种测定法评估NK细胞活性：非放射性细胞毒性测定法、测量IFN-γ产生的酶联免疫吸附测定法（ELISA）和评估CD107a表达的流式细胞术测定法。
阻断NKG2D降低了NK细胞的细胞毒性、IFN-γ产生和CD107a表达。此外，阻断TGF-β和IL-10显著增加了NK细胞与CD4⁺CD25⁺Foxp3⁺Treg细胞共培养物中NKG2D的表达。同样，阻断TGF-β和IL-10增强了NK细胞的细胞毒性、IFN-γ产生和CD107a表达；Transwell小室测定法也显示IFN-γ产生以及CD107a和NKG2D表达增加。
CD4⁺CD25⁺Foxp3⁺Treg细胞通过细胞接触依赖性机制以及增加TGF-β和IL-10的产生来抑制外周血中NKG2D介导的NK细胞细胞毒性。
