| Background and ObjectiveRegulatory T(Treg)cells play a crucial role in immunological self-tolerance and suppression on effector cells in an effort to evade the immune system.In general,NK cells have high antitumor activity with no prior stimulation,function as the major immunosurveillance component.NKG2D is an NK cell receptor that mediates the NK cell cytotoxicity by recognizing and binding to NKG2D ligands(NKG2DLs),which are upregulated within the context of cancer.Studies have been reported that the percentage of CD4 CD25+ Treg cells is negatively correlated with NK cell cytotoxicity in metastatic melanoma patients as well as with the expression of NKG2D in peripheral blood.Furthermore,CD4 CD25+ Treg cells may inhibit NKG2D-mediated NK cell cytotoxicity in mice with melanoma and in human squamous cell carcinoma in vitro.Our previous studies showed that the number of CD4+CD25+Treg cells was significantly increased in the peripheral blood of patients with colorectal cancer but that NKG2D expression was significantly decreased.Nonetheless,whether CD4+CD25+ Treg cells can suppress NK cytotoxicity via downregulating NKG2D expression in peripheral blood and the potential underlying mechanism underlying are not well understood.Various cytokines,such as TGF-? and IL-10,and a cell contact-dependent mechanism mediate the suppressive effect of Treg cells.TGF-? is regarded as an inhibitory cytokine that regulates Treg-mediated suppression.In cervical cancer patients and a mouse melanoma model,Treg cells can abrogate NK cell cytotoxicity via the TGF-? pathway,and IL-10,a key anti-inflammatory cytokine produced by activated Treg cells,may modulate NKG2D ligand expression on melanoma cells and NK cell cytotoxicity in vitro.Surface molecules on Treg cells,such as TGF-?,directly bind to corresponding receptors on the target cell,and studies have shown that CD4+CD25+Treg cells inhibit the cytotoxic activity of NK lymphocytes via direct cell-to-cell interactions in vitro.However,the exact mechanism by which CD4+CD25+Treg cells inhibit NKG2D on NK cells(i.e.,via cell-to-cell interaction or upregulated TGF-? and IL-10 production)remains unclear.We herein aimed to explore whether CD4+CD25+ Treg cells suppress NKG2D-mediated NK cell cytotoxicity in peripheral blood and elucidate the exact mechanism underlying this phenomenon.Methods1.Regulatory function study of NKG2D:Anti-NKG2D mAbs or control IgG was added to purified NK cells and targeted cells(HT29 cells or K562 cells)cocultures to block NKG2D expression on NK cell surface.NK cell cytotoxicity(detected by non-radioactive cytotoxicity assay),IFN-y(detected by ELISA)and CD107a(detected by flow cytometry)expression were performed to assess NK cell activity of anti-NKG2D group and control group.2.Regulatory Function study of TGF-? and IL-10:TGF-?-,IL-10-blocking antibody or control IgG was added to purified CD4+CD25+Treg cells,purified NK cells and targeted cells(HT29 cells or K562 cells)co-cultures(defined as anti-TGF-? group,anti-IL-10 group and control IgG group).NK cell cytotoxicity,IFN-? and CD107a expression were evaluated after 24 hours of co-culture,meanwhile,NKG2D expression on the NK cell surface were detected by flow cytometry.3.Cell-contact mechanism study:CD4+CD25+ Treg cells were placed in the upper chamber,while NK cells and targeted cells(HT29 cells or K562 cells)were in the lower chamber(Transwell group),meanwhile,CD4+CD25+ Treg cells,NK cells and targeted cells(HT29 cells or K562 cells)were placed together in the lower chamber in Transwell control group.NK cytotoxicity and NKG2D expression were measured after 24 hours of co-culture.Results1.Decreased NKG2D expression downregulates NK cell activity.NKG2D expression in anti-NKG2D group was significantly lower than that in the control group(HT29:51.37±3.93%vs 21.13±2.32%,p=0.003;K562:42.66±3.54%vs 25.59±2.56%,p=0.021),indicating the anti-NKG2D mAbs worked.NK cell cytotoxicity and IFN-y concentration in anti-NKG2D group were significantly lower than that in the control group(HT29:P=0.003,0.003;K562;P=0.001,0.015).CD 107a expression in anti-NKG2D group was significantly decreased,and it was statistically different from the control group(HT29:32.40±3.33%vs 19.37±2.59%,p=0.037;K562:26.95±1.69%vs 19.70±0.99%,p=0.021).2.Blocking TGF-? increases NKG2D expression on NK cells downregulated by CD4+CD25+Treg cells.TGF-? concentration in anti-TGF-? group was significantly lower than that in the control IgG group.It was indicated that TGF-? expression was inhibited.NK cell cytotoxicity and IFN-y concentration in anti-TGF-? group were significantly higher than that in control IgG group,and the difference was statistically significant(HT29:p =0.01,<0.001;K562:p=0.001,0.002).CD107a expression in anti-TGF-? group was significantly increased,and it was statistically different from the control IgG group(HT29:12.29±2.02%vs 5.31±0.47%,p=0.028;K562:17.29±0.70%vs 8.32±0.82%,p=0.001).Meanwhile,NKG2D expression in anti-TGF-? group was significantly increased,and it was statistically different from the control IgG group(HT29:19.57±1.59%vs 12.80±1.33%,p=0.031;K562:22.84±1.82%vs 13.85?1.43%,p=0.01 8)?3.Blocking IL-10 enhances NKG2D expression on NK cells downregulated by CD4+CD25+ Treg cells.IL-10 production in anti-IL-10 group was lower than that in control IgG group which indicated the anti-IL-10 mAbs worked.NK cytotoxicity was upregulated in anti-IL-10 group,and NK cells exhibited increased IFN-? production,which was statistically different from the control IgG group(HT29:p=0.08,<0.0001;K562:p=0.001,p=0.030).NKG2D expression in anti-IL-10 group was higher than that in control IgG group(HT29:18.97±1.00%vs 12.80±1.33%,p=0.02;K562:18.14±0.32%vs 13.85±1.43%,p=0.043).4.CD4+CD25+ Treg cells inhibit NKG2D expression on NK cells via a cell contact-dependent mechanism.Transwell experiments showed that IFN-y concentration in Transwell group was higher than that in Transwell control group(HT29:p=0.022;K562:p=0.005).CD107a expression was significantly increased in Transwell group,which was statistically different from Transwell control group(HT29:5.94±0.61%vs 3.43±0.40%,p=0.025;K562:10.37±0.88%vs 6.05±1.07%,p=0.036).And NKG2D expression in Transwell group was higher than that in Transwell control group(HT29:17.27±1.11%vs 11.10±0.80%,p=0.011;K562:16.1 8±0.29%vs 11.0±1.40%,p=0.022).ConclusionThe activating receptor NKG2D mediates NK cell cytotoxicity in peripheral blood.CD4+CD25+ Treg cells suppress NKG2D-mediated NK cell cytotoxicity via a cell contact-dependent mechanism and increased TGF-? and IL-10 production. |
PDF Full Text Request