The Effect Of T-reg On The Anti-breast Cancer Cell Induced By NK Cells In Vitro

Objective To analyze the expression of T-reg in peripheral blood from patients with breast cancer and its relationship with NK cell and the subset of T cell; explore the effect of T-reg cell on cytotoxicity of NK cell against breast cancer cell in vitro and analyze the possible underlying mechanism; explore the role of NKG2D-MICA on cytotoxicity of NK cell against breast cancer cell; observe the effect on the NKG2D-MICA by T-reg cell.Methods 1. Flow cytometry(FCM)were evaluated the proportion of T-reg,NK cell and the subset of T cell;and detect the expression of Membrane-bound TGF-β1 on T-reg. 2. LDH assay was manipulated to test the cytotoxicity of NK cell on breast cancer cells.3. ELISA was performed to examine the level of IFN-γsecreted by NK cell in supernatant and TGF-β1 secreted by T-reg cell.4. RQ-PCR was used to identify the expression of MICA-mRNA on breast cancer cell lines. 5. FCM were performed to identify the expression of MICA on the cell surface and intracell of breast cancer cell lines,Immunohistochemistry(IH) and immunofluorescence (IF) were used to detect the distribution of MICA on breast cancer cell lines. 6. FCM were used to detect the expression of NKG2D on NK cells.Results 1. The expression of T-reg in patients with breast cancer:The proportion of T-reg in patients with breast cancer was higher than that of volunteers ((7.5±3.0)% Vs(5.1±1.5)% ) P<0.01. 2. The effect of T-reg on cytotoxicity of NK cells:when the ratio of NK and breast cancer cell was 10:1,the cytotoxicity of NK cell was (40.7±3.0)%,(15.5±3.1)%,(29.7±1.8)% and (59.1±2.3)% on MCF-7,MDA-MB-435s,SKBr-3 and MDA-MB-231 respectively;the cytotoxicity of NK cell decreased to(1.9±0.2)%,(5.7±0.4)%,(3.6±2.8)% and(3.9±0.2)% respectively while co-culture with 1/5 T-reg cell;and (7.6±1.6)%,(7.3±1.0)%,(6.6±2.5)% and(19.7±1.1)% respectively while co-culture with 1/10 T-reg cell.3.ELISA detect IFN-γsecreted by NK cell display: when the ratio of NK and breast cancer cell was 10:1, the concentration of IFN-γwas (221.5±2.6)pg/ml,(264.4±5.5)pg/ml,(754.6±19.0)pg/ml and(423.0±14.6)pg/ml on MCF-7,MDA-MB-435s,SKBr-3 and MDA-MB-231 respectively; the concentration of IFN-γdecreased to (70.7±8.5)pg/ml,(14.6±0.9)pg/ml,(128.3±4.0)pg/ml and(6.0±1.0)pg/ml respectively while co-culture with 1/5 T-reg cell;the concentration of IFN-γchange to(216.6±4.8)pg/ml,(251.7±2.2)pg/ml,(535.0±8.5)pg/ml and(52.57±3.4)pg/ml respectively while co-culture with 1/10 T-reg cell.4. ELISA detect TGF-β1 secreted by T-reg cell in the supernatant display: when the ratio of NK and breast cancer cell was 10:1, the concentration of TGF-β1 was(13.4±0.2)pg/ml,(11.3±0.8)pg/ml,(62.9±1.3)pg/ml and(6.2±0.5)pg/ml on MCF-7,MDA-MB-435s,SKBr-3 and MDA-MB-231 respectively; the concentration of TGF-β1 increased to (27.2±0.3)ng/ml,(12.2±1.0)ng/ml,(77.6±1.0)ng/ml and(9.1±0.1)ng/ml respectively while co-culture with 1/5 T-reg cell; the concentration of TGF-β1 increse to(46.8±1.6)ng/ml,(15.8±0.9)ng/ml,(87.8±2.3)ng/ml and(10.9±0.6)ng/ml respectively while co-culture with 1/10 T-reg cell.5.The distribution and expressin of MICA:(1)RQ-PCR discover: Relative content of MICA mRNA of MDA-MB-231,SKBr-3,MCF-7 and MDA-MB-435s was 5.56×10-3,2.53×10-3,1.68×10-3and 5.99×10-5 respectively;(2) MICA was not detected on the surface of MDA-MB-435s and SKBr-3,(61.5±4.5)% on MDA-MB-231 and(56.5±4.7)% on MCF-7 by FCM;after cell perforating,the percentage of MICA was (26.4±2.5)% and(20.7±1.3)% on the MDA-MB-435s and SKBr-3 respectively, but the percentage of MICA had not changes for the MDA-MB-231 and MCF-7; (3)Immunohistochemistry(IH) expreriments confirm that MICA express on the membrane and in the cytoplasm of the MDA-MB-231 and SKBr-3, MICA express in the cytoplasm and nuclear membrane of the MCF-7, and in the cytoplasm of the MDA-MB-435s;Immunofluorescence(IF) expreriments confirm that MICA express on the membrane and in the cytoplasm of the MDA-MB-231 and SKBr-3, MICA express on the membrane of the MCF-7,and in the cytoplasm of the MDA-MB-435s.With the different location of MICA,cytotoxicity of NK cells is different.Conclusion T-reg cells may inhibit the cytotoxicity of NK cell, which was associated with the secretion of TGF-β1. The membrane MICA could increase the cytotoxicity of NK cell on breast cancer cell.