Reciprocal Immunoediting Between Allogeneic NK Cells And Human Epithelial Ovarian Cancer Cells

Objectives:we will build the immunoediting model of NK cells andepithelial ovarian cancer.we planned to investigate the expression of NKG2Dand MICA,and then discover the effect and significance of immunoedting toNK and cancer cells.At the same time,to discuss the clinical significance ofNKG2D-NKG2DL on anti-tumor immunityMethods:1Human ovarian cancer cell SKOV3and SKOV3/CDDP resuscitativeand culture,observed cells morphological by inverted microscope2Test the IC50of the drug-resistant ovarian cancer cell SKOV3/CDDPto SKOV3through MTT,and calculate resistant index3NK cells were obtained from10heathy persons’ peripheral bloodmononuclear cells(PBMCs) by CD56antibody magnetic isolation.The purityof NK cells is measured by flow cytometry.Two groups were designed intonon-edited Allo-NK cells group(obtained by cultivated with100U/m1rhIL-2for24h),edited Allo-NK cell group(obtained by mixed with SKOV3cells orSKOV3/CDDP cells at the effector-to-target ratio of10:1with100U/m1rhIL-2for4h,24h,48).Build the immunoediting model in vitro4Isolate the RNA and protein of the4groups of SKOV3cells andSKOV3/CDDP cells.Measure the expression of the MICA,which express onovarian cell surface,at the mRNA and protein level by real-time RT-PCR andwestern blot5Expression of the active receptor NKG2D of NK cells was analyzed byflow cytometryResult:1The IC50for cisplatin of SKOV3and SKOV3/CDDP was13.78and47.68mg/L.The resistance multiple was3.46 2Purity of CD3-CD16+CD56+cells isolated from10healthy persons bymagnetic bead selection is (91.07士2.18)%3Expression of MICA mRNA on the surface of SKOV3cells andSKOV3/CDDP cells at different time of immunoediting.Take2-ΔΔCTrepresentsrelative quantity of MICA and make the nonedited SKOV3cells’ expressionas contrast.The expression mass of0h,4h,24h,48h group as follows:1.00±0.00,1.40±0.16,2.25±0.37,3.02±0.10;the difference between groups wassignificant (F=57.210,P=0.000<0.05),and the difference within groups wasalso significant.similarly,to SKOV3/CDDP cells,the expression of betweengroups was obvious different at different time points of immunoediting(0.067±0.011,0.065±0.004,0.048±0.009,0.042±0.007,F=7.3717,P=0.011<0.05)However, within group comparison,there were no statistical differences in0hwith4h groups as well as24h with48h groups.Our experiment shows:MICAmRNA increased in edited SKOV3cells,and increased in SKOV3/CDDP after24h of immunoedting4Expression of MICA protein on the surface of SKOV3cells andSKOV3/CDDP cells at different time of immunoediting.Take gray value ofMICA/beta-tubulin represents relative quantity of MICA.The expression massof0h4h24h48h group as follows:0.202±0.017,0.170±0.010,0.106±0.005,0.094±0.005;the difference between groups was significant(F=71.410,P=0.000<0.05).However,there was no statistical differences in0h with4h groupswithin group comparison.Similarly,to SKOV3/CDDP cells,the expression ofbetween groups was obvious different at different time points of immu-noediting (0.067±0.011,0.065±0.004,0.048±0.009,0.042±0.007,F=7.3717,P=0.011<0.05).However,within group comparison,there were no statisticaldifferences in0h with4h groups as well as24h with48h groups.Ourexperiment shows:compare to MICA mRNA,MICA protein decreased inedited SKOV3cells,and decreased in SKOV3/CDDP after24h of immuno-editing5Expression of the active receptor NKG2D of NK cells through flowcytometry:in the groups that immunoediting with SKOV3cells,there was statistical differences of the between groups comparison (F=117.454,P=0.00<0.05).Meanwhile,in the groups that immunoediting with SKOV3/CDDP cells,there was statistical differences between group comparison(F=82.406，P=0.00<0.05).Conclusion:Our findings unveiled that the existence of reciprocal-immunoed-iting between Allo-NK cells and SKOV3cells and SKOV3/CDDPcells,which basic is the NKG2D-NKG2DL signal pathways and specificity ofligand-receptor engagement.That is the decrease expression of MICA andNKG2D of edited cells.Furthermore confirmed that regulating this signalpathways could boost edited Allo-NK cells function and enhance sensitivity ofedited SKOV3cells or SKOV3/CDDP cells to Allo-NK cells cytotoxicity.