ABSTRACT
Objective To investigate the regulatory effect of CsA on the expression of NK cell inhibitory receptor ILT4 and cytotoxicity of NK cells.Methods NK cells treated with CsA ( 10 mg/L) or DMSO for 12,24 and 36 h were chosen as three experimental groups and control groups respectively.RTqPCR and flow cytometry were performed to detect the alteration of ILT4 at the mRNA and protein level respectively.The expression of HLA-G in human gastric cancer cell line BGC-823 and human placental choriocarcinoma cell line JEG-3 were measured at the same time,and then the cytolytic activity of the untreated NK cells and NK cells treated with CsA for 36 h against BGC-823 and JEG-3 cells was determined with MTT.One-way analysis of variance was employed to compare the different ILT4 expression at different time points after medication; Dunnett test was performed to carry out the pairwise comparison between each mean.The difference of HLA-G expression between JEG-3 cells and BGC-823 cells,and the difference of NK cell cytolytic activity against JEG-3 cells and BGC-823 cells were analyzed by student's t-test.Results RT-qPCR assay indicated that the relative levels of ILT4 mRNA in NK cells treated with CsA for 12,24 and 36 h in turn were 0.99 ± 0.27,1.79 ± 0.29,6.79 ± 0.64,and those of their contrast groups treated with DMSO were 0.86 ±0.11,0.94 ±0.12,1.06 ±0.17.The expression of ILT4 in NK cells treated with CsA for 24 h or 36 h was higher than that in NK cells of their contrast groups respectively ( t value of 4.69,14.99,P ＜0.05,respectively),but there was no significant difference between the two groups of NK cells treated for 12 h ( t =0.78,P ＞0.05 ).Through flow cytometry,the positive rates of ILT4 protein expression in NK cells treated with CsA for 12,24 and 36 h [(5.16 ± 0.42 ) ％,( 6.23 ± 0.48 ) ％,( 23.8 ± 1.5 ) ％]were higher than those in NK cells after treatment with DMSO for 12,24 and 36 h respectively[(3.08 ±0.19)％,(3.35 ±0.12)％,(3.36 ±0.21 )％ ;t value of 7.70,10.06,20.72,P＜0.01,respectively].The expression of ILT4 in NK cells treated for 36 h was much higher than that in NK cells for 12 and 24 h at the mRNA and protein level (t value of 16.38,14.12 ;21.81,20.56,P ＜ 0.01,respectively).Meanwhile the killing rates of NK cells treated with 10∶1 effector-target ratio CsA on BGC-823 cells (low HLA-G expression) were ( 8 1.96 ± 2.80 ) ％ ( before treatment) and ( 60.23 ± 1.57 ) ％ ( after treatment),which were higher than those on JEG-3 cells (HLA-G-overexpression) [(53.46 ±2.21 )％ ( before treatment),(28.30 ± 1.85 ) ％ ( after treatment)].The changes of cytotoxicity of NK cells treated with CsA against target cells showed that CsA inhibited the killing activity of NK cells to BGC-823 and JEG-3 cells (t value of 11.74,15.16,P＜0.01,respectively),and the inhibitory rates were (26.48 ±2.42)％ and (47.10 ±1.59 ) ％ respectively.CsA had a higher killing rate inhibition on JEG-3 than on BGC-823 ( t =12.31,P ＜0.01 ).Conclusion CsA induces upregulation of ILT4 in NK cells,and the cytotoxicity of NK cells to tumor cells can be affected by interaction of ILT4 and HLA-G.