Effect of NKG2D Expression on NK Cells by Recombinant Soluble MICA Protein
|Keywords||sMICA NK cell NKG2D receptor cytotoxicity|
Objective:NK cells are the body’s innate immune effector cells. NKG2D receptor and its ligand MICA recognition play an important role in the immune surveillance of leukemia. But leukemia cells can still escape the NK cell immune surveillance, considering the soluble MICA shedding from acute leukemia cell surface could damage the anti-leukemia effect of the NKG2D-MICA. To study the effects of recombinant soluble MICA protein (sMICA) on the NKG2D expression, cytotoxicity, secretion of IFN-γ, so as to search a new approach for the development of leukemia immunotherapy.Method:Peripheral blood mononuclear cells (PBMNC) were isolated on a Ficoll with lympho-prep density. NK cells were separated from PBMNC by MACS. Purification of NK cells after immunomagnetic isolation were detected by flow cytometer. NK cells were divided into control and experimental groups. Control group were treated by RPMI-1640complete medium of an equal volume, experimental groups were stimulated by recombinant soluble MICA protein (sMICA) at200μg/L,500μg/L.800μg/L concentration. After24hours of stimulation, the NKG2D receptor was detected by the flow cytometry, the cytotoxicity of NK cell on target cells was detected by LDH release assay, the supemant was collected to determine the concentration of IFN-γ by ELISA.Results：Purification of NK cells after immunomagnetic isolation was over90percent. Compared with blank control group, sMICA-treated groups down-regulated NKG2D expression on NK cells, inhibited the cytotoxicity of NK cells and decreased the secretion of IFN-γ (P<0.05). With sMICA concentration increasing, NKG2D expression level of NK cells, the cytotoxicity, the secretion of IFN-γ were Gradually decreased, the difference was statistically significant (P<005).Conclusion:Recombinant soluble MICA protein (sMICA) down-regulated the expression of NKG2D receptor on NK cells, inhibited the cytotoxicity of NK cells and decreased the secretion of IFN-γ. The soluble MICA shedding from acute leukemia cell surface could damage the anti-leukemia effect of the NKG2D-MICA, which leads to one of the acute leukemia immune escape mechanisms. The methods of enhancing immune therapy based on NKG2D-MICA worthy of further study.