Cloning and expression of HLA-G1-G4 molecule in JAR cells and its effects on NK cell function / 中华微生物学和免疫学杂志

ABSTRACT

Objective To establish the expression of membrane-bound HLA-G1-G4 isoforms in choriocarcinoma cell line JAR and to investigate its roles in NK cytotoxicity in vitro. Methods Stable expression of HLA-G1, -G2, -G3 and -G4 in JAR cells was established by gene cloning and transfection.HLA-Gtranscripts and protein expression in the transfected JAR cells was tested by RT-PCR, flow cytometry, Western blot and immunocytochemistry, respectively. High-affinity peptide KIPAQFYIL pulsing was performed to evaluate its effects on HLA-G expression. Effects of HLA-G1-G4 isoforms on NK cytotoxicity was performed with lactic dehydrogenase (LDH) releasing method. Results RT-PCR, Western blot and immunocytochemistry results showed that exogenous HLA-G1-G4 gene were successfully transfected and proteins were stably expressed in the HLA-G negative JAR cells; Flow cytometry data showed that only HLAG1, but not HLA-G2-G4 isoform was detectable in those transfected JAR cells and the peptide pulsing did not affect their expression status. However, all HLA-G1-G4 isoform expressed JAR cells could significantly decreased the NK cell cytotoxicity (P＜0.05). Conclusion HLA-G1-G4 isoform expression could dramatically inhibit NK-92 cell lysis, indicating that membrane-bound HLA-G isoforms are importantly immunotolerant and may play immune regulation roles in various physio-pathological situations.