Expression And Function Of HLA-G In HTLV-1 Positive T Cells

BackgroundHuman T-lymphocytic leukemia type 1 virus（HTLV-1）is a retrovirus that can be transmitted through blood transfusion,injection or sexual contact,or vertically through the maternal-infant route such as the birth canal,placenta or breastfeeding,causing diseases such as adult T-cell leukemia（ATL）,HTLV-1 associated myelopathy/tropical spastic paralysis（HAM/TSP）and other diseases.The incubation period of HTLV-1 virus infection is long,up to several decades,and approximately 2% to 5% of asymptomatic carriers can develop ATL,which has a poor clinical prognosis and high mortality rate.In order to investigate the pathological mechanism of HTLV-1 virus infection and pathogenesis,some scholars have attempted to restrain viral replication and transcription from the perspective of molecular immunosuppression.Our previous results showed that the immune tolerance molecule human leukocyte antigen-G（HLA-G）was highly expressed in He La cells after HTLV-1 virus infection,and HLA-G promoted the high expression of HTLV-1 Tax protein,which may be due to the fact that the binding of HLA-G molecules to immune cells such as CD8+ T cells effectively inhibited their killing activity and contributed to the HTLV-1 virus replication.Based on this,we intend to explore the specific role of HLA-G in HTLV-1 virusinfected T cells and provide new ideas for the diagnosis and treatment of HTLV-1-related diseases.ObjectiveTo observe the expression of HLA-G in T lymphocytes and explore the specific function of HLA-G in the occurrence and development of adult T lymphocyte leukemia type 1 virus（HTLV-1）infection.Methods1.The expression changes of HLA-G molecules in HTLV-1 virus-positive T cell lines and HTLV-1 virus-negative T cell lines were determined by immunoblotting（Western blot） and fluorescence real-time quantitative PCR（Real-time PCR）.2.The cell model of HTLV-1 virus infection was established by MT4 Jurkat cell co culture system.MT4 cells were co cultured with Jurkat cells for 24 hours.The changes of HLA-G expression of Jurkat cells before and after virus infection were detected by Western blot.3.Three combinations were designed to silence HLA-G si RNA,and the group with the best silencing effect in MT2 and MT4 cells was selected through Western blot experiment.Then,the control si RNA with fluorescent Cy3 label was used to determine the transfection efficiency of liposomes.4.The screened si RNAs were used to silence HLA-G in MT2 and MT4 cells,and the effects of HLA-G molecules on the expression levels of HTLV-1-associated proteins Tax and P19 were observed at the m RNA and protein levels.5.At the same time,real-time PCR was used to monitor the expression of antiviral cytokines in MT2 and MT4 cells after HLA-G gene silencing at the RNA level.6.CCK8 cell proliferation experiment was used to observe the changes of proliferation ability of leukemia cells MT2 and MT4 before and after HLA-G knockdown.7.After silencing HLA-G in HTLV-1 positive T cells,the changes of STAT3 pathway related proteins were detected using Western blot and Real-time PCR.Results1.HTLV-1 virus-positive T cells highly expressed HLA-G molecules: compared with MOLT4 and Jurkat（HTLV-1 virus-negative）,MT2 and MT4 cells（HTLV-1 virus-positive）highly expressed HLA-G molecules at both m RNA and protein levels;and after using MT4 cells co-cultured with Jurkat cells for 24 h,it was found that HTLV-1 virus infection induced abnormal HLA-G protein expression in Jurkat cells.2.The sieved si RNA transfection was effective,and the interference effect of si（3）-HLA-G was the best;about 70% of MT2 cells were seen to emit red fluorescence under fluorescence microscope 10 x,which showed that the si RNA transfection was more successful.3.HLA-G silencing inhibited the expression of HTLV-1 viral protein: after HLA-G gene silencing,the expression of Tax and P19 at m RNA and protein levels in MT2 and MT4 cells showed a decreasing trend.4.Silencing HLA-G can affect its cytokine expression: The expression of IFN-γ and TNF-α increased and the expression of IL-6 decreased in MT2 and MT4 cells after HLA-G gene silencing.5.Silencing HLA-G suppressed the proliferative capacity of leukemia cells: after knocking down the HLA-G gene in HTLV-1-positive leukemia cells MT2 and MT4,the proliferative capacity of the cells was significantly reduced.6.Silencing HLA-G suppressed STAT3-related pathway protein expression: STAT3 phosphorylation levels were significantly reduced after HLA-G gene silencing in HTLV-1-positive T cells,while total STAT3 levels did not change significantly.ConclusionHTLV-1 virus induces T cells to highly express the immune tolerance molecule HLAG.Inhibition of HLA-G expression promotes the production of antiviral factors,reduce the expression of IL-6 and the phosphorylation level of STAT3,and inhibit cell proliferation,which may effectively inhibit the replication and spread of HTLV-1 virus.