A Preliminary Study On The Relationship And Mechanism Of CTLA-4 In The Pathogenesis Of AIDS In Chinese Rhesus Monkeys

Objective:After HIV-1/SIV virus infection,the direct or indirect action of the virus will lead to the activation of the body’s immune system.Cellular immunity plays an important role in controlling virus replication in patients.Immune checkpoint molecules play a negative regulatory role in the immune response,but its mechanism of action in AIDS is still unclear.Therefore,this study used the Chinese macaque AIDS animal model as the research object to explore the relationship between the expression of the immune checkpoint molecule CTLA-4 and the progression of AIDS in Chinese macaques,and preliminarily explore the mechanism of CTLA-4upregulation in SIV infected Chinese macaque.Methods:This study is based on the SIV-infected Chinese rhesus monkey AIDS animal model.Flow cytometry was used to count CD4+T and CD8+T cells in whole blood,detect the expression levels of cell surface molecules HLA-DR and CD38,and detect intracellular CTLA-4 and Ki67.Expression level detection,using real-time fluorescent quantitative polymerase chain reaction（qPCR）to detect plasma viral load.Analyze the correlation between CTLA-4 expression and the number of CD4+T and CD8+T cells,immunological indicators and virological indicators,in order to explore the relationship between CTLA-4 expression and the progression of AIDS.Secondly,establish a cell model of oxidative stress,detect the expression level of CTLA-4 by flow cytometry,and explore the possible regulation of the expression of CTLA-4 by reactive oxygen species（ROS）.The levels of ROS and CTLA-4 were detected by virus infection of human T lymphocyte line and extracellular H₂O₂ stimulation,and the effects of virus infection and extracellular oxidative stimulation on the expression of ROS and CTLA-4 were analyzed.Cyclic adenosine monophosphate and other compounds were used to stimulate PBMC cells,the expression of CTLA-4 was detected at gene and protein levels,and the enzyme-linked immunosorbent assay was used to detect protein kinase A（PKA）in peripheral blood mononuclear cells（PBMC）of rhesus macaques（PBMC）that were stimulated by compounds and infected with SIV.The content and activation of CTLA-4 were further analyzed,and the result was further verified by PKA inhibitor treatment to detect the expression of CTLA-4.qPCR was used to detect the effect of phosphodiesterase（PDEs）and cyclic adenosine monophosphate（AC）gene expression levels on CTLA-4 expression levels,so as to clarify the mechanism of CTLA-4 upregulation after SIV infection in rhesus monkeys.Results:After SIVmac239 infects Chinese macaques,the expression of CTLA-4 in CD4+T cells was significantly up-regulated and changed with the progress of the infection.The expression of CTLA-4 in CD4+T cells was significantly positively correlated with the number of CD8+T cells,was significantly negatively correlated with the number of CD4+T cells and the CD4/CD8 ratio,was significantly positively correlated with proliferation and activation,and was correlated with the log of viral load There is no significant correlation.The study of the mechanism of up-regulation of CTLA-4 expression found that H₂O₂ stimulation did not lead to up-regulation of intracellular CTLA-4 expression.Studies on the mechanism of the up-regulation of CTLA-4 expression have found that viral infection and H₂O₂ stimulation increase the level of intracellular ROS,but neither will lead to the up-regulation of intracellular CTLA-4 expression.Further study of its mechanism found that IBMX,dbc AMP and Forskolin can all induce the up-regulation of CTLA-4 expression;the intracellular cAMP concentration increased after compound stimulation,and the PKA activity was significantly increased.When PAK inhibitors were added,CTLA-4 was significantly down-regulated;SIV infection in the acute phase,intracellular cAMP concentration increased significantly,PKA was significantly activated,AC6 and AC7 types were significantly up-regulated,while PDE3,PDE4 and PDE5 types were significantly down-regulated.Conclusion:The expression level of CTLA-4 in CD4+T cells is related to the progression of AIDS in Chinese macaques.The mechanism of the up-regulation of CTLA-4 expression in infected Chinese macaques is not caused by the increase in the level of oxidative stress.It may be caused by the increase in intracellular cAMP levels caused by virus infection,which enhances the activation of PKA.It shows that oxidative stress is not the main factor affecting the up-regulation of CTLA-4expression,and the up-regulation of CTLA-4 expression may be regulated by the cAMP-PKA pathway.