Correlation Of CD3~+ And CD8~+ T Cell Activation And Exhaustion In HIV-1 Infected Patients

Objective:In order to investigate the effects of activation and exhaustion of CD3~+T cells and CD8~+T cells at different stages of infection on the course of HIV-1 infection,By detecting the difference of CD38,HLA-DR,CD95 and PD-1 expression levels in peripheral blood CD3~+T cells and CD8~+T cells of HIV-1 infected patients at different stages of infection,as well as the difference of PD-1 and CD95 expression levels in CD38~+HLA-DR~+CD3~+T/CD8~+T cells,The difference of LAT phosphorylation level in T cells was analyzed by flow cytometry.Methods:To investigate the levels of molecular activation and depletion of CD3~+T cells and CD8~+T cells in HIV-1 infected patients,96 HIV-1 infected patients without antiviral treatment were collected and divided into low count group,medium count group and high count group according to the CD4 cell count.At the same time,27 infected patients receiving HIV-1 antiviral therapy were collected,and 30 healthy donors were selected as negative control group.All the subjects mentioned above were excluded from co-infection of hepatitis B,hepatitis C and other viruses,and no other infectious diseases were found in clinical examination.Blood was extracted to separate Peripheral blood mononuclear cells(PBMC),and the cell concentration was adjusted to 1×10~7cells/m L after counting under microscope.The Pacific Blue Mouse anti-human CD3,BV510 Mouse anti-human CD8,percp-Cy 5.5 Mouse anti-human CD38,and PE Mouse were absorbed by micropipette Anti-human CD95,APC Mouse Anti-human CD279,Alexa Flour TM 700 Mouse Anti-human HLA-DR were stained and incubated.After washing,the samples were fixed with 4%paraformaldehyde solution and tested on the machine.Graph Pad Prism 8.0statistical software was used for statistical analysis.All data measurement data were represented as mean±standard deviation((?)±s),and comparisons were made by Ordinary one-wayanova method.The expression differences of CD38,CD95,HLA-DR and PD-1 in HIV-1 untreated group,HIV-1 treated group and healthy control group were compared by unpaired t-test.The difference between the two groups was considered statistically significant at P<0.05.The correlations of CD38,CD95,HLA-DR and PD-1 expression frequencies among the same sample parameters were analyzed by Pearson correlation analysis P<0.05 indicates that the difference is statistically significant.To explore the phosphorylation level of LAT in CD8~+T cells,81 untreated patients were collected and divided into three groups according to their CD4cell count:48 patients with Typical AIDS progression(TPs);16 patients with Chronic HIV-1 infected persons(CPs)were included;A total of 17 patients with HIV Carrys(CRs)were included;24 patients receiving antiviral therapy were collected,and 29 Healthy donors were selected as the negative control group.All the above subjects were excluded from co-infection with other viruses such as hepatitis B and C,and no other infectious diseases were found in clinical examination.The peripheral venous blood was extracted to isolate the PBMCs,and the concentration of the cells was adjusted to 1×107/m L by microscope counting,and the excitants were added to the Purified anti-human CD3 and anti-human CD28 for incubation.After fixing the broken membrane,LAT antibody staining was performed.After washing,4%paraformaldehyde was added to fix the cells,and the data were detected on the machine and recorded.Graph Pad Prism 8.0 statistical software was used for statistical analysis.All data measurement data were expressed as mean±standard deviation((?)±s)and compared by Ordinary one-wayanova method.LAT phosphorylation levels of CD8~+T cells in peripheral blood of TPs group,CPs group,CRs group,Treated group and Heathys group were compared by unpaired t test.P<0.05 indicated that the difference between groups was statistically significant.Pearson correlation analysis was used to analyze the correlation between the expression frequency of the same sample parameters.P<0.05 indicates that the difference is statistically significant.Results:1.(1)The expressions of CD38,HLA-DR,CD95 and PD-1 in peripheral blood CD3~+T cells of HIV-1 infected patients were higher than those of healthy controls;(2)The expression of PD-1 in peripheral blood CD3~+T cells of untreated HIV-1 patients was negatively correlated with CD4 cell count;(3)The expressions of PD-1 and CD95 in peripheral blood CD38~+HLA-DR~+CD3~+T cells of HIV-1 infected patients were higher than those of healthy controls;(4)The expression of CD38~+HLA-DR~+CD3~+T cells in the peripheral blood of untreated HIV-1 patients was negatively correlated with the CD4 cell count,and the expression of CD95 was negatively correlated with the CD4 cell count.2.(1)The expressions of CD38,HLA-DR,CD95 and PD-1 in peripheral blood CD8~+T cells of HIV-1 infected patients were higher than those of healthy controls;(2)Expression of PD-1 in peripheral blood CD8~+T cells of untreated HIV-1 patients was negatively correlated with CD4 cell count;(3)The expressions of PD-1 and CD95 in peripheral blood CD38~+HLA-DR~+CD8~+T cells of HIV-1 infected patients were higher than those of healthy controls;(4)The expression of PD-1 in peripheral blood CD38~+HLA-DR~+CD8~+T cells was negatively correlated with CD4 cell count,and the expression of CD95 was negatively correlated with CD4 cell count in untreated HIV-1 patients.3.The expression of LAT in peripheral blood CD8~+T cells in TPS group was higher than that in CRS group;The expression of LAT in peripheral blood CD8~+T cells of HIV-1 infected patients in CRS group was higher than that in Healthy group.The expression of LAT in CD8~+T cells in untreated HIV-1 patients was negatively correlated with CD4 count.Conclusion:During the course of chronic HIV-1 infection,the expression of activated molecules CD38 and HLA-DR and apoptotic depleted molecules CD95 and PD-1 are up-regulated on CD3~+and CD8~+T cells.The co-expression of PD-1 and CD95 in CD38~+HLA-DR~+CD3~+/CD8~+T cells suggested that the overactivation of T cells would lead to functional depletion with the progression of the disease.The progression of HIV-1 disease is related to the change of LAT phosphorylation level of CD8~+T cells,and with the progression of HIV-1 infection with CD8~+T cells,the function of HIV-1 infection is activated,thus promoting the immune killing effect of the body.