Effect Of PD-1 Expression On Src And SHP2 Phosphorylation In PBMCs,CD3+T Cells And CD8+T Cells Of HIV-1 Infected Individuals

Objective:In this study,we plan to quest the expression of PD-1(Programmed Death),protein tyrosine kinase Src,protein tyrosine phosphatase 2(SHP2)on PBMCs(Peripheral blood mononuclear cells),CD3+T cells and CD8+T cells during HIV-1(human immunodeficiency virus-1)infection and seek the correlation of PD-1 and the phosphorylation levels of Src and SHP2.Our destination is to explore the mechanism of action of PD-1 impact on phosphorylation of Src and SHP2 signal pathway of PBMCs,CD3+T cells,and CD8+T cells during HIV-1 infection.Method:1.62 cases of HIV-1 infection were recruited in Guang Xi Center for Disease Prevention and Control according to the?Diagnostic Criteria of AIDS and HIV Infections?of The People's Republic of China.All of patients were divided into chronic HIV-1 infected persons(CPs)33 cases and typical AIDS progressors(TPs)29 cases according to CD4 cell count(inclusion criteria:CPs refers to CD4+T cell count?350/?l,not receiving antiviral treatment;Tps refers to infection to study time is not more than 10 years,CD4+T cell count<350/?l,not receiving antiviral treatment);Blood from 18 healthy donors(HDs)was collected as a control group.All above researchers excluded the possibility of hepatitis B virus(HBV)and hepatitis C virus(HCV)co-infection.2.Peripheral blood of HIV-1 infected patients and healthy donors was collected in a quiet and empty stomach state in the morning and placed in a vacuum blood collection vessel.RPMI-1640 culture solution with the same volume as peripheral blood was added and mixed uniformly.PBMCs were isolated by Ficoll-paque lymphocyte separation solution by density gradient centrifugation method and washed twice with RPMI-1640.PBMCs was counted and adjusted to 1×10~7cells/ml.3.Adding proper amount of fluorescein labeled anti-human monoclonal antibody Pacific blue mouse anti-human CD3,BV510 mouse anti-human CD8,APC anti-human PD-1 for incubation,adding purified anti-human CD3 and purified anti-human CD28stimulant,stimulating in 37?incubator for 15 minutes,fixing and breaking the membrane with Fixation buffer and Perm buffer ? respectively,adding proper amount of fluorescein-labeled anti-human monoclonal antibody PE mouse anti-human SHP2 and PE mouse anti-human Src.4.The cells were fixed and filtered by paraformaldehyde and detected by flow cytometry.The lymphocyte population was determined by FSC-SSC,and the expression levels of Src,SHP2 and PD-1 on PBMCs,CD3+T cells and CD8+T cells were detected respectively.5.Prism 6.0 statistical software was used for analysis and processing.The measurement data are expressed in (?).The expression levels of PD-1,Src and SHP2 in CPs group,TPs group and HDs group were compared by unpaired t test.The difference was statistically significant(P<0.05).Pearson correlation analysis was used to analyze the correlation between Src,SHP2 and PD-1 expression levels of the same sample parameters.P<0.05 indicates that there is a correlation between the two parameters,and r value indicates the correlation coefficient.Results:1.Expression levels of PD-1,Src and SHP2 on PBMCs of HIV-1infection:The expression frequency of PD-1 on PBMCs in TPs group was higher than that in CPs group and HDs group(P=0.0415,P<0.0001).The frequency of Src expression on PBMCs in TPs group and CPs group was lower than that in HDs group(P=0.0048,P=0.0107).The frequency of SHP2expression on PBMCs in TPs group and CPs group was higher than that in HDs group(P=0.0015,P=0.0012).CD4 count was negatively correlated with PD-1(P=0.0006),positively correlated with Src(P=0.0038),and negatively correlated with SHP2(P=0.0487).The expression of PD-1 and Src was negatively correlated(P=0.0422)and positively correlated with SHP2(P=0.0354)in HIV-1 infected patients.2.Expression levels of PD-1,Src and SHP2 on CD3+T cells of HIV-1infection:The expression frequency of PD-1 on CD3+T cells in TPs group was higher than that in CPs group and HDs group(P=0.0171,P<0.0001).The frequency of Src expression on CD3+T cells in TPs group and CPs group was lower than that in HDs group(P=0.0035,P=0.0012).The frequency of SHP2expression on CD3+T cells in TPs group and CPs group was higher than that in HDs group(P=0.0017,P=0.0022).CD4 count was negatively correlated with PD-1(P=0.0028),positively correlated with Src(P=0.0020),and negatively correlated with SHP2(P=0.0218).The expression of PD-1 and Src was negatively correlated(P=0.0499)and positively correlated with SHP2(P=0.0155)in HIV-1 infected patients.3.Expression levels of PD-1,Src and SHP2 on CD8+T cells of HIV-1infection:The expression frequency of PD-1 on CD8+T cells in TPs group was higher than that in CPs group and HDs group(P=0.0451,P<0.0001).The frequency of Src expression on CD8+T cells in TPs group and CPs group was lower than that in HDs group(P=0.0271,P=0.0499).The frequency of SHP2expression on CD8+T cells in TPs group and CPs group was higher than that in HDs group(P=0.0023,P=0.0025).CD4 count was negatively correlated with PD-1(P=0.0285),positively correlated with Src(P=0.0299),and negatively correlated with SHP2(P=0.0425).The expression of PD-1 and Src was negatively correlated(P=0.0036)and positively correlated with SHP2(P=0.0212)in HIV-1 infected patients.Conclusion:Expression levels of PD-1 in HIV-1 infected patients was significantly higher than that in healthy controls and increased with the course of disease.PD-1 may affect the activation process of immune cells by negatively regulating Src phosphorylation level and positively regulating SHP2 phosphorylation level during HIV-1 infection.