Effect Of TCF1 And T Cell Self-renewal On Disease Progression In HIV-infected Patients

Objective:Human Immunodeficiency Virus(HIV)infection is seriously endangering human health and life worldwide.Humans show different disease processes after HIV infection.About 10%of HIV-infected people develop HIV Rapid Progressor(RP)within 3 years of infection.However,about 5%of HIV-infected people can show no clinical symptoms for up to 10 years without receiving highly effective antiretroviral therapy(HAART).The CD4~+T lymphocyte count remains at a normal level,which is called Long term non-progressor(LTNP).Compared with RP,the risk of LTNP mortality and morbidity is significantly reduced.A variety of factors are known to affect the disease progression of LTNP,but the mechanism is not yet clear.Therefore,clarifying the mechanism by which maintains disease progression is of great significance for the immune recovery of HIV-infected patients.In HIV infection,the number of memory T cells(TM)affects the progression of HIV disease,which depends on the self-renewal of memory T cells.Memory T cells(TM)have the ability to self-renew and maintain life-long immunity and expose pathogens.Memory CD4~+and CD8~+T cells are organized in a layered subset and have gradually increasing effector characteristics,including central memory(TCM),effector memory(TEM),and effector memory CD45RA positive(TEMRA).We found that the transcription factor TCF1 can inhibit HIV replication and promote T cell development and self-renewal.TCF1+cells self-renewal through asymmetric divisions to produce TCF1~+cell progeny,active TCF1~+progenitor cells are constantly re-implanted into the effector cell bank,which may be periodically replaced by recruiting resting TCM cells.At present,the researches on TCF1 mostly focus on T cells of tumor diseases,but the expression of TCF1 in CD4+T and CD8+T cell subsets of HIV-infected patients has not been reported.T-cell factor 1(TCF1)is a sequence-specific transcription factor that introduces?-catenin into Wnt target genes and plays a role in the Wnt/?-catenin signaling pathway.TCF1 expression plays an important role in the immune system,nervous system diseases and tumor pathogenesis.In HIV infection,the expression of TCF1 in monocytes and monocyte-derived macrophages(Mdms)is related to HIV virus replication,but the relationship between TCF1 expression in T lymphocytes and the progression of LTNP disease is not known report.In this study,the correlation between the expression level of TCF1 in LTNPs and RPs in CD4+T cells,CD8+T cells and their subpopulations,CD4+T cell count,and viral load was detected by using real-time quantitative PCR technology and flow cytometry.To clarify the mechanism by which TCF1 regulates the self-renewal of CD4+T and CD8+T cell subsets in HIV infection to maintain the progression of LTNP disease.Method:1.Study subjects:56 HIV-infected persons who did not receive antiretroviral therapy were selected from this study.They were from Liaoning,Henan and other places.All patients were diagnosed with HIV-1 infection by immunoblotting.Among them,there are 15 cases of HIV long-term non-progressor group(LTNP group).The selection criteria is HIV infection?10 years,mainly transmitted by blood collection and supply,and HIV infection in blood collection sites without ART treatment.?l,the absolute value of CD4~+T cell count is 748.53±222.89/?l;20 cases of HIV rapid progressor group(RP group),the inclusion criteria is one year after HIV infection,CD4+T count<350 cells/?l,CD4+T The absolute cell count was248.13±67.78 cells/?l;21 cases of HIV-infected untreated patients,6 of them had CD4+T counts<350 cells/?l,and the absolute CD4+T cell count was 193.33±62.20cells/?l.The CD4~+T count of 15 HIV-infected patients was>500 cells/?l,and the absolute CD4+T-cell count was 590.73±66.19 cells/?l.The 29 non-infection controls(normal control,NC group)were randomly selected and not exposed to HIV,HIV-negative healthy adults were male.All subjects were approved by the ethics committee and signed informed consent.2.Extraction of peripheral blood mononuclear cells(PBMC).Lymphocyte separation fluid(Ficoll)was used to separate peripheral blood mononuclear cells by low density gradient centrifugation.3.Negative selection of CD4~+T,CD8~+T cells.CD4~+T and CD8~+T cell enrichment kit(Stem Cell)was used to select CD4~+T and CD8~+T cells negatively in PBMC.PBMCs were resuspended at a concentration of 5×107 cells/ml using 1 mM EDTA and 2%Fetal bovine serum(FBS)and PBS without magnesium and calcium ions.Add 50?l Enrichment Cocktail to each ml of PBMC suspension and mix well.Incubate at room temperature for 10 minutes.Then add 100?l Magnetic Particles to each ml of PBMC suspension and mix well.Incubate at room temperature for 5 minutes.The PBMC suspension was made up to 2.5 ml with a medium,and the flow tube was placed in a magnetic pole of Stem Cell,and the supernatant obtained after being left at room temperature for 5 minutes was a CD4~+T,CD8~+T cell suspension.4.Determination of TCF1/LEF1 expression levels in RP,LTNP and NC.RPMC,LTNP and NC were isolated by low density gradient centrifugation.CD4~+T and CD8~+T cells were sorted using BD FACSAria.The RNA of CD4+T and CD8+T cells was extracted with RNeasy Micro Kit.The PrimeScriptTM RT reagent Kit kit was configured with reverse transcription system,and the RNA was reverse transcribed into cDNA by PCR.GAPDH was used as the internal reference to configure the real-time quantification system for cDNA inoculation into 96-well plates.Roche LightCycler?480 was used to analyze CT values.5.Detection of TCF1 expression in HIV-infected and normal human T cell subsets.PBMCs from untreated HIV-infected patients and normal humans were stained with flow surface antibodies in the dark at 4?for 30 min.Cytofix/Cytoperm reagent was used to perform cell rupture experiments.The cells were placed at 4?and 40 min with the flow cytometry antibody in the dark together,and the cells were resuspended with FACS buffer.Then,the TCF1 expression of T cell subsets was analyzed by flow cytometry FACSCanto II.6.CD4~+T,CD8~+T cell proliferation.Cell Trace?Violet Cell Proliferation Kit was used to detect T cell proliferation.Add 1?l Violet to PBMCs of HIV-infected patients and incubate for 30min at 37?and 5%CO2.Collect primary cell peaks and transfer the cells to 96-well plates.Add 0.6?l anti-?CD3/CD28 Dynabeads to each well and continue to culture To 5 days.The cells were washed with FACS buffer(PBS containing 1%PenStrep and 2%FBS),and then the cells were stained with flow cytometry antibodies for 4min at 4?for 30 min.Wash twice with FACS buffer,permeabilize and fix PBMC with Cytofix/Cytoperm reagent,place the cells and flow cytometry antibodies in the dark at 4?,40 min,and resuspend the cells with FACS buffer,then Flow cytometry FACSCanto?was used to analyze cell proliferation and T cell subsets.Data were analyzed using Flow jo software.7.Detection of HIV viral load.The RT-PCR method was used to detect the viral load of HIV using the Roche CoBAS TaqMan System.8.Absolute detection of CD4~+T cells.The absolute value of CD4~+T cells was obtained by flow cytometry BD FACS Calibur detection.9.Statistical analysis.The data of all groups were processed by SPSS19.0 software for statistical data processing.Mann-Whitney U test software was used to compare data between groups,and the obtained data was expressed as mean±standard deviation;Spearman was used to analyze rank correlation data;when P<0.05,the difference was statistically significant.Use graphpad prism 7.0 software for mapping.Result:1.The expression level of TCF1/LEF1 in PBMC and its correlation with CD4~+T cell count and viral load in people with long-term HIV infection.With the progress of HIV disease,the expression level of TCF1/LEF1 gradually increases,and the progressive increase of TCF1 expression is closely related to disease progression.The expression level of LEF1 is positively correlated with the number of CD4~+T cells and negatively correlated with viral load Trend but not statistically significant.2.The expression level of TCF1 in CD4+T,CD8+T cells and its correlation with CD4~+T cell count and viral load in HIV infection patients who have long term non-progressor as the expression level of TCF1 in CD4~+T and CD8~+T cells gradually increased,the number of CD4~+T cells also gradually increased,and the viral load gradually decreased.The expression level of TCF1 was positively correlated with the number of CD4+T cells,and was positively correlated with the viral load The amount was negatively correlated.3.The expression level of TCF1 in CD4~+T cell subgroups in HIV infection patients who have long term non-progressor.PBMCs from untreated patients and healthy adults infected with HIV for the first time were detected to determine the expression of TCF1 in CD4~+T cell subsets under different immune states.4.The expression level of TCF1 in CD8+T cell subgroups in people who have long term non-progressor.To determine the expression of TCF1 in CD8~+T cell subsets under different immune states,PBMCs from healthy and HIV-infected untreated individuals were tested for the first time.5.Self-renewal of TCF1 in T cell subgroups by people who have long term non-progressor.TCF1 regulates the self-renewal of CD4~+T and CD8~+T cells in HIV infection and maintains the disease progression of LTNP.Conclusion:1.The correlations between the expression levels of LTNP,RP and TCF1/LEF1 in PBMC and CD4+T cell count and viral load were determined.2.The correlations between the expression levels of TCF1 in LTNP and RP in CD4~+T cells,CD8~+T cells and their subgroups,and the CD4~+T cell count and viral load were clarified.3.It is clear that TCF1 regulates the self-renewal of CD4+T and CD8+T cell subsets in HIV infection to maintain the disease progression of LTNP.