Study On The Contribution Of CD4~+CD38~+ Central Memory T Lymphocyte To Virus Reservoir For Long-term HIV Infected Patients

Objective:Infection with the human immunodeficiency virus(HIV)leads to a significant decrease in the number of CD4~+T lymphocytes,eventually leading to acquired immunodeficiency syndrome(AIDS).When anti-retroviral therapy(ART)appears,it can rapidly reduce the VL in patients and effectively prolong the life of the patients.However,once ART is stopped,the VL in the patient quickly rebounds and the goal of a functional HIV cure is never achieved.A large number of documents indicate that the main reason that the HIV virus cannot be completely eradicated is the existence of a virus reservoir.A variety of tissues and cells in the human body are involved in the establishment and maintenance of reservoir,including na?ve T cells(TNAs),central memory T cells(TCM),effector memory T cell(TEM),stem cell-like memory T cell(TSCM),transitional memory T cell(TTM)in peripheral blood,lymph nodes,central nervous system and intestinal mucosa-associated tissues.Because of the long-term survival of resting memory CD4~+T cells and their ability to silence HIV transcription in them,they become the largest HIV reservoir in the body.CD38 and HLA-DR have been regarded as the activation markers of HIV.The current resting memory T cells of HIV-infected individuals are mainly defined by CD45RA~-/RO~+and HLA-DR~-.Our previous analysis of activated subpopulations found that the expression of CD38 on the CD4~+TCM of HIV infected people was significantly higher than that of HLA-DR.Further studies showed that the expression of CD38 and HLA-DR subpopulations in NA?VE,TCM and TEM cells were quite different.HLA-DR was not expressed on the surface of non-activated NA?VE cells,but CD38was highly expressed,and the proportion of CD38~+HLA-DR~-expression in CD4~+TCM is higher.Previous studies have also shown that CD38 is highly expressed on TCM.CD38,known as cyclic ADP ribose hydrolase or T10,is a single-stranded transmembrane glycoprotein.The expression of CD8CD38~+in HIV is used as a marker of cell activation.However,subsequent studies found that the expression of CD38 in CD4~+T cells and CD8~+T cells is different,CD8~+CD38~+subsets are activated cells,and CD4~+CD38~+subsets represent immature cells.Studies have shown that CD38~+cells express viral reservoir markers and that CD38~+can also promote tumor cell proliferation and inhibit its apoptosis.The long-term survival and homeostatic proliferation is also a major feature of the reservoir cells.In 2014,it was reported that HIV-infected patients had no difference in the expression of HIV total DNA,2-LTR and HIV integrate DNA after 1 year of ART treatment on CD38~+and CD38~-,and found that after 12 weeks of treatment,The virus half-life of CD38~+cells relative to HLA-DR~-CD38~-is longer.How characteristic of CD38 expression in TCM cells and whether it can be used as a long-term viral reservoir has not been reported yet.Antiviral treatment of 1-2 years mainly clear virus in the activated CD4~+T cells,and the inhibition of the reservoir cells was weak.The study showed that after 7 years of follow-up,there was some degree of dynamic change in the HIV reservoir size and the virus half-life was found to be shorter in patients treated for more than 3 years.Long-term treatment is crucial to clearing the reservoir.However,there is no study of the contribution of CD38 to the long-term therapeutic reservoir for HIV infection.This study intends to further study the specific features of CD4~+CD38~+TCM cells and its effect on the virus reservoir,providing new ideas for the mechanism of HIV virus reservoir and the cure of HIV.Methods:Research object.Thirty subjects with HIV-1 who received ART:ART>5years,CD4~+T cells>350 and VL<50 copies/ml.Experimental methods include:1,T lymphocytes count the absolute value.Add 20?L of CD4/CD8/CD3 Tri TEST Reagent to each tube.Add 50?L of anticoagulant solution by reversed-phase and incubate at room temperature for 15 minutes.Add 450?L of no-wash hemolysin and keep at room temperature for 15 minutes.FACS Calibur flow cytometry samples and use MultiSET software testing for automatic analysis,calculate the CD4~+T,CD8~+T,CD3~+T cell absolute value and the corresponding ratio.2,HIV viral load determination.The COBAS?TaqMan?system was used for automated PCR reaction preparation and viral load testing using the COBAS?AmpliPrep?/COBAS?TaqMan?HIV-1 Test,v2.0 kit from Roche.The detection limit is 1 copy/mL.3,density gradient centrifugation of peripheral blood mononuclear cells.Peripheral blood of HIV-1infected persons was drawn.The fresh peripheral blood was thoroughly mixed with PBS according to the ratio of 1:1.The mixture was slowly and uniformly added to the whole blood Lymphocyte separation liquid level,and density gradient centrifugation.After density gradient centrifugation,carefully draw the white coat.Washed twice with PBS,discard the supernatant,which is PBMCs.4.CD4~+T Cell sorting.Fresh PBMCs of HIV-infected persons were extracted and CD4~+T cells were negatively selected by Stem Cell's CD4~+T cell enrichment kit;5.Cell sorting.CD4~+TCMCD38~+and CD4~+TCMCD38~-cell sorting using a FACS Aria flow cytometer.6,Flow cytometry CD4~+T features.Extracted peripheral blood mononuclear cells were surface-stained:CD3-PE-CY7,CD4-APC-CY7,CD38-PE,HLA-DR-APC,CD45RA-FITC,CCR7-Percp-CY5,CD25-PE-CY7,CD69-APC,PD-1-FITC,CD127-APC and Ki-67-Violet,FBS wash,300g,10min,5 drop 5,4 deg.Centrifuge,discard supernatant,250ul PBS resuspended cells,BD LSR?flow cytometry and BD FACSDiva TM software to detect and analyze the activation level of CD4~+CD38~+TCM cells feature.7,DNA extraction.Sorted CD4~+T cells and CD4~+TCMCD38~+and CD4~+TCMCD38~-DNA were extracted using a QIAamp blood DNA mini kit according to the manufacturer's instructions and dissolved in 50?l of enzyme-free water.8,digital drop PCR detection of HIV total DNA.The extracted DNA samples,using digital droplet PCR system configuration and reflect the specific procedures for testing.RPP30 as a quantitative and quantitative total RNA internal control.9,knocking out CD38 CD4~+T lymphocyte function testing.CD4~+T lymphocyte CD38 knockdown,96-well U-bottom plate were untreated group,control group and experimental group,each well cells were resuspended with 100ul serum-free 1640 medium,si-CD38 and si-Control The final transfection concentration was 20 nM.The specific transfection process:100?l of serum-free 1640 medium was dissolved in si-CD38 and si-Control,gently agitated,and then added with 1ul/0.2 million cells of transfection reagent Lipofectamine?RNAi MAX,Incubate for 20 minutes at room temperature.si-CD38 and negative controls were then added to the cell wells.After 48 hours in the incubator,knockout efficiency was measured.Apoptosis of si-CD38 and negative control(Annexin V and7-AAD)was measured at 96 hours.Five days later,proliferation of si-CD38 and negative control was examined.Violet Cell Trace(5?M)was labeled with overnight differentiated si-CD38 and si-Control cells and stimulated with CD3/CD28beads(1ug/ml).Co-cultured for 4 days.BD LSR II was then used to detect CD4~+T cell proliferation.10,statistical analysis.SPSS17.0 statistical software and Graphpad Prism5.0 software for statistical analysis,the relevant comparison between the two groups using two samples paired T test,P<0.05 was considered statistically significant.Results:1,After long-term treatment of HIV-infected CD4~+memory T cells CD38,HLA-DR expression is different.The expression of CD38 on TCM and NAIVE were highly,and CD38 on NA?VE,TCM and TEM were higher than that on HLA-DR(P<0.001,P<0.001,P=0.009).The expression of CD38 and HLA-DR subgroups on NA?VE,TCM and TEM were also significantly different,and the percentage of CD38~+HLA-DR~-expressing on CD4~+TCM was higher.CD4~+CD38~+has similar TCM expression to CD4~+HLA-DR~-.2,CD4~+CD38~+TCM has a reservoir of phenotypic markers.CD4~+CD38~+TCM low expression of activated molecules CD25,CD69,and similar with the classical reservoir of CD4~+HLA-DR~-TCM,but has significant difference with activated CD4~+HLA-DR~+(P=0.028,P=0.015,Figure 2 A-C).The expression of PD-1 and KI-67 on CD4~+CD38~+TCM markers were higher than CD4~+HLA-DR~-TCM(P=0.009,P=0.040)but with activated CD4~+HLA-DR~+no significant difference.CD127 expression was higher in CD4~+CD38~+TCM than in CD4~+HLA-DR~+(P=0.010)but lower in CD4~+HLA-DR~-TCM(P=0.003).3,CD4~+CD38~+TCM is associated with HIV total DNA in CD4 cells.There was a positive correlation between CD4~+CD38~+TCM and HIV total DNA in CD4~+T cells(r=0.505,P=0.033).There was no correlation between CD4~+CD38~-TCM and HIV total DNA in CD4~+T cells.We also used a negative two-item regression model to evaluate the relationship between HIV total DNA and CD4~+CD38~+TCM/CD4~+CD38~-TCM and found that CD4~+CD38~+TCM was able to predict HIV total DNA content in CD4 cells(P=0.018).Corrected with current CD4~+T cells and baseline CD4~+T cells were still predictive(P=0.009,P=014).4,CD38~+contributes more to TCM as a reservoir.We detected the expression of CD38~+and CD38~-on TCM using flow cytometry and found that the expression of CD38~+was higher than that of CD38~-(P<0.001).Then we detected the total DNA of these two groups by digital drop PCR and found that The total DNA content of CD4~+TCMCD38~+was higher than that of CD4~+TCMCD38~-(P=0.0358).5,CD38 can promote the proliferation of HIV-infected CD4~+T cells and inhibit their survival and apoptosis.We first knockdown the CD38 of HIV-infected CD4~+T cells and found that the proliferation of si-CD38 group was significantly lower than that of si-Control group(P=0.011).The 7-AAD and Annexin V of si-CD38 group was significantly higher than that of si-Control group(P=0.003,P=0.004).Conclusion:1,CD4~+CD38~+TCM has a reservoir feature and has an important contribution to the HIV reservoir.2,CD38 can play a role in HIV important reservoir TCM by reducing the apoptosis ability of CD4~+T cells and increasing its proliferation and survival ability.