The Study On The Effect Of CD38 Expression To T Cell Function And Disease Outcome After HIV Antiretroviral Therapy

Objective:Acquired immunodeficiency syndrome(AIDS)is an infectious disease caused by human immunodeficiency virus(HIV)infection,which can impair and even destroy the human immune system.With the emergence of HIV antiretroviral therapy(ART),the morbidity and mortality of HIV infection have been greatly reduced,but ART can not completely restore immune health,some infected patients have poor immune function recovery and clinical outcome.These infected individuals have clinically relevant metabolic disorders and end-organ damage.The reason for this phenomenon has not yet been concluded.Current research believes that it may be due to chronic inflammation,oxidative stress,mitochondrial dysfunction,immune deficiency,and side effects of antiretroviral drugs.Therefore,exploring the mechanism of different disease outcomes of HIV-infected patients after ART is helpful to control the disease process and provide a new strategy for the functional cure of HIV.Early studies found that a large number of lymphocytes in the peripheral blood of AIDS patients highly express CD38(Cluster of Differentiation 38),and there is a strong correlation between the CD38 expression of T cells in patients and the progression of HIV disease.As a marker of T cell activation,CD38 can independently and effectively predict the progression of the disease.Compared with several other activation markers such as inflammatory mediators,the expression of CD38 is stronger for disease prediction.CD38 expression in patients with poor immune recovery is higher,which cannot be recovered by ART.As a classic activation marker,CD38 is the result of chronic inflammation caused by HIV infection,but it is not clear how it affects the function of T cells and the prognosis of HIV-infected patients after ART.CD38 is the human body's main NAD hydrolase(NADase),which catalyzes Nicotinamide Adenine Dinucleotide(NAD)to produce Adenosine Diphosphate Ribose(ADPR)and Cyclic ADP-ribose,c ADPR).NAD is an important coenzyme in the process of cell metabolism.It acts as a carrier of hydrogen ions in glycolysis,tricarboxylic acid cycle and mitochondrial respiratory chain and is responsible for transferring electrons to assist the synthesis of adenine nucleoside triphosphate(ATP).The homeostasis of NAD plays an important role in regulating cell metabolism and mitochondrial function.NAD is also the only substrate of the Sirtuin(SIRT)family,which are important intracellular histone deacetylases.Intracellular NAD concentration can significantly affect the enzyme activity of SIRT and then influence cell protein modification,transcriptional regulation,metabolism,inflammation,aging and even circadian rhythm through a variety of mechanisms.The strong predictive ability of CD38 expression level for HIV disease suggests that it may play an important role in the disease process of infected patients.However,how CD38 affects T cell function,senescence,and immune recovery in infected patients after ART has not been elucidated.Exploring its mechanism can provide clues for improving immune recovery and clinical outcome of patients.As a central regulator of metabolism,NAD homeostasis is particularly important for the maintenance of human health.This highlights the potential of using CD38 as a drug target to increase the level of NAD,improve immune function and delay cell aging.The transmembrane structure and multifaceted functions of CD38 provide many opportunities to design drugs that can inhibit the enzyme.Some drugs have been developed and used to improve cardiovascular disease,diabetes,Alzheimer's disease,and obesity-related diseases.Further exploring the effects of CD38-related inhibitory drugs on T cell function,metabolism and aging can provide new therapeutic strategies for improving immune function and clinical outcome of HIV infected patients.Methods:1.Patient selection.63 HIV-infected patients who had received effective ART for more than 2 years,9HIV-infected patients who had not received ART,and 25 HIV-negative healthy people without other related diseases were recruited for this study.The peripheral blood samples of the above population were collected from The First Affiliated Hospital of China Medical University and obtained ethical approval from China Medical University.All participants were told that their peripheral blood samples were used in this study and signed an informed consent form before collecting the samples.2.Cell sortingPeripheral blood mononuclear cells(PBMCs)of whole blood were extracted by density gradient centrifugation for the next step of flow staining and sorting.T cells were sorted by magnetic beads using the Stemcell T cell Isolation kit and used in subsequent experiments.PBMCs were labeled by flow cytometry antibodies CD3-PE-Cy7 and CD38-PE into CD38~+T cells and CD38~-T cell subpopulations,and the two populations of cells were sorted by flow cytometry.The sorted cells were used for subsequent experiments such as transcriptome detection.3.Flow Cytometry of peripheral whole blood cells.Flow cytometry antibodies were used to label peripheral whole blood cells.For detecting the relationship between CD38 expression and aging,the staining scheme is:CD3-PE-Cy7,CD4-APC-Cy7,CD8-FITC,CD38-BV421,CD57-BV510.For detecting the relationship between CD38 expression and PD-1,the staining scheme is:CD3-PE-Cy7,CD4-APC-Cy7,CD8-Per CP-Cy5.5,CD38-BV421,PD-1-FITC.BD CANTO II flow cytometer and Flowjo v10.3 software were used for the detection and analysis of peripheral blood cell surface markers.4.Flow cytometry of intracellular cytokine.After T cell culture,flow cytometry antibodies CD4-APC-Cy7 and CD8-Per CP-Cy5.5 were used to label cell surface markers.After cell permeabilization,INF-?-APC/Alexa Flour 647 and IL-2 Alexa Flour 488 flow cytometry antibodies were used for intracellular staining.BD LSRII flow cytometer and Flowjo v10.3 software were used for flow cytometric detection and analysis of intracellular cytokine secretion.5.Lentiviral transfection and culture of Jurkat T cell line.The CD38 sh RNA lentivirus(CD38i)and the control lentivirus(NC)were transfected into Jurkat cells with a lentiviral transfection reagent at a transfection concentration of 20MOI.After 16 hours of transfection,the medium was changed,and the cells continued to be cultured.After 4 days,puromycin was added at a final concentration of 0.5?g/ml to select cells for subsequent experiments.6.RNA extraction and quantitative real-time PCRAccording to the kit manual,RNeasy Plus Mini Kit or RNeasy Plus Micro Kit was used to extract total RNA.In the extraction process,the g DNA Eliminator provided in the kit has been used to remove DNA to prevent genomic DNA contamination.Primpscript RT reagent kit was used for RNA reverse transcription,and TB Green(?)Advantage was used for rt-PCR.The relative quantification of m RNA was calculated by2-??Ct.7.NAD concentration and SIRT enzyme activity detection.For NAD concentration detection:cell permeabilization buffer were added to cells and then centrifuged to remove the culture medium,cell permeabilization buffer was used to permeabilize the cells according to the reagent manual.After the permeabilization was completed,the supernatant was collected by centrifugation,and the NAD concentration was detected by Amplite(?)Fluorimetric NAD Assay Kit and automatic enzyme immunoassay according to the kit manual.For SIRT activity:Nuclear Extraction Kit was used to extract nuclear proteins.The Universal SIRT Activity Assay kit was used to detect SIRT enzyme activity according to the kit manual.8.Seahorse Extracellular Flux Analysis.Seahorse XFp Cell Mito Stress Test Kit was used to detect oxidative respiratory pressure.The drugs added to each dosing point were:1.5?M oligomycin(Oligo),0.5?M carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone(FCCP),0.5?M rotenone/Antimycin A(R&aa).The adjusted cells were added to the cell culture plate which treated with cell-tak,and then detected after equilibrating at 37°C.9.Flow cytometric detection of cell mitochondrial related markers and?-galactosidase activityMito Tracker(?)Orange CMTMRos and Mito Tracker(?)Green FM were used to label mitochondrial membrane potential(MMP)and mitochondrial mass(MM),Cell ROX(?)Deep red kit is used to label cytoplasmic reactive oxygen species(ROS),Mito SOX(?)Red was used to label mitochondrial ROS,and Senescence?-Galactosidase Activity Assay Kit was used to detect cell?-galactosidase activity.10.Gene Set Enrichment AnalysisGSEA v3.0 software(Broad Institute software)was used for transcriptome GSEA(Gene Set Enrichment Analysis)analysis.The GO ontologies-biological process(BP)data set in the MSig DB data set was used for enrichment analysis,and q-value<0.05 was statistically significant.11.Statistical analysis.Graph Pad Prism v8.0 software was used to perform statistical analysis.Spearman correlation test was used to analyze the correlation between CD38 and various surface markers.The differences in CD38 expression between HC,untreated and ART samples were analyzed by Mann Whitney test.The difference in CD38 expression between PD-1~+/~-and CD57~+/~-groups,the difference in cytokine secretion of CD38~+/~-T cells,and the difference in the detection indicators between the drug treatment group and the control group were analyzed using paired t test,P<0.05 was statistically significant.Results:1.CD38 expression on the surface of T cells increased significantly after HIV infection,and ART could not fully restore CD38 expressionWe found that the percentage and fluorescence intensity of CD38 expressed on CD4~+T cells and CD8~+T cells in HIV-infected patients were significantly increased when they had not received ART,and CD38 expression levels could not be fully recovered after ART,which was still higher than the healthy control group.We also proved that before and after HIV-infected patients received ART,the CD38 expression level of CD4~+T cells was negatively correlated with CD4 T cell count.HIV-infected patients with CD4~+T cell counts less than 450 cells/?l have higher CD38 expression on CD4~+T cells.2.CD38 expression is associated with senescence and exhaustion of T cellsAfter detecting the expression of T cell senescence marker CD57 and exhaustion marker PD-1 by flow cytometry,we found that the senescence level of CD8~+T cells in the peripheral blood of HIV-infected patients was positively correlated with the expression ratio of CD38 after 2 years of ART treatment,and the ratio of CD38 expression on CD8~+CD57~+T cells was higher.On the other hand,CD38 expression on CD8~+PD-1~+T cells was higher after receiving ART treatment,and CD38 was expressed at higher levels on both CD4~+PD-1~+T cells and CD8~+PD-1~+T cells before ART treatment.3.CD38 affects the cytokine secretion ability of T cellsCD38~+T cells and CD38~-T cells were sorted by a flow cytometer,and the IFN-?secretion level of the sorted cells was detected by flow cytometry after 5 hours of PMA/ionomycin stimulation.We found that the IFN-?secretion capacity of CD4~+T cells and CD8~+T cells in the sorted CD38~+T cells were significantly lower.4.NAD concentration,SIRT activity and cellular oxidation respiration increased after CD38 knocking downWe knocked down the expression of CD38 by transfecting the CD38 sh RNA lentivirus into the Jurkat cell line,then detected the intracellular NAD concentration and nuclear SIRT enzyme activity.We found after CD38 knocking down,the NAD concentration and the nuclear SIRT enzyme activity level significantly increased.Then we detected the mitochondrial oxidative respiration ability of the cells after CD38knocking down,and found that the basal oxygen consumption rate and maximal respiration have been improved to a certain extent.5.CD38 affects genes related to T cell function,development and cytotoxicityCD38~+/CD38~-T cells were sorted by flow cytometry for transcriptome detection and analysis.We found that the expression of T-bet,an important gene that affects IFN-?cytokine secretion and Th1 development,was reduced in CD38~+T cells.In addition,cytotoxicity-related genes GZMK(granzyme K),GNLY(granulysin)and NKG7(natural killer cell granule protein 7)were also significantly down-regulated,suggesting that CD38 can inhibit T cell function,development and cytotoxicity.6.CD38 affects cell mitochondrial function,promotes mitochondrial dysfunction and ROS accumulationThrough transcriptome analysis,we found that CD38~+T cells highly express the mitochondrial fission regulator 2(mitochondrial fission regulator 2)and the autophagy-related gene DEPDC1(DEP domain containing 1).Combined with GSEA analysis,we speculate that CD38 will affect the mitochondrial homeostasis and cell survival of T cells.Through flow cytometry,we proved that CD38~+T cells have high mitochondrial dysfunction,poor mitochondrial function and high ROS levels.7.CD38 inhibitors can increase the NAD concentration and SIRT enzyme activity of T cells in HIV-infected patientsAfter extracting T cells from the peripheral blood of HIV-infected patients receiving ART treatment,the cells were treated with CD38 inhibitor 78c and stimulated with CD3/CD28 beads for 24 hours to detect the intracellular NAD concentration and SIRT enzyme activity.We proved that 78c can significantly increase the intracellular NAD concentration of T cells in infected patients,and the SIRT enzyme activity also shows an upward trend.8.CD38 inhibitors can reduce mitochondrial dysfunction and inhibit T cell senescenceThe mitochondrial mass,mitochondrial membrane potential and senescence marker?-galactosidase of the cells were labeled with flow dyes to detect the mitochondrial function and cell senescence level after treatment with CD38 inhibitor 78c.It was found that CD38 inhibitors can reduce T cell mitochondrial dysfunction and delay aging.We also detected the difference in mitochondrial membrane potential and ROS levels,and found that 78c had no significant effect on mitochondrial potential and ROS.9.CD38 inhibitor can increase the cytokine secretion and proliferation ability of T cellsAfter treating T cells with CD38 inhibitor 78c,the cytokine secretion function and cell proliferation ability were detected,and we found that the CD38 inhibitor can significantly increase the IFN-?secretion but has no significant effect on the level of IL-2,the T cell proliferation ability is also improved.We also proved that NAD precursor supplement drug?-Nicotinamide mononucleotide(NMN)can delay T cell senescence but has no significant effect on cytokine secretion and mitochondrial function.We speculate it may be due to the high expression of CD38 consumes NMN and NAD,making the exogenous NAD supplementary drugs unable to play an effective role.Conclusion:1.In the first part of this study,we clarified the relationship between CD38 and T cell function and senescence after ART in HIV-infected patients.We found that the expression of CD38 was significantly increased after HIV infection,and ART treatment could not fully recover it.The expression of CD38 is negatively correlated with the absolute value of CD4~+T cells in patients.We also found that CD38 is expressed at a high level on exhausted and senescent T cells,and the sorted CD38~+T cells have poor cytokine secretion function,indicating that CD38 may promote the senescence and exhaustion of T cells and inhibit T cell function during HIV infection.2.In the second part of this study,we explored the biological function and mechanism of CD38 in HIV-infected T cells,and found that CD38 can inhibit intracellular NAD concentration,SIRT enzyme activity and mitochondrial respiratory capacity.Through transcriptome analysis,we speculate that CD38 will inhibit the survival,function and cytotoxicity of T cells.We also proved that CD38 affects the mitochondrial homeostasis of T cells,increases mitochondrial dysfunction and ROS levels,and affects cell function and survival.3.In the third part of this study,we first explored the possible intervention methods for the increase of CD38 in HIV-infected patients and their impact on T cells.We found that CD38 inhibitors can increase the NAD concentration and SIRT enzyme activity of T cells in HIV-infected patients.We also found that CD38 inhibitors can reduce T cell mitochondrial dysfunction,delay cell senescence,and can also improve the ability of cytokine secretion and cell proliferation.These results provide new clues for the effects of CD38 inhibitory drugs on T cells,and also provide new strategies for improving the immune recovery and disease outcome of HIV.