Study On Functions And Related Mechanisms Of Tetraspanin CD151 On NK Cells In HIV Infected Individuals

Objective:Human immunodeficiency virus（HIV）can be transmitted among the population through blood transmission,sexual transmission and mother-to-child transmission.The replication of HIV in the body causes a decrease in CD4⁺T cells in the infected individual,thus diminishing their immune system and resulting in Acquired Immune Deficiency Syndrome.Therefore,HIV infection has always been an important public health problem.Natural Killer（NK）cells are lymphocytes that highly express cytotoxic particles and play an important role in antiviral immunity.NK cells can lyse and eliminate HIV-infected cells by releasing cytotoxic molecules such as granzymes and perforin.The effector function of NK cells is regulated by a variety of cell surface transmembrane proteins.Tetraspanin（TSPAN）family is a protein family composed of 33protein members.Attention to the control of tetraspanins on immune cells has been growing;however,the effect of other tetraspanin family members on the antiviral activity of NK cells in HIV-infected individuals is yet to be explored further.The purpose of this study is to clarify which members of the tetraspanin family are differentially expressed between HIV-infected individuals and healthy controls,to explore the related factors affecting their expression,and to explore the correlation between differentially expressed tetraspanin members and the disease progression.Further study of its effects on NK cell proliferation,apoptosis and cytotoxicity and its related mechanisms provides a new understanding of the antiviral effect of NK cells and provides new ideas for the development of new anti-HIV strategies.Methods:1.Research objects:In this study,103 HIV-infected individuals（HIV group）were recruited from the Red Ribbon Clinic of the First Hospital of China Medical University,of which 86 were treated with antiretroviral therapy（ART+）;seventeen HIV-infected individuals were first diagnosed with HIV infection and had not yet started antiretroviral therapy（ART-）.Health control group（HC）included 27 people.The Ethics Committee of the First Hospital of China Medical University has agreed to this study,and the informed consent form has been signed by all subjects.2.Acquisition and analysis of GEO datasets:We downloaded the GSE140713,GSE2171 and GSE102771 from the GEO database（https://www.ncbi.nlm.nih.gov/geo/）,and the data sets were processed and analyzed using the SANGERBOX sequencing data online analysis platform.We first matched the downloaded data expression matrix with the corresponding coding genes and removed duplicates and missing values.Next,we used the R software package limma to obtain differential expressed genes（DEG）between HIV groups and control groups.In the GSE140713 dataset,we obtained DEG between HIV and HC according to the filter conditions adj.P<0.05 and|Log FC|>4.5.In the GSE102771 dataset,we first divided HIV individuals into two groups,CD151 high expression and CD151 low expression,based on the median expression value of CD151,and used R software package UMAP to perform dimensionality reduction clustering to determine whether this grouping standard can distinguish CD151 high and low expression groups.According to the filter conditions adj.P<0.05 and|Log FC|>1.5,we obtained the DEG in two groups.GO,KEGG and GSEA enrichment analysis were further performed.Heat map,volcano map,enrichment result map and Wayne map were completed on the SANGERBOX sequencing data online analysis platform.The expression of CD151 in various cell types of peripheral blood lymphocytes was obtained by querying the human proteomics database.3.Extraction of peripheral blood mononuclear cells（PBMC）:We obtained the PBMC by density gradient centrifugation.4.Detection of CD151 expression on NK cells:The fresh PBMC were stained with flow cytometry to select NK cells and test the proportion and MFI of CD151.FC receptor blockers were used to block FC receptors to reduce non-specific staining.After the infusion of flow antibodies,the cells were exposed to darkness for 20 minutes,then washed and spun.Subsequently,200μL PBS was added,and the cells were re-suspended and assessed by BD Canto II flow cytometry.5.Inducing the expression of CD151 on NK cellsAfter 48 hours of stimulation with ENV,LPS,TLR7/8 agonist,IL-2 and TGF-β,CD151expression on NK cells was detected.To mark dead cells,7AAD was added to the surface staining,mixed,and 200μL PBS was added.Subsequently,the cells were detected.6.Detection of KI-67 and BCL2Obtained from the peripheral blood of HIV-infected individuals,PBMC surface were stained for 30 minutes at 4°C in darkness to distinguish NK cells and CD151 negative and positive cell populations.After washing and discarding the supernatant,the nucleus was broken with Thermo Fisher’s nuclear breaking reagent.After washing,the nucleus was stained and stained at 4°C in dark for 45 minutes,washed,centrifuged,and the cells were resuspended for flow detection.7.Detection of NK cell apoptosisPBMC were cultured for 72 hours,and surface staining with flow antibodies was used to label NK cells and distinguish CD151 negative and positive cell populations.Binding Buffer was diluted 10 times with distilled water,washed cells with 1m L diluted Binding Buffer,centrifuged（350g,speed up 5 speed down 5,5 minutes）.Annexin V was added,mixed well,and stained in dark at room temperature for 15 minutes.After that,7AAD was added,mixed,and re-suspended with 200μL Binding Buffer for flow detection.8.Detection of granzyme B and perforin in NK cellsPBMC were obtained from peripheral blood of HIV-infected individuals.After a 30-minute staining at 4℃in the dark,the cells were washed and then broken with BD Cytofix/Cytoperm membrane Fixation/Permeabilization Kit.After washing and centrifugation,the supernatant was discarded and intracellular staining was performed at4°C for 20 minutes,and the cells were resuspended for flow detection.9.Detection of perforin after CD151 activation antibody stimulationCD151 activation antibody was used to stimulate PBMC for an hour,after which IL-12（10 ng/m L）,IL-15（50 ng/m L）and IL-18（100 ng/m L）were added to activate NK cells.NK cells were cultured in an incubator at 37°C for 24 hours,and perforin was detected by flow cytometry according to the above staining process.10.Detection of NK cell phosphorylationCD151 activation antibody was used to stimulate PBMC for an hour,after which IL-12（10 ng/m L）,IL-15（50 ng/m L）,and IL-18（100 ng/m L）were added to activate NK cells for 4 hours.For thirty minutes,the cells were stained in a dark atmosphere at a temperature of 4℃.The cells,after washing and discarding the supernatant,were then subjected to BD’s phosphorylation buffer for phosphorylation staining.For 30 minutes,the cells were exposed to a dark atmosphere at a temperature of 4℃.After a thorough wash and centrifugation,the cells were assessed.11.Statistical analysisFLOWJO software was used to analyze all flow data.All statistical results were performed using Graphpad Prism 9 software.Mann Whitney test was used to compare HIV group with healthy control group,treatment group and untreated group.Wilcoxon matched-pairs signed-rank test was used to analyze the experimental results of CD151 on the functions and mechanisms of NK cells.The Kruskal-Wallis test is used to compare multiple groups.Pearson correlation analysis was used to study the correlation between the expression of CD151 on NK cells and the development of HIV disease.The standard of significant difference was P<0.05.ns:no significant difference;*:P<0.05;**:P<0.01;***:P<0.001.Results:1.Changes of tetraspanin expression in PBMC of HIV-infected individuals.1.1 Tetraspanin CD151 and TSPAN8 were significantly different between healthy control and HIV-infected individuals.By analyzing the sequencing data of PBMC in the GEO database,a total of 1747 DEG were obtained,of which 602 genes were up-regulated in HIV-infected individuals and1145 genes were down-regulated in HIV-infected individuals.The enrichment of genes revealed that those with increased expression were enriched in multiple leukocyte immune activation processes.Intersection of 1747 DEG and 33 tetraspanins between HIV-infected individuals and healthy controls yielded CD151 and TSPAN8,suggesting that the two were distinct.In comparison to healthy controls,CD151 expression was increased in HIV-infected individuals,while TSPAN8 expression was diminished.1.2 CD151 is mainly expressed by NK cells in peripheral blood lymphocytes.Analysis of sequencing data from the database revealed that CD151 expression in PBMC of HIV-infected individuals receiving antiretroviral therapy was significantly lower than that of untreated HIV-infected individuals,while TSPAN8 expression remained unchanged after treatment,suggesting that CD151 is a more significant factor in HIV infection and prognosis.We analyzed the expression of CD151 in peripheral blood lymphocytes by querying the human proteomics database.The results showed that CD151 was expressed in a variety of lymphocytes,and the highest expression was in NK cells.2.The expression of CD151 on NK cells and the factors affecting its expression.2.1 In HIV-infected individuals,the NK cells displayed a heightened expression of CD151.The expression of CD151 on NK cells was detected.The proportion and mean MFI of CD151⁺NK cells were higher in HIV-infected individuals than in HC.The proportion and MFI of CD151⁺NK cells in ART+group were significantly lower than those in the ART-group.2.2 The expression of CD151 was mainly increased on CD56^dimNK cell subsets.Compared with healthy controls,the percentage and MFI no significant difference in CD151 on CD56^briNK cells was observed in HIV-infected individuals;however,the percentage and MFI of CD151 on CD56^dimNK cells were significantly increased in HIV-infected individuals.In HIV-infected individuals,a marked rise in CD151 on CD56neg NK cells was observed.No significant difference in the fluorescence intensity of CD151on CD56^negNK cells was observed in HIV-infected individuals.2.3 ENV protein of HIV induced the expression of CD151 on NK cells.There was no significant change in the expression of CD151 in NK cells stimulated by LPS,TLR7/8 agonist,IL-2 and TGF-β.The percentage and MFI of CD151 in NK cells were significantly increased after NK cells were stimulated by HIV ENV protein.3.The expression of CD151 on NK cells and the disease progression.3.1 No association was observed between CD151 on NK cells and CD4⁺T cells;no association was observed between CD151 on NK cells and CD4/CD8 ratio in HIV infection.In HIV infection,no significant correlation was found between the proportion and MFI of CD151 on NK cells and CD4⁺T cell counts;no significant correlation was found between the proportion and MFI of CD151 on NK cells and the ratio of CD4/CD8.3.2 The expression of CD151 on NK cells in HIV-infected individuals was negatively associated with viral load.A negative correlation between viral load and the proportion and MFI of CD151 on NK cells in HIV-infected individuas was observed.4.The effects of CD151 on the proliferation,apoptosis and cytotoxicity of NK cells.4.1 Differentially expressed genes and enrichment results of CD151 high and low expression groupsA comparison of CD151 high and low expression groups revealed that 308 genes were increased in the high expression group,while 37 genes were decreased.KEGG enrichment analysis indicated that the DEG were mainly enriched in the cell cycle.4.2 CD151⁺NK cells have stronger proliferation ability.The percentage of Ki-67⁺NK cells in CD151⁺NK cells was greater than that of CD151-NK cells,and the average fluorescence intensity of Ki-67 in CD151⁺NK cells was higher,suggesting that CD151⁺NK cells had a more potent proliferation capacity.4.3 The apoptosis level of CD151⁺NK cells were lower.Compared with CD151^-NK cells,CD151⁺NK cells had lower levels of apoptosis and death.CD151⁺NK cells expressed higher anti-apoptotic protein BCL-2.4.4 CD151⁺NK cells are more cytotoxic.The cytotoxic pathway of NK cells was enriched in NK cells with high expression of CD151.CD151⁺NK cells compared with CD151^-NK cells.5.CD151 enhanced the expression of perforin through PI3K-AKT pathway.5.1 Activation of CD151 enhanced the expression of perforin.Compared with Ig G control,NK cells expressed more perforin after CD151 activation antibody stimulation.5.2 CD151 had no significant effect on the phosphorylation of NF-κB.CD151significantly enhanced the phosphorylation of AKT.Compared with Ig G control,we found no significant differences in the phosphorylation of NF-κB after activation of CD151.The phosphorylation of AKT was significantly increased after CD151 activation antibody stimulation.The phosphorylation of AKT was significantly decreased after activation of CD151 with PI3K inhibitor.Conclusion:1.HIV-infected individuals exhibited a heightened expression of tetraspanin CD151 in their PBMC.2.The expression of CD151 on NK cells increased in HIV-infected individuals and decreased after ART,and the ENV protein of HIV caused CD151 expression to be increased on NK cells.3.No significant correlation was found between the proportion and MFI of CD151 on NK cells in HIV-infected individuals and the amount of CD4⁺T cells or CD4/CD8 ratio,yet a negative relationship was observed with viral load.4.In terms of function,CD151⁺NK cells in HIV-infected individuals displayed a greater proliferation,decreased apoptosis,and a more potent cytotoxicity than CD151^-NK cells in terms of function.5.In terms of mechanism,CD151 enhances the cytotoxicity of NK cells through the PI3K-AKT pathway.