Study On Expression And Mechanism Of CD54 In Immunological Nonresponders After Antiretroviral Therapy

Objective:Some HIV-infected patients who receive antiretroviral therapy（ART）fail to recover the number of CD4⁺T cells to the normal level,which are known as immunological nonresponders（INR）.Their clinical prognosis is poor and the mortality rate is high.The causes of INR are complex,among which the excessive destruction of CD4⁺T cells is one of the important reasons,which is related to the abnormal activation and apoptosis of CD4⁺T cells and the cleavage of CD4⁺T cells by NK cells.CD54 is an adhesion molecule that can be highly expressed in a variety of inflammatory diseases.Our previous study found that CD54 molecules on the surface of CD4⁺T cells can participate in the killing of autologous CD4⁺T cells by NK cells.In this study,we compared the expression of CD54 in CD4⁺T cells between immunological responders（IR）and INR,and further explored the possible role of CD54 in INR.We also explored the possible promoting factors of CD54 on CD4⁺T cells and the interventions to reduce their expression.Methods:1.Study Participant:A total of 82 HIV infected patients with ART therapy longer than 2 years and viral load<20 copies/m L were selected.There were 44 cases of IR（CD4⁺T cell absolute count>500 cells/μL）and INR and 38 cases of INR（CD4⁺T cell absolute count≤350 cells/μL）.There were 70 HIV-negative healthy volunteers.2.Detection of Absolute CD4⁺T Cell Counts:Anticoagulated whole blood was mixed with CD3/CD4/CD8 Tri TEST reagent in an absolute counting tube and then hemolysin was added.BD Calibur flow cytometry was used for detection and Multi SET software was used for automatic analysis.3.HIV viral load test:The whole blood of the subjects was collected and the plasma was isolated and detected by an automatic load analyzer.4.Extraction of peripheral blood mononuclear cells（PBMCs）:PBMCs were extracted by density gradient centrifugation.Peripheral venous blood was collected and mixed with an equal proportion PBS,then slowly added to the Ficoll surface and centrifuged.Carefully draw the cell layer and centrifuge twice with PBS.5.Cell surface marker detection:The extracted PBMCs were added with mixed fluorescent antibodies for 4°C,30 min dark staining.6.Apoptosis experiment:The extracted PBMCs were divided into three parts:one part was stained for CD3-PE-cy7,CD4-APC-cy7,CD54-BV421 surface staining followed by apoptosis staining.The steps were as follows:the cells were washed once with cold PBS,and then washed again with Binding Buffer.After discarding the supernatant,Annexin V and 7-AAD dye were added,incubated in the dark at room temperature,and the Binding Buffer was added and analyzed by flow cytometry within 1hour.The other two were resuspended with R10 and incubated in 96-well plates with Ig G and anti-CD54 antibodies respectively and the cells were collected from incubator for surface staining after 24 hours.7.Cytoplasmic reactive oxygen species（ROS）assay:The cells were mixed with Cell ROX^TM Green Reagent（1.25μM）and incubated in dark at 37°C for 30 min.After washing the cells twice,Live/Dead dye was added to stain the cells in dark at room temperature.8.Negative selection of CD4⁺T cells and NK cells from peripheral blood:Resuspend the concentration of PBMCs to 5×10⁷cells/m L,add the corresponding antibody according to the number of cells and incubate at room temperature,then add the corresponding magnetic beads.Add PBS to 2.5 m L and place the test tube on the magnetic pole and leave it at room temperature.The supernatant is collected to obtain the desired CD4⁺T cells and NK cells.9.NK cells killing of autologous CD4⁺T cells assay:Negatively selected NK cells and CD4⁺T cells were cultured in 96-well plates with R10.NK cells were stimulated with IL12/IL15/IL18,while CD4⁺T cells were divided into two groups,one for control and one for CD3/28 stimulation.After 2 days of separate culture,cells were collected and counted,and co-cultured at the ratio of CD4⁺T cells:NK cells（1:0）or（1:5）.Cells were collected for flow assay.10.Induction of CD54 expression in CD4⁺T cells:（1）HIV-1 Gag,HIV-1 Env,CXCL9,TNF-α,IL6,IL12,IL15 were coincubated with the cells respectively and then subjected to surface staining.（2）CD4⁺T cells after negative selection were co-incubated with IL15 and cells were collected at different time points,or CD4⁺T cells were collected after co-incubation with different concentrations of IL15 for 24 hours for surface staining.（3）Extracted PBMCs were incubated with different concentrations of HIV-1Env and then cells were collected and surface detected by flow cytometry.11.RNA extraction:Cells were lysed using lysis Buffer RL.The g DNA Eraser Spin Column was used to remove impurities and g DNA.RNA Spin Column was used to bind RNA.Buffer RWA and Buffer RWB play a cleaning role.Finally,RNase Free d H2O was added to the center of the RNA Spin Column membrane to elute RNA.12.Reverse transcription and RT-q PCR:According to the Prime Script RT Master Mix reagent instructions,reverse transcription was performed to obtain c DNA.RT-q PCR was performed according to the TB Green?Premix Ex Taq TM II reagent instructions.13.Effect of IL15 on CD54 and ligand LFA-1 in NK cells:Cells were collected after co-incubation of PBMCs with 25 ng/m L IL15 for 48 hours and fluorescent antibody staining with CD3-APC,CD4-FITC,CD14-Percp-cy5.5,CD19-Percp-cy5.5,CD16-APC-cy7,CD56-PE-cy7,CD54-PE,LFA-1-BV421.14.Inhibition of CD54 expression assay:（1）Pathway inhibitors:The extracted PBMCs were incubated with control DMSO,PI3K inhibitor LY294002,AKT inhibitor MK-2206,ERK inhibitor SCH772984 for 1 hour,25 ng/m L IL15 was added,and the cells were collected two days later for surface staining.（2）Glutathione:The extracted PBMCs were divided into two groups.One group was the control group and one group was spiked with 500μM glutathione and the cells were collected after 24 hours of incubation.One was subjected to cytoplasmic ROS staining and the other was stained with CD3-PE-cy7,CD4-Apc-cy7,CD54-PE,7AAD.（3）Silybin:After negative selection,CD4⁺T cells were first incubated with silybin for 1h,followed by addition of 25 ng/m L IL15,and cells were collected after 48 h.PBMCs were first incubated with silybin for 1h,followed by addition of anti-CD3/28 antibody,and cells were collected after 48h.Surface staining was performed for CD3-APC,CD4-FITC,CD54-PE and 7AAD.15.Statistical analysis:Flow Jo v10.6.2 and Graph Pad Prism 9.0 were used to analyze the data and results respectively.For the unpaired two sets of data,if it conforms to the normal distribution,the unpaired test is used,and the Mann-Whitney U test is used if it does not conform.For paired data,Wilcoxon paired rank sum test was used for comparison between the two groups,and Friedman test was used for comparison between multiple groups.Spearman rank correlation test was used for correlation analysis.Results:I.CD54 was highly expressed on the surface of CD4⁺T cells in INR and was negatively correlated with immune recoveryThe percentage and mean fluorescence intensity of CD54 expression on INR CD4⁺T cells were higher than those on IR.In INR,the percentage of CD54 expression on CD4⁺T was negatively correlated with the absolute count of CD4⁺T cells（r=-0.3360,P=0.0392）.II.The role of elevated CD54 on CD4⁺T cells1.Activation increases the expression of CD54,which increases the killing of NK cells on CD4⁺T cellsThe expression of HLA-DR and CD71 on CD54⁺CD4⁺T cells was higher than that on CD54^-CD4⁺T cells.The expression of CD54 on CD4⁺T cells was positively correlated with the expression of HLA-DR in Hiv-Infected patients.The expression of CD54 on the surface of CD4⁺T cells was significantly increased after stimulation with anti-CD3/28antibody.In HIV-Infected patients after ART,the mortality of CD4⁺T cells in co-culture group was higher than that in CD4⁺T cell culture alone group.After removing the natural death rate of CD4⁺T cells during the culture process,we observed the killing effect of NK cells on CD4⁺T cells.It was found that CD4⁺T cells were more killed after CD3/28antibody stimulation.2.CD54 promoted apoptosis of CD4⁺T cellsThe level of early apoptosis of CD54⁺CD4⁺T cells was higher than that of CD54^-CD4⁺T cells.In INR,cytoplasmic ROS in CD4⁺T cells showed a significant positive correlation with CD54 expression.The cytoplasmic ROS of CD4⁺T cells decreased after incubation with PBMCs using anti-CD54 blocking antibody.When the PBMCs were co-cultured with anti-CD54 blocking antibody,the early apoptosis level of CD4⁺T cells was significantly decreased compared with the control antibody group.III.Possible promoting factors for the increased expression of CD54 on CD4⁺T cells1.HIV-1 Env protein effectively induced an increase in CD54 expression on CD4⁺T cells HIV-1 Env protein induced a concentration-dependent increase in CD54 expression on CD4⁺T cells.2.IL15 can induce the increase of CD54 expression on CD4⁺T cellsIL15 was found to be a potent cytokine inducing the expression of CD54 on CD4⁺T cells in a concentration-dependent and time-dependent manner.This effect was also observed at the transcriptional level of CD54 on CD4⁺T cells.IL15 can also induce the expression of CD54 and its ligand LFA-1 on NK cells.3.PI3K,AKT and ERK are involved in IL15-induced CD54 expression on CD4⁺T cells PI3K pathway inhibitor LY294002,AKT pathway inhibitor MK-2206 and EKR pathway inhibitor SCH772984 significantly inhibited IL15-induced CD54 expression in PBMCs S pretreated with inhibitors targeting specific pathway components.IV.Interventions to reduce CD54 expression on the surface of CD4⁺T cells1.The antioxidant glutathione（GSH）can reduce the expression of CD54 in CD4⁺T cells GSH can reduce the content of ROS and the expression of CD54 on the surface ofCD4⁺T cells.2.Silybin decreased the expression of CD54 in CD4⁺T cellsThe optimal concentration of silibinin was selected according to cell survival,and it was found that this concentration could effectively inhibit CD3/28 antibody and IL15stimulated CD4⁺T cells CD54 expression.Conclusion:1.The expression of CD54 on CD4⁺T cells was higher in INR than in IR and was negatively correlated with the absolute count of CD4⁺T cells.2.CD54 on the surface of CD4⁺T cells can reduce the number of CD4⁺T cells by killing CD4⁺T cells by NK cells and promoting apoptosis.3.HIV-1 Env protein and IL15 can effectively induce the expression of CD54 on CD4⁺T cells.4.Antioxidants and silybin can be used as effective means to reduce CD54 on CD4⁺T cells.