Study On The Role And Mechanism Of CD155/TIGIT In The Effect Of Mesenchymal Stem Cells On NK Cell Function In HIV Infected Individuals

Objective:Acquired Immune Deficiency Syndrome（AIDS）is caused by Human Immunodeficiency Virus（HIV）.After HIV infection,persistent antigen stimulation leads to high levels of systemic immune activation and inflammation,resulting in invasion of the human immune system,CD4⁺T cell depletion and immune dysfunction and dysregulation.NK cells are an important part of the innate immune system of human body,showing strong killing activity in the course of anti-virus infection and anti-tumor.The inhibitory receptor TIGIT on the surface of NK cells after binding with its ligand CD155can inhibit NK cells-mediated immune responses.However,during HIV infection,the expression of a variety of activated and inhibitory receptors on the surface of NK cells is dysregulated,and the immune function cannot be performed normally.Mesenchymal Stem Cells（MSCs）are pluripotent stem cells with multi-differentiation potential and immunomodulatory properties,which have been widely used in the treatment of immune-related and inflammatory diseases.It has been shown that in vivo infusion of MSCs can improve the immune reconstitution in HIV-infected individuals,but the effect and related mechanism of MSCs on the function of NK cells are still unknown.In this study,IFN-γpre-stimulated Adipose-derived mesenchymal stem cell stem cells（ADSCs）were co-cultured with IL-12/15/18-activated NK cells from HIV-infected individuals in vitro for the first time,to investigate the effect of ADSCs on the apoptosis,proliferation,cytokine release,degranulation,metabolism and activation of NK cells in HIV-infected individuals,and to explore whether ADSCs exert an immunomodulatory role on NK cells through CD155/TIGIT pathway,which provides a new target and idea for clinical treatment of HIV.Method:1.Research objectsThis study included 88 subjects,48 of whom were HIV-infected individuals from the Red Ribbon Clinic of the First Affiliated Hospital of China Medical University,and 40 were healthy controls（HC）.The healthy controls were HIV-antibody negative,with normal blood routine and liver function test results,without hepatitis B virus and hepatitis C virus infection,and no immune system disease.All of the subjects above signed the informed consent form and passed the approval of the Ethics Committee of the First Affiliated Hospital of China Medical University.2.Culture of ADSCs cellsADSCs were isolated from the fat donated by healthy donors,the cells were confirmed to be in accordance with the International Society for Cellular Therapy（ISCT）criteria.ADSCs were seeded in petri dishes,add high glucose DMEM medium（D15）containing15%fetal bovine protein and 1%penicillin,then culture in the incubator.The cells were passaged when the confluence reached 80%-90%,and P3-P8 cells were used in the follow-up experiments.3.The expression of CD155 on the surface of ADSCs was detected by flow cytometryAfter digestion of ADSCs,adjust the cell concentration to 1million/m L,add fluorescent antibody CD155-PE,vortex,incubate in dark for 30 minutes,wash the cells twice（350g,5 min）to remove the unbound dye,add 200μL PBS and use flow cytometry to detect.4.The expression of CD155 on the surface of ADSCs was detected by confocal microscopyADSCs were seeded into a 12-well plate which was placed with coverslips,and when the cell confluence reaches 80-90%,wash the cells with cold d PBS twice,add 400μL 4%paraformaldehyde to soak for 30 minutes to fix the cells.Wash the cells three times,then add 200μL d PBS and incubate overnight at 4℃.After washing the cells,add 0.5%Triton X-100 for 15 minutes.After permeation,add 100μL blocking solution for 30 minutes.CD155-PE and CD90-FITC were added and incubated overnight at 4℃,wash with 200μL Tween-20-d PBS solution 3 times.Add DAPI dye for 3 minutes,wash the cells 5 times,add one drop of sealing medium,and observe under the microscope.5.Peripheral blood mononuclear cells were extracted by density gradient centrifugationEDTA anticoagulant blood was collected,dilute the blood with PBS,fully mix it,then add it to the upper layer of 12m L lymphocyte separation solution.After centrifugation,the liquid level was divided into plasma layer,PBMCs layer,lymphocyte separation layer and erythrocyte layer from top to bottom.Suck out the PBMCs layer and transfer it into a 50m L centrifuge tube,wash twice with PBS to obtain peripheral blood mononuclear cells（PBMCs）,then count.6.Detection the expression of TIGIT on the surface of immune cells and subgroups PBMCs were surface-stained,the following fluorescent antibodies were added:CD3-Per CP,CD4-APC/Cy7,CD8-PE/Cy7,CD19-BV510,CD56-PE/Cy7,CD16-APC/Cy7,lin1-FITC,HL-DR-Per CP and TIGIT-APC,incubate in dark for 30 minutes,wash cells,then use flow cytometry to detect.7.Negative selection and purity determination of NK cellsThe extracted PBMCs were re-suspended with 2%FBS-d PBS to a concentration of 50million/m L,NK cells were negatively selected and counted with Easy Sep^TM Human NK⁺T Cell Enrichment Kit,then count.0.02 million cells were taken for purity detection,the remaining cells were cultured in RPMI 1640 medium containing 10%fetal bovine protein and 1%penicillin（R10）,IL-12,IL-15 and IL-18 were added to stimulate NK cells.Add fluorescent antibodies 7AAD-Per CP,CD56-PE/Cy7 and CD16-APC/Cy7,incubate in dark for 30 minutes,use flow cytometry to detect after washing the cells.8.NK cells and ADSCs co-culture system was establishedDigest ADSCs and count,add 0.015million into the 48-well plate,and put into the incubator.After 24h,use IFN-γpre-stimulate ADSCs.After 48h,ADSCs can reach0.04million.After wash the well plate with PBS,add NK cells,0.2million,to the corresponding wells according to the ratio of ADSCs:NK=1:5.Finally,use R10 medium to supplement the volume of each well to 500μL,then put it into the incubator.9.Detection of NK cell apoptosisAfter co-culture for 48h,NK cells were absorbed into the flow tube,wash cells with Annexin V Binding Buffer,and re-suspend cells with 100μL.Add Annexin V-PE,incubate away from light for 15 minutes,add 7AAD-Per CP,incubate for 5 minutes.Add 200μL Binding Buffer,then use flow cytometry to detect.10.Detection of proliferation ability of NK cellsAfter co-culture for 120h,NK cells were absorbed into the flow tube,add Live/Dead-aqua dye,incubate in dark for 30 minutes,wash cells twice.Then,nuclear-breaking staining was performed:add Foxp3 fixed/membrane breaking working solution,vortex,incubate in dark for 45 minutes,and wash cells with washing solution（500g,5min）.After discarding the supernatant and re-suspending,add fluorescent antibody Ki67-BV421,stain 45minutes away from light,wash twice with membrane breaking solution,then detect by flow cytometry.11.Detection of the secretion of IFN-γ,TNF-αand degranulation function（CD107a）of NK cellsCo-culture at 18h,add CD107a-PE into the corresponding well,and Golgistop 6h before the end of culture.At 48h,NK cells were absorbed into the flow tubes,add Live/Dead-aqua dye,incubate in dark for 30 minutes,wash cells twice.Then conduct membrane breaking staining:add perm solution,incubate for 20 minutes away from light and centrifuge（500g,5min）.After centrifugation,discard supernatant and resuspend,wash cells twice with membrane breaking lotion.Add fluorescent antibodies:IFN-γ-BV711 and TNF-α-FITC,incubate at 4℃for 30 minutes without light,wash two times with membrane breaking solution.Then add 1%paraformaldehyde to fix the cells,use flow cytometry to detect.12.Detection of the expression of metabolism-related surface transporter receptor on NK cellsAfter co-culture for 48h,NK cells were absorbed into the flow tubes,add fluorescent antibodies:Glut-1-PE,CD36-APC,CD71-APC and CD98-PE,incubate in dark for 30minutes.After washing cells,flow cytometry was used for detection.13.Detection of the glucose uptake capacity of NK cells（2-NBDG）After co-culture for 48h,NK cells were absorbed into the flow tubes,resuspend cells in 1m L sugar-free medium,add fluorescent antibody 2-NBDG-FITC,incubate at 37℃for 30minutes,wash cells,add Live/Dead-aqua dye,incubate in dark for 30 minutes,and then use flow cytometry to detected after washing cells.14.Detection of NK cells surface activation markersAfter co-culture for 48h,NK cells were absorbed into the flow tube,add fluorescent antibodies:CD25-APC/Cy7,CD69-PE/Cy7,CD38-BV711,HLA-DR-APC,Live/Dead-aqua,dye in dark for 30 minutes,wash cells and use flow cytometry to detect.15.Monoclonal antibody blocked CD155 and TIGIT experimentsBefore co-culture,add 10μg/m L anti-CD155 and 2.5μg/m L anti-TIGIT into the corresponding wells in 96-well plates,and incubate for 1h to block the CD155 on the surface of ADSCs and TIGIT on the surface of NK cells.After incubation,0.2million NK cells were transferred into the corresponding wells of 48-well plate according to the ratio of ADSCs:NK=1:5,and the volume of each well was supplemented to 500μL with R10medium,then cultured in the incubator.The following steps,such as staining,are the same as above.16.Statistical analysisUse Flow Jo software v 10.6.2 to analyze the results obtained by flow cytometry.Use Graph Pad Prism 8 software to analyze the experimental data and generate statistical charts.Non-parametric Mann-Whitney U test was used to compare the two groups of quantitative data that did not conform to the normal distribution.Wilcoxon paired test was used to compare and analyze the same sample before and after treatment.A p-value<0.05 was considered that the difference was statistically significant.Results:（一）Detection of expression of immunomodulatory molecules on the surface of NK cells and MSCs1.Detection of TIGIT expression on the surface of NK cells and four subsets in HIV-infected individuals.The study found that the percentage and the mean fluorescence intensity of TIGIT expression on the NK cells’surface in HIV-infected individuals were significantly higher than that of healthy controls.The expression of TIGIT on the four subsets of NK cells:CD56^dimCD16⁺NK cells,CD56^dimCD16^-NK cells,CD56^-CD16⁺NK cells and CD56^brightNK cells was also significantly increased.2.ADSCs express CD155.CD155,a ligand of the inhibitory receptor TIGIT on the surface of NK cells,was found to be expressed on the surface of ADSCs by flow cytometry detection,this result was verified by confocal microscopy.（二）Effects of ADSCs on apoptosis,proliferation,cytokine release,degranulation function,metabolism and activation of NK cells in HIV-infected individuals.1.Effects of ADSCs on apoptosis and proliferation of NK cells in HIV-infected individuals.After co-culture of ADSCs with NK cells,it was found that ADSCs can promote the apoptosis of NK cells in healthy controls and HIV-infected individuals（HC:p=0.0039;HIV:p<0.0001）and inhibited the expression of Ki67（HC:p=0.0156;HIV:p=0.0005）.2.Effects of ADSCs on the expression of IFN-γ,TNF-αand degranulation of NK cells in HIV-infected individuals.ADSCs can promote the secretion of IFN-γand TNF-αby NK cells in HIV-infected individuals,as well as promote the expression of the degranulation marker CD107a（IFN-γ:p=0.0313;TNF-α:p=0.0313;CD107a:p=0.0059）.3.Effects of ADSCs on the expression of metabolism-related transporter receptors on NK cells in HIV-infected individuals.ADSCs can promote the expression of Glut-1,CD36 and CD98 on NK cells（Glut-1:p=0.0078;CD36:p=0.0156;CD98:p=0.0353）as well as glucose uptake（2-NBDG:p=0.0469）in HIV-infected individuals.4.Effects of ADSCs on activation of NK cells in HIV-infected individuals.After co-culture of ADSCs and NK cells,the effects of ADSCs on activation level of NK cells in HIV-infected individuals were studied by detecting CD25,CD69,CD38 and HLA-DR.The results showed that ADSCs can promote the expression of CD25,CD38 and HLA-DR⁺CD38⁺on NK cells in HIV-infected individuals（CD25:p=0.0391;CD38:p=0.0195;HLA-DR⁺CD38⁺:p=0.0039）.（三）Effects of ADSCs on the secretion of cytokines and apoptosis of NK cells through CD155/TIGIT pathway in HIV-infected individuals.1.Effects of ADSCs on the expression of TIGIT on the surface of NK cells in HIV-infected individuals.After co-cultured with NK cells for 48 hours,ADSCs could down-regulate the expression of the inhibitory receptor TIGIT on the surface of NK cells in HIV-infected individuals（p=0.0215）.2.Effects of ADSCs on the secretion of cytokines by NK cells after using antibodies blocking CD155-TIGIT.When TIGIT on the surface of NK cells is blocked with antibodies or co-blocked with CD155 on the surface of ADSCs,the secretion of IFN-γby NK cells was further elevated in HIV-infected individuals（anti-TIGIT:p=0.0313;anti-CD155/TIGIT:p=0.0313）,however,the secretion of TNF-αdidn’t change significantly.3.Effects of ADSCs on apoptosis of NK cells after using antibodies blocking CD155-TIGIT.When TIGIT on the surface of NK cells is blocked with antibodies or co-blocked with CD155 on the surface of ADSCs,the pro-apoptotic effect of ADSCs on NK cells of HIV-infected persons and healthy controls can be reduced（HC group:anti-TIGIT:p=0.0273;anti-CD155/TIGIT:p=0.0039;HIV group:anti-TIGIT:p=0.0156;anti-CD155/TIGIT:p=0.0020）.Conclusion:In this study,the co-culture system of IFN-γpre-stimulated ADSCs and IL-12/15/18-activated NK cells from peripheral blood of HIV-infected people was used for the first time to explore the effects and mechanisms of ADSCs on apoptosis,proliferation,cytokine release,degranulation,metabolism and activation of NK cells in HIV-infected individuals,it was found that ADSCs can promote the apoptosis and inhibit the proliferation of NK cells in HIV-infected individuals.ADSCs can promote the expression of IFN-γ,TNF-αand CD107a,as well as CD25,CD38 and HLA-DR⁺CD38⁺,the results suggested that ADSCs can improve the function of NK cells in HIV-infected individuals and promote their activation level;ADSCs promote the expression of Glut-1,CD36 and CD98 on NK cells and glucose uptake（2-NBDG）in HIV-infected individuals,suggesting that ADSCs could promote the metabolism of glucose,fatty acid and amino acids of NK cells.ADSCs could down-regulate the expression of TIGIT on NK cells in HIV-infected individuals.After blocking CD155-TIGIT with antibodies,ADSCs can further promote the secretion of IFN-γ,and attenuate the proapoptotic effect on NK cells,suggesting that ADSCs may partially inhibit the secretion of IFN-γand promote NK cell apoptosis through CD155/TIGIT pathway.This study provides an important data support for exploring the immunomodulatory effect of ADSCs on NK cells in HIV infected individuals through CD155/TIGIT pathway.