Study On Functions And Mechanisms Of Immune Checkpoint Tigit On NK Cells In HIV Infected Individuals

Objective:Natural killer（NK）cells are important for maintenance of innate immune system stability and serve as a first line of defense against tumors and virus infections.After tumor or chronic viral infection,innate immune NK cells and adaptive immune T cells exert immune functions to fight tumors and infections.However,because the immune system cannot quickly eradicate the disease,the immune cells are in an over-activated state,and the high expression of immune checkpoint makes cells cannot exert normal immune functions.More and more immune checkpoints and their inhibitory pathways have been confirmed in tumor diseases.However,the significance of the expression of these immune checkpoints after HIV infection and the clinical outcome is rarely studied.In this study,by detecting and analyzing a variety of immune checkpoints on the surface of NK cells,we obtained the key immune checkpoint which the most closely related to the progression of HIV disease—TIGIT（Paper 1）;For the first time to explore the effects of TIGIT on the function of NK cells in HIV-infected individuals and to intervene（Paper 2）;For the first time to explore the effect of TIGIT on the glucose metabolism of NK cells in HIV-infected individuals and related mechanisms（Paper 3）.Overall,these results highlight the important role of TIGIT in NK cell function and suggest a potential new avenue for the development of therapeutic strategies toward a functional cure for HIV.Methods:1.Study ParticipantsThis study included a total of 203 individuals,among which 96 were HIV-infected and had never been treated with antiretroviral therapy;16 were ART-treated patients;91 were HIV-negative healthy controls.All individuals were matched in age and gender.The results of routine blood tests in healthy controls were normal and there was no immune system disease.Patients with HIV were recruited at the Red Ribbon Clinic in the First Affiliated Hospital of China Medical University,and the healthy controls were all healthy volunteers recruited by the AIDS Institute of the First Affiliated Hospital of China Medical University.All individuals signed informed consent forms approved by the Research and Ethics Committee of The First Affiliated Hospital of China Medical University（Shenyang,China）.2.Detection of Absolute CD4⁺T Cell CountsAnticoagulant-treated whole blood samples（50μL）and 20μL Tri TEST anti-CD4-FITC/CD8-PE/CD3-Per CP（BD Biosciences）reagents were added into Trucount tubes（Becton Dickinson）.A single-platform lyse-no-wash procedure was performed and cells detected using a BD FACS Calibur flow cytometer.Data were analyzed using Multi SET software.3.Measurement of Plasma HIV RNA LevelsReverse transcription polymerase chain reaction（RT-PCR）was used to determine plasma HIV RNA levels.The process was performed using the COBAS R Ampli Prep R/COBAS Taqman system（Roche Diagnostic Systems,Indianapolis,IN,USA）;the detection range of this assay is 20–10,000,000 copies/m L.The manufacturer’s reference standards were used to calculate HIV RNA copy number.4.Multiple immune checkpiont expression detectionCell phenotyping was detected by flow cytometry using fresh peripheral blood mononuclear cell（PBMC）samples,which were isolated by Hypaque-Ficoll（GE Healthcare,Uppsala,SE）density gradient centrifugation.The expression of TIGIT,PD-1,Tim-3 on the surface of T cells were detected in PBMC samples using the following immune fluorescent antibodies:CD3-PE-Cy7,CD4-APC-Cy7,CD8-FITC,Tim-3-PE,TIGIT-APC,PD-1-BV421;The expression of Lag-3,CTLA-4,2B4 on the surface of T cells were detected in PBMC samples using the following immune fluorescent antibodies:CD3-PE-Cy7,CD4-APC-Cy7,CD8-FITC,Lag-3-PE,CTLA-4-APC,2B4-BV421;The expression of TIGIT,PD-1,Tim-3 on the surface of NK cells were detected in PBMC samples using the following immune fluorescent antibodies:CD3-FITC,CD14-FITC,CD19-FITC,CD56-PE-Cy7,CD16-APC-Cy7,Tim-3-PE,TIGIT-APC,PD-1-BV421;The expression of Lag-3,CTLA-4,2B4 on the surface of T cells were detected in PBMC samples using the following immune fluorescent antibodies:CD3-FITC,CD14-FITC,CD19-FITC,CD56-PE-Cy7,CD16-APC-Cy7,Lag-3-PE,CTLA-4-APC,2B4-BV421.After adding the corresponding fluorescent antibody,stain for 30 min at 4°C and avoid light.Wash the cells twice（300g,10min）with PBS（containing 2%FBS）to remove unbound dye.Discard the supernatant Vortex and mix the cells,then add 200μL PBS（containing 2%FBS）to resuspend,and add 10μL 7AAD to each tube,use flow cytometry to detect T cells and NK cells surface TIGIT,Tim-3,PD-1.The expression of CTLA-4,2B4 and Lag-3.The results obtained by the flow cytometer were analyzed with Flow Jo software v10 software.5.Detection the expression of TIGIT on NK cell subsets and CD226 expression in total NK cells.The expression of TIGIT on NK cell subsets and CD226 expression in total NK cells were detected in PBMC samples using the following immune fluorescent antibodies:CD3-Per Cp-Cy5.5,CD56-PE-Cy7,CD16-APC-Cy7,TIGIT-APC,CD226-FITC,Live/Dead-BV510.6.Detection the expression of CD155 on CD4⁺T cell.CD155 ligand expression was also determined on live CD4⁺T cells in PBMC samples,using the following immune fluorescent antibodies:anti-CD3-Per CP,Anti-CD4-APC-Cy7,anti-CD155-BV421,BV421 mouse Ig G1,κisotype control,and Fixable Viability Stain 510.7.Detection of interferon-Gamma（IFN-γ）production and degranulation of NK cells（CD107a）.IFN-γrelease and cytotoxic molecule degranulation（CD107a）levels were assessed by flow cytometry.For staining,PBMCs were seeded in 96-well round-bottom plates（50,000–60,000 per well）and stimulated with a cocktail of cytokines（IL-12,IL-15,and IL-18 at 10,20,and 100ng/m L,respectively）.Unstimulated cells were used as negative controls.Cells were incubated in RPMI media containing 10%fetal bovine serum（FBS）and 1%penicillin/streptomycin（PS）for 24 h at 37℃with 5%CO₂in the presence of CD107a-PE antibody,in a total volume of 200μL.Golgi Stop（1μL）was added during the final 4 h of culture.Cells were harvested,washed,and stained with anti-CD3-FITC,anti-CD56-PE-Cy7,anti-CD16-APC-Cy7,and anti-TIGIT-Per CP/Cy5.5 in staining buffer on ice in the dark for 20 min.APC mouse Ig G1,κisotype control,PE mouse Ig G1,κisotype control,and Per CP/Cy5.5 Mouse Ig G1,κIsotype control were used to define gates.Cells were then fixed with Fixation/Permeabilization solution at room temperature in the dark for 20 min,washed twice with Perm/Wash buffer,and intracellularly stained with anti-IFN-γ-APC for 20 min.Then cells were washed and re-suspended in staining buffer and analyzed in the BD FACS Canto II cytometer.8.Detection of changes in TIGIT expression levels on CD226⁺NK cells treated with different stimuli.Changes in TIGIT expression levels on CD226⁺NK cells were detected by flow cytometry.For staining,PBMCs were seeded in 96-well round-bottom plates（50,000–60,000 per well）and stimulated with IL-10（10ng/m L）,IL-12+IL-15（10ng/m L and 50ng/m L,respectively）,or transforming growth factor-beta（TGF-β）（50ng/m L）respectively.Unstimulated cells were used as negative controls.Cells were incubated in RPMI media containing 10%FBS and 1%PS for 24 h at 37?C with 5%CO₂in a total volume of 200μL,then harvested,washed,and stained with anti-CD3-Per CP,anti-CD56-PE-Cy7,anti-CD16-APC-Cy7,and anti-TIGIT-APC in staining buffer on ice in the dark for 20 min.APC mouse Ig G1,κisotype control was used to define gates.Cells were then washed twice with PBS containing 2%FBS,re-suspended in staining buffer,and analyzed using a BD FACS Canto II cytometer.9.TIGIT,CD226,and CD155 blockade assays.The effects of blocking TIGIT,CD226,or CD155 on NK cell functions were assessed by preincubating PBMCs in the presence of functional grade purified anti-human TIGIT antibody（5μg/m L）,purified anti-human CD226 antibody（20μg/m L）,purified anti-human CD155 antibody（20μg/m L）,or purified mouse Ig G1,κisotype control（5μg/m L or 20μg/m L）for 1h before stimulation with IL-12,IL-15,and IL-18（10,20,and 100 ng/m L,respectively）for 24h at 37?C and 5%CO₂.After an incubation,IFN-γrelease and CD107a were evaluated as“7”described above.Recombinant human TIGIT Fc Chimera Protein（r TIGIT;25,50,and 100 ng/m L）and LEAFTM Purified Mouse Ig G1,κIsotype control（25,50,and 100 ng/m L）were added to pre-treated PBMCs for 1h before stimulation with IL-12,IL-15,and IL-18（10,20,and 100 ng/m L,respectively）for24h at 5%CO₂and 37℃.IFN-γrelease and CD107a were evaluated as“7”described above.10.Enrichment of NK cells from PBMCs.Counting the cell to adjust the concentration to 5?10⁷/m L.Add 50μL/m L negative selection reagent and incubate at room temperature for 10 minutes;at the same time,prepare Magnetic Particles.After votexing the beads,add Magnetic Particles to the cells and incubate at room temperature for 5 minutes.PBS was replenished to 2.5m L and aspirate the upper foam,mix the magnetic pole upside down,place it in the magnetic pole for 5 minutes,and aspirate NK cells for subsequent experiments.11.Detection of the expression of metabolism-related receptors such as Glut-1 on the surface of NK cells.The sorted NK cells were divided into two groups for staining:the first group was added with TIGIT-BV421,Glut-1-PE,CD36-APC,Live/Dead-BV510 fluorescent antibody,and the second group was added with TIGIT-BV421,CD98-PE,CD71-APC,Live/Dead-BV510 fluorescent antibody,stain for 30 min at 4?C in the dark,wash the cells twice（300 g,10 min）with PBS（containing 2%FBS）to remove unbound dye.The supernatant Vortex was discarded and the cells were mixed and resuspended in 200L PBS（containing 2%FBS）.Flow cytometry was used to detect the expression of NK cell Glut-1 and other metabolism-related receptors and TIGIT.12.TIGIT^-/+NK cell glucose uptake capacity（2-NBDG）test.First,fully dissolve 5mg 2-NBDG powder with 500?L DMSO,resuspend the sorted NK cells in 1m L glucose-free medium,add 3.4?L of dissolved 2-NBDG solution to it,incubate at 37℃for 30min and wash（300 g,10 min）.Next,perform surface staining:add TIGIT-BV421 dye to the detection tube,stain for 30 min at 4?C in the dark,wash the cells twice（300 g,10 min）with PBS（containing 2%FBS）to remove unbound dye.The supernatant Vortex was discarded and the cells were mixed and resuspended in 200?L PBS（containing 2%FBS）.Flow cytometry was used to detect the glucose uptake capacity of TIGIT^-/+NK cells.13.The effect of exogenous glucose on TIGIT⁺NK cells.（1）Detection of the influence of exogenous glucose on IFN-γand CD107a of TIGIT⁺NK cells.Collect 10m L of whole blood for peripheral blood mononuclear cell extraction and sort NK cells.After counting the cells,use R10（RPMI 1640+10%FBS）to resuspend and plant them in a 96-well plate to ensure 50,000-60,000 cells per well.The cells were stimulated with a cytokine mixture of 10ng/m L IL-12,20ng/m L IL-15,and 100ng/m L IL-18.The unstimulating cells were used as a negative control.Add 10mmol/L glucose to the glucose detection well,and then the steps of cell culture,surface staining,membrane rupture and intracellular staining are the same as those described in“7”.（2）Detection of the effect of exogenous glucose on the expression of Glut-1 in TIGIT⁺NK cells.Collect 10 ml of whole blood for peripheral blood mononuclear cell extraction,sort NK cells,count the cells and resuspend them in a 96-well plate with R10（RPMI 1640+10%FBS）to guarantee 50,000-60,000 cells per well.The cells were stimulated with a cytokine mixture of 10ng/m L IL-12,20ng/m L IL-15,and 100ng/m L IL-18.The unstimulating cells were used as a negative control.Add 10mmol/L glucose to the glucose detection well,and then the steps of cell culture and surface staining are the same as those described in“11”.14.The experiment of Fc CD155 activating TIGIT signal.After NK cell sorting,the cells were placed in a 96-well culture plate.First,5μg/m L Fc CD155 was added to the corresponding wells to pre-culture the cells for 1h.After the culture was completed,10ng/m L IL-12,20ng/m L IL-15 and 100ng/m L IL-18 cytokine mixture to stimulate the cells.The following steps of cell culture,surface staining,membrane rupture and intracellular staining are the same as those described in“7”and“11”.15.PI3K-Akt-m TOR signaling pathway phosphorylation level detection.After sorting,NK cells were divided into flow cytometry tubes,and the cells in each tube were re-suspended in 200μL sugar-free medium,corresponding to 5μg/m L Fc CD155and 10mmol/L glucose.Heat Phosflow?Fix Buffer I to 37?C,and cool BD Phosflow?Perm Buffer III to-20?C for later use.After pretreatment,NK cells were stimulated with a cytokine mixture of 10ng/m L IL-12,20ng/m L IL-15 and 100ng/m L IL-18 for 15minutes.At the end of the treatment,immediately put a volume of warm BD Phosflow?Fix Buffer I is mixed with one volume of cell suspension（one volume in this experiment=200μL）.After mixing well,incubate the tube in a 37?C water baths for 10 minutes.After the incubation,the cells were centrifuged at a speed of 250g for 10 minutes,and then the supernatant was aspirated.Washing the cells once with BD Pharmingen?Stain Buffer（2%FBS in PBS）,centrifuge at 350g for 5 minutes and remove the supernatant;vortex to loosen the cells.Permeability cells by slowly adding 100μL of cold BD Phosflow?Perm Buffer III during vortexing,incubate on ice for 30 minutes,then vortex the cells and wash the cells with 3 m L BD Pharmingen?Stain Buffer（2%FBS in PBS）Twice（250g 10min）.Resuspend the cells in BD Pharmingen?Stain Buffer（2%FBS in PBS）at a concentration of 1×10⁷/m L,and aliquot 100μL into each flow tube to continue phosphorylation staining:add to the corresponding detection tube m TOR-PE,4EBP1-FITC,s6k-PE-Cy7,Akt-APC and Erk-Per CP dyes,stain for 30 min at 4?C in the dark,wash the cells twice（300g,10 min）with PBS（containing 2%FBS）Remove unbound dye.Discard the supernatant Vortex,mix the cells,add 200μL PBS（containing2%FBS）to resuspend,and use flow cytometry to detect.16.Signal pathway blocking.After NK cell sorting,plant the cells in a 96-well culture plate,and add 5mmol/L 2-DG,10μmol/L MHY1485,200nmol/L Torin 1,50μmol/L CMK,50μmol/L LY294002.The cells were pre-cultured with 20μmol/L SC79 and 10μmol/L MK-2206 for 1h.After the culture,the cells were stimulated with a cytokine mixture of 10ng/m L IL-12,20ng/m L IL-15,and 100ng/m L IL-18.The following steps of cell culture,surface staining,membrane rupture and intracellular staining are the same as those described in“7”.17.Statistical AnalysisThe results obtained by flow cytometry were plotted using Flow Jo software v7.5.2.Graph Pad Prism 7 and SPSS 22.0 software were used to graph and analyze the experimental results.In the process of statistical analysis,the data was first tested for normal distribution,and it was found that all the data conformed to the non-parametric distribution.The non-parametric Mann-Whitney U test is used to compare the difference in quantitative data between two groups,the Kruskal Wallis test is used for multiple group comparisons,the Wilcoxon paired test is used for paired rank sum test between two groups,and the Friedman test is used for multiple groups.For paired comparison,Spearman is used to analyze the correlation between the two groups,and the correlation analysis between the immune regulatory receptors uses the Pearson correlation test.The tests are all two-sided tests,and p<0.05 is considered a significant statistical difference.Results:（一）Study on the expression of six immune checkpoints on the surface of NK cells and T cells in HIV-infected individuals.1.TIGIT is the highest immune checkpoint on the surface of NK cells in HIV-infected individuals.By using multicolor flow cytometry to detect the expression of six immune checkpoints on the surface CD4⁺T,CD8⁺T,and NK cells in HIV-infected individuals,it was found that each immune checkpoint increased to varying degrees,and TIGIT is found to be the most significant immune checkpoint on the surface of NK cells in HIV-infected individuals.2.The level of TIGIT on the surface of NK cells is related to the progression of HIV disease.Further we analyzed the association of the six immune checkpoints with CD4⁺T cell counts and plasma viral loads.We found that the PD-1 level on the surface of T cells in HIV-infected individuals is related to both CD4⁺T cell counts and plasma viral loads.For the first time,we found that the level of TIGIT on the surface of NK cells is related to both CD4⁺T cell counts and plasma viral loads.3.TIGIT is the key immune checkpoint on NK cells during HIV infection.Further analysis of the expression of the levels of six immune checkpoints in immune reconstitution after ART treatment,we found that the level of TIGIT on the surface of NK cells is elevated in HIV-infected individuals with poor immune reconstitution.（二）Study on functions and mechanisms of immune checkpoint TIGIT on NK cell in HIV infected individuals.1.The expression level of TIGIT on NK cell subsets changed significantly in HIV infected individuals.NK cells can be divided into four distinct subgroups based on their surface expression of CD56 and CD16,we found that TIGIT levels were elevated in CD56^-CD16⁺and CD56^dimCD16⁺NK cells in the HIV group.2.The expression of TIGIT limits the production of IFN-γin NK cells.HIV disease progression may be related to impaired NK cell function,we explored whether NK cell IFN-γproduction levels were affected by TIGIT expression.We observed a higher level of IFN-γproduction in TIGIT^-NK cells compared with TIGIT⁺NK cells.Furthermore,there was a negative correlation between the percentage of TIGIT⁺NK cells and IFN-γ⁺NK cells.3.TIGIT is mainly expressed on CD226⁺NK cells in HIV-infected individuals.Compared with those of the HC group,the expression levels of CD155 on CD4⁺T cells were significantly higher in the HIV-infected group.we found a higher level of TIGIT receptor expression on CD226⁺NK cells in the HIV group.Furthermore,we found that stimulation with a combination of r IL-12 and r IL-15,or with r IL-10,led to significant increases in TIGIT expression on CD226⁺NK cells from HIV-infected individuals;however,no effects on these cells were observed on stimulation with r TGF-β.4.Inhibition of TIGIT can restore the function of NK cells in HIV-infected individuals.We further explored whether NK cell function could be enhanced by treating PBMCs from HIV-infected individuals with anti-TIGIT antibody or r TIGIT,we found that both anti-TIGIT antibody and r TIGIT were able to reverse the inhibitory effect of TIGIT on NK cell function.（三）Study on the glucose metabolism effect and mechanism of TIGIT on NK cell in HIV infected individuals.1.The level of Glut-1 on the surface of NK cells in HIV-infected individuals was decreases and negatively correlated with the level of TIGIT.Given that TIGIT can inhibit NK cell function,we further explored whether TIGIT affects cell function by inhibiting NK cell-related metabolic pathways.We tested the expression of four representative indicators related to metabolism on the surface of NK cells in HIV-infected patients.It was found that the level of Glut-1 on the surface of NK cells in HIV-infected patients decreased and was negatively correlated with the level of TIGIT.2.The glucose metabolism activity of TIGIT⁺NK cells decrease,and exogenous glucose can reverse the expression of Glut-1 and IFN-γin TIGIT⁺NK cells.We further explored the changes in glucose metabolism of TIGIT⁺NK cells and the effect of glucose metabolism on the function of TIGIT⁺NK cells.For the first time,we found that the level of Glut-1 on the surface of TIGIT⁺NK cells decreased,the glucose uptake ability decreased,and the inhibition of glucose metabolism led to the decline of TIGIT⁺NK cell function.To further study whether exogenous glucose can reverse the exhaustion of NK cells and abnormal glucose metabolism levels related to TIGIT,we found for the first time that HIV-infected TIGIT⁺NK cells Glut-1 and IFN-γlevels can be reversed by exogenous glucose,but the production of CD107a cannot be reversed.3.TIGIT/CD155 signal inhibits Glut-1 expression in NK cells.In order to study whether TIGIT/CD155 signal is involved in NK cell function and glucose metabolism abnormality after HIV infection,we constructed a CD155/TIGIT signal model in vitro.We found that when the TIGIT/CD155 signaling pathway is activated,the expression of Glut-1 on the surface of NK cells decreases and the ability to secrete IFN-γalso decreases.Blocking TIGIT can restore the realization of related functions.4.TIGIT inhibits the function of NK cells in HIV-infected individuals by inhibiting the PI3K-Akt-m TOR-m TORC1（s6k）signaling pathway.Through the exploration of the metabolic mechanism,we found that TIGIT/CD155signal can inhibit the phosphorylation of m TOR and s6k in the PI3K-Akt-m TOR signaling pathway.Using m TORC1（s6k）inhibitor CMK to further verify that s6k is the main protein site affected by TIGIT,so that TIGIT can inhibit the ability of NK cells to secrete IFN-γ.Conclusion:1.TIGIT is the key immune checkpoint on NK cells during HIV infection.2.The higher expression of TIGIT limits the production of IFN-γin NK cells,inhibition of TIGIT can restore the function of NK cells in HIV-infected individuals.3.The higher expression of TIGIT affects the activity of NK cell glucose metabolism in HIV-infected individuals.4.The higher expression of TIGIT inhibits the phosphorylation level of m TORC1 in the PI3K-Akt-m TOR signaling pathway,thereby inhibiting the ability of NK cells to secrete IFN-γ.