Study Of The Relationship Between NK Cell Metabolism Receptors And CD47 And HIV Reservoir In HIV Infected Individuals

Objective:With the application of highly active anti-retroviral therapy（HAART）,viremia in HIV（Human immunodeficiency virus）patients can be inhibited below the viral detection limit for a long time.Despite this,HIV is not completely cleared from the patient’s body,and the HIV reservoir persists,with viral load rebounding exponentially once treatment is stopped.In order to achieve the goal of an HIV cure,we are in dire need of finding a factor that can regulate the size of the HIV reservoir.Natural killer（NK）cells have been shown to have an important antiviral role in HIV infection,but their effect on HIV reservoir has been less studied.NK cells rely on multiple mechanisms to perform cellular functions and thus control HIV infection,and cellular metabolism plays a key role in influencing cellular functions,but the relationship between NK cell metabolism and HIV reservoir has not been reported.NK cell function is impacted by multiple immune activating and inhibitory receptors on the NK cell surface,after HIV infection,the expression of immune checkpoints on the NK cell surface is elevated,and this change inhibits the killing function of NK cells.The immune checkpoint CD47 has been shown to have a major role in tumors and other chronic infections,but the association of NK cell metabolism-related receptors with it and whether CD47 has an effect on reservoirs have not been reported.In this study,we explored for the first time the relationship between NK cell metabolic markers and HIV DNA,us RNA and ms RNA,and the association between NK cell metabolic markers and NK cell surface immune activating and inhibitory receptors.We observed some properties of the immune checkpoint CD47 of interest,so we further explored the relationship between CD47 expression and HIV reservoirs and its effects on NK cell function in HIV-infected individuals,investigated for the first time the changes in the cellular potential of NK cells to eradicate HIV reservoirs after blocking CD47 signaling,and finally explored drug interventions on CD47 expression and provide new strategies and basis for HIV reservoir eradication.Methods:1.Study ParticipantsA total of 132 subjects were included in this study.The study population was composed of126 HIV-infected individuals who had received ART for≥2 years and had a post-treatment viral load<20copies/m L,and 6 healthy individuals with normal routine blood tests,with no detectable major immune system-related diseases as well as HCV or HBV infection.All infected individuals participating in this study were recruited by the Red Ribbon Clinic of the First Hospital of China Medical University,and all healthy individuals were recruited through the Key Laboratory of AIDS Immunology of the Ministry of Health,and all subjects were aware of their participation in this study and also signed an informed consent form,which was approved by the Ethics Committee of the First Hospital of China Medical University.2.Detection of the expression of metabolism-related receptors such as Glut-1,CD36 and CD98 on the surface of NK cellsPBMCs were withdrawn from the blood of the subjects,and the cells were labeled with flow-through fluorescent antibodies against CD3,CD14,CD19,CD16,CD56,Glut-1,CD36,CD98,and then left to stand at 4°C protected from light,followed by washing the unbound dye with PBS.The supernatant was discarded and 200μL of PBS was added to each flow tube,the cells were shaken with Vortex,and then collected by flow cytometry to detect the expression level.3.Detection of CD47 expression on NK cell surfacePBMCs were withdrawn from the blood of the subjects,and the cells were labeled with flow-through fluorescent antibodies against CD3,CD14,CD19,CD16,CD56,CD47,and then incubated at 4°C and protected from light,followed by washing the cells with PBS to wash the unbound dye.The supernatant was removed and 200μL of PBS was added to each flow tube,the cells were shaken with Vortex,and then collected by flow cytometry to detect the expression level.4.Detection of IFN-γsecretion levels by NK cells after inhibition of CD47PBMCs were withdrawn from the subject’s blood,resuspended with R10（RPMI 1640+10%FBS+1%penicillin）,and placed in 96-well plates.Anti-CD47 was added to the wells for the corresponding treatment,and the cells were co-stimulated with IL-12 and IL-15after 1 h.Unstimulated cells were used as comparison.Golgi-stop was added 6 h before cell collection.24 h later,cells were collected,distinguish dead surviving cells by Live/Dead and then incubated at 4°C and protected from light,and then washed with PBS to remove unbound dye.The supernatant was discarded,and the cells were labeled with flow-through fluorescent antibodies CD3,CD14,CD19,CD16,CD56,incubated at 4°C and incubated with PBS to wash the unbound dye,the supernatant was discarded,and the perm agent was added,the cells were shaken with Vortex and the membrane was broken at 4°C and protected from light.The perm agent was removed by direct centrifugation,the supernatant was discarded,and 1×perm buffer was added and centrifuged.After discarding the supernatant and labeling IFN-γwith dye,the cells were shaken with Vortex and incubated at 4°C with protection from light.The unbound dye was washed by adding 1×perm buffer and then centrifuged.The supernatant was discarded and 200μL of PBS was added to each flow tube,and the cells were shaken well by Vortex and collected by flow cytometry to detect IFN-γlevels.5.Enrichment of CD4⁺T and NK cells from PBMCs.PBMCs were withdrawn from the subject’s blood and the cells were resuspended in PBS to a concentration of 50millon/m L.Place the cells into a new flow tube,add the negative selection antibody solution,and let it stand at room temperature for 5 min.Mix the matching magnetic beads,put the same volume of magnetic beads as the above antibody into the flow tube,shake well,and let it stand at room temperature for 5 min.Replenish the tube with PBS to a volume of 2.5 m L,place it into the magnetic pole negative selection,and divert the negatively selected cells into a new flow tube.6.DNA and RNA extractionPBMCs were extracted from the whole blood,CD4⁺T cells were sorted with the Easy Sep^TM Human CD4⁺T Cell Isolation Kit,and cells were counted（cell number<5million）.Divide the sorted CD4⁺T cells into two parts,add 20μL of proteinase K and 200μL of Buffer AL,and then extract DNA with QIAamp DNA Blood Mini Kit;The other part was added with 350μL of Buffer RTL Plus,RNA was extracted using the RNeasy Plus Mini Kit.After extraction,the purity and concentration of the sample was tested with a micro ultraviolet spectrophotometer.7.RNA reverse transcriptionThe extracted RNA was reverse-transcribed into c DNA using i Script advanced c DNA synthesis kit and performed in a PCR machine at 42℃,30min;85℃,5min;4℃,∞condition.8.HIV reservoir detection with Digital Droplet PCR（dd PCR）The probe,primers and DNA/c DNA are mixed according to the system configuration,and the mixture is placed into the Sample wells of DG8 Catridge,and the droplet generation oil is placed into the Oil wells.At the end of the procedure,the generated droplets are transferred to the matching 96-well plate,and after the operation is completed,the aluminum film is placed on the surface of the 96-well plate and the plate is sealed in the preheated PX1 heat sealer.After sealing,the plate is placed in the PCR amplification machine.After amplification,the results are read by QX200^TM Droplet Reader.9.NK cells clearance of latently infected CD4⁺T cellsAbout 30m L of the subject’s whole blood was collected and PBMCs were extracted,CD4⁺T cells and NK cells were sorted and counted separately with the Easy Sep^TM Human CD4⁺T Cell Isolation Kit and the Easy Sep^TM Human NK Cell Isolation Kit.Cells were resuspended with R10 at a 2:1 target ratio and planted in 96-well plates.Add 1μL of anti-CD47,1μL of anti-Ig G to the corresponding wells.After 1 hour,CD4⁺T cells were stimulated with CD3/28 magnetic beads（25μL/million cells）,and NK cells were incubated for 48 hours at 37°C and 5%CO₂ condition after combined stimulation with IL-12 and IL-15,and unstimulated wells were used as negative controls.After 48 hours,the stimulus was washed off,and CD4⁺T cells and NK cells were co-cultured for 5 hours.After culture,collect the cells,centrifuge at 350 g for 5 min,discard the supernatant,and the dead surviving cells were separated by Live/Dead,incubate at 4°C in the dark,and wash the cells with PBS to remove the unbound dye.After discarding the supernatant,cells were labeled with flow-through fluorescent antibodies against CD3 and CD4,incubated at 4°C protected from light,and cells were washed with PBS to remove unbound dyes.The through-breaking operation is the same as 4.After discarding the supernatant,add P24,vortex and incubate cells at 4°C protected from light.Add 1m L of 1×perm buffer and centrifuge to wash off the unbound dye.After discarding the supernatant,add 200μL of PBS,vortex cells and collects them with flow cytometry to detect P24 levels.10.Detection of metabolism-related receptor levels after stimulation of PBMC HIV Env proteinsPBMCs from health persons were withdrawn,resuspended with 200μL R10,and then placed in 96-well plates,and the cells in the wells were stimulated with HIV Env protein,and the unstimulated cells were used as negative controls.Incubated cells at 37°C with 5%CO₂ for 48 h.After the culture,the cells were collected and incubated with Live/Dead to distinguish dead surviving cells,incubated at 4℃and protected from light,and then washed with PBS without bound dye.The supernatant was discarded,and the cells were labeled with flow-through fluorescent antibodies CD3,CD14,CD19,CD16,CD56,Glut-1,CD36,CD98,then incubate cells at 4°C and protected from light,and then washed with PBS with unbound dye.After discarding the supernatant,200μL of PBS was added,and the cells were shaken with Vortex and collected by flow cytometry to detect the expression levels.11.Detection of CD47 levels after metabolic inhibitor treatment of PBMCPBMCs from HIV-infected individuals were withdrawn,resuspended with 200μL R10,placed in 96-well plates,and the cells in the wells were stimulated with IL-12 and IL-15,followed by incubation at 37℃and 5%CO₂ for 1 h.Drugs 2-DG,BCH,DON and Etomoxir were added to the treated wells after 1 h.Unstimulated cells were used as negative controls.Cells were collected at the end of incubation,distinguished from dead surviving cells by Live/Dead,incubated at 4°C protected from light,and washed with PBS for unbound dye.The supernatant was discarded,and the cells were labeled with flow-through fluorescent antibodies CD3,CD14,CD19,CD16,CD56,CD47,incubated at 4°C and protected from light,and then washed with PBS to remove the unbound dye.The supernatant was discarded and 200μL of PBS was added,and the cells were shaken with Vortex and collected by flow cytometry to detect the expression level.Result:I.Relationship between NK cell metabolism-related receptors and reservoirs in HIV-infected individuals.1.The expression level of Glut-1 in NK cells of HIV-infected individuals was positively correlated with the reservoir.NK cell Glut-1 expression levels were positively correlated with HIV DNA and HIV ms RNA copies.HIV-infected individuals NK cells Glut-1 is mainly expressed on CD56^bright NK cells,with higher expression levels than both subsets of CD56^dim NK cells and CD56^-CD16⁺NK cells.Glut-1 expression levels of CD56^bright NK cells were not correlated with any of the three indicators of the reservoir.CD56^dim NK cells Glut-1expression levels were positively correlated with HIV DNA,HIV us RNA,and HIV ms RNA copies.CD56^-CD16⁺NK cells Glut-1 expression levels were positively correlated with HIV us RNA and HIV ms RNA copies.2.The expression level of CD36 in CD56^bright NK cells of HIV-infected individuals was positively correlated with the reservoir.Total NK cell CD36 expression levels were positively correlated with HIV ms RNA copies.HIV-infected individuals NK cells CD36 was mainly expressed on CD56^-CD16⁺NK cells,with higher expression levels than both subsets of CD56^bright NK cells and CD56^dim NK cells.CD56^bright NK cells were positively correlated with HIV DNA,us RNA,and HIV ms RNA copies.The expression levels of CD36 on CD56^dim NK cells and CD56^-CD16⁺NK cells did not correlate with any of the three indicators of reservoir.3.The expression level of CD98 in NK cells of HIV-infected individuals was positively correlated with the reservoir.NK cell CD98 expression level was positively correlated with HIV us RNA and HIV ms RNA copies.NK cell CD98 was mainly expressed on CD56^-CD16⁺NK cells in HIV-infected individuals,and the expression level was higher than the two subsets of CD56^brightNK cells and CD56^dim NK cells.CD56^bright NK cell CD98 expression levels were positively correlated with HIV us RNA and HIV ms RNA copies and showed a positive tendency with HIV DNA copies.CD56^dim NK cells CD98 expression level was positively correlated with HIV us RNA,HIV ms RNA copies.CD56^-CD16⁺NK cells CD98 expression level was positively correlated with HIV ms RNA copies.4.Elevated expression of metabolism-related receptors after activation of NK cells by HIV protein.Elevated expression levels of Glut-1,CD36 and CD98 after activation of NK cells by HIV Env protein in vitro.II.Expression of NK cell metabolism-related receptors in relation to immune activation and inhibitory receptors.1.Relationship between NK cell metabolism-related receptors and immune-activated receptors.Further exploring the association between NK cell metabolism-related receptors and NK cell surface immune activating and inhibiting receptors,we found that NK cell metabolism-related receptors were not significantly associated with the levels of CD226and NKG2C on the surface of NK cells.2.Relationship between NK cell metabolism-related receptors and immune checkpoints.Among the immune checkpoints examined,we found that the expression levels of NK cell Glut-1 and CD98 were positively correlated with the expression level of immune checkpoint CD47.3.Inhibition of NK cell metabolism-related receptors decreased CD47 expression.A significant decrease in CD47 levels was observed after simultaneous treatment of cells with 2-DG,BCH,DON and Etomoxir.III.The expression level of CD47 in NK cells in HIV-infected individuals was positively correlated with the reservoir.NK cell CD47 expression levels were positively correlated with HIV DNA,HIV us RNA and HIV ms RNA copies.CD47 expression in HIV-infected individuals NK cells was mainly on CD56^bright NK cells,followed by CD56^dim NK cells,and least on CD56^-CD16⁺NK cells.CD56^bright NK cell CD47 expression levels showed a positive tendency with HIV DNA copies and a positive correlation with HIV us RNA and HIV ms RNA copies.CD56^dimNK cell CD47 expression levels were positively correlated with HIV DNA,HIV us RNA,and HIV ms RNA copies.IV.Inhibit NK cell CD47 to promote NK cell function.To investigate the effect of CD47 on NK cell function,we treated NK cells with Anti-CD47.We found that the addition of Anti-CD47 blocked CD47 signaling in NK cells restored the level of IFN-γsecretion.V.Blocking NK cell CD47 signaling enhances the ability of NK cells to eradicate reactivated latently infected CD4⁺T cells.The intracellular P24 expression level increased of HIV-infected individuals latently infected CD4⁺T cells after stimulation with CD3/28,decreased after co-culture with their own NK cells,and decreased again after co-culture with their own NK cells after blocking of CD47.VI.Metformin can reduce the expression level of CD47 on the surface of NK cells.The addition of Metformin was able to reduce the expression level of CD47 on the surface of NK cells after IL-12 and 15 stimulations.Conclusion:In this study,we investigated the relationship between the metabolic levels of NK cells and HIV reservoir in HIV-infected individuals,and found for the first time that the levels of NK cells Glut-1,CD36 and CD98 in HIV-infected individuals showed positive correlation with the levels of HIV reservoir,HIV Env protein upregulates Glut-1,CD36 and CD98levels in NK cells;the relationship between the expression of NK cell metabolism-related receptors and immune activating receptors and checkpoints was explored for the first time,and the levels of NK cell Glut-1 and CD98 were found to be significantly positively correlated with the expression of CD47,inhibition of multiple NK cell metabolism reduces CD47 expression;it was further demonstrated that the levels of CD47 in HIV-infected patients showed a significant positive correlation with the levels of HIV reservoirs;blocking NK cell CD47 could promote NK cell function;it was first demonstrated that blocking NK cell CD47 signaling enhances the ability of NK cells to eradicate reactivated latently infected CD4⁺T cells;metformin can be used as an intervention to reduce the level of CD47 expression on the surface of NK cells.The results demonstrate that the cellular metabolism and CD47 expression of NK cells are closely related to the level of HIV reservoir,providing a new potential strategy for the clearance of HIV reservoir.