Study On The Effect Of NK Cell Function On HIV Reservoir In HIV Infected Individuals

Objective:Antiretroviral therapy can make HIV-infected individuals reach a state of viremia suppression,but ART alone cannot completely eliminate HIV because there is a reservoir of latent virus.Once treatment is interrupted,new viremia will be induced,we urgently need to find some factors that affect the HIV reservoirs.Natural killer cells as an important lymphocyte of the body’s anti-viral immunity,plays an important role in controlling HIV disease progression,but about the influence of NK cells to the reservoir research rarely.After infection,the expression of immune checkpoints on NK cells is up-regulated and inhibits cell function through various mechanisms,whether the expression of immune checkpoints on NK cells has an effect on the reservoir has not been studied.Although some studies have reported the influence of the expression of certain activated receptors on NK cells on the reservoir,NKG2C and CD226 are important receptors that regulate NK activation,whether their expression on NK cells also affects the reservoir has not been studied.In addition,NK cells can kill target cells in both cytotoxic and non-toxic ways,but the relationship between NK cell toxicity and reservoirs has not been reported.HIV us RNA and ms RNA represent a reservoir of active transcription that may have adverse effects on the body,the effect of NK cells on transcriptionally active reservoirs has not yet been studied.This experiment aims to explore the influence of the percentage of total NK cells and NK subsets,the expression of immune checkpoints and activated receptors on NK cells,and the relationship between the cytotoxic and non-toxic functions of NK cells of HIV-infected individuals and the reservoir at DNA level and transcriptionally active reservoir,and the ability of NK cells to kill reactivated reservoir cells.In order to provide new ideas for clinical HIV reservoir cure strategies.Methods:1.Study Patients:In this study,33 HIV-infected individuals from the Red Ribbon Clinic of the First Affiliated Hospital of China Medical University were selected.All of them received ART treatment for≥2 years and had viral loads after treatment<20 copies/ml.2.Detection of immune checkpoints on the surface of NK cells:PBMC was extracted from whole blood,and then Percp-CD3,Percp-CD14,Percp-CD19,7AAD,APC-CY7-CD16,PE-CY7-CD56,APC-TIGIT,and PE-LAG-3 were added in the PBMC,and the cells were incubated at 4℃for 20min without light.Wash cells with PBS and detected by flow cytometry.3.Detection of activated receptors on the surface of NK cells:PBMC was extracted from whole blood,and then Percp-CD3,Percp-CD14,Percp-CD19,7AAD,APC-CY7-CD16,PE-CY7-CD56,PE-NKG2C or PE-NKG2D,BV711-CD226 were added in the PBMC,and the cells were incubated at 4℃for 20min without light.Wash cells with PBS and detected by flow cytometry.4.Detection of Granzyme B and Perforin stored in NK Cells:PBMC was extracted from whole blood and added with Live/Dead.The cells were incubated at 4℃for 30min without light,wash cells with PBS.Then Percp-CD3,Percp-CD14,Percp-CD19,APC-CY7-CD16,PE-CY7-CD56 were added in the cells and the cells were incubated at4℃for 20min without light,wash cells with PBS.The perm agent was added in the cells and the cells were incubated at 4℃for 30min without light,then use the perm buffer to wash the cells.FITC-perforin,PE-Granzyme B were added,and the cells were incubated at 4℃for 20min without light.The cells were washed with perm buffer and detected by flow cytometry.5.Detection of IFN-γsecretion and degranulation function of NK cells:PBMC was extracted from whole blood and stimulated with IL-12（10ng/ml）,IL-15（50ng/ml）and IL-18（100ng/ml）.PE-CD107A was added in the cells.The cells were cultured for 24h,and Golgi-stop was added 6 hours before the cells were collected.After the culture,cells were collected and added with Live/Dead,the cells were incubated at 4℃for 30min without light,wash cells with PBS.Then Percp-CD3,Percp-CD14,Percp-CD19,APC-CY7-CD16,PE-CY7-CD56 were added and cells were incubated at 4℃for 20min without light.The perm agent was added in the cells and the cells were incubated at 4℃for 30min without light,then use the perm buffer to wash the cells.APC-IFN-γwas added in the cells and the cells were incubated at 4℃for 20min without light.The cells were washed with perm buffer and detected by flow cytometry.6.Nucleic acid extraction and RNA reverse transcription:PBMC was extracted from whole blood,CD4⁺T cells were negatively selected with the isolation kit,and cells were counted（cell numbers<5million）,DNA was extracted with QIAamp DNA Blood Mini Kit,RNA was extracted with RNeasy Plus Mini Kit,and the concentration and purity of each sample were measured.After that,the RNA was reverse-transcribed into c DNA using the i Script Advanced c DNA Synthesis Kit.7.Detection HIV reservoir with Digital droplet PCR:The QX200 digital droplet PCR system was used to detect HIV total DNA,HIV us RNA and HIV ms RNA,respectively.Add DNA or c DNA template into PCR reaction system,then add the mixture into 8sample wells of DG8 cartridge and put 70μl droplet generating oil into the oil wells,put the cartridge into QX200 droplet generator,then transfer the droplet into 96-well plate for PCR amplification,then transfer the 96-well plate into QX200^TMdroplet reader for analysis.8.Latent infected CD4⁺T cells clearance assay:PBMC was extracted from whole blood,CD4⁺T and NK cells were negatively selected with the isolation kit,and cells were counted.CD4⁺T cells was stimulated with CD3/CD28 magnetic beads for 48 hours,and the NK cells was stimulated with IL-12（10ng/ml）,IL-15（50ng/ml）and IL-18（100ng/ml）for 48h.Wash the cells after 48h,and co-culture the activated CD4⁺T cells alone or with activated NK cells at a ratio of 1:2 for 5h.After the end of culture.After the culture,cells were collected and added with Live/Dead,the cells were incubated at 4℃for 30min without light,wash cells with PBS.Then Percp-CD3,APC-CY7-CD4 were added and cells were incubated at 4℃for 20min without light.The perm agent was added in the cells and the cells were incubated at 4℃for 30min without light,then use the perm buffer to wash the cells.APC-P24 was added in the cells and the cells were incubated at4℃for 20min without light.The cells were washed with perm buffer and detected by flow cytometry.Result:1.The percentage of CD56^-CD16⁺NK cells in HIV-infected individuals were positively correlated with the reservoirsHIV-infected individuals with high CD56^dim percentage had lower HIV DNA copies（p=0.0139）,there was a tendency to show a negative correlation between the percentage of CD56^dim and HIV DNA copies（r=0.3569,p=0.0676）.HIV-infected individuals with high CD56^bright percentage had lower HIV us RNA copies（p=0.0268）,there was a tendency to show a negative correlation between the percentage of CD56^bright and HIV us RNA copies（r=-0.4061,p=0.0545）.HIV-infected individuals with high CD56^-CD16⁺percentage had higher HIV DNA copies（p=0.0332）,the percentage of CD56^-CD16⁺NK are positively correlated with HIV DNA（r=0.4567,p=0.0166）.2.The expression level of TIGIT on NK cells of HIV-infected individuals are positively correlated with the reservoirsThe percentage of TIGIT⁺NK cells are positively correlated with HIV DNA,HIV us RNA and HIV ms RNA copies（p=0.023,p=0.0219,p=0.0404）,and the correlation coefficients are 0.4360,0.4753,0.4400.The percentage of TIGIT⁺CD56^dim NK cells are positively correlated with HIV DNA and HIV ms RNA copies（p=0.0414,p=0.0331）,and the correlation coefficients are 0.3950 and 0.4557,there was a tendency to show a positive correlation between the percentage of TIGIT⁺CD56^dimNK cells and HIV us RNA copies（r=0.3715,p=0.0809）.The percentage of TIGIT⁺CD56^-CD16⁺NK cells are positively correlated with HIV DNA,HIV us RNA and HIV ms RNA copies（p=0.0204,p=0.0181,p=0.0346）,and the correlation coefficients are 0.4438,0.4881,0.4523.3.The expression level of NKG2C on NK cells of HIV-infected individuals are negatively correlated with the reservoirsThe percentage of NKG2C⁺NK cells are negatively correlated with HIV DNA copies（r=-0.3957,p=0.0411）,and HIV-infected individuals with high NKG2C expression on NK cells had lower HIV DNA copies（p=0.0469）.4.The level of IFN-γsecreted by NK cells of HIV-infected individuals are negatively correlated with the reservoirsThe percentage of IFN-γ⁺NK cells are negatively correlated with HIV DNA copies（r=-0.4113,p=0.0458）,and HIV-infected individuals with high IFN-γsecretion of NK cells had lower HIV DNA copies（p=0.0387）.Although there was no significant difference in the correlation between IFN-γ⁺NK cells and HIV ms RNA copies（r=-0.4208,p=0.0575）,there was a tendency to show a negative correlation.HIV-infected individuals with high IFN-γsecretion of CD56^dimNK cells had lower HIV DNA copies（p=0.0387）,HIV-infected individuals with high IFN-γsecretion of CD56^-CD16⁺NK cells had lower HIV DNA and us RNA copies（p=0.0145,p=0.0473）.5.HIV-infected individuals with lower percentage of TIGIT⁺NK cells and higher percentage of NKG2C⁺,CD226⁺or IFN-γ⁺NK cells had smaller reservoirsHIV-infected individuals with low TIGIT⁺and high NKG2C⁺or CD226⁺NK cells had lower HIV DNA and us RNA copies（p=0.0164,p=0.0289,p=0.0402,p=0.0140）,there is a trend that HIV-infected individuals with low TIGIT⁺and high CD226⁺NK cells had lower HIV ms RNA copies（p=0.0649）.HIV-infected individuals with low TIGIT⁺and high IFN-γ⁺NK cells had lower HIV DNA,HIV us RNA and HIV ms RNA copies（p=0.0041,p=0.0262,p=0.0140）.HIV-infected individuals with high NKG2C⁺and high CD226⁺or high IFN-γ⁺NK cells had lower HIV DNA copies（p=0.0140,p=0.0079）,HIV-infected individuals with high CD226⁺and high IFN-γ⁺NK cells had lower HIV DNA copies（p=0.0159）.6.NK cells can kill reactive latently infected CD4⁺T cells in vitroThe percentage of P24⁺CD4⁺T cells in HIV-infected individuals was significantly increased（P=0.0313）after the activation of CD3/CD28 magnetic beads in vitro,and the percentage of P24⁺CD4⁺T cells was significantly decreased after co-incubate with autologous activated NK cells（P=0.0313）.Conclusion:By studying the relationship between the percentage of NK subsets,the expression of NK cell immune checkpoints and activated receptors and NK cell function and the HIV reservoir,we found for the first time that HIV-infected individuals with a high percentage of CD56^dim NK cells and a low percentage of CD56^-CD16⁺NK cells have a smaller reservoir;the expression level of TIGIT on NK,CD56^dim and CD56^-CD16⁺NK cells are positively correlated with the reservoir;the expression level of NKG2C on NK cells are negatively correlated with the reservoir.The secretion level of IFN-γof NK cells are negatively correlated with HIV DNA,and have a negative correlation trend with HIV ms RNA.The expression of TIGIT and the secretion of IFN-γon NK cells are important factors affecting the HIV reservoir,NK cells can kill reactive latently infected CD4⁺T cells in vitro,which directly affects the size of the reservoir and has the potential to reduce the reservoir.