Characteristics Of Adaptive NK Cells In HIV-infected Individuals

Objective: The characteristics of adaptive immune cells: adaptive cells have a stronger immune response in contact with certain pathogens or antigens attack again;these memory cells are long-lived without the persistence of antigens.The traditional view is that only T cells and B cells are adaptive immune cells.Natural killer(NK)cells are an important part of the innate immune system.They can directly kill certain tumor cells and virus-infected cells without antigen sensitization.However,emerging studies have shown that NK cells possess memory-like,antigen-specific,long-lived adaptive immune responses.Studies on human cytomegalovirus(CMV),hepatitis B virus(HBV)and malaria showed that adaptive NK cell were identified with high expression of NKG2 C,CD57 and CD2,and low expression of NKG2 A,Siglec-7,PLZF,Syk and Fc?RI?.NK cells expressing one or several memory markers are called adaptive NK cells.In addition,cell functions and metabolisms of adaptive NK cells were different from canonical NK cells.Acquired Immune Deficiency Syndrome(AIDS)is a serious global infectious disease caused by Human Immunodeficiency Virus(HIV).NK cells play an important role in antiviral defense,which may provide a new direction for HIV immunotherapy by virtue of their innate and adaptive characteristics.Few studies were reported on the adaptive NK cells of HIV infections.Only Peppa reported several memory markers in PLZFadaptive NK cells,but lack of the analysis of the relationship of memory markers and disease progression.The functional properties of adaptive NK cells and the metabolic characteristics of adaptive NK cells in HIV-infected individuals have not been reported.In this study,we explored the expression of memory markers of adaptive NK cells in HIV-infected individuals and the relationship between memory markers and disease progression(Paper 1),the functional characteristics of adaptive NK cells in HIV-infected individuals(Paper 2),and the metabolic characteristics of adaptive NK cells in HIV-infected individuals(Paper 3).The study provides new directions for immunotherapy of HIV infection.Methods: 1.Research participants A total of 139 subjects were used in this study,including 27 healthy controls,37 untreated HIV-infected patients and 75 HIV-infected patients receiving anti-retroviral therapy(ART).Healthy controls are HIV antibody-negative healthy individuals whose age is matched with HIV-infected individuals;untreated HIV-infected individuals are HIV-positive individuals who have not received ART;HIV-infected individuals receiving ART have been treated for at least 1 year and with viral load less than 20 copies/m L.This study was approved by the Ethics Committee of the First Affiliated Hospital of China Medical University,and all the subjects signed informed consents.2.CMV Ig G concentration detection The separated subjects' plasma was cryopreserved at-80 ?.According to the instructions of the CMV Ig G enzyme-linked immunoassay kit,the plasma samples of healthy controls and patients were tested by ELISA method.3.Multicolor flow cytometry to detect adaptive NK cell phenotype CD3-APC-CY7,CD56-PE-Cy7,BV650-CD16,CD14/CD19-Percp--Cy5.5,NKG2C^-PE,CD57-BV421,CD2-FITC,Siglec-7-AF700,NKG2A-BV786 and CD85j-APC antibodies were used for cell surface staining.Cytofix/Cytoperm Fixation/Permeabilization Kit was used for intracellular staining.Fixation and membrane rupture were carried out according to the instructions of the reagents.Fc?R1r-FITC,APC-Syk,PLZF-PE-CF594 antibodies were added for intracellular staining.BD LSR ? flow cytometer was used to detect the cells and FSC Diva software and Flow Jo software were used to analyze the flow cytometry data.4.Detection of NK cell proliferation and anti-apoptosis ability Add CD3-APC-Cy7,CD56-PE-Cy7,BV786-CD16,CD14/CD19-Percp-Cy5.5,NKG2C^-PE and CD57-BV421 antibodies to stain the cell surface;add Ki-67-APC and FITC-Bcl-2 antibodies for intranuclear staining;detect the cells by flow cytometry.5.Detection of IFN-? secretion and degranulation function of NK cells Phorbol 12-myristate 13-acetate(PMA)and ionomycin stimulation: Cell Activation Cocktail(with Brefeldin A)was used to stimulate the NK cells for 6 hours,and then IFN-? were detected by flow cytometric.Infected CD4⁺ T stimulation: HIV NLENG virus infected CD4⁺ T cells were cultured with NK cells for 5 hours and then IFN-? was detected by flow cytometry.K562-E-peptide stimulation: K562 cells were transfected with HLA-E plasmid and cultured overnight with HIV-derived peptides GPEVNIVTD or AISPRTLNA,and then cocultured with NK cells for 5 hours.Cells were harvested and CD107 a was detected by flow cytometry.6.Negative selection of NK,CD4⁺ T and CD8⁺ T cells Sorting kits were used to negatively select NK,CD4⁺ T and CD8⁺ T cells.Resuspend PBMC to a concentration of 5×10⁷ cells/m L with sorting buffer,add 50 ?L/m L antibody and incubate at room temperature for 5 minutes;add 50 ?L/m L magnetic beads and add sorting buffer to 2.5 m L;put the tube in the magnetic pole and leave it at room temperature for 5 minutes;pour out the supernatant which contains target cells.7.Co-immunoprecipitation and liquid chromatography-mass spectrometry tandem analysis of the eluted HLA-E bound peptides Virus infected CD4⁺ T cells were amplified and then lysed.HLA-E antibody and Protein A/G beads were used to elute HLA-E and peptides.The peptides were analyzed by liquid chromatography-mass spectrometry and analyze with Mascot software to analyze.8.Mitochondrial function test Mito Tracker^TM Green FM reagent was used for mitochondrial mass(MM)detection,and Mito Tracker^TM Orange CMTMRos reagent was used for mitochondrial membrane potential(MMP)detection.NK cells were added with 100 n M prewarmed MM and MMP staining reagent and stained for 30 minutes at 37 ?.Wash cells with PBS containing 2% FBS,and then stain the cells with flow cytometry antibodies.9.Sugar intake experiment Resuspend the cells with 500 ?L sugar-free R10,add 100 ?M glucose analogue 2-NBDG,incubate at 37°C for 30 minutes,then wash with PBS and stain the surface.10.Detection of nutrient transporters expression level PBMC were stained with CD3/CD14/CD19-Percp-Cy5.5,CD56-PE-Cy7,CD16-BV786,NKG2C^-PE,CD36-APC-Cy7,CD98-FITC and CD71-BV421 antibodies and detected by flow cytometry.11.Detection of CD39 levels of NK cells PBMC were stained with CD3-FITC,CD14/CD19-Percp–Cy5.5,CD56-PE-Cy7,CD16-BV786,CD39-PE and CD38-BV421 antibodies and detected by flow cytometry.12.NK cell extracellular nucleotide enzyme function detection NK cells were stimulated with IL-12(10 ng/m L),IL-15(50 ng/m L)and IL-18(100 ng/m L)for 18 h.Cells were collected and stained for the detection of IL-10 and TGF-?.13.NK cells inhibit the proliferation of CD4⁺ T and CD8⁺ T cells Sorted NK cells were stimulated with CD38 inhibitor 78 c,CD39 inhibitor ARL67155,CD73 inhibitor AB680,and adenosine deaminase ADA inhibitor EHNA for 30 minutes.Sorted CD4⁺ and CD8⁺ T cells were stained with proliferation dyes respectively.Then NK cells were cultured with CD4⁺ T or CD8⁺ T cells for 5 days.The proliferation was tested by flow cytometry.14.Statistical analysis Statistical analysis was performed with SPSS 22.0 and Graph Pad Prism 8.0 software.The comparison between the two groups was performed by the non-parametric Mann-Whitney U test,and the non-parametric Wilcoxon paired signed rank test was used for the comparison of paired data.The comparison between the three groups was performed by Kruskal-Wallis test;Spearman test was used for correlation analysis.p < 0.05(two-tailed)is considered statistically significant.Results : 1.Phenotypic characteristics of adaptive NK cells in HIV-infected individuals a.Memory markers expression of NK cells in HIV-infected individuals NK cells of HIV-infected individuals expressed high levels of NKG2 C,CD57 and CD2,and low levels of NKG2 A,Siglec-7,PLZF,Syk and Fc?RI?.b.Correlations of the levels of memory markers on NK cells in HIV-infected individuals Correlation analysis showed that the expression of NKG2 C on CD56 dim NK cells of HIV-infected individuals was positively correlated with CD57 and CD2 levels respectively,and negatively correlated with Syk levels;CD57 was positively correlated with CD2 and CD85 j expression levels respectively,but was negatively correlated with Fc?RI?;The expression levels of PLZF were positively correlated with Siglec-7 and Syk respectively,but negatively correlated with the levels of CD2 and Fc?RI?;Syk was negatively correlated with the expression levels of NKG2 A.c.Associations of NK memory marker levels and HIV disease progression The analysis of co-expression of NKG2 C and other memory markers on CD56 dim NK cells showed that the percentage of adaptive NK cells in HIV-infected patients was significantly higher than that of healthy people.The percentage of NKG2C⁺ Syk-on CD56 dim NK cells of HIV-infected patients is positively correlated with CD4⁺ T cell counts.2.Functional characteristics of adaptive NK cells in HIV-infected patients a.Lower proliferation and stronger anti-apoptosis ability of adaptive NK cells in HIV-infected patients Compared with NKG2C^-CD56 dim NK cells,NKG2C⁺ CD56 dim NK cells in HIV-infected patients expressed low levels of Ki-67 and high levels of Bcl-2.b.Higher IFN-? secretion of adaptive NK cells in HIV-infected patients IFN-? produced by adaptive NK cells was significantly higher than that of canonical NK cells with the stimulus of PMA and ionomycin.or NLENG virus infected CD4⁺ T cells.c.HLA-E,the ligand of NKG2 C,bound HIV-specific peptide GPEVNIVTD The HLA-E binding peptides on CD4⁺ T cells infected by isolates HIV virus were detected and analyzed.GPEVNIVTD,a peptide derived from the HIV pol region had a high mass spectrum score.NKG2C⁺ NK cells had a stronger response to K562-E cells binding with GPEVNIVTD peptide.3.Metabolic characteristics of adaptive NK cells in HIV-infected patients a.High mitochondrial function of adaptive NK cell in HIV-infected patients The mitochondrial mass and mitochondrial membrane potential of adaptive NK cells were significantly higher than those of canonical NK cells,while the level of mitochondrial depolarization was lower.b.High ability of glucose uptake but low expression of short-chain fatty acid transporter of adaptive NK cell in HIV-infected patients The ability of NKG2C⁺ CD56 dim NK cells to take up glucose was stronger than that of NKG2C^-CD56 dim NK cells.The levels of CD36 and CD71 of NKG2C⁺ CD56 dim NK cells in HIV-infected patients were significantly lower than those of NKG2C^-CD56 dim NK cells.c.Low expression of CD39 on adaptive NK cells of HIV-infected individuals The CD39 expression of NKG2C⁺ CD56 dim NK cells was significantly lower than that of NKG2C^-CD56 dim NK cells.Compared to healthy controls,the expression levels of CD38 and CD39 on NK cells of HIV infected individuals were increased.The percentages of CD38+ NK cells and CD39+ NK cells were negatively correlated with CD4⁺ T cell counts and the ratio of CD4/CD8 counts.d.Negative regulation function by extracellular nucleotidases on NK cells in HIV-infected individuals When IL-12/IL-15/IL-18 stimulated HIV-infected NK cells,CD39+ NK cells produced more IL-10 and TGF-?.NK cells inhibited the proliferation of CD4⁺ T or CD8⁺ T cells;addition of CD38,CD39 or CD73 inhibitors restored the proliferation of CD4⁺ T or CD8⁺ T cells;addition of adenosine deaminase inhibitor exacerbated the inhibitory effect on CD4⁺ T cell proliferation.Conclusion: 1.The phenotype of adaptive NK cells in HIV-infected patients: high expression levels of activation receptor NKG2 C,maturation marker CD57,costimulatory molecule CD2 and inhibitory receptor CD85 j,and low levels of inhibitory receptors Siglec-7,NKG2 A,transcription factors PLZF and important signaling molecules Syk and Fc?RI?;the expression levels of these memory markers are highly correlated with disease progression.2.Functional characteristics of adaptive NK cells in HIV-infected individuals: adaptive NK cells have weak proliferation and strong anti-apoptotic ability;when stimulated by PMA and ionomycin or stimulated by HIV NLENG infected CD4⁺ T cells,adaptive NK cells have a stronger response;the specific HIV peptide sequence bound by NKG2 C ligand HLA-E is GPEVNIVTD,which can stimulate robust response of NKG2C⁺ NK cells.3.Metabolic characteristics of adaptive NK cells in HIV-infected individuals: adaptive NK cells have stronger mitochondrial functions and sugar uptake capacity;adaptive NK cells express low levels of short-chain fatty acid transporter CD36 and transferrin receptor CD71;CD39+ NK cells secrete more negative regulators and can inhibit the proliferation of CD4⁺ T and CD8⁺ T cells,while adaptive NK cells express low levels of CD39.