| Objective:NK cell is a large granular lymphocyte in the innate immune system,which mainly express CD56 and or CD16 molecules,but not CD3 molecules.They play an important role in anti-tumor and antiviral responses.NK cells can be divided into different subsets according to cluster of differentiation.Different subsets play different roles,including secreting a large number of cytokines and chemokines,killing target cells and negative regulation.Single-cell sequencing is an emerging technology that is mainly used in cell type identification,cell heterogeneity,differential response of signaling pathways,and molecular regulatory networks.HIV infection can cause some changes in the distribution and function of NK cell subsets.However,it has not been seen that this technology is applied to NK cells in HIV infection.For human CD56+NK cells,there has been a new way to divide them according to CD11b and CD27 molecules.Whlie in the study of HIV infection,there is no report about the new way to define CD56+NK cell subpopulation and function.HIV infection leads to an increase in the number of CD56-CD16+subset in NK cells,and the killing function and cytokine secretion function of this subset are weakened.However,whether CD56-CD16+subset play an immunomodulatory function is unknown.This study explored the total NK cells?CD56+NK cells and CD56-NK cells?of HIV-infected individuals through single-cell sequencing technology to reveal the changes in the characteristics and distribution of NK cell subsets;discussed the function and regulation of each subset of CD56+NK cells after using CD11bCD27 definition;explored the changes and regulation of CD56-NK cells in HIV-infected individuals,providing new ideas and directions for HIV immunotherapy.Methods:1.1.Study ParticipantsAll the samples are from the Red Ribbon Clinic and the cohort of man who have sex with man in the First Affiliated Hospital of China Medical University.The normal controls were healthy men with negative HIV antibody,hepatitis B and C.We collected 59untreated HIV infected-individuals,28 ART-treated HIV infected-individuals and 39normal controls.All the research subjects signed the informed consent and obtained the ethics of the First Affiliated Hospital of China Medical University Committee approval.2.CD4+T cell countsAbsolute numbers of CD4+T cells were determined in whole blood samples from study participants using a clinical kit.A single-platform,lyse-no-wash procedure was carried out using Trucount tubes,with a known number of control beads and TriTEST anti CD4-FITC/CD8-PE/CD3-PerCP reagents.The absolute numbers of CD4+T cell subsets were obtained by multiplying the total number of CD4+T cells by the subset percentage,determined by flow cytometry data?FACS Calibur;BD Biosciences?analyzed using MultiSET software.3.Extraction of peripheral blood mononuclear cellsDilute and mix the whole blood collected in the negative pressure vacuum blood collection tube with PBS,using a pipette and add it to the Ficoll surface slowly.After density gradient centrifugation,draw the cell layer and wash the cells twice with PBS.4.Flow cytometry analysis and antibodiesThe flow cytometer was calibrated using UltraComp eBeads?Compensation Beads.Inbrief,freshly isolated PBMCs were washed twice,resuspended in PBS that contained 2%FBS,and labeled at 4?C with the corresponding antibody?Ab?cocktail for 30 min.Anti-CD3-PerCp-Cy5.5/FITC?CD56-PE-Cy7?CD16-BV510?CD11b-APC?CD27-PE?NKG2A-PE?NKG2D-PE?NKG2C-PE?CD11c-PE?CD57-PE?Tigit-PE?CCR7-Percp?CD8-APC-Cy7,CD279?PD-1?-BV421 and the matched isotype control were used in these experiments.5.Detection of NK surface metabolitesAnti-CD3-PerCp-Cy5.5/FITC?CD56-PE-Cy7?CD27-BV510?CD11b-APC?Glut-1-PE?CD98-FITC?CD36-APC-Cy7 were used in the experiments and the PBS contained2%FBS were used to wash the PBMCs twice.Then,cells were acquired using BD LSRII and analyzed with FlowJo software.6.Enrichment of NK cells and the NK subsetsAfter extracting PBMC,use NK negative selection kit?EasySep?Human NK CellEnrichment Kit?to enrich NK cells in PBMC.Sorting of NK subsets:put 1-2ml R10 into flow tube one day in advance,make R10 fully wet the tube wall,and then put it into 4?refrigerator overnight.After PBMC was extracted the next day,cells were washed twice with PBS containing EDTA,300 g,10 min.Anti-CD3-PerCP-Cy5.5,CD56-PE-Cy7?CD16-APC?CD8-APC-Cy7?CD11b-APC?CD27-PE were used to stain the cells,After discarding the supernatant,resuspend the cells to 2-3ml with PBS containing EDTA,and sort using a BD FACS Aria II.7.Detection of intracellular cytokines by multicolor flow cytometryAfter counting the cells,resuspend them with R10 and add them to a 96-well U-bottom plate to ensure that each well contains 0.2-0.5 million PBMC.Add 200ul PBS to the wells around the PBMC to ensure the humidity of the target well.Add 10ng/ml r IL-12?50 ng/m L rIL-15 and 100 ng/m L rIL-18 to stain the cells for 24h,meanwhile added anti-CD107a-APC-Cy7 and the matched isotype.Before the 6h collection of cells,added the 1ul Golgistop for each well.Wash twice the cells and stained with CD3-PerCP-Cy5.5,CD56-PE-Cy7,CD16-BV510 for 30min in 4?,Following processing using the Fix/Perm kit for permeabilization,cells were stained with anti-IL-10-APC?TGF-?-PE?IFN-?-FITC and washed with Perm/Wash Buffer.Then,cells were acquired using the BD LSRII and analyzed with BD FlowJo software.8.Detection of Granzyme B and Perforin in NK cell subsetsIn the experiment,K562 cell line was used to stimulate NK cells.After washing K562cell line with PBS,Cell-Trace-Violet was added according to the ratio of 2 ul/5 million for labeling K562 cell line,37?,5%CO2,20min,protected from light.After staining,use R10 with FBS to stop the staining for 5-10min,wash the cells with R10,300g,10min,repeat twice.Count the cells,adjust the cell ratio,and the effect-target ratio?PBMC:K562=5:1?so that the cell number is 0.2-0.5 million/200ul,and put them into a 96-well plate for 6 hours.After the incubation,transfer the cells in the culture plate into the labeled flow tube,and wash the well plate repeatedly with 200ul PBS.Wash the cells with 2ml 2%FBS,300g,10min,repeat twice,discard the supernatant waste liquid,add7AAD,Vortex to mix the cells,using the BD LSRII to acquire the cells within 15min.9.Inhibition of CD4+T cell proliferationCD4+T cells and CD11b-CD27-NK subsets and CD11b-CD27+NK subsets were selected using flow sorting cytometry.Cell-Trace-Violet was used to label CD4+T cells for 20 min at 37?.According to the ratio of NK:CD4+T cells is 1:1,which is seeded into 96-well plates,stimulate cells with 1 million cells/25ul anti-CD3/28 beads,and add10 ng/m L rIL-12,rIL-15 50 ng/m L,100 ng/m L rIL-18.After 72 hours of incubation,the cells were collected in a tube.The cells were washed twice with PBS,the supernatant was discarded and the cells were labeled with 1ul live/dead/million cells.After staining for 10 minutes at room temperature,wash twice with PBS,resuspend the cells to 200ul.10.Inhibition of CD8+T cells secreting IFN-?Using R10 to resuspend the sorted NK cells and labeled CD8+T cells,put them into a96-well plate at a 1:1 ratio,add 10 ng/mL rIL-12,rIL-15 50 ng/m L,100 ng/m L r IL-18,5?g/m L phytohemagglutinin,and 200 U/mL r IL-2,incubate for 24 hours,add 1ul of Golgistop 6 hours before the end of the culture,pipette the cells and move into labeled flow tubes.Wash the culture wells with 200ul of PBS and repeat twice.Add 2ml PBS,300g,10 min to the flow tube and wash twice.Following processing using the Fix/Perm kit for permeabilization,cells were stained with anti-IFN-?-FITC and washed with Perm/Wash Buffer.Then,cells were acquired using the BD LSRII and analyzed with BD FlowJo software.11.Blocking IL-10,TGF-?and PD-L1For TGF-?,IL-10,and PD-L1 blocking assays,25 ng/ml human IL-10 Ab,5?g/ml human TGF-?Ab,0.25?g human PD-L1 Ab,and 5?g mouse IgG isotype were used.Following processing using the Fix/Perm kit for permeabilization,cells were stained with anti-IFN-?-FITC/PE and washed with Perm/Wash Buffer.Then,cells were acquired using the BD LSRII and analyzed with BD FlowJo software.12.Statistical AnalysisGraphPad Prism v6.0 and SPSS 20.0?US IBM?were used for statistical analyses.The Mann–Whitney U-test was employed for comparisons between 2 groups.The Wilcoxon signedrank test was used for comparisons between paired groups.P<0.05 was considered to be significant.Results:??Single-cell sequencing reveals the characteristics of CD56-and CD56+NK cell subsets in HIV-infected individuals1.Distribution characteristics of NK cell gene level in normal controlsAfter eliminating the interference of non-NK cells such as T cells and B cells,it was found that NK cells can be divided into more detailed 10 subsets at the single cell gene level.The genes that are highly expressed in the cluster C0 are COL6A2,CD3G,CD3D,PTGDS,CD3E,IL32,KLRC2,PRDX2,GZMH;The top 10 genes that are highly expressed in the cluster C1 are FCER1G,CMC1,KLRC1,CD7,KLRB1,AKR1C3,CD160,SPON2,CLIC3,C1orf162;cluster C2 are CD3E,LAG3,TNFRSF18,CLIC3,IL32 CD52,GZMH,KLRC2,GTF3C1,TRG-AS1;cluster C3 are S100B,SELL,CD2,CLECL1,CD3D,PRSS57,CD3G,CLEC2D,CD3E,CD52;cluster C4 are IGFBP7,ALOX5AP,KIR2DL1,GZMH,MIAT,KLRC2,FGFBP2,LAG3,PFKP,ADGRG1;cluster C5 are ZFP36,FOS,CD69,PPP1R15A,NFKB1A,DUSP1,CCL3,JUN,JUNB,ZNF331;cluster C6 are JUN,NFKB1A,CD69,IER3,TNF,PPP1R15A,CCL4,NFKBIZ,BTG2,ZC3H12A;cluster C7 are FOS,DUSP1,ZFP36,GZMK,CMC1,XCL1,JUN,PPP1R15A,CD69,LTB;cluster C8 are C1orf56,HNRNPH1,MDM4,TMEM120B,CDC42SE1,C16orf54,B4GALT1,APOBEC3C,PHKG1,B3GNT7;cluster C9 are STMN1,PCNA,MCM7,TYMS,DUT,TUBA1B,PCLAF,MCM5,MCM3,GINS2.The enriched functions of each cluster are different.2.The correlation of NK cells in normal controls and HIV-infected individualsThere were significant differences in the number and subset distribution of NK cells between normal controls and HIV-infected individuals.3.HIV infection cause the changes in NK cell functionIn the analysis of clusters with a relatively large number of cells in HIV-infected individuals,it was found that the genes down-regulated by HIV-infected individuals in CC0 are mostly concentrated in the type I interferon signaling pathway,the response to viruses,and the regulation of immune responses.In CC3,the down-regulated genes are concentrated in the type I interferon signaling pathway,T cell co-stimulation,and immune response regulation.The down-regulated genes in the cluster CC5 are also enriched in the type I interferon signaling pathway,responding to viruses and regulating immunity reaction.However,in those HIV-infected individuals,it was found that the genes enriched in CC1 are also involved in IFN-?-mediated signaling pathways.The genes of CC2 and CC4 are also involved in the transcription of viruses.CC8-CC9down-regulated genes were involved Virus defense response.??Study on the function and regulation of CD56+NK cell subsets defined by CD11bCD27 in HIV-infected individuals1.Changes in CD56+NK cell subsets defined by CD11bCD27 in HIV-infected individualsCompared with normal controls,the subsets of CD56+NK cells defined by CD11bCD27in untreated HIV-infected individuals were significantly different from those in normal controls.The percentage of CD11b+CD27-subsets in untreated HIV-infected individuals decreased significantly,while the percentage of CD11b-CD27-subsets increased significantly.2.The expression of activating receptors in CD56+NK cell defined by CD11bCD27 was increasedThe expressions of activating receptors NKp44 and NKp46 in HIV-infected individuals were higher in CD11bCD27 defined subsets of NK cells than in normal controls,and the expression of activating receptors in CD11b-CD27-subset was the lowest among the four subsets.It shows that NK cells show a highly activated state after HIV infection.3.The CD11b-CD27-NK subset presents a naive phenotypic featureIn the study of the maturity of each subset,the CD11b-CD27-NK subset of HIV-infected individuals or normal controls has a low expression of mature marker CD11c,and the expression of CD11c in HIV-infected individuals in all four subsets are higher than normal controls;HIV-infected individuals CD11b-CD27-NK subset have a low expression of aging marker—CD57,CD11b-CD27-NK subset naive marker—CCR7 is higher than CD11b+CD27-NK subsets.4.The checkpoint level of the NK subset defined by CD11bCD27 was increasedThe expression of Tigit in each subset of HIV-infected individuals was significantly higher than that of normal controls.The highest expression of Tigit in the four subsets was CD11b+CD27-NK subset;the PD-1 ligand PD-L1 has the lowest expression of CD11b-CD27-subset in HIV-infected individuals.5.Metabolic characteristics of NK cell subsets defined by CD11bCD27The CD11b-CD27-NK subset of HIV-infected individuals expresses the glucose transporter Glut-1,and there is no significant difference between the two cohorts;the CD11b-CD27-NK subset expresses the lipid receptor CD36,while the amino acid receptor CD98 expresses higher than CD11b+CD27-NK subet.6.CD11b-CD27-NK subset secretes IFN-?and CD107a degranulation dysfunctionUsing cytokines stimulated NK cells to detect the degranulation of IFN-?and CD107a secreted by each subgroup.Among the four subsets,the CD11b+CD27+NK subset highly expressed IFN-?and CD107a,followed by the CD11b-CD27+NK subset,CD11b+CD27-NK subset is lower than the first two subsets,and the CD11b-CD27-NK subset is the lowest.7.The storage capacity of CD11b-CD27-NK subset Granzyme B and perforin is good,and the release ability is impairedAmong the four subsets of HIV-infected individuals,the CD11b-CD27-NK subset had the highest Granzyme B and Perforin,while the Perforin stored in the CD11b-CD27-NK subset decreased significantly after stimulation.8.CD11b-CD27-NK subset secretes negative regulatory cytokinesAfter sorting,each subset was stimulated by cytokines,and the culture supernatant was collected for ELISA to detect IL-10 and TGF-?.It was found that the CD11b-CD27-NK subset could secrete a large amount of IL-10 and TGF-?.9.CD11b-CD27-NK subset inhibits the proliferation of CD4+T cellsSorting the CD11b-CD27-NK subset and CD11b+CD27-NK subset,co-culture them with autologous CD4+T cells,and after the TCR stimulation,it was found that CD11b-CD27-NK subset can inhibit the proliferation of CD4+T cells.??Study on the changes and regulation of CD56-NK cells in HIV-infected individuals1.The percentage of CD56-CD16+NK subset in HIV-infected individuals increases significantlyThe percentage of CD56-CD16+NK subset in HIV-infected individuals was significantly higher than that in normal controls.Although the percentage decreased after ART treatment,it did not fall to normal levels;the percentage of CD56dimCD16+NK subset in HIV-infected individuals was significantly lower than normal controls.The percentage of CD56dimCD16+NK subset in the ART group has rebounded but has not yet reached the level of the normal controls.2.CD56-CD16+NK cells secrete high levels of IL-10 and TGF-?in HIV-infected individualsComparing IL-10 and TGF-?expressed by CD56-CD16+NK subset and CD56dimCD16+NK cells in HIV-infected individuals,it was found that CD56-CD16+NK subset highly expressed IL-10 and TGF-?,and there was no difference in the expression of IL-10 and TGF-?between CD56-CD16+NK subset of untreatedv and ART individuals.3.HIV-infected individuals CD56-CD16+NK cells inhibit CD8+T cell functionAfter co-cultivating CD56-CD16+NK subset and CD56dimCD16+NK subset with autologous CD8+T cells,it was found that CD56-CD16+NK subset can inhibit the IFN-?secretion of CD8+T cells,and the inhibition rate is proportional to CD56-CD16+NK subset was positively correlated.4.Blocking IL-10 and TGF-?secreted by CD56-CD16+NK cells can partially restore the function of CD8+T cellsAfter adding the antibodies of IL-10 and TGF-?to the culture system,the IFN-?secreted by CD8+T cells was partially restored,and the inhibition rate decreased significantly.5.CD56-CD16+NK subset inhibit CD8+T cell function through direct contactSeparation and culture experiments confirmed that CD56-CD16+NK subset can inhibitCD8+T cell secretion of IFN-?through direct contact in addition to indirect contact withautologous CD8+T cell function.6.CD8+T cells highly express PD-1 in HIV-infected individuals,and CD56-CD16+NK subset highly express their ligand PD-L1Compared with CD8+T cells in healthy controls,CD8+T cells in HIV-infected individuals express PD-1,and PD-L1 on CD56-CD16+NK subset in HIV-infected individuals compared to CD56dimCD16+NK subset.Increased,indicating that CD56-CD16+NK subset may exert an inhibitory effect through the high expression of PD-1/PD-L1.7.Blocking PD-L1 can partially restore the function of CD8+T cellsAfter using antibody to block PD-L1 on CD56-CD16+NK cells,the inhibitory effect of this subset on autologous CD8+T cells was significantly weakened.Conclusion:1.Single-cell RNA sequencing revealed that NK cells can be divided into 10 subgroups.The type 1 interferon signaling pathway genes of NK cells in HIV infection were down-regulated,which affected the antiviral effect of NK cells.2.CD56+CD11b-CD27-NK subset was increased in HIV infection and the expression of activating receptor and mature marker is low.Glut1 expression is lower than CD3-CD56+CD11b+CD27-NK cells but the CD98 and CD36 is higher than that.The function of this subset is not good and it could secrete negative cytokines.3.CD56-CD16+NK subsets could inhibit the function of CD8+T cells.Besides it could secrete amount of IL-10 and TGF-?and have the high level of PD-L1.After blocking PD-L1 and the secretion of IL-10 and TGF-?,the function of CD8+T cells could be restored. |
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