Studies On Inhibitory Natural Killer Cell Receptor (iNKR) And Activating Receptor Of Natural Killer Cells In HIV-infected Individuals

ObjectiveHuman immunodeficiency virus(HIV) majorly infects CD4+ T cells of the acquired immune system,resulting in the decreased function of immune system, eventually causes death due to a variety of opportunistic infections or tumors.In the past two decades,immunology research HIV-infected patients mainly concentrated on the acquired immunodeficiency,but has not made a breakthrough in the research.Some recent studies have shown that the innate immunity may play an important role in HIV infections.Natural killer cells(NK) are CD3 negative and CD56 positive or CD16 positive lymphocytes,which are important components of the natural immune system, comprising approximately 10%to 15%of the mononuclear cells in normal peripheral blood.NK cells are functionally classified into three subsets by the density of CD56 and CD16.The majority,approximately 80-90%,of human NK cells are the cytotoxic subset(CD3^neg CD56^dim CD16^pos) and mediate ADCC,antibody-dependent cellular cytotoxicity.Whereas a minority,approximately 2-10%,are the cytokine produced subset(CD3^negCD56^briCD16^pos/neg).In addition,CD3^neg CD56^neg CD16^posNK cells are the highly dysfunctional subset,which is less than 10%.The function of NK cells for the targets is determined by the balance between inhibitory signals and activating signals, which induced by the combination between a series of inhibitory natural killer cell receptors(iNKR) and the activating receptors and corresponding ligands on the surface of target cells.Studies have shown that HIV infection makes changes of receptors expression on the NK cell surface.However,studies are only for activating receptors or inhibitory receptors.There is not any report on whether activating receptors and inhibitory receptors express simultaneously on the same cell or separately express on different cells.As an inhibitory receptor,NKG2A interact with CD94,forming CD94/NKG2A heterodimer,which recognize complexes of non-classical HLA-E. Their combination induce inhibitory signals and transmit inside the cells.Inhibitory receptor KIR3DL1 is one of KIR(killer cell immunoglobulin-like receptor) family, belonging totype-â… transmembrane glycoprotein.It mainly recognizes products of HLA-A and HLA-B alleles,inducing inhibitory signals transmissed inside the cells, and then inhibiting the activity of NK cells.Activating receptor NKG2D expresses on almost human NK cells,and associates noncovalently with the DAP 10 adapter protein, at last recognize corresponding ligands on the targets,producing activating signal to induce NK cells to activated.Some reports suggest that the level of inhibitory receptor KIR3DL1 on NK cells of viremic patients increased compared with healthy donors. While the expression of inhibitory receptor NKG2A decreased.After effective HAART (highly active anti-retroviral therapy),levels of KIR3DL1 and NKG2A on NK cells were restored to near normal.In another study,the percentage of NKG2A+ cytotoxic NK cells(CD56^dimCD16⁺) in AIDS patients were significantly higher compared with healthy donors,and reverse associations between the percentage of NKG2A in cytotoxic NK subset and CD4+T cells in AIDS group also had been found.While the percentage of NKG2A+ dysfunctional NK cells(CD56^-CD16⁺) in AIDS patients were significantly lower than NCs.In the studies,we apply multi-colors flow cytometry analysis to study simultaneously the expression of activating receptor NKG2D and inhibitory receptor NKG2A and KIR3DL1 on NK subsets of 34 treatment na(i|¨)ve HIV/AIDS patients and 20 HIV-infected individuals receiving HAART,defining the relationship between the expression to disease progression.Methods1.Study objectsA total of 34 treatment na(i|¨)ve HIV-infected patients from clinic service of Red Ribbon of the First Hospital of China Medical University were enrolled.There were 24 HIV infected individuals who had CD4+T-cell counts between 200 and 500 cells/μl, and no AIDS-defining condition(18 males,6 females;median age is 42,the age distribution from 28 to 58.There were 10 AIDS patients(CD4+ T-cells less than 200 cells/μl,presence or previous opportunistic infections or HIV-related neoplasms,6 males and 4 females;median age is 48,the age distribution from 35 to 68.HIV-infected subjects were classified into 2 stages.20 HIV-infected individuals receiving HAART continually for 12 months were enrolled(HIV RNA≤50copies/ml,200×10⁶/L≤CD4+ T-cell counts＜500×10⁶/L,12 males,8 females;median age is 42,the age distribution from 31-56.Normal control(NC) was enrolled according to HIV negative,never exposed to HIV,and the age and gender are comparable with the HIV-infected,and there were 16males and 6 females.Blood was drawn by venipuncture from each subject in EDTA tubes(Becton Dickinson) for FACS analysis and the detection of viral load.2.The absolute counts of T cellThe 20μl TriTEST reagents CD4/CD8/CD3 were added to the CD4 absolute vials and 50μ1 whole blood was added then.After 15-min incubation at dark at room temperature,the erythrocytes were lysed by using FACS lysing solution.After 15-min incubation at room temperature and dark again,the vials were taken to perform FACS analysis by using Multiset software.The absolute count and the percentage of CD3+CD4+,CD3+CDS+ and CD3+ T cells were calculated automatically.3.Determination of NKG2D,NKG2A and KIR3DL1 levels of NK cell subsetsWhole blood samples were incubated with the following monoclonal antibody combinations anti-CD3-FITC 6μl / anti-CD16-Percpcy5.5 6μl / anti-CD56-PE-Cy7 3μl / anti-NKG2D-APC 51μl / anti-NKG2A-PE 10μl or anti-KIR3DL1-PE 10μl for 30 minutes at room temperature and dark.After lysis of red blood cells by FACS lysis buffer(Becton Dickinson) for 8 min,then the samples centrifugate(300g) for 5min and discard the supernatant cells were washed twice with PBS,finally fixed with 1% paraformaldehyde,accquired and analyzed with the FACSAria.Firstly,lymphocytes were identified by light FSC(forward scatter) profile and light SSC(side scatter) profile.Then CD3 negaitive lymphocytes were identified gating based on lymphocytes. CD3^negCD56^negCD16^ppos NK,CD3^negCD56^briCD16P^pos/neg NK and CD3^negCD56^dim CD16^pos NK comprised total NK cells were defined gating based on CD3 negaitive lymphocytes.Percentage of each NK subsets were analyzed,and then activating receptor and inhibitory receptor were detected simultaneously.4.Determination of viral loadHIV RNA extracted from plasma samples was amplified by a standardized reverse transcription PCR assay according to the manufacturer's recommendations(COBAS Amplicor,HIV-1 Monitor Test Version 1.5,Roche Diagnostics,USA).The detection level in plasma was defined as 400 HIV-1 RNA copies/ml.5.Statistical analysisSPSS 11.5 software was used to make the Statistical analysis.Geometric means were determined for log-distributed variables.Comparisons of means for different groups were done using one-way ANOVA.Spearman rank correlations were used as a measure of correlations between CD4 and other variables,and between viral load and the variables.Results1.Subsets Changes of NK cells of HIV/AIDS patients and HIV-infected individuals treated with HAARTWe found that the percentage of CD56^-CD16⁺ of HIV and AIDS groups were both significantly higher than NCs,while the percentage of CD56^dimCD16⁺ of HIV and AIDS groups were significantly lower than NCs,and the percentage of CD56^briCD16^+/- of HIV and AIDS were also significantly lower than NCs.The percentage of the three subsets between HIV and AIDS had no significant differences.The percentage of HAART group was also no statistics significance compared with HIV and AIDS groups.2.Research activating receptor or inhibitory receptor alone on NK subsets(1) The expression of NKG2D,NKG2A and KIR3DL1 on CD56^dimCD16⁺ NK ceilsThe expression of NKG2D had no evident difference between HIV group and NCs.The percentage of AIDS was lower than NCs significantly,and also lower than HIV group.The percentage of HAART was significantly higher than AIDS group,but didn't have significantly difference compared to the NCs.The expression of NKG2A and KIR3DL1 had no significant difference between every groups.(2) The expression of NKG2D,NKG2A and KIR3DL1 on CD56^briCD16^+/-NK cells The expression of KIR3DL1 +,the percentage of HIV group was lower remarkably than NCs.There was no significantly difference between other groups.The expression of NKG2D and NKG2A had no significant difference between every groups.(3) The expression of NKG2D,NKG2A and KIR3DL1 on CD56^-CD16⁺ NK cellsThe expression of NKG2A,the percentage of HIV group was significantly higher than NCs.The differences between other groups had no significance.The expression of KIR3DL1,the percentage of HAART group was lower remarkably than AIDS group.There was no significantly difference between other groups.Among groups,there was no obvious difference about the expression percentage of NKG2D.3.To explore expression of activating receptor and inhibitory receptor at equal pace on NK subsets.(1) Expression on CD56^dimCD16⁺ NK cells.The expression of NKG2D+KIR3DLI+ NK,the percentage of HIV group and AIDS group were both lower than NCs significantly.There was no significant difference between HIV group and AIDS group.There was also no significant difference between HAART and other groups.The percentage of NKG2D+NKG2A- in HIV group and AIDS group were significant lower than NCs(p＜0.01),and the percentage of AIDS group was lower than HIV significantly(p＜0.05).The expression in HAART was significant higher than AIDS(p＜0.05),but there was no significant difference compared with health control and HIV group.The expression of NKG2D-NKG2A+ NK in HIV and AIDS were significantly higher than NCs,and the percentage of AIDS was higher than HIV significantly.The percentage of HAART was significant lower than AIDS,but there was no significant difference compared with NCs(between HAART,HIV and NCs.The percentage of NKG2D+KIR3DL1- NK in AIDS was significant lower than NCs and HIV group.The percentage of HAART was significant higher than AIDS,and there was no significant difference compared with NCs. The expression of NKG2D-KIR3DL1+ NK in AIDS was significant higher than NCs and HIV group.The percentage of HAART was significant lower than AIDS,and there was no significant difference compared with NCs.There was no significant difference about the percentage of NKG2D+NKG2A+ NK between each group.(2) Expression on CD56^briCD16^+/- NK cells.The percentage of NKG2D+KIR3DL1- NK in HIV was significant higher than NCs(p＜0.01) and AIDS groups(p＜0.05).The expression of NKG2D+NKG2A+,NKG2D+KIR3DL1 +,NKG2D+NKG2A-, NKG2D-NKG2A+ and NKG2D-KIR3DLI+ had no significant differences between each group.(3) Expression on CD56^-CD16⁺ NK cells.The percentage of NKG2D+NKG2A+ in HIV group was lower obviously than NCs and AIDS.The percentage of HAART was significant lower than HIV,but there was no significant difference compared with NCs and AIDS.AIDS were lower remarkably than HIV.The percentage of NKG2D+NKG2A- in HIV group was significantly higher than AIDS,but there was no significant difference compared with NCs.The percentage of HAART was significant lower than AIDS,and had no significant difference compared with NCs.And there was no significant difference between HIV,AIDS and NCs.The percentage of NKG2D+KIR3DL1- NK in AIDS group was significant lower than NCs and HIV,but the differences between HIV and NCs had no statistics significance.HARRT,NCs and HIV was no significant difference,but higher remarkably than AIDS.AIDS were significantly lower than HIV.Among groups,there was no obvious difference in the percentage of NKG2D-NKG2A+,NKG2D-KIR3DL1+and NKG2D+KIR3DL1+NK.4.The Correlation between level of activating receptor and inhibitory receptor on NK cells,the absolute count of CD4+T cell and viral load(VL)Analysis the correlation between activating receptor or inhibitory receptor on CD56^dimCD16⁺ NK,the absolute count of CD4+T cell and VL.(1) The expression of NKG2D-NKG2A+ on CD56^dimCD16⁺ NK cells in HIV/AIDS patients had negative correlation with absolute count CD4(r=-0.42,P＜0.05), had positive correlation with VL(r=0.37,P＜0.05).(2) The expression of NKG2D-KIR3DLI+ on CD56^dimCD16⁺ NK cells in HIV/AIDS patients had negative correlation with absolute count CD4(r=-0.55,P＜0.05), had positive correlation with VL(r=-0.55,P＜0.05).Conclusion1.HIV-1 infection was associated with a depletion of CD56^dimCD16⁺ NK cells with a paralleled increase of CD56^-CD16⁺ NK cells and CD56^briCD16^+/- NK cells in HIV/AIDS patients.2.After HIV infection,the expression of activating receptor and inhibitory receptor on the CD56^dimCD16⁺ NK cells surface changed.The NKG2D+KIR3DL1+ NK cells in HIV group,AIDS group was significantly lower than NCs.The NKG2D+NKG2A- the NK cells levels in HIV group,AIDS group was markedly lower than NCs and AIDS group was lower than HIV group.The NKG2D- NKG2A+ NK cells levels in HIV group and AIDS group were significantly higher than NCs,AIDS group was significantly higher than HIV group.The NKG2D+KIR3DL1- NK cells,the level of AIDS group was lower than NCs,AIDS group was lower than HIV group.The NKG2D-KIR3DL1+ NK cells in AIDS group was remarkably higher than the level of NCs,AIDS group was markedly higher than HIV group.With HIV-infected patients entered the AIDS phage,the NK surface activating receptor was reduced and inhibitory receptor was increased even more significantly.3.The expression of NKG2D+NKG2A-,NKG2D-NKG2A+,NKG2D+KIR3DL1-and NKG2D-KIR3DL1+ on CD56^dimCD16⁺NK cells can be restored after effective HAART.