The Study Of NK Cell Surface Markers And Functions Of Immune Non-responders Who Have Received ART

Objective:Currently,the majority of HIV-infected individuals have received antiretroviral therapy?ART?.The CD4⁺T cell counts of the majority of HIV-infected patients can be restored by ART,however,about 1/3 of individuals still cannot restore CD4⁺T cell counts to the normal level by ART.Therefore,HIV-infected individuals who have received ART are divided into immunological responder?IR?and immunological non-responder?INR?according to their CD4⁺T cell counts.At present,it is considered that the thymus output function of INR is decreased,T cell apoptosis is increased,accompanying with mitochondrial dysfunction and a series of other changes.Another important cell in the immune system--NK cell plays an important role in innate immunity which affects anti-tumor,antiviral infection and immune regulation processes,however,studies on NK cells of INR are relatively rare,especially on NK cell functions.This research intends to study IR and INR individuals who have received ART for more than 2 years,to identify the differences of the expression of NK cell surface markers and for the first time exploring NK cell functions among different immune responders,in the meanwhile investigating the relationship between NK cell immune functions and HIV disease progression.Methods:1.The detection of NK cell surface markersPercp-Cy5.5-CD3,PE-Cy7-CD56,APC-Cy7-CD16,BB515-NKG2D,APC-LFA-1,PE-PD-1,BV786-CD69,BV421-TIGIT were added in the PBMC,and the cells were dyed at 4?for 30 minutes without light.The cells were washed with PBS?350g,5min?and detected by flow cytometry.2.The detection of NK cell intracellular perforin and granzymeBPerCp-Cy5.5-CD3,PE-Cy7-CD56 and APC-Cy7-CD16 were added in the PBMC,and the cells were dyed at 4?for 30 minutes without light.The perm agent was added in the cells and the cells were incubated for 20min,then the perm buffer was used to wash the cells?500g,5min?.PE-perforin and APC-granzymeB were added in the cells,and stained for 30 minutes at 4?in dark.The cells were washed with perm buffer and detected by flow cytometry.3.The detection of NK cell secretion and degranulation functionIL-12?10ng/ml?,IL-15?50ng/ml?and IL-18?100ng/ml?were used to stimulate the PBMC,and PE-CD107a was added in the cells.Cells were cultured for 24 hours,and Golgi-stop was added 6 hours before the end of culture.Live/Dead staining was added in the cells and the cells were stained for 20min without light,then Percp-Cy5.5-CD3,PE-Cy7-CD56 and Apc-CD16 were added and the cells were dyed at 4?for 30minutes in dark.The perm agent was added in the cells and the cells were incubated for20min,then the perm buffer was used to wash the cells?500g,5min?.FITC-IFN-?was added in the cells,and the cells were stained for 30 minutes at 4?in dark.The cells were washed with perm buffer and detected by flow cytometry.4.The detection of NK cell proliferation capacityNK cells were negatively selected by magnetic beads.Violet staining was added in the cells and the cells were dyed at 37?for 30min on shaker.The cells were suspended by R10 and then added in a 96-well culture plate.The cells were stimulated by IL-12?10ng/ml?,IL-15?50ng/ml?and IL-18?100ng/ml?for 5 days.The cells were collected after culture,and 7AAD was added staining for 4 minutes.The cells were detected by flow cytometry.5.The detection of NK cell migration capacityNK cells were negatively selected by magnetic beads.The cells were suspended by RPMI1640.In the lower chamber of Transwell plate,600?l SDF-1?100ng/ml?was added,and 100?l cells were added in the upper chamber.After incubation at 37?for3 hours,the cells were liquated from the lower chamber,and the total number of migrated cells was counted by the cell counter.6.The detection of NK cell actin and cofilinNK cells were negatively selected by magnetic beads.The perm agent was added in half of the cells and the cells were incubated for 20min,then the perm buffer was used to wash the cells?500g,5min?.Phalloidin was added in the cells,and the cells were stained for 30 minutes at 4?in dark.The cells were washed with perm buffer and detected by flow cytometry.The remaining half of the cells were added with 1.5%paraformaldehyde and incubated at room temperature for 10min.Then the cells were add iced methanol and incubate at 4?for 10min.The cells were added with p-cofilin antibody and incubated at room temperature for.Then the cells were added second antibody and incubated at room temperature out of light for 30 minutes.The cells were detected by flow cytometry.Results:1.The analysis of NK cell subpopulation and surface markers1.1 The analysis of NK cell subpopulationIn INR,CD56^dimCD16⁺NK cells were significantly lower than that in NC?P<0.01?.The proportion of CD56brightCD16^+/-is basically same in the three groups.While the proportion of CD56^-CD16⁺in INR was significantly higher than that in NC and IR?P<0.01,P<0.05?,and the proportion of CD56^-CD16⁺NK cells was negatively correlated with CD4⁺T cell count?r=0.4769,P<0.01??figure 1B,C?.1.2 The analysis of NK cell surface markersThe expression of CD69 in INR was much higher than that in NC and IR?P<0.01,P<0.01??figure 1D?.The INR expressed more LFA-1?P<0.01,P<0.05?,while the IR expressed the same level as NC?figure 1E?.The expression level of PD-1 and TIGIT on NK cells were significantly higher in INR than in NC?P<0.001,P<0.001;P<0.05,P<0.05?,especially the differences showed on PD-1?figure 1F,G?.There was no significant difference in NKG2D among the three groups?figure 1H?.2.The correlation analysis of NK cells surface markers and disease progressionThe expression of CD69,LFA-1 and PD-1 were negatively correlated with CD4⁺T cell count?P<0.01,P<0.01,P<0.001?,and the r values were-0.4426,-0.5859,-0.5333,respectively?figure 2A,B,C?.Although there was no significant difference in the correlation between TIGIT and CD4⁺T cell count,there was a clear tendency to show a negative correlation?figure 2D?.3.The analysis of NK cells cellular perforin and granzymeBNK cell intracellular perforin in INR was significantly lower than that in NC and IR?P<0.01,P<0.05?,while the level of IR intracellular perforin almost recovered to the level of NC?figure 3A,B?.There was a clear tendency showing that INR intracellular granzymeB was lower than that in NC and IR?figure 3C,D?.4.The analysis of NK cells IFN-?and CD107a after stimulationThe proportion of IFN-?⁺NK cells in INR was significantly lower than that in NC and IR?P<0.01,P<0.01?,and the proportion of IFN-?⁺NK cells in IR and INR were positively correlated with CD4⁺T cell count?r=0.5969,P<0.01??figure 4B,C?.After stimulation,the expression level of CD107a was significantly lower in INR than that in NC and IR?P<0.05,P<0.01?,and the proportion of CD107a⁺NK cells was positively correlated with the CD4⁺T cell count?r=0.5515,P<0.05??figure 4E,F?.5.The analysis of NK cells proliferationNK cells scarcely proliferated without stimulation,however,it proliferated obviously when stimulated by interleukin?figure 5A?.The proportions of proliferated NK cells were approximately identical among the three groups,only the median proliferation rate of NC was slightly higher than that of IR and INR?figure 5B?.6.The analysis of NK cells migration and involved mechanismWith the increasing concentration of SDF-1,the total number of migrated NK cells gradually increased?figure 6A?.Under the chemotaxis induction of 100ng/ml SDF-1,the total number of migrated NK cells in NC was significantly higher than that in HIV-infected patients?IR:P<0.05,INR:P<0.01?,and there were also significant differences between IR and INR?P<0.05??figure 6B?.F-actin and P-cofilin of INR were significantly decreased than that of NC?P<0.05,P<0.05??figure 6C-F?.Correlation analysis between F-actin MFI and P-cofilin MFI in the IR and INR showed a significant positive correlation?r=0.4691,P<0.05??figure 6G?.Conclusions:By the study of surface markers and functions changes of NK cells among different immune responders,we considered that disordered subpopulation of NK cells,highly expressed inflammatory receptors and immune checkpoints,impaired cytotoxicity capacity,and weakened chemotaxis will all have negatively effects on immune reconstruction.