| Objective:Human immunodeficiency virus(HIV)infects the human body to cause a progressive decrease in the number of CD4+T lymphocytes,and ultimately triggers acquired immunodeficiency syndrome(AIDS).Continuous anti-retroviral therapy(ART)can inhibit viral replication in HIV-infected patients,and increase the number of CD4+T cells in patients.Those with CD4+T cells greater than 500 cells/μl are called immunological responders(IR),but there are still a small number of patients whose CD4+T cell levels cannot be effectively recovered,less than 350 cells/μl which are called immunological non-responders(INR).Because of poor recovery of CD4+T cells,INR patients cannot obtain sufficient immune reconstitution.The body is in a state of continuous chronic immune activation,and the risk of combined opportunistic infections is higher.At present,there are very limited interventions that can effectively promote INR immune reconstitution.Therefore,it is urgent to investigate the reasons why the immune function of INR patients cannot be fully recovered,and to find new interventions that can promote INR immune recovery.This is an urgent problem in the current HIV research field..The growth,development and physiological functions of CD4+T cells are closely related to their metabolic activity,and HIV infection of CD4+T cells is also accompanied by changes in their metabolic activity.However,there are currently few studies related to the metabolism of CD4+T cells in INR patients,especially the mitochondrial function,glucose metabolism and its related regulatory factors.This thesis studies the expression of nutrient transporters,glucose uptake capacity,and glucose metabolism-related regulatory factor levels of CD4+T cells in INR and IR patients;further studies mitochondrial function,cell proliferation and cytokine secretion,etc.of CD4+T cell,providing laboratory data reference to promote exploration interventions for INR immune reconstruction.Methods:1.Density gradient centrifugation to extract peripheral blood mononuclear cells(PBMCs).2.CD4+T cell surface marker detection:the extracted PBMCs were stained on the surface,incubated at 4℃ for 30 minutes in the dark,washed with 2%FBS washing solution and centrifuged,discarded the supernatant,resuspended in 200ul PBS,and detected by BD Canto Ⅱ flow cytometer.3.Detection of nuclear markers in CD4+T cells:resuspend the extracted PBMCs into lml PBS,add 1μl LIVE/DEAD,incubate at 4℃ for 30 minutes in the dark,wash the cells and then stain the surface,and finally permeation was performed with Thermo Fisher’s permeabilization reagent,and then the nuclear markers were added separately,incubated for 30 min at 4℃ in the dark,washed the cells with washing buffer,resuspended and tested on the machine.4.Detection of phosphorylation markers in CD4+T cells:resuspend the extracted PBMCs into 1ml PBS,add 1μl LIVE/DEAD,incubate at 4℃ for 30 minutes in the dark,wash the cells and then stain the surface,and finally treat the cells with BD’s phosphorylation reagent,then add p-mTOR dye,incubated for 30 min at 4℃ in the dark,washed the cells with 2%FBS solution,resuspended and tested on the machine.5.Negative selection of CD4+T cells:extract PBMCs from research objects,and negatively select the required CD4+T cells with Stem Cell’s CD4+T cell enrichment kit.6.CD4+T cell migration ability detection:the CD4+T cells obtained by negative selection were resuspended to 0.5 Million/100μl with RPMI1640,100μl cell suspension was gently added to the upper chamber of the 5.0μm-Transwell plate,and 600μl chemokine SDF-1(100ng/ml)was added to the lower chamber.After incubating in 37℃ for 3 hours,count the number of cells in the lower chamber.7.CD4+T cell proliferation ability detection:take 0.5 Million negatively selected CD4+T cells and resuspend them in PBS to a volume of 1ml,add 1μl Cell Trace Violet dye,and incubate at 37℃ for 30min in the dark.Wash the cells and discard the supernatant.Then add 200μl R10 to resuspend the cells and transfer the cells to the 96-well plate and add CD3/CD28 beads(added 1:1 with the number of cells)at the same time to stimulate the cells,place them in a 37℃ incubator,change the medium on the third day and continue the culture to the fifth day,collect the cells and use 7-AAD staining,test on the machine within 5 minutes.8.Detection of the ability of CD4+T cells to secrete cytokines:resuspend the negatively selected CD4+T cells with R10 to 0.5 Million/200μl,then transfer the cells to a 96-well plate(200μl per well),add CD3/CD28 beads stimulus and CD107a-PE dye,gently mix and incubate in 37℃ for 24 hours,and add Golgistop 6 hours before the end of the culture.After collecting the cells,add LIVE/DEAD staining,incubate at 4℃ for 30 minutes in the dark,then perform surface staining(CD4-APC-Cy7),incubate at 4℃for 30 minutes in the dark,and then incubate for 20 minutes with permeabilization reagent at 4℃ in the dark.Then the cells are washed with washing solution,and then stained with IL-2-APC and TNFα-PE-Cy7,incubated at 4℃ for 30 minutes in the dark,and the cells are washed with washing solution and tested on the machine.9.CD4+T cell glucose uptake function detection:resuspend PBMCs in glucose-free R10 medium to 1ml,add 2-NBDG(100μM)to mix,and incubate at 37℃ for 30 minutes in the dark.Wash the cells twice and stain the surface(CD3-PE-Cy7,CD4-APC-Cy7),and finally add 7-AAD and tested on the machine within 5 minutes.10.CD4+T cell mitochondrial function and ROS level detection:resuspend PBMCs into 1ml with PBS preheated to 37℃,add MM(12.5nM),MMP(12.5nM),ROS(1.25μM)dyes and mix well,incubate at 37℃ in the dark for 30 minutes,wash the cells twice,then surface staining(CD3-PE-Cy7,CD4-APC-Cy7),finally 7-AAD is added,and tested on the machine within 5 minutes.Results:1.The expression of CD71 on CD4+T cells of INR was significantly higher than that of IR.There was no significant difference in the expression of Glut1 and CD98 among the three groups(Figure 1A-D).The CD36 expression of CD4+T cells in the IR group was significantly higher than that in the NC group(P<0.01,Figure 1E,F).The expression level(percentage and MFI)of CD71 on CD4+T cells in the INR group was significantly higher than that of the NC and IR groups(P<0.01,P<0.01,P<0.01,P<0.01,Figure 1G-J).2.The expression level of CD71 on CD4+T cells in HIV-infected patients is significantly negatively correlated with the number of CD4+T cells.The percentage and MFI of CD71 was significantly negatively correlated with the number of CD4+T cells in infected patients(r=-0.6285,r=-0.4891,Figure 2A,B).3.The CD4+T cell glucose metabolism level of INR is significantly different from that of NC group.The MFI of 2-NBDG in CD4+T cells in the IR and INR group was significantly higher than that in the NC group(P<0.01,P<0.01),but there was no significant difference between IR and INR(Figure 3A,B).The level of p-mTOR(MFI)in CD4+T cells in the INR group was significantly lower than that in the NC group(P<0.01),but there was no significant difference between the IR group and the NC group(Figure 3C,D).The expression level(MFI)of HIF-la in CD4+T cells in the IR and INR group was significantly higher than that in the NC group(P<0.01,P<0.01),but there was no significant difference between IR and INR(Figure 3 E,F);c-Myc expression level(MFI)has no significant difference among the three groups of NC,IR,and INR(Figure 3G,H).4.The level of ROS in CD4+T cells of INR is significantly higher than that of IR.There was no significant difference in the expression level of MM in CD4+T cells between the NC,IR,and INR groups(Figure 4A,B),while the expression level of MMP in CD4+T cells in the IR and INR group was significantly higher than that in the NC group(P<0.01,P<0.01),but there was also no significant difference between the IR and INR(Figure 4C,D).The ROS levels(percentage and MFI)of CD4+T cells in the INR group were significantly higher than those in the NC and IR groups(P<0.01,P<0.01,P<0.01,P<0.01,Figure 4E-H).5.The level of ROS in CD4+T cells of HIV-infected patients is significantly negatively correlated with the number of CD4+T cells.The level of ROS in CD4+T cells(percentage and MFI)was significantly negatively correlated with the number of CD4+T cells in infected patients(r=-0.7607,r=-0.7250,Figure 5A,B).The expression level of CD71 on the surface of CD4+T cells was significantly positively correlated with the intracellular ROS level(percentage and MFI)(r=0.7619,r=0.9280,Figure 5C,D).6.The CD4+T cell proliferation capacity of INR is not lower than that of IR.The proportion of CD4+T cell death in the INR group was significantly higher than that in the NC group(P<0.01),but there was no significant difference between the IR and the NC(Figure 6A).The expression level of Ki-67 in CD4+T cells in the INR group was significantly higher than the NC and IR groups(P<0.01,P<0.01),and the IR group was also significantly higher than the NC group(P<0.01,Figure 6B,C).And the expression level of Ki-67 in CD4+T cells was significantly negatively correlated with the number of CD4+T cells in infected patients(r=-0.6937,Figure 6D).There was no significant difference in the proliferation and fission ability of the three groups(Figure 6E,F).7.The CD4+T cell activation and apoptosis levels of INR are significantly higher than those of IR.The CD4+T cell activation(CD38+HLA-DR+)level of INR was significantly higher than that of IR and NC groups(P<0.01,P<0.01,Figure 7A,B).The CD4+T cell senescence(CD57+)level of IR and INR group was significantly higher than that of NC group(P<0.01,P<0.01,Figure 7 C,D).At the same time,the early apoptosis(Annexin V+7-AAD-)and overall apoptosis(Annexin V+)level of INR CD4+T cells were significantly higher than the IR and NC groups(P<0.01,P<0.01,P<0.01,P<0.01,Figure 7 E,F).The activation(r=-0.5504,P<0.01)and apoptosis(r=-0.3567,P=0.011)levels of CD4+T cells in HIV-infected patients were significantly negatively correlated with the number of CD4+T cells in HIV-infected patients(Figure 7G,I).8.The ability of INR CD4+T cells to secrete IL-2 is significantly reduced.There was no significant difference in the migration ability of CD4+T cells among the three groups(Figure 8A).The level of CD4+T cell degranulation(CD 107a)and the level of TNF-α secretion were not significantly different between the three groups(Figure 8B-E),while the level of IL-2 secreted by CD4+T cells in the INR group was significantly lower than that of NC Group and IR group(P<0.01,P<0.01,Figure 8F,G).Conclusion:There was no significant difference in the expression levels of Glut1,CD98,and CD36 on CD4+T cells in INR and IR patients,but the expression level of CD71 in the INR patients was significantly higher,and the CD71 level was significantly negatively correlated with the CD4+T cells of the patients;the CD4+T cell glucose metabolism level of INR is significantly different from that of NC group;the MMP level of CD4+T cells in the INR and IR groups was significantly higher than that of the NC group.The cytoplasmic ROS level of CD4+T cells in the INR group was significantly increased,and the ROS level was significantly negatively correlated with the patient’s CD4+T cells.The CD71 level of CD4+T cells is significantly positively correlated with the level of ROS;the proliferation capacity of CD4+T cells in INR patients is not lower than that in IR patients,but their activation and apoptosis levels are significantly higher;and the level of IL-2 secreted by CD4+T cells in INR patients is significant decreased;this paper provides laboratory data reference for exploring interventions to promote immune reconstitution in INR patients. |
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