| Objectives:Follicular helper T(Tfh)cells can be divided into germinal center-Tfh(GCTfh)cells and circulating Tfh(cTfh)cells according to their distribution in anatomical position.GC-Tfh cells located in lymphoid tissues are necessary for the formation of the germinal centers and the proliferation of B cells,assisting in the production of antibodies and maintaining the humoral immunity.cTfh cells are a group of memory CD4+T cells expressing CXCR5 in peripheral blood,which also express the basic functional phenotypes of GC-Tfh cells,such as ICOS,PD-1,secrete IL-21,IL-4,and other characteristic cytokines,which can help B cells produce antibodies.Since secondary lymphoid tissue is not readily available,it’s difficult to study Tfh cells in humans.As a result,most studies used cTfh cells to study the functions and mechanisms of human Tfh cells in vaccine responses and diseases.The pathogen of Acquired Immunodeficiency Syndrome(AIDS)is the human immunodeficiency virus(HIV),which gradually attacks and destroys the immune system,causing various opportunistic infections and tumors.Cellular metabolism is closely related to the differentiation and effector function of Tfh cells,however,studies on the metabolism of Tfh cells focused on autoimmune diseases.Whether the metabolism of Tfh cells changes or their function can be affected during HIV infection has not been investigated.In addition,Tfh cells had higher levels of HIV than others in CD4+T cells,which is named by non-Tfh cells,it is not yet known whether metabolism plays a role in the infection of Tfh cells.The objective of this study was to detect the metabolic changes of cTfh cells in HIV-infected individuals comprehensively,analyze their relationship with disease progression and clinical outcome,and find the cell metabolism that changed obviously and played an important role(Section 1).Then we studied the effect of metabolism on the function of cTfh cells(Section 2),exploring the role and potential mechanisms of metabolism on the susceptibility of HIV in cTfh cells(Section 3).Our investigation provided new ideas and directions for the recovery of humoral immunity and clearance of viruses in HIV-infected individuals.Methods:1.Study Participants This study included 68 HIV-negative healthy controls and 113 HIV-positive individuals,among which 58 were patients who had never accepted antiretroviral therapy,and 55 were ART-treated individuals.All participants were matched in age and gender and recruited at the Red Ribbon Clinic.All the research subjects signed informed consent and passed the approval of the Ethics Committee.2.Detection of Plasma HIV RNA LevelsPlasma samples were isolated from whole blood collected from study subjects.Real-time quantitative PCR was adopted to determine plasma HIV RNA levels.The procession was performed using the COB AS AMPLICOR automatic loader of Roche Company.Results below the lower detection limit of 20 copies/ml are shown as<20.3.Measurement of Absolute CD4+T Cell CountsPeripheral blood was collected from the study subjects.CD4/CD8/CD3 TriTEST reagents whole blood was added to the Trucount tubes.A single-platform lyse-no-wash procedure was performed and cells were detected using a BD FACS Calibur flow cytometer.Data were analyzed using Multi SET software.4.Detection of Multiple Nutrient TransportersPeripheral blood mononuclear cells(PBMCs)were extracted from the whole blood of study subjects.Human TruStain FcXTM was added to the tube containing PBMCs as an Fc segment blocker to reduce non-specific stains.The process of cell surface stain was performed using fluorescent dyes including CD3-PE-Cy7,CD4-APC-Cy7,CD45RABV510,CXCR5-BV711,Glutl-PE,CD36-APC,FATP-1-Alexa Fluor 647,CD71-APC,CD98-PE,CXCR3-PE-Cy5,CCR6-PE-Cy7,7-AAD.BD FACS LSR Ⅱ flow cytometer was used to detect the expression levels of nutrient transporters.5.Measurement of Mitochondrial Marker and Reactive Oxygen SpeciesPre-warmed PBS at 37℃ was added to the tube containing PBMCs.MitoTracker Green,CMTMRos,and CellROX reagents were added and followed by incubation at 37℃ for 30 min.Cells were washed twice with PBS and the expression was detected by BD FACS LSR II flow cytometer.6.Measurement of Cellular ApoptosisCD4+T cells were negatively selected using EasySepTM Human CD4+T Cell Isolation Kit from PBMCs,then cultured at 37℃,5%CO2 for 24h.Collect and wash CD4+T cells,followed by adding Annexin-V and 7-AAD to the tube.Cells were detected by BD FACS LSRⅡ flow cytometer without being washed.7.Detection of Effector Function Molecules and Cytokines of cTfh cellsCD4+T cells were stained using Fixable Viability Stain 620 for excluding dead cells.Use Fixation/Permeabilization Solution Kit to rupture the cell membrane,then add IL-21BV421,and IL-4-APC to the tube.ICOS can be detected directly by surface stain,and CD40L needs to be stimulated by DynabeadsTM CD3/CD28 T-Activator.Unstimulated cells acted as a control group.8.Detection of Intranuclear Factors Ki67 and Bcl-2The nuclear membrane of PBMC was ruptured by using eBioscienceTM Foxp3/Transcription Factor Staining Buffer Set.Ki67-PE and Bcl-2-FITC were added to the tube.Cells were washed and then measured by BD FACS LSR Ⅱ flow cytometer.9.Experiment of Intervention on Cell MetabolismInhibitor reagents of metabolism including 2-DG(5mM),Sodium Oxamate(20mM),BCH(lOmM),and DON(2μM)were added to the wells containing CD4+T cells of untreated HIV-infected individuals.Add the same volume of PBS to the control group,then cells were cultured at 37℃,5%CO2 for 24h.Detection of ICOS expression was activated using Dynabeads CD3/CD28 T-Activator to amplify the effect of the intervention on cell metabolism.10.Measurement of Phenotype about Cell Activation and Co-inhibitory ReceptorsPBMCs were stained by the fluorescent antibodies including CD3-Percp-Cy5.5,CD4APC-Cy7,CD45RA-BV510,CXCR5-BV711,CD25-PE-Cy7,CD69-APC,HLA-DR-PE,TIGIT-BV421,PD-1-PE-Cy7.The staining method was described above.11.Detection of Glucose UptakeAdd pre-warmed glucose-free 1640 medium at 37℃ to washed PBMCs,followed by adding 100 μM 2-NBDG.Then cells were incubated at 37℃ for 30 min and washed twice with PBS.The expression level of 2-NBDG was detected by the BD FACS LSR Ⅱ flow cytometer.12.Detection of Signal Pathway Related to MetabolismPBMCs were fixed using eBioscienceTM Intracellular Fixation&Permeabilization Buffer Set.The cell membrane was ruptured by adding pre-cooled 50%methanol.p-S6-APC and p-Akt-Alexa Fluor 488 were added to the tube and cells were washed after 40min at 4℃.The expression level of p-S6 and p-Akt were detected by the BD FACS LSR Ⅱ flow cytometer.c-myc and HIF-1α are transcription factors in the cellular nucleus,and the method has been described above.13.Production of HIV reporter viruses293T cells were cultured for 12-24h the day before transfection so that cells covered 80%90%district of the plate.pNLENG-IRES and Lipo 2000 are mixed in the medium of DMEM.Aspirate the cultural supernatant in the 293T cells plate and then add 5 ml DMEM to the plate.The mixture was carefully added to the 293T cell culture plate which was incubated at 37℃,5%CO2 for 5 h.Aspirate the culture supernatant and add DMEM+10%FBS to the plate followed by incubation for 48 h.Supernatants were collected and aliquoted with cryopreservation tubes,then stored at-156 0 C for later use.14.HIV Reporter Viruses and Infection ModelCD4+T cells of healthy controls were cultured in a medium containing IL-2 overnight.ADD PHA the next day to activate cells and continue the culture for 3 days.After disaggregation,CD4+T cells were spinoculated with either GFP reporter virus for 2 h at 1,200g at room temperature.Then cells were washed to remove unbound virus and media,and cultured for 3 days at 37℃ with 5%CO2.Cells were collected and stained by Fixable Viability Stain 620,and the procedure of surface stain was performed using CD45ROBV510 and CXCR5-BV711 to discriminate cTfh and non-cTfh cells.After adding 1%paraformaldehyde to fix the cells,followed by detecting the expression level of GFP by BD FACS LSR II flow cytometer to evaluate the effect of infection.15.Experiment of Intervention on Cell Metabolism and signal pathway in Infection Model Relevant reagents of intervention on cell metabolism and signal pathway were introduced in the infection model,including STF-31(100nM),2-DG(5mM),UK5099(25μM),3-PO(1μM),Sodium Oxamate(20mM),Oligomycin(2μg/ml),BCH(10mM),DON(2μM),Rapamycin(200 nM),CCT007093(10 μM),Mycro-3(10 μM),BAY 87(10 μM),which were added to culture tubes 2h before activating the cells.Add the same volume of PBS or 1%DMSO as a control group.After the centrifugation infection was completed,the reagents above were added back to continue the culture.16.Statistical analysis GraphPad Prism 7 and SPSS 22.0 software were used to graph and analyze the experimental results.The non-parametric Mann-Whitney U test is used to compare the difference in quantitative data between two groups,the Kruskal Wallis test is used for multiple group comparisons,and the Wilcoxon paired test is used for the paired rank-sum test between two groups.Spearman is used to analyze the correlation between the two groups for paired comparison.The tests are all two-sided tests,and p<0.05 is considered a significant statistical difference.Results:Ⅰ.Study on metabolic changes in circulating follicular helper T cells in HIV-infected individuals1.The expression levels of nutrient transporter related to the metabolism of glucose metabolism,amino acid,and iron in cTfh cells were significantly increased in HIV-infected individuals.Compared to healthy controls,the expression levels of glucose transporters Glutl,transferrin receptor CD71,and amino acid transporter CD98 on cTfh cells were significantly increased in untreated HIV-infected individuals,and the levels of Glutl and CD71 on cTfh cells of treated patients were lower than that in untreated patients.The expression of CD98 in patients’ cTfh cells after treatment was still significantly higher than that in healthy controls.There was no significant difference in the expression of lipid metabolism-related transporters CD36 and FATP-1 between healthy people and HIVinfected patients.2.There was no significant difference in the mitochondrial metabolism of cTfh cells in HIV-infected individuals compared with healthy peopleWe used transcellular membrane dyes to stain intracellular organelles and found that there was no significant difference in the expression level of mitochondrial mass,mitochondrial membrane potential,and intracellular reactive oxygen species between healthy people and HIV-infected people through flow cytometry.3.There were significant differences in metabolic index expression between cTfh cell subsets.According to the expression of CXCR3 and CCR6,cTfh cells can be divided into 4 subsets,including cTfh1,cTfh2,and cTfh17 subsets.The proportion of cTfh subsets did not change significantly between healthy controls and HIV-infected individuals.The levels of Glut1,CD71,and CD98 expressed on the cTfh2 subset were significantly lower than those of the cTfh1 and cTfh17 subsets,and the expression levels of CD71 on the cTfh1 subset were higher than those of the cTfh17 subset,and the level of CD98 was lower than those of the cTfh17 subset.4.The expression levels of multiple metabolic indicators of cTfh cells are associated with disease progression.The levels of Glut1,CD36,CD71,CD98,mitochondrial mass,and reactive oxygen species of cTfh cells were negatively correlated with CD4+T cell counts in HIV-infected patients.The levels of CD98,mitochondrial mass,and reactive oxygen species were positively correlated with viral load,and FATP-1 was negatively correlated with viral load.5.Glut1,CD71,and CD98 on cTfh cells may be important nutrient transporters related to metabolism for the clinical outcome of HIV.Based on the results of all the data,we found that Glut1,CD71,and CD98 of cTfh cells were significantly elevated in HIV-infected patients and closely related to disease progression of AIDS and recovery of the immune system.Ⅱ.Study on the relationship between the function of circulating follicular helper T cells and metabolic changes in HIV-infected individuals1.The number of cTfh cells in HIV-infected patients decreased,but the proliferation ability was not weakenedCompared with healthy controls,the percentage of cTfh cells in CD4+T cells was significantly reduced in untreated HIV-infected patients,and the percentage and absolute counts of cTfh cells in treated patients were significantly higher than those of untreated HIV-infected patients.Although the number of cTfh cells decreased,the proliferation ability was not significantly weakened,which was manifested by the increased expression level of the proliferation index Ki67 in the cTfh cells of untreated HIV-infected individuals.Healthy controls and treated patients had significantly higher levels of Ki67 in cTfh cells than those in non-cTfh cells.2.The level of apoptosis in cTfh cells was increased in HIV-infected individuals,and correlated negatively with CD4+T cell counts.The reduction of cTfh cell counts is related to apoptosis,which was manifested by a significant increase in the proportion of early apoptosis and late apoptosis in cTfh cells of untreated HIV-infected patients compared to healthy people.The percentage of apoptotic cells was negatively correlated with CD4+T cell counts.Besides,the proportion of early apoptosis and late apoptosis in cTfh cells was significantly higher than those in non-cTfh cells.3.The expression levels of ICOS and IL-21 of cTfh cells were elevated in HIV-infected individuals,which is associated with disease progression.Compared with healthy controls,the effector function molecules of cTfh cells,ICOS,and IL-21 were significantly increased in HIV-infected patients and were negatively correlated with the CD4+T cell counts.The expression level of IL-21 was positively correlated with viral load;the levels of CD40L and IL-4 were not significantly changed.4.The expression levels of multiple functional phenotypes of cTfh cells were significantly correlated with the levels of nutrient transporters related to metabolism.The percentage of early apoptotic cells in cTfh cells was positively correlated with the expression level of Glut1,and the percentage of late apoptotic cells was significantly positively correlated with the expression level of CD98.The percentage of ICOS was positively correlated with the expression levels of Glut1 and CD98.The intracellular cytokine IL-21 was positively correlated with the level of CD98.5.Inhibition of glucose metabolism and amino acid metabolism will affect the levels of effector molecules and apoptosis in cTfh cellsAs an analog of glucose,2-DG can competitively inhibit glucose uptake of cells.Inhibition of glucose metabolism by 2-DG of cTfh cells in untreated HIV-infected patients,the level of ICOS was significantly reduced,but the proportion of apoptotic cells was increased.BCH and DON can inhibit the expression of amino acid transporter proteins on the cell surface.Using BCH or DON to inhibit amino acid metabolism of cTfh cells in untreated HIV-infected patients,we found a significant reduction in the proportion of apoptosis in cTfh cells.Ⅲ.Study on the role of metabolism and related mechanisms on the susceptibility of HIV in circulating follicular helper T cells.1.Compared with non-cTfh cells,cTfh showed susceptibility to HIV,while expressing a higher level of co-inhibitory receptors,which were positively correlated with the expression levels of Glut1,CD71,and CD98.Plasmid NLENG was used to generate viral particles with GFP,which can infect CD4+T cells in vitro.It was found that the proportion of GFP in cTfh cells was significantly higher than that in non-cTfh cells,indicating that cTfh cells are more susceptible to HIV infection compared with non-cTfh cells.The expression levels of the co-inhibitory receptors TIGIT and PD-1 were significantly higher in cTfh cells than those in non-cTfh cells and were significantly positively correlated with the levels of nutrient transporters Glut1,CD71,and CD98.2.Compared with non-cTfh cells,the expression levels of multiple metabolic markers cells were higher in cTfh cells.The expression levels of Glut 1,CD98,2-NBDG,an indicator of glucose uptake,mitochondrial mass,mitochondrial membrane potential,and reactive oxygen species were significantly higher than that in non-cTfh cells.While the expression level of the transferrin receptor CD71 was higher in non-cTfh cells.3.The level of cell activation markers in cTfh cells was significantly lower than that in non-cTfh cellsCD25,CD69,and HLA-DR are commonly used as indicators to assess the status of cell activation.Through flow cytometry,we found that the percentage of CD69 on the surface of cTfh cells did not change significantly,but the percentages of CD25 and HLA-DR decreased distinctly compared with non-cTfh cells.4.The levels of signal pathways related to metabolism in cTfh cells were higher than those in non-cTfh cellsS6 and Akt are the downstream molecules of subunits,mTORCl,and mTORC2,of the metabolic signal pathway mTOR,respectively.Phosphorylation of S6 and Akt indicated that they are in the activated state.In addition,c-myc and HIF-1α are also involved in the regulation of cellular metabolism.The results showed that the levels of p-S6,p-Akt,and c-myc in the cTfh cells were significantly higher than those in non-cTfh cells,and the expression of HIF-1α was not significantly different between cTfh and non-cTfh cells.5.Inhibition of glucose metabolism and amino acid metabolism restrained infection of cTfh cells by HIV.Inhibitors of multiple nutrients or transporters were introduced in infection experiments in vitro to detect the effect of metabolism on the susceptibility of cTfh cells to HIV.The results showed that 2-DG restrained the infectious ability of HIV on cTfh cells by inhibiting glucose uptake,and BCH and DON inhibited the infectious ability of HIV on cTfh cells by inhibiting amino acid metabolism,which showed that the proportion of GFP on cTfh cells in 2-DG,BCH,and DON treatment groups were significantly reduced compared with control.6.Blocking the signal pathways related to metabolism affected the impact of HIV infection on cTfh cells.We continue to introduce inhibitors of metabolic pathways in infection experiments in vitro.Rapamycin targets the inhibition of mTORC1;Mycro-3 is a selective inhibitor of MycMax dimerization that can effectively suppress c-myc;BAY 87 can reduce HIF-1α levels.The results showed that the proportion of GFP in cTfh cells in the Rapamycin-treated group was significantly reduced compared with control,while the proportion of GFP in the Rapamycin-treated group was significantly elevated compared with control in cTfh cells.Conclusion:1.The expression level of multiple nutrient transporters on cTfh cells increased in HIVinfected patients,and had also been different between cTfh subsets.In addition,the various metabolic indicators of cTfh cells were correlated with HIV disease progression and immune recovery.2.The counts of cTfh cells decreased significantly in untreated HIV-infected patients,but the ability of proliferation was not weakened,while the proportion of apoptotic cells increased.The level of apoptosis,ICOS,and IL-21 in cTfh cells was significantly correlated with nutrient transporters’ expression.Inhibition of amino acid metabolism in untreated HIV-infected individuals can reduce the apoptosis levels of cTfh cells,while inhibition of glucose metabolism will promote apoptosis,but can suppress the expression of ICOS on the surface of cTfh cells.3.Compared with non-cTfh cells,cTfh cells are more susceptible to HIV infection.The expression of co-inhibitory receptors on cTfh cells is related to the expression of nutrient transporters.Compared with non-cTfh cells,cTfh cells are in a state of higher degrees of glucose,amino acid,and mitochondrial metabolism,which is not related to the level of activation but is involved in its signal pathway related to metabolism.Inhibition of glucose metabolism,amino acid metabolism,or metabolic signal pathway mTORCl can inhibit the infection on cTfh cells by HIV. |
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