Study On The Role Of T Helpler 17 Cells In Supporting HIV-1 Persistence During Antiretroviral Therapy

Objective:Acquired Immune Deficiency Syndrome?AIDS?,caused by Human Immunodeficiency Virus?HIV?,is a severe infectious disease threatening the global health.It's characterized by the countinuously detrimental infection of CD4⁺T cells by HIV-1.At present,effective vaccine is vacant in HIV-1 prevention and eradication.Highly Active Antiretroviral Therapy?HAART?can effectively suppress HIV-1replication and clinical HIV-1 disease manifestations,resulting in the restoration of immune function to some extent and the improvement of life quality.Although HAART could inhibit the viral burden in peripheral plasma of HIV-1 infected patients,which couldn't be detected using custom technique,however,viruses will rebound once the treatment is stopped typically in a few days,which indicates HIV-1 persists along with therapy.To identify and clear the residue virus under therapy will be helpful in HIV-1function cure or sterilizing cure.The existence of HIV-1 cellular reservoir is the main cause of viral persistence during therapy.CD4⁺T cells with more susceptibility are infected by HIV-1,which leads to a small pool of HIV-1 latently-infected resting memory CD4⁺T cells carrying integrated HIV-1 genomes settle down,which was initially found in 1995.These cells do not produce infectious virus in the resting state,but can do so upon cellular activation.This latent reservoir in resting CD4⁺T cells persists even in patients on HAART who have no clinically detectable viremia.Mathematical model on longitudinal analysis demonstrated that the time to eradication of the pool of latently infected cells in patients on HAART,would be more than 60 years,which means that the patients will not get rid of the burden of therapy and take the risk of side effect of the drugs for life-long time.This latent reservoir is now represented as the major barrier to HIV-1 eradication.Thus,identifying the functional and phenotypic characteristics of cell subsets that harbor replication-competent virus during suppressive antiretroviral therapy is a critical step for developing interventional strategies to target residual viral reservoirs.Most available evidence supports the notion that latently infected CD4⁺T cells containing chromosomally-integrated but transcriptionally silent HIV-1 DNA represent the most dominant cell population responsible for HIV-1 persistence despite treatment.However,it is increasingly recognized that such cells represent a phenotypically diverse cell population that consists of a variety of different cell subsets with distinct developmental profiles and functional properties,divergent levels of permissiveness to HIV-1 infection and latency,and,most likely,different susceptibilities to clinical strategies or pharmaceutical agents aiming at reversing viral latency and HIV-1 eradication.Memory CD4⁺T cell is a subset of CD4⁺T cell which have encountered antigen during prior infection and possess the ability to mount a fast and stronger immune response upon the second infection.From a developmental perspective,memory CD4⁺T cell evolution can be described as a hierarchical process during which immature,long-lived cells undergo progressive commitment to more differentiated cell types.Recent data suggest that the initial,most immature population of memory CD4⁺T cells consists of a small number of extremely long-lasting cells that phenotypically express a mix of na?ve and memory cell markers,and display superior abilities to self-renew through homeostatic proliferation,while replenishing the pool of more mature memory cells in a stem cell-like fashion.These cells,termed T memory stem cells(T_SCM),give then rise to central-memory cells(T_CM),which typically represent the largest compartment within the CD4⁺T cell pool and can serve as precursor cells for effector-memory cells(T_EM)specializing in direct antimicrobial immune defense.In addition to this developmental hierarchy,CD4⁺T cells also differ from a perspective of functional polarization.Polarization of CD4⁺T cells occurs under the influence of cytokines that induce specific functional profiles characterized by expression of signature cytokines,transcription factors and phenotypic surface markers.Functional polarizations described so far include Th1 cells,which secrete the antiviral cytokine IFN?under transcriptional control of T-bet,and Th2 cells with roles in antihelmintic and antiparasitic immune defense mediated by interleukin-4?IL-4?and regulated by The transcription factor Gata-3.Th17 cells represent a more recently discovered population of helper cells with critical functions for maintaining mucosal antimicrobial immune defense through the production of interleukin-17?IL-17?;These cells express ROR?t as a master transcriptional regulator.Expressing CCR6,Th17 cells are not homogeneous,containing many different subsets such as CXCR3-CCR4+CCR6+cells?Th17 cells?,CXCR3+CCR4-CCR6+cells?Th1/17Th1/17 cells?,and the newly discovered CCR6+CD95+naive-like T cells?also called Th17 precursors or stem cell memory Th17cells?.T follicular helper cells?Tfh?express The transcription factors Bcl-6 and represent a CD4⁺T cell population specializing in supporting B cell development and function,mostly by secreting IL-21;These cells uniformly express CXCR5 and are localized in the germinal center of lymph node B cell follicles,but populations of circulating CD4⁺T cells with phenotypic and functional characteristics similar to Tfh have also been described.Recent studies report that Th17 cells possess the capacity of self-renew and homeostatical proliferation,resembling the signature of T_SCM cells,thus surviving long as a population of effector memory cells.In addition,circulating Th cells include highly and low CXCR5 expressing compartments of different Th cells.How different Th cells including CXCR5+or CXCR5-cells contribute to HIV-1 reservoir pool is poorly known.Importantly,recent studies suggest that the functional commitment to individual patterns of CD4⁺T cell polarization also affects developmental and maturational aspects,including the ability to survive long term,to proliferate homeostatically and to serve as precursor cells for effector cell populations.This is particularly obvious in the context of Th17 cells,which represent a long-lasting cell population with increased abilities to self-renew and to repopulate pools of terminally-differentiated helper cells of the same,and possibly of other polarizations.Together,Identifying new populations especially cells with homeostatic proliferation is necessary in deeply exploring strategies for HIV-1 eradication.In this study,we aim to perform a detailed analysis to investigate the ability of CD4⁺T cells with different polarization to serve as long-term reservoirs for HIV-1 during antiretroviral treatment.First,we analyzed the susceptibility of different Th cells to HIV strains in vitro and the possibly intrinsic mechanism.We subsequently recruited the HIV-1 infected patients under long-term therapy.Different Th cells were sorted including Tfh-like and non-Tfh-like Th1,Th2,Th17 and Th1/17 cells and relevant reservoir size and their contribution to HIV-1 reservoir pool were analyzed.The phylogenetic analysis on HIV-1proviral sequence compare with short-term and long-term HAART was then generated to give clues on HIV-1 persistence under long-term therapy,which is meaningful in strategies for HIV-1 eradication.Methods:1.Donors and patientsFor the study on cellular susceptiblity to HIV-1,20 healthy donors were recruited.Study participants from HIV-1 treated patients since acute and chronic phases were recruited under the following criteria.Acute treated patients?n=8?:HIV-1 viral load<50cps/ml;Samples were collected around 1-2 year and over 5 years upon treatment.Chronic treated patients?n=9?:HIV-1 viral load<50cps/ml;Samples were collected around 5 years upon treatment.Study subjects gave written informed consent to participate and were approved by the ethic Review Board.2.Sample preparationThe whole blood was collected with EDTA-anticoagulation.Plasma was separated within 6 hours and preserved in-80?freezers.PBMCs were isolated by density gradient centrifugation and frozen down.3.Cell sorting and flow cytometryCD4⁺Tcell populations were stained with monoclonal antibodies to CD3,CD4,CD45RA,CCR4,CCR6,CXCR3 and CXCR5.After 20 minutes at dark in 4?freezers,cells were washed with 2%FBS-PBS.After 10 mins of 300g centrifugation,cells were suspended in 2%FBS-PBS.Indicated cell populations were live-sorted in a specifically designated biosafety cabinet?Baker Hood?,using a FACS Aria cell sorter?BD Biosciences?at 70 pounds per square inch.Cell sorting was performed and resulted in isolation of live lymphocytes with the defined phenotypic characteristics of>95%purity.For phenotypic characterization,cells were additionally stained with CCR5 and CXCR4,and acquired on a LSRII flow cytometer?BD Biosciences?.Data were analyzed using FlowJo software?Treestar?.4.In vitro HIV-1 infection assaysUnselected PBMCs from HIV-1 negative donors were cultured in RPMI medium supplemented with 10%FCS and 50 U/ml of rh IL-2.A total of 10x10⁶ PBMCs were infected with a GFP-encoding VSV-G-pseudotyped virus?MOI=1,unless otherwise indicated?or GFP-encoding R5-?Ba-L?or X4?NL4-3?-tropic viral strains?both at an MOI=1?.All isolates were kindly provided by Dr.Littman,New York University,New York.After three hours,cells were washed twice with PBS and cultured for 5 days.On day 5,cells were stained with surface antibodies to identify individual CD4 T cell subsets,washed and analyzed on a LSRII flow cytometer.5.Gene expression analysisGene expression analysis included the detection of transcriptional factors of different Th cells?T-bet,Gata-3,ROR?t and Bcl-6?and restriction factor?SAMHD1,TRIM5?,CDKN1A and BST2?.Sorted CD4⁺T cell populations were activated with CD3/CD28 beads?Dynabeads?for 3 days.RNA was then extracted after the beads were removed from cells and reverse transcripted to cDNA.Expression of selected gene transcripts in individual Th cell subsets was analyzed by semiquantitative PCR using Taqman gene expression assays with customed standardized primers/probes,and normalized to the expression of the housekeeping gene Actb?encoding-actin?in each CD4⁺T cell population.For the detection of gene expression,real-time PCR was employed with the following condition:94?for 15 seconds,60?for 60 seconds for 40cycles?Relative quantitative 2^-??Ct method was used to analyze the gene expression difference of distinct Th cells.6.Assessment of cell-associated HIV-1 DNASorted CD4⁺T cell populations were digested as previously described to extract cell lysates.We amplified total HIV-1 DNA using digital droplet PCR,using primers and probes described previously.PCR was performed using the following program:95?for10 min,45 cycles of 94?for 30s and 60?for 1 min,98?for 10 min.The droplets were subsequently obtained by QX100 droplet reader and data were analyzed using QuantaSoft software?Bio-Rad?.7.HIV-1 proviral sequencingCell lysates from sorted T cell populations were used for single-genome HIV-1envelope sequencing encompassing the V3 region.According to the HIV-1 DNA concentration of cell lysates from each CD4⁺T subsets?copies/?l?as assessed by droplet digital PCR,appropriate amount of cell lysates was diluted in 48 wells or 96 wells aiming to get less than 30%of wells yielding amplification products.Nested RT-PCR reaction was performed in triplicates using outer primers envA/LA11.PCR products were used as a template to generate an amplicon by nested PCR with inner primers LA12and LA13.Amplification products were then purified and sequenced using standard procedures.Sequences were aligned with an HXB2 reference sequence using BioEdit V7.1.9.A neighbour-joining method,as implemented in MEGA4,was used to construct phylogenetic trees with phylogenetically informative HIV-1 nucleotide sequences.8.StatisticsData are summarized as individual data plots with horizontal lines reflecting the median,or as box and whisker plots indicating the median,interquartile range,and minimum and maximum values.Differences were tested for statistical significance using Wilcoxon rank sum paired tests,Mann-Whitney U test,Kruskal-Wallis test,followed by Bonferroni correction for multiple comparisons where applicable.Spearman's correlation coefficient was calculated to analyze correlations.#p<0.05 represents significance using Wilcoxon signed rank test.*p<0.05,**p<0.01 and***p<0.005 represent Wilcoxon signed rank test after Bonferroni correction for multiple comparisons.Results:1.Identification of different Th cells by cell sorting and transcriptional factor analysisIn each of the CXCR5+and CXCR5-populations,cells were subsequently classified into four distinct subsets:A population of CXCR3+CCR4-CCR6-cells enriched for Th1-polarized cells upregulating the transcription factor t-bet,a CXCR3-CCR4+CCR6-population enriched for Th2 cells overexpressing the transcription factor Gata-3,a CXCR3-CCR4+CCR6+population enriched for Th17 cells with enhanced expression of the transcription factor ROR?t,and a population of CXCR3+CCR4-CCR6+population with Th1/17 polarization expressing higher levels of both t-bet and ROR?t.Expression of the Tfh-like transcription factor Bcl-6 was slightly higher in the CXCR5+CD4 T cell compartment.2.The susceptibility of Th17 cells to HIV-1 and the relevant mechanisms1)We found that CXCR5+CD4 T cells generally tended to be more susceptible to HIV-1than their CXCR5-counterparts,and was most pronounced in the context of R5-tropic infection.Among CXCR5-cells,lowest susceptibilities to R5-,X4-and VSV-pseudotyped infection occurred within cells enriched for Th2 polarization,while Th1,Th17 and Th1/17 polarized cells seemed to have similar levels of susceptibility to all three viruses tested.Within CXCR5+cells,Th1 enriched cells were least susceptible to R5-and X4-tropic infection,and highest levels of ex-vivo infection was noted in Th17and Th1/17 cells.2)Expression of CCR5 was highest in cells with preferential Th1/17 polarization,followed by those with Th1 and Th17 polarization;lowest levels of CCR5 expression occurred in cells enriched for Th2 polarization.CXCR4 expression was highest in Th1polarized cells,while levels in Th17,Th1/17 and Th2 cells were relatively similar.These patterns were consistent in both the CXCR5+and the CXCR5-CD4 T cell populations,but CXCR4 was more strongly expressed in CXCR5+cells,independently of further subclassification of cells.3)No significant differences were seen in expression levels of the host restriction factors SAMHD1,Trim5?,BST2 and CDKN1a among the studied cell populations.3.The contribution of Th17 and Th1/17-polarized cells to the viral reservoir1)In a cohort of HIV-1 patients who had been on suppressive antiretroviral therapy for a median of 7.25 years,lowest levels of HIV-1 DNA were detected in cells enriched for Th2 polarization in both CXCR5+and CXCR5-CD4⁺T cells.Highest HIV-1 DNA levels within CXCR5-CD4⁺T cells occurred in cells with Th1/17 polarization,while highest HIV-1 DNA levels in CXCR5+CD4⁺T cells were observed in cells enriched for Th17polarization.A discrepancy between those parameters were noted for cells enriched for a Th17 polarization,which made a disproportionately larger contribution to the viral reservoir relative to their contribution to the memory CD4 T cell pool,both within the CXCR5+and the CXCR5-cell compartment.2)In a cohort of six HIV-1 patients who initiated HIV-1 treatment during early HIV-1infection,followed by continuous,uninterrupted antiretroviral therapy over the next4.5-6.7 years.CD4 T cell subsets enriched for different polarizations were sorted after short-term antiretroviral therapy?1.5-1.9 year?and after long-term antiretroviral therapy,followed by analysis of HIV-1 DNA.HIV-1 DNA was strongly decreasing over time in the Th1 cell populations in most patients,both within the CXCR5+and the CXCR5-cell subset.The contribution of Th1 polarized CD4 T cells to the viral reservoir declined over time,while the contribution of these cells to the memory CD4 T cell pool remained fairly constant.Th17 and Th1/17 polarized cells showed an opposite pattern;their contribution to the viral reservoir substantially increased during prolonged antiretroviral therapy,in the background of a largely stable contribution to the memory CD4 T cell pool.The contribution of CXCR5+CD4 T cells to the memory CD4 T cell pool and to the viral reservoir in memory CD4 T cells declined over,although such changes did not reach statistical significance.4.Long-term persistence of viral sequences from Th17 and Th1/17 CD4 T cellsIn two patients who had remained on suppressive antiretroviral therapy,cells after short-term?1-2 years?and long-term[78 months?patient 1?and 56 months?patient 2?respectively]antiretroviral therapy were collected.CD4⁺T cells with different functional polarizations were sorted and HIV env sequences were amplified from genomic DNA.Interestingly,we noted identical viral sequences that were amplified from Th17 and Th1/17,consistent with prolonged persistence of HIV-1 in these cell populations.In contrast,identical viral sequences after short-and long-term antiretroviral therapy in Th1and Th2 cells were only noted in one of the two study subjects.Moreover,we observed that in several cases,Th17 and Th1/17 cells from early antiretroviral treatment stages harbored viral sequences identical to those detected in Th1 and Th2 cells several years later.Conclusion:1.In Th17 and Th1/17 cells,both with or without expression of the Tfh marker CXCR5,have increased susceptibility to HIV-1,suggesting that they may serve as preferential reservoirs for HIV-1 in ART-treated HIV-1 patients.Higher susceptibility to HIV-1 of these populations is associated with higher expression of HIV-1 coreceptor CCR5.No correlation were found in expression levels of the host restriction factors SAMHD1,Trim5?,BST2 and CDKN1a among different Th populations.2.Th17 and Th1/17 represent a very durable component of the viral reservoir that becomes increasingly important with prolonged duration of antiretroviral therapy.3.Viral sequence analysis supports the role of Th17 and Th1/17 cells as a long-lasting cellular reservoir that can promote viral persistence during antiretroviral therapy.