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CD4+CD25+CD127 regulatory cells play multiple roles in maintaining HIV-1 p24 production in patients on long-term treatment: HIV-1 p24-producing cells and suppression of anti-HIV immunity

Open AccessPublished:June 18, 2015DOI:https://doi.org/10.1016/j.ijid.2015.06.008

      Highlights

      • Depletion of regulatory T (Treg) cells may reduce HIV p24-producing cells and increase HIV-specific cytolytic T lymphocytes (CTL).

      Summary

      Background

      A major question when attempting to eradicate and treat HIV-1 infection is how to reactivate latent proviruses. Stimulating HIV-1-specific cytolytic T lymphocytes (CTL) has been shown to facilitate the elimination of the latent viral reservoir after viral reactivation. Regulatory T (Treg) cells are known to be capable of lowering both HIV-specific immunoreactions and general immune activation during HIV-1 infection. It was hypothesized that the depletion of Treg cells could increase the HIV-1-specific cytolytic T lymphocyte response and reactivate HIV-1 p24 production.

      Methods

      Treg cells were isolated by isolation kit according to the surface marker of Treg cells. Real-time PCR method was used to quantify HIV-1 DNA. P24 antigens in the cell culture supernatant was done by ELISA. Cells activation and HIV specific HIV-1 CD8+ T cells were analyses using a FACSCalibur flow cytometer and CELLQUEST software.

      Results

      This study included both HIV-infected patients who were antiviral treatment-naïve and patients with sustained viral responses to antiretroviral therapy (ART) for 1 or 5 years. It was found that the HIV-DNA levels in Treg cells were approximately 10-fold higher than those in non-Treg CD4+ cells and that the depletion of Treg cells could enhance the frequency of HIV-1-specific CTL and immune activation after 5 years of effective ART.

      Conclusions

      CD4+CD25+CD127 regulatory cells play multiple roles in maintaining HIV-1 p24 production in long-term ART patients. Treg cells may be a target for eliminating the latent HIV reservoir after effective long-term ART.

      Keywords

      1. Introduction

      Since the discovery of HIV, many classes of antiviral drugs that can limit viral replication to an undetectable level have been developed. These effectively prevent the occurrence of AIDS-related symptoms in infected individuals. Although current antiretroviral therapies can control viral replication, the persistence of latent HIV is a major obstacle to eradicating the virus from an infected individual. In latently infected cells, the integrated provirus is transcriptionally silent,
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      Stimulation of HIV-1-specific cytolytic T lymphocytes facilitates elimination of latent viral reservoir after virus reactivation.
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      Several studies have reported that during HIV infection, Treg cells contribute to HIV-specific immune dysfunction by limiting HIV-specific immunoreaction,
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      The majority of studies involving patients who were HIV-infected long-term non-progressors (LTNPs) or elite controllers, have reported decreased Treg cell percentages in peripheral blood
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      and in vivo.
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      These data suggest that Treg cells constitute a part of the viral reservoir.
      It is important to determine whether the depletion of Treg cells would assist in the eradication of HIV-1 production. It was hypothesized that the depletion of Treg cells would contribute to elevating the frequency of HIV-specific CTL and HIV-1 production, which may benefit latent viral eradication in patients on prolonged ART. To assess this hypothesis, treatment-naïve HIV-infected patients and HIV-infected patients who had received ART for 1 or 5 years with sustained viral responses were studied. It was found that Treg cells constitute an important factor in HIV-1 p24 production and that the depletion of Treg cells enhanced the frequency of HIV-1-specific CTL, immune activation, and the depletion of p24 expression after 5 years of effective ART treatment.

      2. Materials and methods

      2.1 Patients

      Thirty adults who were chronically infected with HIV-1 (cared for by the Center for Infectious Diseases, Beijing You’an Hospital, Capital Medical University) were enrolled in this study. No enrolled patient was co-infected with hepatitis B virus (HBV) or hepatitis C virus (HCV). The patients were divided into three groups: 10 AIDS patients who were ART-naïve (Table 1), 10 patients who had received ART for 1 year (ART1) and who exhibited persistent viral suppression with plasma HIV-1 RNA levels below 50 copies/ml after 12 weeks of ART (Table 1), and 10 patients who had received ART for 5 years (ART5) and who exhibited persistent viral suppression with plasma HIV-1 RNA levels below 50 copies/ml after 12 weeks of ART (Table 1). The ART regimen included two nucleoside reverse-transcriptase inhibitors (NRTIs) plus one non-nucleoside reverse-transcriptase inhibitor (NNRTI). CD4+ T cell numbers were <200 cells/μl before ART treatment for the ART-treatment patients.
      Table 1Characteristics of patients in this study
      PatientAge (years)GenderCD4 before ART (cells/ml)CD4 after ART (cells/ml)Viral load before ART (copies/ml)Time on ART (years)
      138M50-60 7350
      228M259-13 2840
      341M225-23 5970
      430M226-47 9070
      529M271-70 0000
      630M267-90430
      728M202-86 0000
      843M153-91 0000
      950M256-19 0000
      1049M260-19 0000
      1128M13631281921
      1228F30434811 0001
      1324M32137313 0001
      1426M22729585001
      1529F32738612 0001
      1635M33447610 0001
      1725M23632762001
      1832M19821825 0001
      1933F24530212 0001
      2026F2212617001
      2143M11335027 0005
      2227M31372034 0005
      2324M23843037 0005
      2426M21139821 0005
      2530M17638911 0005
      2627F22041015 0005
      2725F257415190 0005
      2824M22440922 0005
      2937M17337922005
      3036M106350140 0005
      ART, antiretroviral therapy; M, male; F, female.
      The exclusion criteria included pregnancy, active tuberculosis (TB; defined as suspected TB or being in the first 2 months of anti-TB therapy), and moribund status.
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      • Barugahare B.
      • Jones N.
      • Okello M.
      • Mutalya S.
      • Kityo C.
      • et al.
      Depletion of regulatory T cells in HIV infection is associated with immune activation.
      The study protocol was approved by the Beijing You’an Ethics Committee, and written informed consent was obtained from each subject. All of the HIV-infected subjects were serologically identified as having the human leukocyte antigen (HLA)-A2+ genotype, which was further confirmed by PCR.
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      PD-1 up-regulation is correlated with HIV-specific memory CD8+ T-cell exhaustion in typical progressors but not in long-term nonprogressors.

      2.2 Primers, probes, and M13 bacteriophages containing target sequences were used as standards for real-time PCR (qPCR)

      The primer and probe sequences for the qPCR assays were optimized and designed to be as similar as possible to both previous reports.
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      The chemokine receptor CCR5 was chosen as a surrogate for estimating the number of cells, because each cell contains only a single copy of CCR5, as shown previously.
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      2.3 qPCR quantification of HIV-1 DNA and cellular CCR5

      Total DNA from the target cells was extracted using the QIAamp DNA Blood Mini Kit (Qiagen). qPCR was performed in a 25-μl volume containing 2.5 μl DNA target, 12.5 μl Gene Expression Master Mix (Applied Biosystems, 4369016), 1 μM primers, and 0.2 μM probe. The reaction consisted of the following conditions using an ABI 7500 PCR machine (Applied Biosystems): 95 °C for 10 min, followed by 95 °C for 15 s and 60 °C for 1 min (40 cycles). A single round of qPCR was sufficient for total HIV-1 DNA and the CCR5 gene using the reaction conditions indicated previously.
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      For each run, a standard curve was created in a 7-log-unit range by 1:10 serial dilutions of the M13 bacteriophage standard containing the appropriate gene; all samples were performed in duplicate.

      2.4 Cell isolation

      Peripheral blood mononuclear cells (PBMCs) were isolated using Ficoll–Hypaque density gradient centrifugation of heparinized blood samples. CD4+CD25+CD127 Treg cells were isolated from PBMCs by CD4 negative selection followed first by CD127 negative selection and then by CD25 positive selection using a CD4+CD25+CD127dim/− isolation kit (Miltenyi Biotech) with MidiMACS and MiniMACS separator units (Miltenyi Biotech), according to the manufacturer's instructions. The purity of CD4+CD25+CD127dim/− Treg cells was >90% for each subject group, as determined by flow cytometry analysis (Figure 1a ).
      Figure thumbnail gr1
      Figure 1HIV-DNA levels are higher in Treg cells than in non-Treg CD4+ cells. (a) Dot plots of flow cytometry results obtained for purified CD4+CD25+CD127 regulatory cells. (b) Comparison of HIV-DNA levels (per 106 cells) between Treg and non-Treg CD4+ cells in treatment-naïve, ART1, and ART5 patients.

      2.5 Cell culture

      PBMCs (1 × 106), PBMCs depleted of Treg cells (1 × 106), and Treg cells (2.5 × 105) were maintained in RPMI 1640 medium (Gibco, Grand Island, NY, USA) supplemented with 10% heat-inactivated human AB serum, 2 mM l-glutamine, 20 mM HEPES, 100 U/ml penicillin, 100 g/ml streptomycin, and 5 × 105 M 2-mercaptoethanol. After incubation at 37 °C in a 5% CO2 atmosphere for 72 h, the cells were washed, stained, and analyzed.

      2.6 Flow cytometry analysis

      All antibodies were purchased from BD Pharmingen (San Diego, CA, USA), except anti-HIV-1 gag p24 KC57-FITC, which was from Beckman Coulter (Fullerton, CA, USA), and phycoerythrin (PE)-labeled HLA-A2 pentamer complexes loaded with the HIV-1 gag p17 epitope (77–85, SL9, SLYNTVATL), which were from ProImmune (Oxford, UK). PBMCs were isolated from freshly heparinized blood by Ficoll–Hypaque centrifugation (Pharmacia, Uppsala, Sweden).
      After 72 h of cell culture, the detection of CD4 cell activation was performed by immunolabeling with PE-anti-HLA-DR, FITC-anti-CD38, PerCP-anti-CD3, and APC-anti-CD4.
      For the staining of virus-specific CD8+ T cells, the cells were simultaneously stained after 72 h of culture with anti-CD3-PerCP, anti-CD8-allophycocyanin, pentamer-PE, or the corresponding isotype. For the assessment of intracellular p24 gag expression in CD4+ T cells, the cells were harvested after 72 h of cell culture and were first stained with PerCP-anti-CD3 and APC-anti-CD4, and then fixed and stained using the Fix and Perm kit (Caltag) and anti-p24 KC7-FITC antibody, as described previously.
      • Geiss G.K.
      • Bumgarner R.E.
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      • Agy M.B.
      • van’t Wout A.B.
      • Hammersmark E.
      • et al.
      Large-scale monitoring of host cell gene expression during HIV-1 infection using cDNA microarrays.
      • van’t Wout A.B.
      • Lehrman G.K.
      • Mikheeva S.A.
      • O’Keeffe G.C.
      • Katze M.G.
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      Cellular gene expression upon human immunodeficiency virus type 1 infection of CD4(+)-T-cell lines.
      After staining, four-color flow cytometry analyses were performed using a FACSCalibur flow cytometer and CELLQUEST software (Becton-Dickinson, San Jose, CA, USA).

      2.7 ELISA

      Detection of p24 antigens in the cell culture supernatant was done by ELISA. In brief, 96-well MaxiSorp plates (Nunc, Denmark) were coated with 0.5 mg/ml of anti-Gag p24 antibodies in 15 mM Na2CO3, 35 mM NaHCO3 (pH 9.7), overnight at 4 °C. The plates were blocked in phosphate-buffered saline (PBS) containing 2% (w/v) skimmed milk, and sera were added in serial dilutions. Specific antibodies were detected following incubation with rabbit anti-human IgG1 or IgG2a conjugated to horseradish peroxidase (Zymed/Invitrogen). Development and absorbance reading was done as described above.

      2.8 Assays for CD4 T cell counts and plasma HIV-1 RNA levels

      T cell counts were determined by three-color flow cytometry using anti-CD3-APC, anti-CD4-FITC, and anti-CD8-PE monoclonal antibodies (BD Bioscience San Diego, CA, USA). The analysis was performed using a BD FACSCount flow cytometer in accordance with the Chinese Center for Disease Control and Prevention (CDC) guidelines. Plasma HIV RNA was quantified by real-time PCR (Roche, Germany). The detection sensitivity of this assay is 40 copies/ml.

      2.9 Statistical analysis

      Comparisons were performed using non-parametric independent sample tests, and all reported p-values were two-sided and considered significant at p < 0.05. All data were analyzed using SPSS statistical software, version 16.0 (SPSS Inc., Chicago, IL, USA).

      3. Results

      3.1 HIV persistence in Treg cells

      To determine whether Treg cells are an important site of HIV persistence, the HIV-DNA levels in Treg and non-Treg CD4+ cells from direct material from 30 HIV-infected patients (10 treatment-naïve, 10 ART1, and 10 ART5 patients) were examined. The results showed a higher level of HIV-DNA in Treg cells than in non-Treg CD4+ cells in all three groups of patients (Figure 1b, p < 0.001), with an approximately 10-fold higher HIV-DNA level in Treg cells than in non-Treg cells (Figure 1b). Further, the intracellular HIV-1 p24 levels in PBMCs, PBCMs depleted of Treg cells, and Treg cells after 72 h of cell culture and the levels of p24 antigen in the cell culture supernatant were examined. It was found that there were more p24 antigens in Treg cells than in the other cell types (Figure 2a–c), as well as in Treg cell culture supernatant (Figure 2d, e).
      Figure thumbnail gr2
      Figure 2There is more p24 in Treg cells than in non-Treg cell culture supernatant. (a) Representative plots of the percentage of p24 in the PBMC, non-Treg PBMC, and Treg cells after 72 h of culture. Comparison of p24 levels in PBMC, non-Treg PBMC, and Treg cells from (b) ART1 and (c) ART5 patients after 72 h of culture. Comparison of p24 levels in cell culture supernatant of PBMC, non-Treg PBMC, and Treg cells from (d) ART1 and (e) ART5 patients after 72 h of culture. Patients are numbered P11 through P30.

      3.2 Depletion of Treg cells facilitates CD4+ T cell activation

      Because Treg cells can suppress immune activation, whether the depletion of Treg cells facilitates CD4+ T cell activation after certain periods of ART was next examined (Figure 3a). The results indicated that the depletion of Treg cells induced CD4+ T cell activation in all patients after 72 h of cell culture, regardless of the treatment period (Figure 3b–d). These results support the notion that Treg cell depletion facilitates CD4+ T cell activation.
      Figure thumbnail gr3
      Figure 3Depletion of Tregs enhances the activation of CD4+ T cells in patients on prolonged ART. (a) Representative plots of CD38 and HLA-DR expression in CD4+ T cells. Changes in the ratios of the percentages of HLADR+CD38+ T cells over total CD4+ cell counts in PBMCs (dots on the left) and depleted Treg cells (dots on the right) in (b) treatment-naïve, (c) ART1, and (d) ART5 patients. The y-axis represents the ratios of the percentage of HLADR+CD38+ T cells over the total CD4+ cell counts. The patients are numbered P1 through P30.

      3.3 Depletion of Treg cells increases the frequency of HIV-specific CD8+ T cells with prolonged ART

      Because depletion of Treg cells in HIV-infected individuals would induce immune cell activation, whether the depletion of Treg cells would increase the frequency of HIV-specific CTL after different time periods of ART and thereby improve the control of viral persistence was investigated (Figure 4a). As shown in Figure 4b, four of the 10 treatment-naive patients exhibited an elevated frequency of HIV-specific CTL after depletion of Treg cells, whereas the other six patients exhibited a reduced frequency of HIV-specific CTL after depletion of Treg cells after 72 h of cell culture. The frequencies of HIV-specific CTL were elevated in six of the 10 ART1 patients and all of the ART5 patients upon the depletion of Treg cells after 72 h of cell culture (Figure 4c, d).
      Figure thumbnail gr4
      Figure 4Depletion of Treg cells increases the frequency of HIV-specific CD8+ T cells in patients on prolonged ART. (a) Representative plots of HIV-specific CD8+ T cells. Changes in the ratios of the percentages of HIV-specific CD8+ T cells over total CD8+ cell counts in PBMCs (dots on the left) and depleted Treg cells (dots on the right) in (b) treatment-naïve, (c) ART1, and (d) ART5 patients. The y-axis represents the ratios of the percentage of HIV-specific CD8+ T cells over total CD8+ cell counts. The patients are numbered P1 through P30.

      4. Discussion

      An increasing body of evidence has demonstrated that Treg cells can reduce both HIV-specific immunoreactions
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      Human immunodeficiency virus-driven expansion of CD4+CD25+ regulatory T cells, which suppress HIV-specific CD4 T cell responses in HIV-infected patients.
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      and immune activation during HIV-1 infection.
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      The decrease of regulatory T cells correlates with excessive activation and apoptosis of CD8+ T cells in HIV-1-infected typical progressors, but not in long-term non-progressors.
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      • Jones N.
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      Depletion of regulatory T cells in HIV infection is associated with immune activation.
      • Oswald-Richter K.
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      HIV infection of naturally occurring and genetically reprogrammed human regulatory T-cells.
      It has also been demonstrated that stimulating HIV-1-specific CTLs and reactivating latent HIV-1 may be essential for the successful eradication of the latent viral reservoir.
      • Shan L.
      • Deng K.
      • Shroff N.S.
      • Durand C.M.
      • Rabi S.A.
      • Yang H.C.
      • et al.
      Stimulation of HIV-1-specific cytolytic T lymphocytes facilitates elimination of latent viral reservoir after virus reactivation.
      Accordingly, it was hypothesized that the depletion of Treg cells may be beneficial for reducing the HIV-1 DNA levels and decreasing HIV-1 p24 production. In this study, the results showed that the HIV-DNA levels were approximately 10-fold higher in Treg cells than in non-Treg CD4+ cells and that the depletion of Treg cells increased CD4+ cell activation after 5 years of ART. Because HIV should exist primarily as a latent viral reservoir after 5 years of sustained viral response to ART, these findings of higher levels of p24 antigen in Treg cells and in Treg cell culture supernatants indicate that Treg cell depletion may reduce the viral reservoir. Therefore, Treg cell depletion may facilitate the elimination of latent HIV by reducing the viral reservoir and elevating the frequency of HIV-specific CD8+ T cells. Although the results of the present study demonstrate that Treg cells can increase the level of CD4+ cell activation, these data do not indicate that Treg cell depletion can reactivate latent virus. Further studies should be performed to confirm whether Treg cell depletion can reactivate latent virus.
      In addition, it has become increasingly clear that the severity of AIDS is associated with chronic immune hyperactivation. Because the immune activation was high in the non-ART and ART1 patients, the depletion of Treg cells may have caused an enhancement of this excessive immune activation.
      • Jiao Y.
      • Fu J.
      • Xing S.
      • Fu B.
      • Zhang Z.
      • Shi M.
      • et al.
      The decrease of regulatory T cells correlates with excessive activation and apoptosis of CD8+ T cells in HIV-1-infected typical progressors, but not in long-term non-progressors.
      However, in the 5-year ART patients, HIV had been suppressed for a very long time, and the excessive immune activation had been controlled to some extent. These results are consistent with those of previous reports that have shown that a low Treg response may contribute to both viral control and generalized immune activation in HIV controllers.
      • Owen R.E.
      • Heitman J.W.
      • Hirschkorn D.F.
      • Lanteri M.C.
      • Biswas H.H.
      • Martin J.N.
      • et al.
      NIAID Center for HIV/AIDS Vaccine Immunology: HIV+ elite controllers have low HIV-specific T-cell activation yet maintain strong, polyfunctional T-cell responses.
      • Schulze Zur Wiesch J.
      • Thomssen A.
      • Hartjen P.
      • Tóth I.
      • Lehmann C.
      • Meyer-Olson D.
      • et al.
      Comprehensive analysis of frequency and phenotype of T regulatory cells in HIV infection: CD39 expression of FoxP3+ T regulatory cells correlates with progressive disease.
      • Shaw J.M.
      • Hunt P.W.
      • Critchfield J.W.
      • McConnell D.H.
      • Garcia J.C.
      • Pollard R.B.
      • et al.
      Increased frequency of regulatory T cells accompanies increased immune activation in rectal mucosae of HIV-positive noncontrollers.
      • Hunt P.W.
      • Landay A.L.
      • Sinclair E.
      • Martinson J.A.
      • Hatano H.
      • Emu B.
      • et al.
      A low T regulatory cell response may contribute to both viral control and generalized immune activation in HIV controllers.
      Treg cells are Foxp3-positive cells. Bettelli et al. initially described Foxp3 as functionally interacting with and inhibiting the transactivation of the transcription factors nuclear factor of activated T cells (NFAT) and nuclear factor kappa B (NF-κB).
      • Bettelli E.
      • Dastrange M.
      • Oukka M.
      Foxp3 interacts with nuclear factor of activated T cells and NF-kappa B to repress cytokine gene expression and effector functions of T helper cells.
      Several groups have reported changes in the histone acetylation of certain promoters regulated by Foxp3.
      • Holmes D.
      • Knudsen G.
      • Mackey-Cushman S.
      • Su L.
      FoxP3 enhances HIV-1 gene expression by modulating NFkappaB occupancy at the long terminal repeat in human T cells.
      • Wu Y.
      • Borde M.
      • Heissmeyer V.
      • Feuerer M.
      • Lapan A.D.
      • Stroud J.C.
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      FOXP3 controls regulatory T cell function through cooperation with NFAT.
      • Chen C.
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      • Thomas R.M.
      • Hancock W.W.
      • Wells A.D.
      Transcriptional regulation by Foxp3 is associated with direct promoter occupancy and modulation of histone acetylation.
      Thus, the mechanism by which Treg cell depletion could reactivate latent HIV needs further study.
      In this study, it was found that the depletion of Treg cells elevated the frequency of HIV-1-specific CTL, which is consistent with previous publications. A notable report showed that HIV-specific CD8+ T cells carrying protective HLA alleles (HLA B*27 and B*57) were able to evade Treg suppression,
      • Elahi S.
      • Dinges W.L.
      • Lejarcegui N.
      • Laing K.J.
      • Collier A.C.
      • Koelle D.M.
      • et al.
      Protective HIV-specific CD8+ T cells evade Treg cell suppression.
      which suggested that the suppressive activity of Treg cells may be a major mechanism that impairs the development of protective effector responses. It was also reported that a high perforin-to-Foxp3 ratio was associated with non-progressive disease.
      • Nilsson J.
      • Boasso A.
      • Velilla P.A.
      • Zhang R.
      • Vaccari M.
      • Franchini G.
      • et al.
      HIV-1-driven regulatory T-cell accumulation in lymphoid tissues is associated with disease progression in HIV/AIDS.
      Other reports have shown the ex vivo depletion of Tregs from PBMCs or lymphoid cell suspensions to enhance T cell responses to HIV or SIV antigens.
      • Weiss L.
      • Donkova-Petrini V.
      • Caccavelli L.
      • Balbo M.
      • Carbonneil C.
      • Levy Y.
      Human immunodeficiency virus-driven expansion of CD4+CD25+ regulatory T cells, which suppress HIV-specific CD4 T cell responses in HIV-infected patients.
      • Kinter A.L.
      • Hennessey M.
      • Bell A.
      • Kern S.
      • Lin Y.
      • Daucher M.
      • et al.
      CD25(+)CD4(+) regulatory T cells from the peripheral blood of asymptomatic HIV-infected individuals regulate CD4(+) and CD8(+) HIV-specific T cell immune responses in vitro and are associated with favorable clinical markers of disease status.
      • Nilsson J.
      • Boasso A.
      • Velilla P.A.
      • Zhang R.
      • Vaccari M.
      • Franchini G.
      • et al.
      HIV-1-driven regulatory T-cell accumulation in lymphoid tissues is associated with disease progression in HIV/AIDS.
      • Aandahl E.M.
      • Michaelsson J.
      • Moretto W.J.
      • Hecht F.M.
      • Nixon D.F.
      Human CD4+ CD25+ regulatory T cells control T-cell responses to human immunodeficiency virus and cytomegalovirus antigens.
      • Hryniewicz A.
      • Boasso A.
      • Edghill-Smith Y.
      • Vaccari M.
      • Fuchs D.
      • Venzon D.
      • et al.
      CTLA-4 blockade decreases TGF-beta, IDO, and viral RNA expression in tissues of SIVmac251-infected macaques.
      • Kared H.
      • Lelièvre J.D.
      • Donkova-Petrini V.
      • Aouba A.
      • Melica G.
      • Balbo M.
      • et al.
      HIV-specific regulatory T cells are associated with higher CD4 cell counts in primary infection.
      • Nikolova M.
      • Carriere M.
      • Jenabian M.A.
      • Limou S.
      • Younas M.
      • Kök A.
      • et al.
      CD39/adenosine pathway is involved in AIDS progression.
      Furthermore, the stimulation of HIV-1-specific CTLs and the reactivation of latent HIV-1 may be essential for the successful eradication of the latent viral reservoir.
      • Shan L.
      • Deng K.
      • Shroff N.S.
      • Durand C.M.
      • Rabi S.A.
      • Yang H.C.
      • et al.
      Stimulation of HIV-1-specific cytolytic T lymphocytes facilitates elimination of latent viral reservoir after virus reactivation.
      In this study, it was found that Treg cells are important HIV-1 p24-producing cells and the depletion of Treg cells may increase the frequency of HIV-1-specific CTLs and reactivate CD4+ cells, thereby enhancing the eradication of the latent viral reservoir. In addition, because only the HLA-A2-positive phenotype can be detected by flow cytometry, cells from HLA-A2-positive phenotype patients were examined by flow cytometry and the CTL detected; however, this does not exclude the possibility that individuals with other HLA phenotypes respond in the same way.
      It was found that the HIV-DNA levels were higher in Treg cells than in non-Treg CD4+ cells, which is consistent with the report that HIV-DNA-harboring cells appeared to be more abundant in the Treg subset than among non-Treg cells in patients on prolonged ART.
      • Tran T.A.
      • de Goër de Herve M.G.
      • Hendel-Chavez H.
      • Dembele B.
      • Le Névot E.
      • Abbed K.
      • et al.
      Resting regulatory CD4 T cells: a site of HIV persistence in patients on long-term effective antiretroviral therapy.
      It was found that the HIV-DNA levels were 10-fold higher in Treg cells than in non-Treg CD4+ cells; in a previous report, this difference ranged from 1.5- to 8-fold higher.
      • Tran T.A.
      • de Goër de Herve M.G.
      • Hendel-Chavez H.
      • Dembele B.
      • Le Névot E.
      • Abbed K.
      • et al.
      Resting regulatory CD4 T cells: a site of HIV persistence in patients on long-term effective antiretroviral therapy.
      This variation may be due to racial differences and the different surface markers used. Because the results of the present study, along with those reported in a number of previous publications, suggest that Treg cells could constitute part of the viral reservoir, the depletion of Treg cells may reduce the size of this viral reservoir.
      The latent reservoir is established at the earliest stage of infection. Furthermore, ART in acutely infected macaques increases SIV-specific viral responses and the capacity of the immune response.
      • Lori F.
      • Lewis M.G.
      • Xu J.
      • Varga G.
      • Zinn Jr., D.E.
      • Crabbs C.
      • et al.
      Control of SIV rebound through structured treatment interruptions during early infection.
      The transient depletion of Treg cells in transgenic mice reactivated virus-specific CD8+ T cells and reduced the retroviral set points.
      • Dietze K.K.
      • Zelinskyy G.
      • Gibbert K.
      • Schimmer S.
      • Francois S.
      • Myers L.
      • et al.
      Transient depletion of regulatory T cells in transgenic mice reactivates virus-specific CD8+ T cells and reduces chronic retroviral set points.
      Thus, early ART in combination with Treg cell depletion may help preserve immune responses and eliminate the viral reservoir; however, this hypothesis requires further study for confirmation.
      In conclusion, CD4+CD25+CD127 regulatory cells play multiple roles in maintaining HIV-1 p24 production in long-term ART patients. The results of this study indicate that the depletion of Treg cells may facilitate the elimination of HIV-1 p24 production by depleting those cells that produce p24 and by increasing the frequency of HIV-specific CD8+ T cells. Whether Treg cell depletion can reactivate latent virus needs further examination. Indeed, Treg cells may be a good target for strategies to eliminate the latent HIV reservoir.

      Acknowledgements

      This study was supported in part by the National Natural Science Foundation of China (81371803), Beijing Natural Science Foundation (7152072, 7132108), the National 12th Five-Year Major Projects of China (2012ZX10001-003, 2012ZX10001-006), Beijing AIDS clinical data and sample repository development and applied research (D131100005313005), and High levels of health technical personnel plan in Beijing city health system (Yan-Mei Jiao), Capital health research and development special fund (2011-2018-01). Beijing Municipal Administration of Hospitals Clinical Medicine Development of Special Funding Support (ZYLX201511).
      Conflict of interest: The authors declare that they have no competing interests.

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