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CD4+CD25+ regulatory T cells (Tregs) have been demonstrated to play a crucial role in mediating immunotolerance and suppressing the activation and proliferation of innate and adaptive immunocytes (21-23). It has also been implicated in controlling excessive immune responses to chronic pathogens and in limiting immunopathology (4). In human, the T cell subset is identified by high expression of IL-2Rα chain (CD25) and the forkhead/winged helix trans-cription factor (FoxP3), which has been demonstrated to be a unique mark restricted to the T regulatory cells (3, 19).
Human immunodeficiency virus type-1 (HIV-1) in-fection is characterized by a progressive loss of CD4+ T cells and a wide array of immune dysfunc-tions, including chronic immune activation and paradoxical anergy of immunocytes (6, 14). Emerging evidence support the hypothesis that pathogenesis of virus chronic infection may be directly related to the levels of circulating CD4+CD25+ Treg or to the balance of the Treg versus effector T cells. In hepatitis C virus (HCV) and hepatitis B virus (HBV) infected subjects, CD4+CD25+ Treg may contribute to the persist infections by down-regulating antigen-specific T cell response (5, 25). But in HIV infection, how Treg regulates immune responses and correlates with disease progression in chronic HIV infection remains unknown (2, 8, 12, 16, 24). A recent study suggested that Tregs are generally depleted in HIV infection and their loss may facilitate the immune hyperactivation (7). But some other studies supported the notion that Tregs contribute to HIV-specific immune dysfunction by limiting immunoreactions (10, 24). However, whether circulating Treg was increased or not in HIV infection is not very clear.
In this study, we hypothesized that functional Tregs might mediate the immune dysregulation in chronic HIV infection. It was found that with the disease progression, the circulating Treg frequency was significantly increased; while Treg absolute counts were decreased. And there was a negative correlation between circulating Treg frequency and CD4 counts. More important, our data indicated Treg could inhibit both CD4+ and CD8+ T cell proliferation in vitro, and then might result in decreasing CD4 and CD8 T cell counts. These findings suggested that the alteration of Tregs may act as an important prognostic marker for the disease progression of chronic HIV infection.
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A total of 75 HIV-1 infected antiretroviral-na ve individuals were enrolled in this study. HIV seroposi-tivity was determinated by enzyme-linked immuno sorbent assay (ELISA) and confirmed by Western blot analysis. All these individuals were paid blood donors and infected during the period of 1994-1995. No evi-dence of active opportunistic infections and tumors were found for all of HIV-infected subjects at the time of blood sampling. Further exclusion criteria included pregnancy, active tuberculosis (TB; defined as sus-pected TB or in the first 2 mo of anti-TB therapy), or moribund status (7). Thirty healthy subjects with age and gender-matched were employed as normal con-trols (NC). The basic characteristics of these individuals were shown in Table 1. The study protocol was approved by the Ethics Committee of our unit, and written informed consent was obtained from each subject.
Table 1. Characteristics of subjects in the study
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Peripheral blood mononuclear cells (PBMC) were isolated by Ficoll-Hypaque density gradient centrifu-gation from heparinised blood sample. CD4+CD25+ Treg were isolated from PBMC by CD4 negative selection followed by CD25 positive selection, using CD4+CD25+ T-cell isolation kit (Miltenyi Biotech, Bergisch-Gladbach, Germany) according to the manu-facturer's instructions. Treg-removed PBMCs were collected for next experiments. Treg frequency in Treg-removed PBMCs was < 0.5%, as determined by flow cytometric analysis.
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All antibodies were purchased from BD Phar-Mingen (San Diego, USA), except for anti-FoxP3 antibody from eBiosciences (San Diego, USA). For staining of Treg, the cells were first stained with PerCP-anti-CD3, APC-anti-CD4 and PE-anti-CD25 Abs, then permeabilized and fixed using eBioscience fix/perm (eBiosciences, San Diego, USA) according to the manufacturer's instructions. After 30-minute permeabilization, FITC-anti-FoxP3 was added for another 30 minutes. Four-color flow cytometric analysis was performed using FACSCalibur and CELLQuest software (Becton Dickinson, San Jose, USA).
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The proliferation of PBMCs was analyzed via CFSE [5-(and 6-)carboxyl-fluorescein diacetate, suc-cinimidyl ester] labeling assay as described previously (11, 27). In brief, PBMCs and Treg-removed PBMCs were incubated in PBS containing 0.1% BSA with 5 μmol/L CFSE for 10 min at 37 ℃. Then labeling was quenched with RPMI1640 containing 10% FCS on ice for 5 min and cells were washed twice with PBS. The CFSE-labeled cells were seeded at 5×105 cells/mL in 96-well plates. PBMCs were stimulated with anti-CD3 and anti-CD28 (1 μg/mL) for 96 h in vitro. Proli-feration was analyzed using FACSCalibur (Becton Dickinson, San Jose, USA).
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The 7900HT Sequence Detection System (Applied Biosystems) was used to quantify HIV-1 RNA levels in plasma samples in our laboratory. The cut-off value was 500 copies/mL. The protocol was previously described (13).
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Data were analyzed using SPSS 13.0 for Windows software (SPSS Inc., Chicago, USA) and expressed as mean and standard deviation. Multiple comparisons Kruskal-Wallis H nonparametric test was applied with Bonferroni step down (Holm) correction. Mann-Whi-tney nonparametric U test was used for difference between two groups. Wilcoxon signed ranks test was used for two-related-samples test. Spearman cor-relation analysis was performed between two para-meters. P < 0.05 is considered as a significant diffe-rence.