HIV-1-derived immunodeficiency is simultaneously associated with T cell activation, which has been recognized as a pivotal predictor for clinical disease progression in HIV-1 infected patients.1,2 Recent studies showed that aberrant immune activation at least partly led to CD4+ or CD8+ T cell depletion through activation-induced cell death or enhancing viral replication.3-6 Thus, suppressing hyperactivation of T cells may contribute to slow down the disease progress during HIV-1 infection. Accumulating evidences have revealed that regulatory T cells (Tregs) are endowed with unique suppressing properties to modulate the homeostasis of reactive T cells in HIV-1 infected subjects.7,8
Tregs were initially characterized by the constitutive expression of interleukin 2 (IL-2) receptor (R) alpha (α) chain (CD25),9 and further studies have reported that some critical markers such as forkhead box p3 (Foxp3), IL-7 receptor (R) alpha (α) chain (CD127), glucocorticoid- induced tumor necrosis factor receptor family-related gene (GITR), cytotoxic T lymphocyte- associated antigen-4 (CTLA-4) may be necessary for defining the phenotype of Tregs.10-13 CD4+CD25+Foxp3+ T cells have been documented as the accurately characterized Treg cell population. However, the requirement of intracellular staining for Foxp3 has restrained its extensive usage. Recently, several groups have provided strong evidences that CD127 expression inversely correlates with Foxp3 on CD4+CD25+ Tregs,11,14,15 therefore, CD4+CD25+CD127lo/- is considered as the best substitute for CD4+CD25+Foxp3+ phenotype.
Tregs are able to negatively regulate CD4+ and CD8+ T cell activation, proliferation, and antigen-reactive function both in vitro and in vivo.16,17 Though the precise mechanisms are not fully understood for Tregs, several mechanisms have been proposed, including the secretion of negative regulatory cytokines of IL-10 and transforming growth factor β (TGF-β) or the direct cell-cell contact to impart negative signal.13,18,19 Recent studies indicated that Tregs might be associated with the pathogenesis of HIV-1 infected patients. Eggena et al8 observed that absolute numbers of Treg cells (defined as CD4+CD25+CD62L+) decreased and was associated with immune activation. Tsunemi et al20 demonstrated that increased Treg frequencies in peripheral blood were associated with low peripheral blood CD4+ T cell counts and the polarization toward Th2 immune responses in HIV-infected patients. In addition, Nillson et at21 showed that therapeutic manipulation of Treg number and/or function could improve immune control of HIV infection.
HLA-DR, a glycoprotein expressing on dendritic cells, monocytes, B cells and activated T cells, is a important indicator for T cell activation during HIV-1 infections.22,23 Previous data have indicated that HLA-DR expressing CD4+CD25high T cells is a functionally distinct subpopulation of Tregs,24 and HLA-DR+CD4+CD25high Tregs is significantly increased during Hashimoto’s thyroiditis.25 However, no observation on this sub-populatio of Tregs has been made during HIV-1 infection, it remains unknown whether this sub-population of Tregs has an association with disease progress and immune activations. In this study, we qualified the expression of HLA-DR on CD4+CD25+CD127lo/- Tregs in HIV-1 infected patients at different clinical stages. In addition, we further examined the association between this subpopulation of Treg with the immune activation of different immune cells.
Eighty-one antiretroviral therapy-naïve HIV-1-infected patients (29 female and 52 male) and twenty-two HIV-1-seronegative subjects were recruited from Shanghai city and Yuncheng city in Shanxi Province in China. Mean age of HIV-1-infected patients was 38 years old (range 21–67 years old), with a range of viral loads (VL) from <50 copies/ml to 190 000 copies/ml. Plasma HIV RNA level was determined, and CD4+ T cell count was qualified at the time of blood draw. All patients were grouped by either VL or CD4+ T-cell counts (Table). The study was approved by Institutional Review Board of Shanghai CDC and all participants in this study signed informed consent.
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Table. Characteristics of subjects in the study
Peripheral blood mononuclear cells (PBMCs) were isolated from freshly heparinized blood by Ficoll-Hypaque (Amersham Biosciences, Piscataway, NJ) density gradient centrifugation and suspended at indicated concentrations in RPMI1640 as previously described.26 The following directly conjugated monoclonal antibodies, including CD3-Pacific Blue (clone UCHT1), CD4-PerCP (clone L200), CD25-FITC or CD25-APC (clone M-A251), CD127-PE (clone HIL-7R-M21), CD279-APC (clone MIH4), and Ki67-FITC (clone B56) with their respective isotype control antibodies, were purchased from BD Biosciences (San Jose, CA); HLA-DR-ECD (clone Immu-357) and its isotype control antibody were purchased from Beckman Counter, Hialeah.
Flow cytometric analysis
Monoclonal antibodies to surface markers, including anti-CD3-Pacific Blue, anti-CD4-PerCP, anti-CD25-FITC, anti-CD127-PE, anti-CD38-PE-Cy7, anti-HLA-DR-ECD, and anti-PD-1-APC, were used to stain PBMCs for quantifying PD-1, CD38, and HLA-DR expression on CD4+CD25+CD127lo/- Tregs. For intracellular staining, monoclonal antibody to surface molecules, including anti-CD3-Pacific Blue, anti-CD4-PerCP, anti-CD25-APC and anti-127-PE were used to stain surface markers and then cells were washed twice with PBS. After further fixation and permeabilization with Cytofix/Cytoperm solution (BD Biosciences) at 4°C for 20 minutes, and washing with Perm Wash Buffer (BD Biosciences), anti-Ki67-FITC was used to stain intracellular level of Ki67. After staining, cells were washed twice with Perm Wash Buffer (BD Biosciences) and fixed in 1% paraformaldehyde. ECD- or FITC-labeled mouse IgG1 isotype controls were applied as needed. Events in the stained cells 1×106 were collected and nanlyzed with a FACSAria flow cytometer (BD).
Plasma HIV-1 RNA testing
Plasma RNA was purified from 1 ml of venous blood derived from the studied subjects. RNA 5 μl and 90 μl primers mixture were added into an Eppendorf tube, followed by an incubation at 65ºC for 2 minutes and 41ºC for 2 minutes. Enzyme mix 5 μl and 45 μl diluent reagent were then added, mixed thoroughly and centrifuged for 2 minutes. The plasma HIV-1 RNA was quantified in a NucliSens EasyQ instrument.
All data were analyzed by Wilcoxon matched pairs test and Mann-Whitney test of GraphPad Prism, Version5.0 software to determine the significance between different groups. Values were expressed as mean ± standard deviation (SD). Correlations between variables were evaluated using the Spearman rank correlation test. P values less than 0.05 were considered statistically significant.
We evaluated the levels of HLA-DR on
Tregs in HIV-seronegative individuals and HIV-1 infected subjects at different clinical stages. As shown in Figure 1, HLA-DR levels were increased on CD4+
Tregs from HIV-seronegative individuals ((18.97±9.18)%) to HIV-infected subjects. The level of HLA-DR was significantly enhanced as VL increased to (27.53±11.02)% for group with VL <2000 copies/ml, (29.89±13.03)% for group with VL at 2000–20 000 copies/ml, (38.96±15.85)% for group with VL >20 000 copies/ml (Figure 1B, all P
<0.05). In addition, this increased expression of HLA-DR was also observed in the patients with a disease progression event as the patients were stratified by CD4+
T cell counts at (27.07±12.06)% for group with CD4 >500 cells/μl, (33.80±13.22)% for group with CD4 at 200–500 cells/μl, and (42.21±17.62)% for group with CD4 <200 cells/μl (Figure 1C). Additionally, we confirmed that HLA-DR was predominantly expressed on CD4+
Tregs but not on CD4+
T cells, furthermore, both the HLA-DR frequency and HLA-DR MFI on CD4+
Tregs (frequency at (32.39±14.30)%; MFI at 2724±761) were significantly higher than that on CD4+
T cells (frequency at (10.16±5.26)%; MFI at 1712±692; both P
<0.01; Figure 1D and 1E).
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HLA-DR expression on CD4+
Tregs is higher in different stages of HIV-1 infected patients than in healthy donors and is associated with progression disease. A:
Representative HLA-DR expression on CD4+
Tregs in the 4 groups, including group with VL <2000 copies/ml, group with VL at 2000−20000 copies/ml, group with VL ≥20000 copies/ml and HIV-1 seronegative subjects. B
Percentage of HLA–DR expressing CD4+
Tregs on different stages of HIV-1 infected patients and HIV-1 seronegative subjects. D
Percentage and mean fluorescence intensity (MFI, relative fluorescence units) of HLA-DR expression on CD4+
Tregs compared with CD4+
T cells. F and G:
Relationship between percentage of HLA-DR expressing CD4+
Tregs and HIV-1 viral load and CD4+
We further examined the association of HLA-DR expression on CD4+CD25+CD127lo/- Tregs with HIV-1 replication and disease progression and observed that the percentages of HLA-DR expressing CD4+CD25+CD127lo/- Tregs were positively associated with VL (r=0.3163, P=0.004; Figure 1F) and negatively with CD4 T cell counts (r= −0.4153, P <0.0001; Figure 1G). Our data suggested that HLA-DR expressing CD4+CD25+CD127lo/- Tregs might be a good marker for HIV-1 disease progression.
Levels of HLA-DR on CD4+CD25+CD127lo/- Tregs were associated with T cell activation markers
The previously published data have demonstrated that HLA-DR, CD38, and Ki67 were important predicators for immune activation during viral infections.22,23,27-29 To test whether HLA-DR expression on Treg is associated with activation markers on other T-cell subpopulations, we then evaluated the association between the levels of HLA-DR on CD4+CD25+CD127lo/- Tregs and the expression of HLA-DR, CD38, and Ki67 on CD4+ and CD8+ T-cell populations. Our data showed that significant associations were observed between HLA-DR expression on CD4+CD25+CD127lo/- Tregs with all activation markers, including R value at 0.6388 with HLA-DR on CD4+ T cells (P <0.0001; Figure 2A), 0.4488 with HLA-DR on CD8+ T cells (P <0.0001; Figure 2B), 0.23 with CD38 expression on CD4+ T cells (P=0.0388; Figure 2C), 0.25 with CD38 expression on CD8+ T cells (P=0.0228; Figure 2D), 0.4158 with Ki67 expression on CD4+ T cells (P <0.0001; Figure 2E) and 0.2268 with Ki67 expression on CD8+ T cells (P=0.0417; Figure 2F). Meanwhile, we also observed that the level of HLA-DR on Treg was also significantly associated with CD38 (r=0.2605, P=0.0189; Figure 2G) and Ki67 (r=0.267, P=0.016; Figure 2H) on CD4+CD25+CD127lo/- Tregs.
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Figure 2. HLA-DR expression on CD4+CD25+CD127lo/- Tregs is associated with immune activation. A and B: There are positive correlation between the percentage of HLA-DR expressing CD4+CD25+CD127lo/- Tregs and CD4+ and CD8+ T cells. C and D: There are positive correlation between the percentage of HLA-DR expressing CD4+CD25+CD127lo/- Tregs and CD38 expressing CD4+, CD8+ T cells. E and F: There are positive correlation between the percentage of HLA-DR expressing CD4+CD25+CD127lo/- Tregs and Ki67 expressing CD4+, CD8+ T cells. G and H: There are positive correlation between HLA-DR expression and CD38, KI67 expression on CD4+CD25+CD127lo/- Tregs.
Previous data from several groups have strongly suggested that Tregs are capable of limiting immune activation.16,30-32 However, the role of Tregs during HIV infection has been controversial. It has been observed that the frequencies of Treg in CD4+ T cells increased during HIV infection and was associated with the disease progression.33-35 It was further rationalized that the increase of Treg may blunt the inhibitory effect of HIV-specific T cells on HIV replication and thereby causes the loss of viral control and leads to disease progression. However, this hypothesis of “favoring disease progression” for Treg during HIV infection is not conciliated with the observation that HIV-specific T cells and even the entire immune system are over activated during HIV infection.36,37 Indeed, the immune activation during HIV infection was shown as a better predictor for disease progression than CD4+ T cell counts.38 Therefore, it is likely that the inhibitory effect of Treg is insufficient to restrain the hyperactivation of immune system during HIV infection instead of over inhibition on HIV-specific T cells or on immune system. Interestingly, one group recently reported that Treg counts actually decreased in parallel with the depletion of CD4+ T cells during HIV infection.39 In accordance with this observation, our unpublished data also demonstrated that Treg counts decreased over HIV infection though the frequencies of Treg in CD4+ T cells increased. Overall, we proposed that dampening inhibitory effect of Treg may result in the over activation of immune system during HIV infection which at least partially accounts for the pathogenesis of immune exhaustion.
Several mechanisms may account for the depletion of immune cells during HIV infection, including the HIV-infection induced death, the killing resulted from HIV-specific T cells on HIV-expressing cells, the Env-induced apoptosis and the activation induced death. As known, the activation induced death could account for the rapid turnover of CD8+ T cells during HIV infection.40
This mechanism also played an important role in the depletion of CD4+ T cells36 and thereby the activation markers are closely associated with disease progression.5,41 Several activation markers have been explored in previous reports, including HLA-DR, CD38 and Ki67.22,23,27-29 Since HLA-DR+ Treg may represent an independent subpopulation of Treg,24 we focused on HLA-DR expression on Treg in our current study.
HLA-DR was characterized as an activation marker of T lymphocytes as early as in 1995, Ramzaoui et al42 observed a significant increasing in the percentage of HLA-DR+CD4+ T cells which closely associated with the activation of CD8+ T cells in HIV-1-infected subjects, which was subsequently further confirmed by Choi et al43 report in 2002. In addition, Kestens et al44 demonstrated that levels of HLA-DR and CD38 on CD8+ T-lymphocyte compartment increased from 8% in controls to 49% in asymptomatic HIV-infected subjects. Han et al45 further proposed that HLA-DR expression on CD8+ T could be used as a predictor for HIV disease progression and HAART outcome in some resource-limited areas of China. All these data rationalized that HLA-DR on Treg might represent a good marker to gauge the activation of Treg and was thereby postulated to associate with the decrease of Tregs. In this study, we observed for the first time that HLA-DR levels on Treg were significantly enhanced during HIV infection and this increase was more manifested on Treg than non Tregs, which was consistent with previous observations in chronic hepatitis B patients in whom Tregs also expressed high levels of HLA-DR in vivo.46 Importantly, the enhanced expression of HLA-DR was coincided with the increase of viral loads and the decrease of CD4+ T cells. These data suggested that the HLA-DR on Treg could be considered as a predictor for the viral replication and disease progression.
We further examined the association between HLA-DR expression and activation of other immune cells. Our data showed that HLA-DR levels on Treg were significantly associated with activation markers, including HLA-DR, CD38 and Ki67, on CD4+ and CD8+ T cells, and also in parallel with CD38 and Ki67 on Treg. These data suggested that HLA-DR on Treg was a good activation marker for Treg and the increase of HLA-DR+ Treg was a part of the activation of entire immune system.
It remains un-determined whether the increased expression of HLA-DR could result in the apoptosis of Tregs though the activation of Treg may lead to the activation induced death and thereby cause the decrease of Tregs as observed in previous studies. A further interesting question is that whether the HLA-DR+ and HLA-DR− Tregs are functionally different and HLA-DR+ Treg represents a functional independent Treg population.
Acknowledgments: We thank all HIV-infected individuals and healthy volunteers in this study for their cooperation.
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