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Proportions of circulating T cells with a regulatory cell phenotype increase with HIV-associated immune activation and remain high on antiretroviral therapy

Lim, Andrewa; Tan, Dinoa; Price, Patriciaa,b; Kamarulzaman, Adeebac; Tan, Hong-Yienc; James, Iand; French, Martyn Aa,b

Author Information

From the aSchool of Surgery and Pathology, University of Western Australia, Western Australia

bClinical Immunology and Immunogenetics, Royal Perth Hospital, Western Australia

cUniversity of Malaya Medical Centre, Kuala Lumpur, Malaysia

dCentre for Clinical Immunology and Biomedical Statistics, Murdoch University and Royal Perth Hospital, Western Australia.

Received 14 December, 2006

Revised 30 April, 2007

Accepted 8 May, 2007

Correspondence to Professor Martyn French, Clinical Immunology and Immunogenetics, Royal Perth Hospital, Wellington Street, Perth 6000, Western Australia. Tel: +618 92242899; fax: +618 92242920; e-mail: Martyn.French@health.wa.gov.au

Requests for reprints: A. Lim, School of Surgery and Pathology, Level 2 MRF Building, Rear 50 Murray Street, Perth 6000, Western Australia. Tel: +618 92240217; fax: +618 92240204; e-mail: alyf82@cyllene.uwa.edu.au

doi: 10.1097/QAD.0b013e32825eab8b
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Abstract

Introduction

HIV may evade protective immune responses by inducing regulatory CD4 T (Treg) cells able to suppress CD4 and CD8 effector T cells. Cells expressing cell-surface CD4 and CD25 and able to suppress lymphoproliferative responses against HIV proteins or peptides have been demonstrated in the blood of HIV-infected patients [1–3]. Proportions of Treg cells and expression of FoxP3 mRNA and protein are also elevated in tonsils and duodenal lymphoid tissue of untreated HIV-infected patients [4,5]. The proportion of circulating CD4 T cells that co-express CD25 and FoxP3 protein are increased in untreated patients with CD4 T-cell counts < 200/μl [6]. Expression of FoxP3 mRNA in tonsillar tissue correlates with plasma HIV RNA levels [4], but the relationship of circulating Treg cells with plasma HIV RNA levels and the effect of antiretroviral therapy (ART) have not been examined.

A key issue is how Treg cell numbers relate to the immune activation associated with HIV infection. This makes the selection of phenotypic markers critical, as CD25 can be expressed on activated non-regulatory T cells. Recent studies show that CD25CD4 T cells expressing low amounts of CD127 (α chain of the interleukin-7 receptor) have the molecular and functional characteristics of Treg cells in healthy control donors and type 1 diabetics [7,8]. Here we characterize CD25CD127loCD4 T cells and correlate proportions of cells with this phenotype with CD4 T-cell activation and plasma HIV RNA levels in antiretroviral-naive HIV-infected patients and in two groups of severely immunodeficient HIV patients beginning ART.

Methods

Subjects

Antiretroviral-naive male HIV-infected patients attending outpatient clinics at Royal Perth Hospital (Perth, Western Australia) and the University of Malaya Medical Centre (Kuala Lumpur, Malaysia) were recruited. Validation of Treg cell staining was undertaken on up to 16 untreated HIV-infected patients with various CD4 T-cell counts and on 10 non-HIV controls. A cross-sectional study was then undertaken on patients from Perth (n = 29) who were stratified by CD4 T-cell counts < 300 cells/μl or > 400 cells/μl (Table 1). These cutoffs were chosen because the count midway between these values (350 cells/μl) is commonly used as an indicator of when to start ART. Six patients with CD4 T-cell counts < 100/μl subsequently commenced ART and donated further samples over 1 year. Twenty age-matched non-HIV control subjects from this site also donated blood (Table 1). Six antiretroviral-naive Chinese HIV-infected patients from Kuala Lumpur with baseline CD4 T-cell counts < 100/μl were also investigated longitudinally from pre-ART for up to 1 year. Twelve Chinese non-HIV control subjects from Kuala Lumpur also donated blood. The study was approved by the Ethics Committees of Royal Perth Hospital and the University of Malaya Medical Centre. Informed consent was obtained from all donors.

Table 1
Table 1:
Cross-sectional analyses show increased proportions of activated and regulatory CD4 T cells in immunodeficient HIV patients.

Measurement of plasma HIV RNA level and CD4 T-cell counts

HIV-1 RNA was measured in plasma collected from EDTA-treated whole blood, using the COBAS Amplicor HIV-1 Monitor Test, v1.0 and/or 1.5 (Roche Diagnostics, Indianapolis, Indiana, USA) at both sites. The cutoff defining undetectable HIV RNA was <50 copies/ml. CD4 T-cell counts were measured in EDTA-treated whole blood by flow cytometry in routine laboratories at both study sites.

Flow cytometric analyses

Peripheral blood mononuclear cells (PBMC) were obtained by Ficoll separation of heparinized whole blood, using techniques standardized between the two laboratories, and cryopreserved in liquid nitrogen. Cryopreserved PBMC were thawed and washed with phosphate buffered saline containing 1% bovine serum albumin, then resuspended at 10 × 106 cells/ml.

The following monoclonal antibodies were used for surface staining of 5 × 105 PBMC: CD4-PECy5, CD8-FITC, CD25-FITC, CD38-FITC, CD127-PE, HLA-DR-FITC, HLA-DR-PE (Coulter Immunotech, Marseille, France), CD25-APC (BD Pharmingen, San Jose, California, USA), and GITR-FITC (R&D Systems, Minneapolis, Minnesota, USA). The following monoclonal antibodies were used for intracellular staining: FoxP3-APC (eBioscience, San Diego, California, USA), CTLA-4-PE (Coulter Immunotech) and CTLA-4-FITC (R&D Systems). Staining of FoxP3 was performed on 1x106 PBMC according to the manufacturer's protocol. To detect intracellular CTLA-4, 5 × 105 PBMC were stained using IntraPrep (Coulter) reagents according to the manufacturer's protocol for combined membrane and intracytoplasmic staining.

All staining was performed in the dark. Acquisition of data was performed on the same day as staining using a Coulter Epics XL-MCL flow cytometer or a Becton Dickinson FACSCalibur flow cytometer for three-colour protocols, or using a Becton Dickinson FACSCanto cytometer for four-colour protocols. 50 000–150 000 events were recorded per tube. Data were analysed using the FlowJo program v5.7.2 (Tree Star, Ashland, Oregon, USA).

Statistical analyses

Mann–Whitney tests were used to compare differences between groups of individuals. Spearman's tests were used to calculate the significance of non-parametric correlation coefficients. Longitudinal data were analysed on a log10 scale using linear mixed models for correlated longitudinal data. Analyses were carried out in S-Plus 7.0 for Windows (Insightful Corporation, Seattle, Washington, USA). Significance levels were determined by Wald and likelihood ratio tests. For all comparisons, P-values < 0.05 were considered to be statistically significant.

Results

CD25CD127loCD4 T cells and CD127loCD4− lymphocytes express FoxP3

In healthy donors, CD25CD4 T cells expressing low levels of CD127 preferentially express FoxP3 and can suppress lymphoproliferation [7,8]. To validate CD25CD127loCD4 as a phenotypic marker of CD4 Treg cells in HIV-infected patients, FoxP3 expression was assessed in 16 untreated HIV-infected patients with CD4 T-cell counts ranging from 6 to 1 073 cells/μl and 10 uninfected controls by co-staining with monoclonal antibodies to CD4, CD25 and CD127 (Fig. 1a). In 25 of the 26 individuals tested, FoxP3 was expressed by 28–89% (median 60%) of CD25CD127loCD4 T cells and 0.7–58% (median 7%) of CD25−CD127loCD4 T cells. FoxP3 was expressed by > 30% of CD25−CD127loCD4 T cells from five of the eight patients with CD4 T-cell counts < 300/μl (compared with < 26% in all other patients and healthy controls, Fig. 1b). CD127hiCD4 T cells did not express FoxP3.

Fig. 1
Fig. 1:
FoxP3 is predominantly expressed in the CD25+CD127lo subset of CD4 T cells. (a) Gating strategy for subsets of CD4 T cells using expression of CD25 and CD127. Cell populations in each region were assessed for the co-expression of (b) FoxP3, (c) HLA-DRhi, (d) CTLA-4 and (e) GITR. Region 1, CD25+CD127lo cells; region 2, CD25−CD127lo cells; region 3, CD127hi cells; ▾, patients with CD4 T-cell counts < 300/μl; ▴, patients with CD4 T-cell counts > 400/μl; •, non-HIV controls.

FoxP3 was not detectable in CD4 T cells from one patient with a CD4 T-cell count of 15/μl. Co-staining with monoclonal antibodies to CD4, CD8, CD127 and FoxP3 revealed that 79% of his FoxP3 lymphocytes were CD8, of which 97% were CD127lo (Fig. 2b, bottom panel). We therefore undertook further assessment of FoxP3+CD4− lymphocytes in the other 25 individuals and in an additional four patients. This showed that all patients and healthy controls possessed CD8 and CD4−CD8− lymphocytes that expressed FoxP3 (Fig. 2b and Fig. 3a), and that at least 68% of FoxP3CD8 and 95% of FoxP3+CD4−CD8− lymphocytes were CD127lo (Fig. 2b). These data support the work of Liu et al.[8], who postulated that FoxP3 may repress CD127 expression.

Fig. 2
Fig. 2:
FoxP3+CD127lo lymphocytes are not restricted to the CD4 subset. FoxP3+ lymphocytes (a) were gated for expression of CD4 and CD8 (b, left column). Cells in the bottom left (CD4−CD8−) and bottom right (CD8) quadrants were subsequently analysed for low expression of CD127 (b, middle and right columns, respectively). Values in the left column of dotplots indicate the percentage of FoxP3+ cells in each quadrant. Values in the middle and right columns of dotplots indicate the percentage of FoxP3+CD4−CD8− or FoxP3+CD8+ cells that are CD127lo. Representative staining of PBMC from an uninfected control and HIV patients with either high or low CD4 T-cell counts are shown. FSC, forward scatter; SSC, side scatter.
Fig. 3
Fig. 3:
Severe untreated HIV disease increases the proportion and number of CD4-negative lymphocytes that express FoxP3. (a) PBMC from healthy control donors (•), patients with CD4 T-cell counts > 400/μl (▴), and patients with CD4 T-cell counts < 300/μl (▾), were gated for FoxP3 lymphocytes and analysed for expression of CD4 and CD8. (b) Absolute numbers of FoxP3+CD4+ and FoxP3+CD4– lymphocytes were determined for both groups of patients. (c) CD4 T-cell counts for both groups of patients. Horizontal bars indicate the median value. *P < 0.05 compared to patients with low CD4 T-cell counts.

We then assessed how absolute numbers of FoxP3+CD4 and FoxP3+CD4− cells changed as CD4 T-cell count declined. Patients with low CD4 T-cell counts had significantly lower numbers of FoxP3+CD4 cells (median, 7 cells/μl) compared to patients with high CD4 T-cell counts (median, 26 cells/μl). Patients with low CD4 T-cell counts also had higher numbers of FoxP3+CD8+ (median, 6 versus 2 cells/μl) and FoxP3+CD4−CD8− lymphocytes (median, 4 versus 1 cells/μl) than patients with high CD4 T-cell counts, but these differences did not reach statistical significance (Fig. 3b). The patients with the five lowest CD4 T-cell counts also had the highest number of FoxP3+CD8+ and FoxP3+CD4−CD8− cells. The total number of FoxP3+ lymphocytes was similar between patients with high and low CD4 T-cell counts (median, 30 versus 26 cells/μl respectively, P = 0.63). The range of CD4 T-cell counts for both groups of patients is provided in Fig. 3c.

HIV disease is associated with increased proportions of Treg cells and their activation

The proportions of CD4 T cells with the phenotypes CD25+CD127lo or HLA-DRhi were then assessed in a larger group of HIV-infected patients and non-HIV controls. Both phenotypes were more common in CD4 T cells from patients with low CD4 T-cell counts (Table 1).

Clear populations of CD25+CD127lo and CD25−CD127lo CD4 T cells were HLA-DRhi in patients with low CD4 T-cell counts (Fig. 1c). The populations were small in non-HIV controls and patients with high CD4 T-cell counts. Expression of HLA-DRhi by CD127loCD4 cells suggests these cells may upregulate markers of activation more readily than CD127hiCD4 T cells in advanced HIV disease.

CD38 staining was performed as a second activation marker in eight patients from the validation group and three controls (data not shown). CD38 was expressed by 27% of CD127loCD4 T cells in one patient. In the remaining patients and healthy controls, < 12% of CD127loCD4 or CD127hiCD4 T cells expressed CD38. No further assessment of CD38 cells was undertaken.

In HIV-infected patients, the proportion of CD25+CD127loCD4 T cells correlated positively with plasma HIV RNA levels and proportions of HLA-DRhiCD4 T cells, but correlated inversely with CD4 T-cell count (Fig. 4). In non-HIV controls, proportions of CD25+CD127loCD4 T cells also correlated with proportions of HLA-DRhiCD4 T cells (r, 0.64, P = 0.002). Hence the proportions of Treg cells and their activation are increased by HIV disease.

Fig. 4
Fig. 4:
Proportions of CD25+CD127loCD4 T cells correlate with plasma HIV RNA levels and activated CD4 T cells, but correlate inversely with CD4 T-cell counts in untreated HIV-infected patients. The proportions of CD25+CD127loCD4 T cells from 29 HIV-infected patients were correlated with their plasma HIV RNA levels (top left), CD4 T-cell counts (bottom left), and the proportions of CD4 T cells that expressed HLA-DRhi (top right). The significance of non-parametric correlation coefficients was determined using Spearman's test.

GITR and CTLA-4 do not mark CD25+CD127loCD4 cells from healthy donors but their expression may be increased by HIV disease

CTLA-4 was expressed by < 10% of CD25+CD127loCD4 T cells from all donors (Fig. 1d). Expression of GITR was similarly low in healthy donors and patients with high CD4 T-cell counts. However, in the eight immunodeficient patients, 5–50% (median, 16%) of CD25+CD127loCD4 T cells and 0–60% (median, 8%) of CD25–CD127loCD4 T cells expressed GITR (compared with 1–8% and 0.3–4% respectively, in the other subjects; Fig. 1e). Hence neither molecule marks the Treg cell population defined as CD25+CD127lo in blood. The correspondence between increased expression of HLA-DRhi and GITR on CD25+CD127loCD4 cells in immunodeficient patients suggests that GITR may mark activated cells, but this was not demonstrated directly.

In the cross-sectional study (Table 1), proportions of CD4 T cells expressing CTLA-4 were highest in HIV-infected patients with low CD4 T-cell counts. There was a weak correlation between proportions of CD25+CD127loCD4 and CTLA-4+CD4+ cells in patients (r, 0.31; P = 0.099), while there was a stronger correlation in control donors (r, 0.58; P = 0.008).

Proportions of CD25+CD127loCD4 T cells remain high on ART despite decreased CD4 T-cell activation

Twelve patients beginning ART with CD4 T-cells counts < 100/μl (6 of the 29 patients from Perth and 6 patients from Kuala Lumpur) were followed during their first year on treatment (Table 2). Most patients achieved and maintained undetectable plasma HIV RNA after 12 weeks of ART. One patient from each site had a plasma HIV RNA level > 1000 copies/ml after 17–19 weeks on ART. Proportions of CD25+CD127loCD4 T cells and activated CD4 T cells are presented in Fig. 5. Linear mixed models for correlated longitudinal data related the log10 of the proportion of cells with each phenotype to time on ART. Proportions of CD25+CD127loCD4 T cells did not decline significantly over time on ART (Fig. 5a). However, proportions of HLA-DRhiCD4 T cells declined significantly (Fig. 5b) and CTLA-4+CD4 T cells showed a marginal decline (Fig. 5c). The slight decline in CD25+CD127loCD4 T cells was significant when the data was analysed after square root transformation (P = 0.04, data not shown), suggesting a small and variable decrease in the proportions of Treg cells during the first 30–40 weeks on ART.

Table 2
Table 2:
Characteristics of the patients studied longitudinally following ART.
Fig. 5
Fig. 5:
Proportions of CD25+CD127loCD4 T cells do not decline at the same rate as activated CD4 T cells in HIV-infected patients receiving ART. Longitudinal profiles of the proportions of CD4 T cells (expressed as log10 values) with the phenotype (a) CD25+CD127lo, (b) HLA-DRhi or (c) CTLA-4+, after patients from Perth (n = 6, solid lines) or KL (n = 6, brown dashed lines) commenced ART. Population-average trend lines are shown for patients from Perth (black dotted line) and Kuala Lumpur (red dotted line) where there are significant or near significant differences. The Perth and Kuala Lumpur data showed similar changes in Treg cells, so the trend line was generated from pooled data (black dotted line). The shaded regions are 95% confidence intervals for the non-HIV control donors – blue for Perth and pink for Kuala Lumpur where they differ and blue overall otherwise.

Patients in Kuala Lumpur retained higher proportions of CD4 T cells expressing HLA-DRhi (P < 0.0001) and CTLA-4 (P = 0.01) on ART, compared with patients from Perth (Fig. 5b, c). There was no significant difference between the population averages for the proportions of CD25+CD127loCD4 T cells in patients from Perth and Kuala Lumpur. Non-HIV controls from Kuala Lumpur also had significantly higher proportions of HLA-DRhiCD4 T cells compared with non-HIV controls from Perth (P = 0.007).

Discussion

Our first goal was to validate Treg cell markers in HIV patients. We showed that CD25+CD127loCD4 T cells from the blood of HIV-infected patients and uninfected controls preferentially express the Treg cell-specific transcription factor FoxP3. We also detected FoxP3 in a high proportion of CD25−CD127loCD4 cells from immunodeficient patients. Limited expression of FoxP3 in CD25–CD127loCD4 T cells from controls and HIV-infected patients with high CD4 T-cell counts was evident, whilst CD127hiCD4 T cells did not express FoxP3 (Fig. 1). Further characterization revealed no preferential expression of GITR or CTLA-4 (< 40% and < 10% respectively) by CD25+CD127loCD4 T cells, with the exception of one immunodeficient patient in whom 50% of these cells expressed GITR. Other studies describe significant co-expression of these markers by CD25+CD4 Treg cells [4,5,9–11]. For example, immunohistochemical staining of lamina propria from non-HIV controls and from treated and untreated HIV-infected patients showed that 60–90% of FoxP3+ cells in the gut mucosa co-expressed GITR and CTLA-4, but not HLA-DR or CD38 [5]. Here expression of HLA-DR and GITR was highest on circulating CD127loCD4 T cells from immunodeficient HIV-infected patients compared with other donors (Fig. 1). Hence blood Treg cells may acquire an activated phenotype in advanced HIV disease.

A novel finding is that FoxP3 is expressed by CD8 and CD4–CD8– lymphocytes in immunodeficient HIV-infected patients. Such cells are CD127lo, consistent with evidence that FoxP3 may repress CD127 expression by binding to the CD127 promoter [8]. Our data suggest that such repression may not be confined to CD4 T cells. Indeed, reduced expression of CD127 on the surface of both CD4 and CD8 T cells is a characteristic of advanced HIV infection [12–14]. In 20 untreated HIV-infected patients used for validation of Treg cell marker staining, the proportion of CD8 and CD4−CD8− lymphocytes that expressed FoxP3 increased as CD4 T-cell counts declined (Fig. 3), with highest expression in patients with CD4 T-cell counts < 50 cells/μl. HIV disease increases the proportion of CD4−CD8− T cells in circulation, possibly caused by the shedding of CD4 by productively infected T cells [15,16]. Downregulation of CD4 expression by CD4 Treg cells or upregulation of FoxP3 by CD4−CD8− cells provide two possible mechanisms for an expanded population of FoxP3+CD4−CD8− cells in immunodeficient patients.

High levels of immune activation in immunodeficient patients could be sufficient to induce and maintain a population of FoxP3+CD8+ cells. One report has described transient upregulation of FoxP3 and CD25 by CD8 T cells after activation, as well as concomitant acquisition of suppressive properties [17]. Numbers of CD4-negative lymphocytes expressing FoxP3 might increase to compensate for the loss of FoxP3+CD4 Treg cells in HIV-infected patients with low CD4 T-cell counts. More recently, Allan et al. demonstrated that transient FoxP3 expression can be induced in proliferating effector CD4 T cells by stimulation with anti-CD3 and anti-CD28, albeit at significantly lower levels than natural Treg cells. Furthermore, activation-induced expression of FoxP3 did not suppress cytokine production, CD127 expression or proliferation [18]. It is therefore possible that expression of FoxP3 by CD8 and CD4–CD8– cells is a normal consequence of cellular activation. It remains to be seen if FoxP3+CD4– cells possess suppressive activity in HIV-infected patients.

We show for the first time that proportions of blood CD4 T cells with the Treg cell immunophenotype CD25+CD127lo are increased in patients with advanced HIV infection (CD4 T-cell counts < 300/μl) and correlate with plasma HIV RNA levels. The proportion of CD4 Treg cells correlated with the proportion of CD4 T cells that were activated (HLA-DRhi), and more Treg cells from immunodeficient donors expressed HLA-DRhi. Whilst the biological significance of the increased proportions of Treg cells is not addressed here, it is pertinent to propose models that would be consistent with our data. Firstly, HIV-associated immune activation may result in an increased proportion of blood CD4 T cells with a Treg cell phenotype. This may follow increases in CD4 Treg cells demonstrated in the lymphoid tissue of humans with early HIV infection [4,5,19], and in macaques with early SIV infection [20]. Secondly, an increase in the proportion of Treg cells in the CD4 T-cell population may limit T-cell effector function required to control HIV or opportunistic infections [1–3]. This would promote immune activation. Additionally, Haase and co-workers associated increased levels of transforming growth factor (TGF)β+ Treg cells during early SIV infection with collagen deposition in lymphoid tissue [21]. Previously they described a correlation between disruption of lymphoid tissue architecture and a reduction in the total and naive CD4 T-cell compartment in HIV-infected individuals [22,23], which adversely affected CD4 T-cell recovery on ART [24]. Increased Treg cells in HIV-infected subjects may therefore contribute to reduced CD4 T-cell counts via TGFβ-mediated fibrosis of lymphoid tissue.

Here, stratification of HIV-infected patients by CD4 T-cell counts shows that the proportion of Treg cells increases as CD4 T-cell count declines. Montes and co-workers presented similar findings after stratifying patients by CD4 T-cell count [6]. If re-calculated as absolute counts, our data would show a decrease in numbers of circulating CD4 Treg cells in parallel with total CD4 T-cell counts, as reported elsewhere [2,6,25]. One report suggested that HIV-associated immune activation is a consequence of decreased total blood Treg cell counts [25]. However, the functional capacity of the residual CD4 T cells in a patient with advanced HIV disease is likely to reflect the ratio of regulatory and effector cells, so our arguments are based on this.

Although ART decreased the proportion of activated blood CD4 T cells in patients with pre-ART CD4 T-cell counts < 100/μl, proportions of CD4 Treg cells remained elevated during the period of observation (12 months). We have previously reported that low antigen-specific CD4 T cell-mediated interferon (IFN)γ responses do not resolve in the first 3 years of ART despite good reconstitution of CD4 T-cell counts [26,27]. Responses of some patients remain low for at least 8 years [27]. Furthermore, we demonstrated that the proportion of CD4 T cells expressing CTLA-4 is increased in HIV-infected patients receiving long-term effective ART [28]. We confirm this finding in Fig. 5c, which shows that proportions of CTLA-4+CD4 T cells remain elevated above the level of non-HIV controls. It is not known if the expression of CTLA-4 in CD4 T cells from patients receiving ART marks cells that are able to limit IFNγ responses. However, CTLA-4 is upregulated in response to cellular activation [29,30], and may simply be expressed by CD4 T cells as a general response to immune activation in HIV-infected patients [31,32].

In conclusion, our data show that a proportion of CD4-negative lymphocytes have the Treg cell phenotype FoxP3+CD127lo in immunodeficient HIV-infected patients. Untreated HIV-infected patients exhibited a direct relationship between proportions of CD4 lymphocytes with a Treg cell phenotype, immune activation and viral load, with a negative correlation with CD4 T-cell count. Finally we showed that the percentage of activated CD4 T cells declined whilst Treg cells remain elevated during a virological response to ART. We suggest that the elevated proportions of blood Treg cells might limit effector responses of CD4 T cells in patients receiving ART, despite control of plasma viraemia. Further studies are needed to determine the long-term effects of immune reconstitution on HIV-associated abnormalities of blood Treg cell populations.

Acknowledgements

The authors thank Dr Graeme Chapman for expert technical assistance with flow cytometry and Steven Roberts for archiving PBMC in Perth. We also thank all the patients involved in this study. This is manuscript 2006-21 for Clinical Immunology and Immunogenetics, Royal Perth Hospital.

Conflicts of Interest: The authors declare that they have no conflicts of interest.

This work was supported by a grant from the National Health and Medical Research Council of Australia (404028 to M.A.F. and P.P.).

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Keywords:

aniretroviral therapy; CD127; FoxP3; HIV; immune activation; regulatory T cells

© 2007 Lippincott Williams & Wilkins, Inc.