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AIDS:
doi: 10.1097/QAD.0b013e3283344895
Basic Science

HIV-1 infection is characterized by profound depletion of CD161+ Th17 cells and gradual decline in regulatory T cells

Prendergast, Andrewa,b; Prado, Julia Ga; Kang, Yu-Hoic; Chen, Fabiand; Riddell, Lynn Ae; Luzzi, Grazf; Goulder, Philipa; Klenerman, Paulb,c

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Author Information

aDepartment of Paediatrics, University of Oxford, UK

bBiomedical Research Centre, John Radcliffe Hospital, UK

cNuffield Department of Medicine, University of Oxford, Oxford, UK

dThe Florey Unit, Department of Genitourinary Medicine, Royal Berkshire NHS Foundation Trust, Reading, UK

eDepartment of Genitourinary Medicine, Northampton General Hospital, Northampton, UK

fDepartment of Genitourinary Medicine, Wycombe Hospital, High Wycombe, UK.

Received 1 June, 2009

Revised 11 September, 2009

Accepted 16 October, 2009

Correspondence to Dr Andrew Prendergast, Department of Paediatrics, University of Oxford, Peter Medawar Building for Pathogen Research, South Parks Road, Oxford OX1 3SY, UK. Tel: +44 1865 271973; fax: +44 1865 281890; e-mail: andrew.prendergast@paediatrics.ox.ac.uk

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Abstract

Objective: CD4+ T-cell depletion is central to HIV pathogenesis. However, the relative impact of HIV on Th17 and regulatory T cell (Treg) subsets remains unclear. CD161+ CD4 cells are a recently identified, gut-homing Th17 precursor population. The balance between pro-inflammatory Th17 and immunoregulatory Tregs may be critical in HIV pathogenesis. This study addressed changes in CD161+, Th17 and Treg subsets during untreated HIV infection.

Methods: Peripheral blood mononuclear cells were isolated from HIV-infected and HIV-uninfected individuals and stained to characterize CD161+ CD4 cells, Th17 cells [by elaboration of interleukin (IL)-17A], Tregs (CD3+CD4+CD25hiFoxP3+ cells) and CD8 activation (CD38+/HLA-DR+ cells). In-vitro infectability of CD161+ and Th17 cells by HIV was assessed in healthy donor CD4 cells by intracellular p24 expression.

Results: Peripheral blood Th17 cells were depleted 10-fold in HIV-infected, compared to HIV-uninfected individuals (P < 0.0001) across a range of disease stages, accompanied by a significant reduction of CD161+ T cells (P = 0.024). Both Th17 cells and CD161+ CD4+ T cells were permissive to HIV replication in vitro. Profound loss of Th17 cells before the onset of advanced disease contrasted with a gradual decline in absolute Tregs during HIV disease progression in untreated individuals followed longitudinally (R = 0.71, P = 0.003). Loss of Tregs was associated with increased immune activation (R = −0.33, P = 0.03).

Conclusion: HIV-infected individuals showed profound loss of Th17 cells, which may impair mucosal immunity, and reduced CD161+ CD4 cells, which may limit Th17 reconstitution. A gradual decline in Tregs during disease progression was associated with increased immune activation.

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Introduction

HIV-1 infection is characterized by an acute phase, during which a massive depletion of CD4+ T cells occurs in the gastrointestinal tract [1–3], followed by a chronic phase, during which a slow decline in CD4+ T cells leads to increased risk of opportunistic infections and onset of AIDS. The speed of disease progression is related to the degree of immune activation [4,5], which may arise in part from microbial translocation following structural and immunological disruption to the gut mucosa during acute infection [6]. CD4+ T cells are, therefore, central to HIV pathogenesis, but the extent to which different subsets are impacted by HIV is not fully understood.

Differentiation of CD4+ T cells into lineages with diverse effector roles is contingent upon expression of specific transcription factors in response to the prevailing cytokine environment. Initially, two subsets were defined: Th1 cells, which produce gamma interferon and mediate protection against intracellular pathogens; and Th2 cells, which produce IL-4, IL-5, IL-9 and IL-13 and provide defence against extracellular pathogens [7]. More recently, two further subsets have been identified: regulatory T cells (Tregs), which have the capacity to impair the activation and proliferation of effector T cells [8,9]; and Th17 cells, which produce IL-17A, IL-17F and IL-22 and appear to confer protection against extracellular bacteria, fungi and mycobacteria [10]. Tregs are characterized as CD4+CD25hiFoxP3+ cells [11,12], whereas Th17 cells express IL-23R, CCR6 and the transcription factor ROR-γt [13–16]. Th17 cells arise exclusively from a population of CD161+CD4+ T cells in the presence of IL-1β and IL-23, and this precursor population has gut-homing potential [17,18].

In this study, we sought to describe the changes that occur within the CD161+, Th17 and Treg subsets of CD4+ T cells during the course of HIV infection. Th17 cells are important in mucosal defence and are depleted from the gut during both HIV and simian immunodeficiency virus (SIV) infection [19,20]. Several previous studies have addressed the role of Tregs in HIV infection, but the results to date are conflicting. Although some report an accumulation of Tregs during HIV disease progression, which may suppress potentially efficacious immune responses [21–26], others report a depletion of Tregs, which may allow generalized immune activation to occur [27–32]. Furthermore, as pro-inflammatory Th17 cells and immunoregulatory Tregs have antagonistic effector functions, the balance between these subsets may be critical in determining the outcome of HIV disease, as has been demonstrated in SIV infection of nonhuman primates [20]. We set out to establish whether changes within these CD4 subpopulations may impact on HIV disease progression in a cohort of untreated, chronically HIV-infected individuals.

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Participants and methods

Study participants

A total of 77 HIV-infected and 36 HIV-uninfected individuals were recruited to this study. HIV-infected individuals were all untreated, chronically infected patients recruited from four clinics after giving written informed consent. Characteristics of the cohort are shown in Table 1. Ethical approval for this study was granted by the Oxfordshire Research Ethics Committee.

Table 1
Table 1
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CD4 cell count determination

CD4 cell count measurement was undertaken by flow cytometry in clinical laboratories at the sites where patients were receiving care, using venous blood taken at the same time as the research sample.

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Blood sampling and peripheral blood mononuclear cell isolation

Venous blood samples were collected in EDTA tubes, transported by courier and processed within 4 h of collection. Peripheral blood mononuclear cells (PBMCs) were isolated from whole blood by density gradient centrifugation using Lymphoprep (Axis Shield plc) and resuspended in R10 medium (RPMI 1640, supplemented with 10% fetal calf serum, L-glutamine, penicillin and streptomycin) before undertaking flow cytometry or intracellular cytokine staining.

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Immunophenotyping and flow cytometry

Fluorochrome-conjugated antibody staining was undertaken on freshly isolated PBMCs for all experiments except analysis of CD161+ cells in HIV-infected individuals, which was undertaken on cryopreserved PBMCs. Panels of antibodies used were anti-CD8-FITC, anti-CD38-PE, anti-CD3-PerCP, anti-HLA-DR-APC (BD Biosciences, Oxford, UK); anti-CD4-PeCy7, anti-CD4-PerCP (BD Pharmingen, Heidelberg, Germany); anti-CD3-FITC/APC, anti-CCR6-FITC, anti-CCR5-APC (R&D Systems, Minneapolis, Minnesota, USA); anti-CD161-APC (Miltenyi Biotech, Bergisch-Galdbach, Germany); anti-IL-17A-PE (eBioscience, San Diego, California, USA); anti-CD161-PE, anti-CD3-ECD (Beckman-Coulter, Fullerton, California, USA).

Treg quantitation was undertaken using the Human Regulatory T Cell Staining Kit 2 (eBioscience), according to manufacturer's instructions. Briefly, fresh PBMCs were stained extracellularly with anti-CD4-FITC, anti-CD25-PE and anti-CD3-PerCP, then fixed, permeabilized, blocked with normal rat serum and stained intracellularly with anti-FoxP3-APC. All samples were acquired on a FACSCalibur flow cytometer (BD Biosciences). Data were analysed using FlowJo software (Tree Star Inc, Ashland, Oregon, USA), with the same gating applied to all samples. Tregs were characterized as CD3+CD4+CD25hiFoxP3+ cells. CD8 activation was measured by co-expression of CD38 and HLA-DR on CD3+CD8+ cells.

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Intracellular cytokine assays

PBMCs were stimulated for 4 h at 37°C in R10 with phorbol 12-myristate 13-acetate (PMA; Sigma, St Louis, Missouri, USA; 50 ng/ml) and ionomycin (Sigma; 500 ng/ml) in the presence of brefeldin A (Sigma; 5 μg/ml). Cells were stained extracellularly with anti-CD4 ± anti-CD161 fluorochrome-conjugated antibody at 4°C for 20 min, washed in phosphate-buffered saline (PBS) and fixed with PBS containing 1% formaldehyde (Sigma). Cells were washed again in PBS and then permeabilized with Fix and Perm B (Caltag Laboratories, Burlingame, California, USA) for 30 min at 4°C and stained intracellularly. Monoclonal antibodies used for intracellular stains were anti-p24-FITC (clone KC57-FITC; Beckman-Coulter), anti-IL-17A-PE (eBioscience; San Diego), anti-IFN-γ-PE (BD Biosciences), anti-IFN-γ-FITC (BD Biosciences) and anti-Ki67-FITC (BD Biosciences). Finally, cells were washed in PBS and resuspended in PBS containing 1% formaldehyde (Sigma). At least 1 million cells were acquired on a FACSCalibur flow cytometer (BD Biosciences) and data analysed using FlowJo software (Tree Star Inc.).

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Gamma-interferon CD4 Elispot assay

CD8+ cells were depleted from fresh PBMCs using anti-CD8 DynaBeads (Invitrogen, UK), following manufacturer's instructions. CD8-depleted PBMCs were plated in 96-well polyvinylidene plates (Millipore, Billerica, Massachusetts, USA) that had been precoated overnight with 100 μl/well anti-IFN-γ (Mabtech, London, UK). Cells were added at a concentration of 200 000 cells/well in 100 μl R10 medium and incubated with Phytohaemagluttinin (PHA) (12.5 μg/ml), cytomegalovirus (CMV) lysate (10 μg/ml; Virusys Corporation, Skyesville, Maryland, USA), Epstein–Barr virus (EBV) lysate (10 μg/ml; Virusys Corporation), Gag peptide pool (2 μg/ml), Pol peptide pool (2 μg/ml), Nef peptide pool (2 μg/ml) or no peptide (negative control). HIV peptide pools were synthesized at the Massachusetts General Hospital Peptide Core Facility and consisted of overlapping peptides (OLP), 17–18 amino acids long and overlapping by 10 amino acids, spanning Gag (66 OLP), Pol (133 OLP) or Nef (97 OLP). Plates were incubated overnight at 37°C in 5% CO2 and developed as previously described [33]. The numbers of spots per well were counted using an automated Elispot plate reader (AID, Germany) and results expressed as spot-forming cells (SFCs)/million PBMCs after subtracting the negative well from test well values.

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Interleukin-17 CD4 Elispot assay

IL-17 Elispot plates were prepared in an identical way to the IFN-γ plates, except plates were precoated with 100 μl/well purified antihuman IL-17 antibody (clone eBio64CAP17; eBioscience), and were incubated for 48 h at 37°C, 5% CO2. Anti-CD2/CD3/CD28 beads were used instead of PHA as a positive control. Plates were developed according to the manufacturer's protocol (Human IL-17A Elispot Ready-Set-Go; eBioscience), using the AEC Elispot Substrate Set (Becton-Dickinson, Franklin Lakes, New Jersey, USA) for the final step. The numbers of spots per well were counted using an automated Elispot plate reader (AID, Germany) and results expressed as SFCs/million PBMCs after subtracting the negative well from test well values.

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In-vitro Th17, Th1 and CD161+CD4+ cell infections

CD4 cells were isolated from HIV-uninfected individuals by negative selection from fresh PBMCs. Briefly, non-CD4 cells were indirectly magnetically labelled with a cocktail of biotin-conjugated monoclonal antibodies, mixed with antibiotin MicroBeads (Miltenyi-Biotech, Bergisch-Gladbach, Germany) and separated on a column (Miltenyi-Biotech) in the field of a magnet, according to the manufacturer's protocol. CD4 cells were resuspended in R10 with 50 IU/ml IL-2 (Roche, Basel, Switzerland) and 3 μg/ml PHA (Sigma) and incubated at 37°C in 5% CO2 for 48 h to activate CD4 cells. After 48 h, cells were counted, split into aliquots of 5 million cells and incubated for 2 h at 37°C with no virus, CCR5-tropic Bal virus or CXCR4-tropic NL4-3 virus (both from NIH AIDS Research and Reference Reagent Program) at an MOI of 0.001–0.01. Cells were then resuspended at 5 million/ml in R10 with 50 IU/ml IL-2 (Roche) and 3 μg/ml PHA (Sigma) and transferred to a 24-well plate. On day 3, 1 ml fresh medium was added to each well. On day 5, cells were harvested and an intracellular cytokine assay was undertaken, as described above, to detect HIV infection (by intracellular p24 staining) of IL-17-producing cells, IFN-γ-producing cells, or CD161+ cells. HIV infection was determined by calculating the proportion of p24-positive cells after subtracting background staining.

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Statistics

Comparisons between groups were made using the Mann–Whitney U-test for nonparametric data and the Student's t-test for parametric data. Nonparametric correlations were analysed using Spearman's correlation. Other statistical tests used are indicated in the text. All statistical analyses were undertaken using GraphPad Prism Version 5.0a (GraphPad Software, La Jolla, California, USA).

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Results

Th17 cells are depleted in HIV infection

We first compared the number of Th17 cells in HIV-infected (n = 20) and HIV-uninfected (n = 13) individuals by stimulating PBMCs with PMA/ionomycin and assessing production of IL-17A by intracellular cytokine staining of CD4+ T cells. HIV-infected individuals had a 10-fold reduction in the proportion of IL-17-producing (Th17) CD4+ T cells compared with HIV-uninfected individuals [median 0.07% (interquartile range [IQR] 0.04–0.17) vs. 0.75% (IQR 0.31–0.99) CD4 cells, respectively; P < 0.0001; Fig. 1a]. To put this finding in context, we also compared the proportion of IFN-γ-producing CD4+ T cells in both groups. HIV-infected individuals similarly had fewer IFN-γ-producing CD4+ T cells compared with HIV-uninfected individuals, although the difference was not as striking as that seen with the IL-17-producing subset [median 0.14% (IQR 0.10–0.42) vs. 0.98% (IQR 0.17–5.40) CD4 cells, respectively; P = 0.04; Fig. 1b]. There was no association between Th17 cell frequency and HIV disease progression, as measured by either number of CD4+ T cells (R = −0.16, P = 0.54) or viral load (R = 0.30, P = 0.19).

Fig. 1
Fig. 1
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Viral-specific Th17 cells are not found in chronic HIV infection

We next sought to determine whether viral-specific Th17 cells were present in 23 chronically HIV-infected individuals, using Elispot assays to determine IL-17 or IFN-γ production by CD8-depleted PBMCs in response to HIV, CMV and EBV. Robust IFN-γ responses were seen following stimulation with HIV Gag (median 100 SFCs/106 CD4 cells), HIV Pol (median 50 SFCs/106 CD4 cells), HIV Nef (median 50 SFCs/106 CD4 cells), CMV lysate (median 230 SFCs/106 CD4 cells) and EBV lysate (median 380 SFCs/106 CD4 cells). In contrast, no IL-17 responses were seen following stimulation with the same viral antigens (Fig. 1c).

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CD161+CD4+ cells are reduced in HIV infection

Given the 10-fold reduction in Th17 cells in HIV-infected individuals, we next looked at the impact of HIV on the CD161+CD4+ T-cell population. CD161 is a C-type lectin-like receptor that has recently been shown to identify a subset of gut-homing CD4+ T cells committed to the Th17 lineage [17,18]. We first confirmed that CD161 identified Th17 cells in our population by stimulating PBMCs from healthy controls with PMA/ionomycin and demonstrating that the vast majority of IL-17-producing CD4+ T cells were CD161+ (Fig. 2a). We then went on to confirm that IL-17-producing CD161+CD4+ T cells co-expressed CCR6, which is a consistent marker of Th17 cells (Fig. 2b). As anticipated, therefore, CD161+CD4+ T cells contained a small population of Th17 cells (confirmed by IL-17A production and CCR6 expression) and a larger population of non-Th17 cells, which have the potential to become Th17 cells [17].

Fig. 2
Fig. 2
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Next, we compared the proportion of CD161+ cells in 15 HIV-infected individuals and 11 healthy controls. There were significantly fewer CD161+CD4+ T cells in HIV-infected individuals than in HIV-uninfected ones [median 15.2% (IQR 8.7–21.0) CD4 cells vs. 20.4% (IQR 18.2–35.3), respectively; P = 0.024; Fig. 2c). There was no relationship between the size of the CD161+ CD4 pool and HIV disease progression, as measured by number of CD4+ T cells (R = −0.35, P = 0.21) or viral load (R = 0.39, P = 0.17).

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Th17 and CD161+ CD4 cells are targets for infection by HIV

We next sought to investigate whether viral infection of Th17 and CD161+CD4+ T cells might explain their reduced frequency in HIV-infected individuals. It has previously been shown that human Th17 cells express CCR5 and CXCR4 [14,34], making them theoretically permissible to HIV infection. As HIV preferentially targets activated CD4 cells, we first assessed the proliferative activity of Th17 CD4+ T cells in healthy controls. A greater proportion of Th17 CD4+ T cells, compared with non-Th17 CD4+ T cells, expressed the proliferation marker Ki67 [median 4.4% (IQR 2.2–8.3) vs. 0.8% (IQR 0.4–1.0) respectively; P = 0.0001), suggesting that Th17 cells are a highly activated, and potentially infectable, subpopulation of CD4 cells.

We, therefore, went on to investigate whether Th17 cells are capable of sustaining HIV replication in vitro. CD4 cells from healthy controls were activated for 48 h with PHA and IL-2 and either infected with CCR5-tropic HIV (Bal), CXCR4-tropic HIV (NL4-3) or left uninfected. After 5 days, cells were harvested, stimulated with PMA/ionomycin for 4 h and stained intracellularly to assess cytokine production (IL-17A or IFN-γ) and HIV p24 content. Both IL-17-producing (Th17) and IFN-γ-producing (Th1) cells were permissible to infection by CCR5-tropic HIV, although only a small proportion of Th1 and Th17 cells were infected in vitro (Fig. 3a and b). Infection of IL-17-producing and IFN-γ-producing cells was not significantly different [median 1.7% (IQR 0.8–3.3) vs. 3.3% (IQR 1.8–13.0); P = 0.14, Mann–Whitney test; Fig. 3c). Th17 cells were also permissible to infection by CXCR4-tropic virus [median 3.4% (IQR 2.4–7.3) infected IL-17-producing cells].

Fig. 3
Fig. 3
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We postulated that direct infection of CD161+CD4+ T cells might explain their reduced frequency in HIV-infected individuals. We, therefore, looked first to see whether CD161+ cells from healthy donors expressed the HIV co-receptor, CCR5. The level of CCR5 expression was higher on CD161+ than in CD161 cells [mean fluorescence intensity (MFI) 6.5 (95% confidence interval [CI] 6.0–7.0) vs. 5.1 (95% CI 4.6–5.6); P = 0.004; Fig. 4a]. We, therefore, went on to compare infection of CD161+ and CD161 CD4 cells. CD4 cells from healthy donors were activated and infected as described above and then co-stained for CD161 expression and intracellular p24. CCR5-tropic virus infected CD161+ and CD161 cells to a similar degree in vitro [median 1.5% (IQR 0.4–2.9) vs. 1.4% (IQR 0.3–3.8) p24+ cells, respectively; P = 1.00; Fig. 4b and 4c).

Fig. 4
Fig. 4
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Taken together, both CD161+CD4+ T cells and Th17 cells are permissible to infection by HIV. Although the reduction in both CD161+ cells and Th17 cells seen in HIV-infected individuals may, in part, be due to direct infection of these CD4 subsets by the virus, neither subset appeared to be infected preferentially.

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Relationship between Th17 and regulatory T cells in HIV infection

As Th17 cells and regulatory T cells have antagonistic effector functions, we hypothesized that changes in one subset may impact the other. Because of the dramatic effects of HIV infection on Th17 cell frequency, we went on to investigate whether reciprocal changes in Treg frequency are seen in HIV-infected individuals. However, we found no relationship between Th17 and Treg frequency in the peripheral blood of 16 HIV-infected individuals for whom data on both CD4 subsets were available, whether expressed as proportions of CD4+ T cells (R = 0.34, P = 0.19) or as absolute numbers (R = 0.11, P = 0.70).

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Changes in Tregs during HIV disease progression

We next explored in more detail changes in peripheral blood Tregs in our untreated, chronically HIV-infected cohort. We first compared Tregs (as a proportion of CD4+ T cells) in 61 HIV-infected and 14 HIV-uninfected individuals and found no significant difference in Treg frequency expressed as a proportion of CD4 cells between HIV-infected and HIV-uninfected individuals [median 2.4% (IQR 1.3–4.0) vs. 2.1% (IQR 1.5–2.7) of CD4+ T cells, respectively; P = 0.22). We next compared Treg frequency as a proportion of CD3 cells and found a significant reduction in HIV-infected, compared to HIV-uninfected, individuals (median 1.4% (IQR 0.9–2.2) vs. 0.8% (IQR 0.5–1.4) of CD3+ T cells, respectively; P = 0.01; Fig. 5a). We next acquired longitudinal data on 15 HIV-infected individuals followed up for median 9.1 months (IQR 6.8–10.9) and analysed changes in Treg frequency over time. In those patients (9/15; 60%) who had a decline in absolute CD4 cell count during the study period, the Treg frequency declined from median 3.5% (IQR 1.4–6.7) CD4 cells at baseline to median 2.8% (IQR 0.9–3.7) CD4 cells at follow-up. By contrast, in those patients (6/15; 40%) who had no CD4 decline during the study period, the Treg frequency increased from median 1.2% (IQR 0.7–1.8) CD4 cells at baseline to median 2.5% (IQR 1.6–4.7) CD4 cells at follow-up. There was, therefore, a strong positive correlation between change in CD4+ T-cell count and change in Treg frequency during the period of follow-up (R = 0.82, P = 0.0005). We next repeated these calculations, using the absolute Treg count, because we reasoned that expressing Tregs as a proportion of CD4 cells may be misleading, as the absolute CD4 cell count declines over time. Again, we found a strong positive correlation between change in absolute Tregs and change in absolute CD4+ T cell count (R = 0.71, P = 0.003; Fig. 5b). Thus, in those untreated individuals who experienced disease progression during the period of follow-up, the Treg frequency (expressed either as percentage of CD4 cells or absolute Tregs) fell. Conversely, in those individuals who had stable HIV disease during the period of follow-up, the Treg frequency was maintained or increased.

Fig. 5
Fig. 5
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Impact of Treg decline on immune activation

The decline in Tregs seen during disease progression in HIV-infected individuals led us to question whether this may impact immune activation. Tregs may play an important role in preventing generalized activation of the peripheral T-cell pool and a decline in Tregs may enable activation to occur. We, therefore, explored the relationship between CD8 activation, measured by co-expression of CD38 and HLA-DR, and absolute Treg count within our cohort. There was a significant negative association between CD8 activation and absolute Treg count (R = −0.33, P = 0.03; Fig. 5c). In addition, there was a trend toward increased CD8 activation as the CD8: Treg ratio increased (R = 0.30, P = 0.056; Fig. 5d). Taken together, these data suggest that as the absolute Treg count declines and the ratio between effector and suppressor populations changes, generalized activation of the CD8 pool increases.

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Discussion

HIV infects and depletes mucosal CD4+ T cells during acute infection, setting the stage for a slow, chronic decline in peripheral CD4+ T cells during chronic infection [1–3,35]. Th17 cells and Tregs are important effector mucosal CD4+ T cells and are, therefore, likely to be impacted by HIV infection, although the changes that occur within these helper T-cell subsets have not hitherto been fully clarified. However, the balance between Th17 cells and Tregs has been shown to be critical in determining outcome in SIV disease [20]. Our data show that there is a disproportionate reduction in Tregs, Th17 cells and CD161+CD4+ T cells in the peripheral blood of HIV-infected individuals, and we hypothesize that this may have important consequences for HIV disease progression.

Few studies to date have explored the impact of HIV infection on Th17 cells. Although one published study reported an increased frequency of Th17 cells in the peripheral blood of HIV-infected individuals [36], another recent study demonstrated that Th17 cells are preferentially depleted from the gut in HIV and pathogenic SIV infection [19]. Our own data show that the proportion of Th17 cells is reduced 10-fold in the blood of chronically HIV-infected individuals than that of HIV-uninfected controls. This profound loss of the Th17 subset of CD4+ T cells occurred in HIV-infected individuals with generally well preserved CD4 cell counts, suggesting that loss of Th17 cells occurs before onset of advanced disease. Loss of Th17 cells in the gastrointestinal tract of HIV/SIV-infected individuals impairs mucosal integrity and immune surveillance. This structural and functional impairment enables microbial translocation to occur, which has been proposed to contribute to systemic immune activation in some studies [6,19], but not in others [37].

Although their full range of effector functions has not yet been elucidated, Th17 cells have been reported to play a role in defence against extracellular bacteria, fungi and mycobacteria [10]. The loss of the majority of Th17 cells in HIV infection may, therefore, reduce the ability of the host to deal effectively with a wide range of pathogens and may contribute to the propensity of HIV-infected individuals to develop recurrent bacterial illnesses, tuberculosis and opportunistic infections, even before the total CD4 cell count has dropped significantly. Whether Th17 cells have an effector function in viral infections remains unclear, although viral-specific Th17 cells have been detected in acute HIV infection and chronic hepatitis C infection [38,39]. We did not find any viral-specific Th17 cells in our study individuals, although all were chronically infected, and HIV-specific Th17 cells may only be found early in the disease [39] or in the context of virological suppression [40]. In addition to classical CD4+ Th17 cells, γδ T cells are a potent source of IL-17 and appear to play an important role in host defence [41]. Expansion of a subset of IL-17-producing γδ T cells has been proposed to contribute to nonprogressive HIV disease and protection against opportunistic infections [42]. There is also an increasing appreciation of the immunoregulatory function of γδ T cells [43–45], although whether they have a regulatory function in HIV disease has not hitherto been investigated. Furthermore, cytotoxic CD8+ T cells that elaborate IL-17 (Tc17 cells) have recently been described and shown to play a role in autoimmunity, cancer and antiviral defence [46,47]. These cells show some developmental and phenotypic overlap with Th17 cells and warrant further investigation in the context of HIV infection.

In addition to the loss of Th17 cells in HIV infection, we showed that the CD161+ CD4 population is significantly reduced in HIV-infected individuals. Whether the reduced frequency of CD161+CD4+ cells in peripheral blood reflects absolute depletion or redistribution to lymphoid tissue is not clear. CD161+ cells express the gut-homing integrin β7 and may, therefore, be lost from the gut mucosa during acute HIV infection, along with Th17 cells [18], or may migrate from blood to gut-associated lymphoid tissue during the course of HIV infection. In addition to its expression on Th17 cells, CD161 identifies CD4+ T cells capable of differentiation along the Th17 lineage [17]. The loss of this precursor population may cause the reduction in Th17 cells, through lack of Th17 differentiation, or may limit the capacity for reconstitution following Th17 depletion. Thus, the combined loss of Th17 cells and CD161+ cells may contribute to impaired mucosal T-cell immunity and microbial translocation.

We wanted to establish whether direct infection of Th17 and CD161+ cells, both important constituents of the mucosal CD4+ T-cell pool, might contribute to their depletion during acute infection. It has previously been shown that human Th17 cells express both CCR5 and CXCR4 chemokine receptors [14,34], and we demonstrated in this study that CD161+ cells express particularly high levels of CCR5. We confirmed, in vitro, that both Th17 cells and CD161+CD4+ T cells are permissive to infection with HIV, although the frequency of infected cells was low and neither subset appeared to be infected preferentially compared with other CD4+ T cells. That Th17 cells are capable of sustaining infection with HIV in vitro is consistent with a previous report demonstrating by quantitative PCR that, in vivo, infection of Th17 cells does occur [19]. However, preferential infection of Th17 cells would not appear to be the mechanism for their dramatic depletion in HIV-infected individuals. Whether a differential sensitivity to activation-induced apoptosis might contribute to the preferential loss of these CD4+ T-cell subsets warrants further investigation. Early loss of Th17 cells from the gut mucosa and inability to repopulate this subset, partly due to the depletion of precursor CD161+CD4+ T cells, may contribute to their reduced frequency. A previous study of HIV-infected children reported preservation of IL-17 production in those individuals with viral suppression, compared with those with persistent viraemia, suggesting that ongoing viral replication might prevent recovery of the Th17 population [40].

Our study demonstrated that Tregs are lost from peripheral blood over time in HIV-infected individuals and that immune activation increases with the decline in Treg count. Loss of Tregs from blood may be due to destruction or relocation to peripheral lymphoid tissue, although it was not possible to distinguish between these mechanisms in the current study. We propose that the inhibitory function of Tregs is important in prevention of immune activation and that loss of Tregs from blood over time contributes to increased immune activation and disease progression. This is in line with the findings of the previous studies that have postulated a protective role for Tregs in preventing the chronic immune activation that eventually leads to CD4 decline in chronic HIV infection [27–32]. It has recently been shown that elite controllers (who maintain undetectable HIV viral loads in the absence of antiretroviral therapy) have preservation of peripheral blood Tregs, in contrast to individuals with progressive HIV infection [28]. Further evidence of the protective capacity of Tregs comes from nonhuman primate models of HIV infection. Nonpathogenic SIV infection in African green monkeys, in contrast to pathogenic infection in macaques, is associated with early induction of an anti-inflammatory milieu through Treg induction, which prevents the chronic immune activation that drives disease progression [48]. Taken together, these studies, and our own, suggest a protective role for Tregs in preventing HIV disease progression by reducing immune activation; conversely, loss of Tregs may enhance disease progression by enabling immune activation and CD4 decline.

There are several potential limitations to our study. First, because of sample availability, we could only assess changes occurring in the peripheral blood of HIV-infected individuals. However, HIV is principally a mucosal disease and events occurring in blood may not reflect events occurring at mucosal surfaces. Second, differences in methodology between studies can impact the findings reported. For example, we characterized Tregs as CD3+CD4+CD25hiFoxP3+ cells, whereas other studies have used different markers to identify Tregs [26,29,31,49]. We believe that FoxP3 remains the best marker currently available to characterize Tregs, but it is important to recognize that not all CD3+CD4+CD25hiFoxP3+ cells are Tregs and that not all Tregs are CD3+CD4+CD25hiFoxP3+ cells [50]. Similarly, characterization of Th17 cells in this study relied on identification of IL-17-producing CD4+ T cells following a 4 h stimulation assay, in line with other published studies [15,18,51,52]. However, other investigators have used longer stimulation times and expressed Th17 cells as a proportion of memory CD4+ T cells, rather than total CD4+ T cells, which will impact the findings [19]. Finally, experiments conducted in vitro may not reflect events occurring in vivo, and our finding that only a small proportion of Th17 and CD161+ CD4 cells are infected with HIV in vitro may not reflect what occurs at mucosal surfaces during acute HIV infection.

In summary, several subsets of CD4 cells appear to be disproportionately depleted from the peripheral blood during HIV infection. Th17 cells appear to be lost relatively early in the disease, as the 10-fold reduction in Th17 cells, compared with HIV-uninfected individuals, occurs even in patients with a high absolute CD4 cell count. CD161+ CD4 cells, which include cells committed to the Th17 lineage, are also reduced and may limit the capacity for Th17 regeneration. Both subsets of CD4 cells are permissible to infection by HIV in vitro and may be depleted during acute infection, especially from mucosal sites, where their loss contributes to systemic immune activation through enhanced microbial translocation. In contrast, the depletion in Tregs appears to occur slowly during the course of HIV infection, but may also contribute to increased immune activation of CD8+ T cells through loss of suppressive mechanisms. Restoration of all these CD4 subsets might be protective in reducing speed of disease progression in HIV-infected individuals.

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Acknowledgements

This work was supported by the Medical Research Council, UK (A.P.), Wellcome Trust (P.G., P.K.), NIHR Biomedical Research Centre Programme and James Martin 21st Century School, University of Oxford.

We thank Mohamed Ghanem, Jane Kemp, Nicola Cook, Sam Walker, Wayne McKenna, and the medical and nursing staff of the Florey Unit, Reading, and Harrison Clinic, Oxford, for help in study subject recruitment.

Study concept and design: A.P., J.G.P., Y.-H.K., P.G. and P.K.

Acquisition of data: A.P., J.G.P. and Y.-H.K.

Analysis and interpretation of data: A.P., J.G.P., Y.-H.K. and P.K.

Drafting of manuscript: A.P. and P.K.

Critical revision of manuscript: J.G.P., Y.-H.K., F.C., L.A.R., G.L. and P.G.

Study recruitment and supervision: F.C., L.A.R. and G.L.

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Keywords:

CD161; CD4; HIV; pathogenesis; Th17

© 2010 Lippincott Williams & Wilkins, Inc.

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