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Keywords:

  • anergy/suppression/tolerance;
  • HIV-1;
  • T cells

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosure
  9. References

Mechanisms by which CD4+ regulatory T cells (Tregs) mediate suppression of virus-specific responses remain poorly defined. Adenosine, mediated via CD39 and CD73, has been shown to play a role in the action of murine Tregs. In this study we investigate the phenotype of Tregs in the context of human immunodeficiency virus (HIV)-1 infection, and the function of these cells in response to HIV-1-Gag and cytomegalovirus (CMV) peptides. Phenotypic data demonstrate a decrease in forkhead box transcription factor 3 (FoxP3+) Treg numbers in the peripheral blood of HIV-1+ individuals compared to healthy controls, which is most pronounced in those with high HIV-1 RNA plasma load. Due to aberrant expression of CD27 and CD127 during HIV-1 disease, these markers are unreliable for Treg identification. The CD3+CD4+CD25hiCD45RO+ phenotype correlated well with FoxP3 expression in both the HIV-1+ and seronegative control cohorts. We observed expression of CD39 but not CD73 on Tregs from HIV-1+ and healthy control cohorts. We demonstrate, through Treg depletion, the suppressive potential of Tregs over anti-CMV responses in the context of HIV-1 infection; however, no recovery of the HIV-1-specific T cell response was observed indicating a preferential loss of HIV-1-specific Treg function. We propose that before immunotherapeutic manipulation of Tregs is considered, the immunoregulatory profile and distribution kinetics of this population in chronic HIV-1 infection must be elucidated fully.


Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosure
  9. References

Human immunodeficiency virus (HIV)-1 infection is characterized by chronic immune activation and functional defects in the T cell population that precede a decline of circulating CD4+ T cell numbers [1]. Mechanisms mediating immune suppression are not fully defined; however, several theories as to the role of regulatory T cells (Tregs) have been proposed. One view is that expanded Tregs are detrimental in HIV-1 infection. Thus, CD4+CD25+ T cells, which are expanded in the peripheral blood of HIV-1-infected patients receiving highly active anti-retroviral therapy (HAART), have been shown to exhibit phenotypic and functional characteristics of Tregs[2]. The apparent loss of HIV-1-specific proliferative responses in HIV-1-infected patients may be related to the suppressive effect of this Treg population, which compete for interleukin (IL)-2 and prevent effector cell interaction with antigen-presenting dendritic cells [2,3]. The second school of thought is that due to the high expression of CCR5, CD4+CD25hiCD45RO+ Tregs are particularly susceptible to HIV-1 infection [4], hence the infection, dysfunction and loss of Tregs could contribute to hyperactivation of HIV-1-specific T cells due to lack of immune regulation. In both scenarios, dysregulation of Tregs may impact upon the quality of the anti-HIV-1 T cell responses during HIV-1 infection. Tregs are enriched in the subset of CD4+ T cells which express the IL-2 receptor α chain (CD25); however, as CD25 is also expressed on activated cells, this CD25+ population will probably be an over-estimate of the number of Tregs. The CD4+CD25hi phenotype, used to overcome the problem caused by the presence of activated cells, is a subset that cannot be distinguished consistently. Therefore, a more reliable phenotype for the identification of Tregs is required if we are to study this population in detail. Alternative combinations of markers have been suggested for the identification of Tregs in HIV-1 seronegative individuals, including the original CD4+CD25hiCD45RO+ panel and subsequently CD4+CD25+CD27+[5], and CD4+CD25+CD127- subsets [6].

Expression of CD127, the α-chain of the IL-7 receptor, in combination with CD25, has been used to discriminate between regulatory and activated CD4+ T cells in blood, lymph nodes and thymus of healthy adults [6]. Regulatory cells were shown to express the IL-2, but not the IL-7, receptor α-chain (CD25+CD127-) [6]. Forkhead box transcription factor 3 (FoxP3) belongs to a family of transcription factors which contain a winged helix-forkhead DNA binding domain. FoxP3 localizes to the nucleus, binds DNA and acts as a transcriptional repressor, playing a vital role in determining the amplitude of the response of CD4+ T cells [7]. The expression of FoxP3 both at the mRNA and protein levels is high in CD4+CD25hi Tregs, and low in both naive and activated cells [8–11]. However, FoxP3 cannot be used as a means of selecting cells for functional assays, as the intranuclear staining process kills the cells.

The mechanisms by which human FoxP3+ Tregs mediate suppression of virus-specific responses remain ill-defined. A role for adenosine in the mediation of immune responses by Tregs has been reported in mouse models [12,13]. Deaglio et al. (2007) reported that CD39 and CD73 are co-expressed on murine FoxP3+ Tregs. CD39 is a surface-bound enzyme that catalyses the conversion of extracellular nucleotides into adenosine monophosphate (AMP), which is then degraded further by CD73 into adenosine, an anti-inflammatory molecule reported to inhibit T cell activation [14]. Here we report on the expression of combinations of FoxP3, CD27, CD39, CD45RO, CD127 and CD73 molecules on CD4+CD25hi T cells in a cohort of 76 HIV-1+ individuals and eight seronegative healthy controls. The suppressive function of Tregs is examined in relation to cytomegalovirus (CMV)- and HIV-1 Gag p24-specific responses, in the context of HIV-1 infection.

Materials and methods

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosure
  9. References

Study population

Blood samples were obtained from 76 HIV-1+ individuals and eight healthy controls, with patients' written informed consent and Ethics Committee approval (RREC1108) for the studies described. Seventy-six HIV-1+ patients, with a median CD4 T cell count of 369 cells/µl blood (range 23–1811), were recruited. HIV-1 plasma load was below the level of detection (50 RNA copies/ml plasma) in 47 of 76 patients (median CD4 T cell count 362 cells/µl blood; range 23–1056) owing to successful HAART, while 29 patients had detectable viraemia (median 6031 copies/ml plasma, range 52–500 000; median CD4 T cell count 371 cells/µl blood, range 30–1811).

Viral load

Patient plasma viral load was determined by the use of the Versant HIV-1 RNA 3·0 branched deoxyribonucleic acid (bDNA) assay (Siemens Healthcare, Camberley, UK). The assay was performed according to the manufacturer's instructions and had a detection range of 50–500 000 HIV-1 RNA copies ml−1 plasma.

Lymphocyte subsets

Murine anti-human monoclonal antibodies to CD3, CD4, CD8, CD56, CD19 and CD45 (Tetra One; Beckman Coulter, High Wycombe, UK) were used to mark lymphocytes within whole blood. Lymphocyte numbers were then evaluated using a Cytomics FC 500 flow cytometer (Beckman Coulter) and Tetra CXP (version 2.2) software.

Antibodies and flow cytometry

Fresh peripheral blood mononuclear cells (PBMC) were separated by density gradient centrifugation. Cells were surface-stained using combinations of CD4-peridinin chlorophyll (PerCP) (SK3), CD25-allophycocyanin (APC) (2A3), CD27-phycoerythrin (PE) (L128), CD45RO-fluorescein isothiocyanate (FITC) (UCHl-1), CD45RO-PE (UCHl-1), CD73-PE (A2), CD127-PE (HIL-712-M21) (all Becton Dickinson, Cowley, UK), CD4-PerCP-Cy5·5 (OKT4), CD25-PE-cyanin 7 (Cy7) (BC96), CD39-FITC (A1), CD127-APC-ALEXA750 (RDR5) (all eBiosciences, San Diego, CA, USA), and subsequently stained for either intracellular FoxP3-FITC (clone PCH101) or FoxP3-APC (clone PCH101) using the FoxP3 buffer set and protocol (all eBiosciences). Appropriate isotype-matched controls were run in parallel for each sample. Staining for flow cytometry was performed as per the manufacturers' instructions. A total of 105 lymphocyte gated events, based on forward- and side-scatter profiles, were collected using an LSRII flow cytometer (Becton Dickinson). Analysis was performed using fluorescence activated cell sorter (FACS) diva version 5·0 (Becton Dickinson).

CD4+CD25+ Treg depletion from PBMC

Removal of the CD4+CD25+ T cells from PBMC was performed using a combination of the Dynal CD4 positive isolation kit and the Dynal CD4+CD25+ Treg kit (both Dynal Biotech ASA, Oslo, Norway). Using the two kits in combination avoided the need to remove cell-bound beads by exposing them to detacher bead, therefore minimizing manipulation of cells prior to culture. Briefly, 1 × 106 PBMC were depleted of CD4+ T cells using the CD4-positive isolation kit to leave bead-free PBMC minus CD4 T cells. From a second 1 × 106 PBMC fraction the Dynal CD4+CD25+ Treg kit was used to produce a bead-free CD4+CD25- fraction. The two bead-free fractions were pooled to give a PBMC minus CD4+CD25+ fraction. Purity of these fractions was checked by flow cytometry.

Proliferation assays

Lymphocyte proliferation was measured over 5 days by [3H]-methyl thymidine (Amersham International, Amersham, UK) incorporation [15]. Briefly, 1 × 104 whole PBMC or 1 × 104 Treg-depleted PBMC were cultured in triplicate with either a pool of 22 overlapping HIV-1-Gag p24 peptides (ARP788·1–22; NIBSC, Potters Bar UK), or a CMV peptide pool (pp65 sequence; strain A169; Becton Dickinson) consisting of 138 peptides; both pools were used at a final concentration of 5 µg/ml of each peptide. Tissue culture medium (TCM; negative control) or phytohaemagglutinin (PHA; mitogen-positive control) were used as described previously [15]. Cell culture supernatant (100 µl/well) was removed carefully and frozen at −80°C for cytokine analysis prior to pulsing with [3H]-methyl thymidine on day 5. Results are expressed as stimulation index (SI): the experimental result divided by the background (PBMC in TCM alone). A positive response was defined as an SI ≥3 providing that the Δ counts per minute (cpm; experimental cpm – background cpm) was ≥600.

Cytometric bead array (CBA)

Measurement of levels of IL-2, IL-4, IL-6, IL-10, interferon (IFN)-γ and tumour necrosis factor (TNF)-α in the tissue culture supernatants were performed using a human T helper type 1 (Th1)/Th2 cytokine kit II (BD Biosciences), according to the manufacturer's instructions, and analysed on the BD LSRII using FACS diva (version 5.0.1) and FCAP Array Software (both BD Biosciences).

IL-7 enzyme-linked immunosorbent assay (ELISA)

Measurement of levels of IL-7 in the tissue culture supernatants were performed using high-sensitivity IL-7 ELISA. Levels were measured in thawed tissue culture supernatants using ELISA kits (R&D Systems, Oxford, UK), according to the manufacturer's instructions. Standard titrations were performed and included in all assays.

Statistical analysis

Due to the non-normal distribution of data, intergroup statistical significance was calculated using a Mann–Whitney U-test. Intragroup statistical significance was calculated by comparing the medians of the paired data, using the Wilcoxon signed-rank test. Medians and ranges are presented with statistical results expressed as P-values, with a 95% confidence interval. Statistical analysis was performed using GraphPad Prism version 5·00 (GraphPad Software Inc., San Diego, CA, USA).

Results

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosure
  9. References

The percentage of CD4+ T lymphocytes that expressed FoxP3 was determined by flow cytometry (Fig. 1a). A median of 2·8% [interquartile range (IQR) 2·2–4·9%] of CD4+ T cells expressed FoxP3 in the HIV-1+ cohort, which was significantly lower than in the healthy control cohort (median 5·8%; IQR 4·3–6·7%; P = 0·0032). The HIV-1+ cohort was split into those with a suppressed HIV-1 plasma load (<50 RNA copies/ml) and those with a detectable viral load (≥50 RNA copies/ml plasma). The number of FoxP3+ cells within the CD4+ T lymphocyte population in the group with detectable viraemia was significantly lower than those with suppressed viral load (Fig. 1b). However, both groups of HIV-1-infected individuals had a lower level of FoxP3+ cells within the CD4+ population than the healthy control group (Fig. 1b). No correlation was observed between current CD4 T cell count and the percentage of CD4+ T cells expressing FoxP3 (Fig. 1c).

image

Figure 1. Expression of forkhead box transcription factor 3 (FoxP3) by CD4+ lymphocytes. Lymphocytes were gated, selected for CD4+ events and used to assess FoxP3 expression (dark grey). A pale grey histogram shows the isotype control plot (a). The percentage of CD4+ lymphocytes expressing FoxP3 in the human immunodeficiency virus 1 (HIV-1+) cohort with HIV-1 RNA plasma load <50 copies/ml plasma (striped bar; n = 47), ≥50 copies/ml plasma (spotted bar; n = 29) and the HIV-1-negative healthy control (HC) cohort (closed bar; n = 8) (B). Relationship between percentage of CD4+ lymphocytes expressing FoxP3 and current CD4 T cell count in the HIV-1+ cohort (C; n = 76). Box plots show the median, 25th and 75th percentile values. Whiskers indicate the 10th and 90th percentiles. Statistics were performed using the Mann–Whitney U-test, with a cut-off for significance of P < 0·05.

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CD3+CD4+CD25hi events were gated and then further gated on the FoxP3+ events to identify the regulatory population (Fig. 2a). The percentage of cells in the CD3+CD4+CD25hiFoxP3+ population which expressed CD27, CD39, CD45RO, CD73 or CD127 was calculated. Gates were set according to appropriate isotype controls (Fig. 2b). Figure 3 shows summary data from HIV-1+ and healthy control cohorts. When events were gated to analyse the total CD3+CD4+CD25hiCD45RO+ population no difference was observed in the number of FoxP3+ cells present within the population in HIV-1+ individuals, compared to that seen in healthy controls (Fig. 3a). In the HIV-1+ cohort CD45RO was expressed on the vast majority of the CD3+CD4+CD25hiFoxP3+ cells, which was comparable to the levels seen in the healthy control cohort (Fig. 3b).

image

Figure 2. Expression of forkhead box transcription factor 3 (FoxP3+) cells within different populations. Representative flow cytograms showing the gating strategy for intracellular FoxP3 staining (dark grey) in human immunodeficiency virus 1 (HIV-1+) individuals. Gates were set using appropriate isotype controls (pale grey) (a). Representative histograms showing the expression of CD27, CD39, CD45RO, CD73 or CD127 on the CD3+CD4+CD25hiFoxP3+ population in dark grey (b). Pale grey histograms show the appropriate isotype controls used to stain the same population. Inset on CD73 plot demonstrates positive staining of whole peripheral blood mononuclear cell (PBMC) population with the anti-CD73 antibody (dark grey) and matching isotype control (pale grey).

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image

Figure 3. Percentage of forkhead box transcription factor 3 (FoxP3+) cells within different CD4 T cell subsets. The percentage of FoxP3+ cells within the CD3+CD4+CD25hi, CD27, CD45RO or CD127 subsets in human immunodeficiency virus 1 (HIV-1+) (open bars; n = 52) and healthy controls (HC) (closed bars; n = 7) cohorts (a). The percentages of the CD3+CD4+CD25hiFoxP3+ cells expressing CD27, CD45RO or CD127 in the HIV-1+ (open bars; n = 52) and HC (closed bars; n = 7) cohorts (b). The percentages of CD3+CD4+CD25hiFoxP3+ cells expressing CD39 or CD73 in the HIV-1+ (open bars; n = 52) and HC (closed bars; n = 7) cohorts (c). Box plots show the median, 25th and 75th percentile values. Whiskers represent the 10th–90th percentiles. Differences were compared between groups using the Mann–Whitney U-test, with a cut-off for significance of P < 0·05.

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Although a wider variation in the percentage of CD3+CD4+CD25hiFoxP3+ cells expressing CD27 was seen across the HIV-1+ cohort compared to the healthy control cohort, these differences were not statistically significant (Fig. 3b). However, when events were gated to analyse the total CD3+CD4+CD25hiCD27+ population a significantly lower number of FoxP3+ cells were present in HIV-1+ individuals compared to that seen in negative controls (Fig. 3a), demonstrating that an elevated number of CD3+CD4+CD25hiCD27+ cells are also present within the FoxP3- population in the HIV-1+ cohort.

As expected, CD127 was expressed on a low percentage of CD3+CD4+CD25hiFoxP3+ cells in healthy controls. However, in the HIV-1+ cohort a significantly greater proportion of the CD3+CD4+CD25hiFoxP3+ cells expressed CD127 (Fig. 3b). Nevertheless, when the cells were gated to analyse the total CD3+CD4+CD25hiCD127- population no difference was observed in the percentage of cells co-expressing FoxP3 in HIV-1+ individuals, compared to that seen in healthy controls (Fig. 3a).

In contrast to the reported evidence in the mouse we did not see expression of CD73 on the CD3+CD4+CD25hiFoxP3+ cells in either the HIV-1+ cohort or the healthy control cohort (Fig. 3c). There was, however, a statistically significant increase in percentage of CD73+ events in the CD3+CD4+CD25+FoxP3- activated T cell population compared to the CD3+CD4+CD25hiFoxP3+ regulatory T cell population (P = 0·0005).

As in the mouse model, both the HIV-1+ and healthy control cohorts expressed CD39 on the CD3+CD4+CD25hiFoxP3+ Treg cells (Fig. 3c). When gated to analyse the total CD3+CD4+CD25hiCD39+ population, no difference was observed between the number of FoxP3+ cells present within the population in HIV-1+ individuals and that seen in healthy controls. Additionally, the level of expression of CD39, as calculated by the geometric mean fluorescent intensity, was significantly higher on the CD3+CD4+CD25hiFoxP3+ Tregs than on the CD3+CD4+CD25+FoxP3- activated T cells (P = 0·0008).

The function of Tregs was investigated in seven HIV-1+ individuals who were receiving successful HAART with undetectable HIV-1 plasma RNA levels (<50 copies/ml plasma) and a current median CD4+ T cell count of 357 cells/µl blood (IQR 98–416). Nadir CD4+ T cell counts ranged from 27 to 416 cells/µl blood (median 190) prior to the initiation of HAART.

To assess the function of Tregs in the HIV-1+ cohort, freshly isolated PBMC were either stimulated directly with pools of HIV-1-Gag or CMV overlapping peptides, or stimulated after the Treg subset was depleted, as described in Materials and methods. Whole PBMC and the purity of the population obtained post-Treg removal were assessed by flow cytometry (Fig. 4a,b, respectively). During optimization of this method a background stimulatory effect was seen when cells were heavily manipulated to remove the isolation beads, and so the protocol was developed to avoid this step. A combination of depletion kits was used to deplete the Tregs from the PBMC, leaving the remaining PBMC population unlabelled after limited manipulation prior to culture. We observed a significant increase in the proliferative response to CMV peptides in the Treg-depleted cultures compared to the whole PBMC fraction (Fig. 5a), with an increase in the anti-CMV proliferative response observed in all seven individuals tested. However, we did not observe a similar increase to levels above the negative cut-off of the assay (SI ≥ 3) in the HIV-1 Gag-specific response in the Treg-depleted cultures (Fig. 5a). The response to CMV was significantly higher than the response to Gag in both the whole PBMC and the Treg-depleted cultures (Fig. 5a, P = 0·0156 for both conditions). This was mirrored in the levels of IL-2 observed in culture supernatants at day 5 (Fig. 5b). There was more IL-2 detected in the CMV-stimulated cultures compared to the TCM (unstimulated) controls for both the PBMC and the Treg-depleted PBMC, although this trend did not reach significance (both P = 0·0625; Fig. 5b). In contrast, this effect was not observed in the HIV-1-Gag-stimulated cultures, where levels of IL-2 were not significantly different from background (Fig. 5b). Levels of IL-4, IL-6, IL-7, IL-10, TNF-α or IFN-γ production showed no statistically significant differences in either the HIV-1-Gag- or CMV-stimulated PBMC, or Treg-depleted PBMC tissue culture supernatants (data not shown).

image

Figure 4. Representative flow cytograms showing cell populations before and after the depletion process, using a combination of depletion kits as detailed in Materials and methods. Briefly, both the Dynal CD4 positive isolation kit and the Dynal CD4+CD25+ regulatory T cell (Treg) kit were used (Dynal Biotech ASA, Oslo, Norway), avoiding the need to remove cell-bound beads with detacher bead, thereby minimizing cell manipulation preculture. Use of the CD4-positive isolation kit on 1 × 106 peripheral blood mononuclear cell (PBMC) left bead-free PBMC minus CD4 T cells. The CD4+CD25+ Treg kit, when used on another 1 × 106 PBMC, resulted in a bead-free CD4+CD25negative fraction. When pooled, the two bead-free fractions contained PBMC minus CD4+CD25+ cells. Fraction purity was checked by flow cytometry, and flow cytometric plots show that the CD4+CD25hi population was (a) present prior to depletion, and (b) absent post-depletion.

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image

Figure 5. Assessment of regulatory T cell function. Proliferative responses to cytomegalovirus (CMV) and human immunodeficiency virus 1 (HIV-1)-Gag peptide pools detected in whole peripheral blood mononuclear cells (PBMC) (striped bars) or in the regulatory T cell-depleted cultures (hatched bars) in the HIV-1+ cohort (a; n = 7). Production of interleukin (IL)-2 in the tissue culture supernatants in response to CMV and HIV-1-Gag peptide pools detected in the whole PBMC (striped bars) or in the regulatory T cell-depleted cultures (hatched bars) in the HIV-1+ cohort (b; n = 7). TCM: tissue culture medium (cells only). Box-plots show median, 25th and 75th percentile values, whiskers show the 10th–90th percentiles. Statistical analysis of paired responses was performed using the Wilcoxon signed-rank test, with a cut-off for significance of P < 0·05.

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Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosure
  9. References

Tregs have been postulated to play an important role in the response to infectious agents and autoimmunity. However, a complete understanding of their role has been hampered by the lack of a definitive phenotype. The impact that Tregs have on HIV-1 disease progression remains to be elucidated fully. Given that effector T cell responses may help to control HIV-1 replication, the action of Tregs in suppressing these responses may be considered detrimental. Conversely, Tregs may be beneficial by helping to regulate the cellular immune activation, which is a hallmark of chronic HIV-1 infection. The first part of this cross-sectional study assessed the phenotype of FoxP3+ Tregs in a cohort including HAART-naive HIV-1+ individuals as well as those receiving successful suppressive HAART, with a variety of CD4 T cell counts and HIV-1 RNA plasma loads.

The percentage of Tregs in the peripheral blood of HIV-1 infected patients was significantly lower than in healthy controls. This difference was most pronounced in the group of HIV-1+ patients with detectable viral load. This concurs with previous findings of decreased Treg cells, as measured by the percentage of CD25hi cells in the CD3+CD4+CD45RO+ subset in the peripheral blood of chronically infected HIV-1+ individuals [16]. Tregs express CD4 and chemokine receptors, such as CCR5, rendering them susceptible to HIV-1 infection [4]. As HIV-1 specific CD4+CD45RO+ memory cells are preferentially infected [17], it is therefore likely that HIV-1-specific Tregs (CD3+CD4+CD25hiCD45RO+FoxP3+) present in the same environment are also infected. The level of FoxP3 in the lamina propria of simian immunodeficiency virus (SIV+) macaques during acute infection has been shown to be significantly lower than in healthy control animals, and a lack of suppressive function of the remaining CD4+ T cells from the gut-associated lymphoid tissue (GALT) of SIV-infected animals indicates a rapid depletion of Tregs from this compartment [18]. However, to complicate this picture, a longitudinal study of HIV-1+ individuals pre- and post-HAART demonstrated that prior to the initiation of HAART, CD4+CD25+FoxP3+ cells migrated from the peripheral blood to the lymphoid tissue during periods of high viraemia, which was reversed by HAART [19], in addition to the reports of redistribution of Tregs from the peripheral blood to the gut in chronically infected SIV+ macaques [20]. Binding of HIV-1 to Tregs has been shown to cause migration to the lymph nodes as well as enhancing Treg function [21]. Patients with undetectable viral load had significantly greater numbers of Tregs within the periphery compared to individuals with detectable viral load (Fig. 1b), indicative of a possible redistribution effect after HAART-induced reduction of viraemia. Furthermore, as the numbers of Tregs seen in the periphery of HIV-1+ individuals were not restored to the levels observed in the HIV-1-negative healthy controls under conditions of HAART-induced viral control, it is possible that some of the cells which were infected were lost during periods of uncontrolled viraemia, or sequestered in lymph nodes or sites of residual viral turnover. In addition to this, FoxP3 – the definitive transcription factor of Tregs – has been shown to enhance gene expression from the HIV-1 long terminal repeat region [22], leading to a paradoxical increase in viral transcription when the suppressive effect of Tregs is initiated.

We dissected the surface phenotype of the CD3+CD4+CD25hiFoxP3+ in the periphery, and observed a wide variation across the HIV-1+ cohort in the percentage of the CD3+CD4+CD25hiFoxP3+ subset that expressed CD27, which was independent of viral load level. Previously this phenotype has been used to identify Tregs expressing FoxP3 in the synovial – another inflammatory setting [5]; however, we did not observe an enrichment for FoxP3 in the CD4+CD25+CD27+ population in peripheral blood of HIV-1+ individuals, indicating that the use of CD27 as a marker may result in over-estimation of the number of Tregs and is therefore not appropriate for detecting circulating Tregs in the context of HIV-1 infection [23,24].

CD127 is the IL-7R α-chain [25], which dimerizes to the common cytokine γ-chain (CD132) [26]. Both CD127 and CD132 are expressed on naive and memory T cells, and in HIV-1 seronegative individuals it was proposed that IL-2R α-chain (CD25) expression in the absence of the IL-7R α-chain (CD127) could be used as a marker for CD4+ Tregs[6], preventing activated cells from contaminating the analysis. Data from healthy controls presented here confirm this observation, as we saw that the majority of the CD4+CD25hiFoxP3+ cells were CD127-. However, in the HIV-1+ cohort presented here, although a high percentage of CD4+CD25hiCD127- cells expressed FoxP3+, this cell-surface phenotype grossly underestimated the total FoxP3+ population. We found significantly more CD3+CD4+FoxP3+ expressing CD127 in the HIV-1+ cohort when compared to the healthy controls, probably indicating dysregulation of IL-7R (CD127) expression in HIV-1+ patients [23]. Unlike recent findings by Del Pozo-Balado et al. [28], we did not observe any significant difference in CD127 expression on CD4+ CD25hiFoxP3+ Treg percentages between the viraemic and aviraemic HIV-1+ cohorts (P = 0·531), and unlike findings by Rallón et al. (2009) CD127 expression was significantly lower in all HIV-1+ individuals compared to healthy controls [27], regardless of HIV-1 plasma load [28]. It has been shown that HIV-1 infection is associated with a progressive loss of CD4+CD127+CD132- T cells, which was evident in both the CD45RO+ and CD45RO- populations. This reduction correlated with CD4+ T cell count [29], providing further evidence of the dysregulation of the IL-7/IL-7R pathway, which may account for the underestimation of the CD4+ Treg population when using CD127 and CD25 as definitive markers.

The correlation of CD4+CD25hiCD45RO+ cells with the FoxP3+ population indicated that the CD4+CD25hiCD45RO+ phenotype is the most consistent surrogate marker for the CD4+ Tregs in the peripheral blood of HIV-1+ individuals at both high and low CD4 counts. Although the majority of cells identified by this phenotype were FoxP3+, there still remains some underestimation of the total CD4+ Treg population using this phenotype. It is of note that CD25 has been indicated as a less reliable as a marker of Tregs in lymph nodes [30].

A role for adenosine in Treg mediation of immune responses in the mouse has been reported [12,13]. Deaglio et al. [13] showed that CD39, a surface-bound enzyme and CD73, which degrades AMP into anti-inflammatory adenosine, are co-expressed on FoxP3+ Tregs in the mouse. Among a large number of effects, adenosine has been shown to inhibit the production of proinflammatory cytokines, desensitize chemokine receptors and inhibit T cell activation [12,31,32]. Here we report the expression of CD39 and CD73 on CD4+CD25hiFoxP3+ T cells in a cohort of HIV-1+ individuals. Previously it has been shown that CD39 expression was significantly higher on CD4+CD45RO+ or CD3+CD25+ than on CD4+CD45RO- or CD3+CD25- populations [33]. We show that this expression is not only higher in cells which express CD25, but is significantly higher in the CD25hi population, indicating a feasible link between CD39 expression and a regulatory phenotype. Although we did not observe the co-expression of CD73 and CD39 on the CD4+CD25hiFoxP3+ regulatory cells, the presence of CD39 provides further evidence for the utilization of adenosine as an immune-suppressor by Tregs. The basis for the low percentage of the CD4+CD25hiFoxP3+CD39+ cells which express CD73 may include: (i) the level of expression of CD73 has been shown previously to be decreased in individuals with cancer or immunodeficiency [34,35], and/or (ii) CD73 expression is regulated on T cells by CD38 [36]. During HIV-1 infection CD38 expression is up-regulated in a high proportion of patients; however, the majority of CD4+CD38+ cells are not CD25+[37], therefore not identified as Tregs. It has also been shown that both laboratory-adapted and clinical isolates of HIV-1 can incorporate biologically active CD39 in their envelope [38], and as such the CD39-bearing viral particles could act as circulating enzymes. Moreover, these particles may be able to affect several physiological processes by manipulating local levels of adenosine triphosphate (ATP), adenosine diphosphate (ADP) and adenosine. Therefore these proteins, both bound to HIV-1 particles and expressed on Tregs, may be contributing to the immunological deficiency seen in HIV-1-infected individuals [38]. This dysregulation of ectoenzymes in HIV-1 infection may lead to disruption of the mechanism of action of the CD25+FoxP3+ Tregsin vivo. Thus, the effects of CD38, HIV-1 bound CD39 and other lymphocyte ectoenzymes on the actions of Tregs in the context of HIV-1 infection need further elucidation.

The function of Tregs on the proliferative response to HIV-1-Gag and CMV overlapping peptide pools was assessed by depletion experiments. We did not see an increase in the magnitude of the HIV-1-Gag proliferative response in Treg-depleted cultures; however, we observed an increase in the CMV-specific response when Tregs were depleted. This effect was supported by IL-2 levels in the tissue culture supernatants. It has been reported previously that CD4+CD25+ T cells isolated from asymptomatic HIV-1+ patients suppress IL-2 production and proliferation of HIV-1-specific T cells [2,39]; however, this was observed only in patients with low viral load and high CD4+ T cell counts.

In the setting of chronic HCV infection, programmed death-1 (PD-1) has been reported to inhibit proliferation of CD4+FoxP3+ Tregs[40]. PD-1 expression is increased in CD4+ naive, central memory and effector T cells, and CD8+ naive T cells of HIV-1+ individuals [41], indicating a possible mechanism of suppression of Treg activity in chronically infected HIV-1+ individuals. We did not observe a suppressive effect of CD4+CD25+ cells on the HIV-1-specific responses in our cohort, although the suppressive effect of this regulatory T cell population on CMV-specific responses is apparent within the same individuals. In support of this finding, no correlation was shown between the level of CD4+CD25+CD127loFoxP3+ T cells and immune activation (measured by CD38 expression) in HIV-1+ patients [42], indicating that these cells were unable to reduce HIV-1-mediated immune hyperactivation. It has been suggested that long-term IL-2 immunotherapy may be necessary to rescue a functional Treg compartment in HIV-1+ individuals [43,44]. Another possible approach may be to deplete Tregsin vivo followed by stimulation with antigen, allowing formation of an effector T cell response without the suppressive effect of Tregs, as has proved successful in the immunotherapeutic treatment of cancer [45].

It is possible that there were no HIV-1-specific Tregs present in the peripheral blood of the patients investigated here, and consequently depletion of the Treg compartment had no apparent effect on HIV-1-specific responses. It has been reported previously that HIV-1-specific Tregs are functionally active at other sites [46], and if suppression of HIV-1-specific effector function in the periphery is not CD4+CD25+ Treg-mediated, then other mechanisms of suppression are implicated [20]. Compartmentalization of Treg function has also been reported, with differential modes of suppression depending on anatomical site and cytokine environment in which Treg activation initially occurred [47].

An apparent reduction of HIV-1-specific Tregs from the periphery and lack of HIV-1-specific Treg activity, as demonstrated here, may contribute to HIV-1-specific immune dysregulation and the state of persistent immune activation observed during periods of high viral load during chronic HIV-1 disease. Future longitudinal studies must aim to determine whether Tregs are truly lost or are ‘hidden’ and remain functional within the lymphoid tissues. Nevertheless, the possible lack of Treg suppression on HIV-1-specific responses demonstrated here may indicate that manipulation or depletion of this subset in vivo, without additional immune-based therapy, may not have any benefit in chronic HIV-1 disease.

Acknowledgements

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosure
  9. References

The authors would like to acknowledge the contribution of the staff and patients of the St Stephen's Centre who took part in this study. This work was funded by the St Stephen's AIDS Trust, Westminster Medical School Research Trust, AVIP EU Programme (grant number LSHP-CT-2004–503487) and MRC (grant number G0501957).

Disclosure

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosure
  9. References

The authors declare no conflict of interest or financial interests.

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  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosure
  9. References
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