Suppressive activity of regulatory T cells correlates with high CD4+ T-cell counts and low T-cell activation during chronic simian immunodeficiency virus infection : AIDS

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Suppressive activity of regulatory T cells correlates with high CD4+ T-cell counts and low T-cell activation during chronic simian immunodeficiency virus infection

Karlsson, Ingrida,b; Malleret, Benoîta,d; Brochard, Patriciaa,c; Delache, Benoîta,c; Calvo, Juliena; Le Grand, Rogera,c; Vaslin, Brunoa,c

Author Information

aCEA, Division of Immuno-Virology, Institute of Emerging Diseases and Innovative Therapies (IMETI), Fontenay-aux-Roses, France

bDepartment of Virology, Statens Serum Institute, Copenhagen, Denmark

cUniversité Paris-Sud, UMR-E01, Orsay, France

dLaboratory of Malaria Immunobiology, Singapore Immunology Network, Agency for Technology and Research (A*STAR), Biopolis, Singapore, Singapore.

Received 1 July, 2010

Revised 17 November, 2010

Accepted 1 December, 2010

Correspondence to Bruno Vaslin, PhD, CEA, Institut des Maladies Emergentes et Thérapies Innovantes (IMETI), Service d'Immuno-Virologie (SIV), 18, route du panorama, BP 6, F-92265 Fontenay-aux-Roses Cedex, France. Tel: +33 1 46 54 94 78; fax: +33 1 46 54 77 26; e-mail: [email protected]

AIDS 25(5):p 585-593, March 13, 2011. | DOI: 10.1097/QAD.0b013e3283437c7b
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Abstract

Introduction

Regulatory T cells, which inhibit the activation and effector functions of T cells, are important regulators of immune responses. Several subsets of regulatory T cells have been described, including natural CD25+ regulatory T cells [1,2], type 1 T-regulatory (Tr1) cells [3] and T-helper 3 (Th3) cells [4]. Natural CD25+ regulatory T cells exert their inhibitory effects principally through a cell–cell contact-dependent mechanism [5], whereas Tr1 and Th3 cells have been reported to suppress the immune response by secreting the immunosuppressive cytokines interleukin (IL)-10 and transforming growth factor-β (TGF-β) [3,4].

Natural regulatory T cells have been studied the most thoroughly. They limit the expansion and activation of CD4+ T-cell populations and have been shown to influence various inflammatory processes, including microbial infections [6] and autoimmune diseases [7]. Natural regulatory T cells are characterized by the expression of various markers. The most widely used of these markers is CD25, both alone and in combination with FoxP3. FoxP3 expression is associated with immune suppression and the expression of FoxP3 may render nonregulatory T cells suppressive [8,9]. Another well described phenotypic and functionally important marker of regulatory T cells is cytotoxic T-lymphocyte antigen (CTLA)-4 [10,11]. The blockade of CTLA-4 has been shown to reverse the suppressive effect of regulatory T cells [11].

CD4+CD25+ regulatory T cells are thought to influence the pathogenesis of HIV infection [12]. The number of peripheral regulatory T cells has been reported to decrease in patients with chronic HIV infection [13–16] and rhesus macaques experimentally infected with SIV [17]. Direct infection and killing have been proposed as possible causes of the decrease in number of these cells [13]. The recovery and/or maintenance of CD4+CD25+ regulatory T cells has been associated with low viral load and low immune activation in rhesus macaques with chronic SIV infection [17] and HIV-1-infected patients with low/undetectable viral load [18]. Several studies have shown that CD4+ regulatory T cells from HIV-infected patients inhibit T-cell responses in vitro[19–23] and that this ability to suppress T-cell responses is negatively correlated with plasma viral load [21]. In contrast to the decrease in the number of peripheral regulatory T cells observed during chronic HIV infection, some studies have shown an accumulation of regulatory T cells in lymphoid tissue during HIV infection in humans [16,24] and SIV infection in rhesus macaques [25]. This increase in regulatory T-cell numbers in lymphoid tissue has been correlated with disease progression of HIV infection [16,24]. However, others and we have not been able to detect an accumulation of regulatory T cells in lymphoid tissue during pathogenic SIV infection of rhesus macaques, pigtailed macaques and cynomolgus macaques [17,26,27].

In a previous study, we characterized CD4+CD25+FoxP3+ T cells in cynomolgus macaques and followed the dynamics of these cells during the course of SIVmac251 infection [26]. The phenotypic characteristics of CD4+CD25+FoxP3+ T cells in cynomolgus macaques were similar to those described in humans expressing markers of memory cells, a high expression of CTLA-4 and low/negative expression of CD127 [26]. They also partly displayed expression of the main HIV/SIV coreceptors, C-C chemokine receptor type 5 (CCR5) and C-X-C chemokine receptor type 4 (CXCR4). During infection of cynomolgus macaques with SIVmac251 the number of CD4+CD25+ regulatory T cells declined in peripheral blood, whereas in peripheral lymph nodes, their proportions remained stable [26]. Here, we have further studied the role of regulatory T cells in the pathogenesis of SIV infection.

We analyzed the in-vitro suppressive capacity of CD25+ regulatory T cells isolated during chronic SIVmac251 infection in cynomolgus macaques. Suppressive capacity of CD25+ cells isolated from peripheral blood and peripheral lymph nodes correlated with sustained CD4+ T cells in these compartments. Moreover, the suppressive capacity of CD25+ cells isolated from peripheral blood correlated with lower activation of peripheral T cells. This suggests that regulatory T-cell function is lost during disease progression and may have controlled T-cell activation. Regulatory T cells may, therefore, participate in the control of HIV/SIV disease progression.

Materials and methods

Animals and infections

We studied 18 young adult male cynomolgus macaques (Macaca fascicularis), each weighing 2.7–4.5 kg, imported from Mauritius and kept according to European guidelines for animal care (‘Journal Officiel des Communautés Européennes’, L358, 18 December 1986). Eighteen macaques were inoculated, via the saphenous vein, with a cell-free virus stock of pathogenic SIVmac251 (kindly provided by A. M Aubertin, Université Louis Pasteur, Strasbourg, France) in 1 ml of PBS. Virions were obtained from the cell-free supernatant of infected rhesus peripheral blood mononuclear cell (PBMC). Cells were infected in vitro with a culture supernatant obtained from a coculture of rhesus PBMC and a spleen homogenate from a rhesus macaque infected with SIVmac251 (provided by R. C. Desrosiers, New England Regional Primate Center, Southborough, Massachusetts, USA). This stock is, therefore, considered close to a primary isolate of SIVmac passaged only on primary cells. The stock was titrated after intravenous, intrarectal and intravaginal inoculation [28,29]. Six animals received 5000 50% animal infectious doses (AID50) of the virus. Twelve animals received 50 AID50, six of which were in addition treated postexposure with azydothymidine (ZDV; 4.5 mg/kg), 2′,3′-dideoxy-3′-thiacytidine (3TC; 2.5 mg/kg) and indinavir (20 mg/kg). Drugs were given orally, twice per day, from 4 h postinfection until 28 days postinfection, as previously described [30]. This cohort allowed animals distributed largely on the progression scale. CD4+ T-cell counts and plasma viral load of the 18 macaques on day 273 postinfection for this single time point cross-sectional analysis are shown in Supplemental Digital Content 1, https://links.lww.com/QAD/A112 and more detailed longitudinal history of these animals is shown in Supplemental Digital Content 2, https://links.lww.com/QAD/A112, as reported previously [26,31]. PBMC samples were taken on day 273 after exposure and lymph node mononuclear cell (LNMC) samples were taken on day 245 postinfection, within clinically asymptomatic phase of chronic infection. LNMC and PBMC samples were also taken before SIV infection.

Determination of plasma simian immunodeficiency virus RNA load

Plasma SIV-RNA load was determined as previously described [26]. All amplifications of RNA extracted from plasma samples and seven order of magnitude standard virus stock dilutions were performed in duplicate. A correlation coefficient of up to 97% was obtained, with a detection level of 100 copies/ml.

CD4+ T-cell counts

CD4+ T-cell counts were determined as previously described [26]. Briefly, 30 μl of whole blood was immunostained with anti-CD3 fluorescein isothiocyanate (FITC; clone FN18; CliniSciences, Montrouge, France) and anti-CD4 PE (clone L200; BD Biosciences, Grenoble, France) antibodies, followed by red blood cell lysis and data acquisition using a BD FACScan instrument (BD Biosciences) and CellQuest software (BD Biosciences). The proportions of CD3+CD4+ cells were determined in the lymphocyte gate, defined in terms of light-scattering properties, using CellQuest software. Absolute counts were calculated from the absolute blood count of lymphocytes obtained by automated cell counting (Coulter MDII; Coultronics, Margency, France).

Cell isolation

PBMCs were isolated from whole blood using CPT tubes (BD Biosciences), according to the manufacturer's recommendations. Residual red blood cells were lysed by hypotonic shock for 5 min, followed by washing in PBS. LNMCs were isolated from whole axillary lymph nodes, mechanically disrupted and passed through a strainer with 40 μm pores (BD Biosciences), and cells were washed in PBS. CD25+ cells were depleted from PBMCs and LNMCs using nonhuman primate anti-CD25-coated microbeads and LD columns according to manufacturer's protocol (Miltenyi Biotec, Paris, France).

Quantification of CD4+ regulatory T cells

Quantification of FoxP3+CD25+ regulatory T cells was performed as previously described [26]. Briefly, PBMCs were immunostained with anti-CD3 FITC, anti-CD4 peridinin-chlorophyll protein (PerCP), anti-CD25 PE (clone 4E3, Miltenyi Biotec) and anti-FoxP3 allophycocyanin (APC; clone PCH101; eBioscience, San Diego, California, USA) antibodies. FoxP3 Fix/Perm buffers were used for the intracellular staining of FoxP3 according to manufacturer's protocol (eBioscience). Data were acquired on a BD LSRI instrument (BD Biosciences), using CellQuest software, and analyzed with FlowJo software (Tree Star, Ashland, Oregon, USA).

Labeling of activation markers

PBMCs were immunostained with anti-CD3 FITC, anti-CD4 PerCP and anti-CD25 PE, anti-human leukocyte antigen (HLA)-DR PE (clone L243, BD Biosciences) or anti-Ki67 PE (clone B56, BD Biosciences) antibodies. Fluorescence activated cell sorter (FACS) lysing and FACS permeabilization solutions (BD Biosciences) were used for the intracellular staining of Ki67 according to manufacturers' protocol. Data were acquired on a BD LSRI instrument, using CellQuest software, and analyzed with FlowJo software.

T-cell proliferation assay

PBMCs (1 × 105) or LNMCs were seeded in flat-bottomed 96-well plates. CD25-depleted (CD25−) and CD25-selected (CD25+) fractions were cultured alone or at a 1: 1 ratio (total 2 × 105 cells). Cells were either cultured alone in media (Roswell Park Memorial Institute (RPMI) medium 1640 supplemented with 10% fetal calf serum and antibiotics) or together with 5 × 104 Dynabeads M450 (Invitrogen, Cergy Pontoise, France) coated with anti-CD3 (10 μg/ml, clone FN18) and anti-CD28 (10 μg/ml, clone 28.2, BD Biosciences) during 5 days. One microcurie of [3H]thymidine (Amersham Biosciences, Orsay, France) was added during the final 8 h of culture. Cells were harvested onto glass fiber filters using a cell harvester (SKATRON, Lier, Norway). [3H]Thymidine incorporation was measured in a Microbeta 1450 Liquid Scintillation Counter (EG&G Wallac, Turku, Finland). Results are expressed as stimulation indexes by dividing the mean counts/min of triplicate anti-CD3+ anti-CD28-stimulated activated wells by the mean counts/min for the triplicate negative control wells. The background proliferation differed between samples, but was generally less than 1.000 counts/min.

Statistical analysis

The Mann–Whitney U-test was used to compare the plasma viral load, CD4+ T-cell counts and proportions of CD25+FoxP3+ cells of different groups of macaques. Spearman's rank correlation test was used for correlations of T-cell activation and clinical parameters. Wilcoxon signed-rank test was used for comparing proliferative activity between total PBMC (or LNMC) and CD25− PBMC (or LNMC). Statview software (SAS Institute Inc., Cary, North Carolina, USA) was used for statistical analysis.

Results

Identification and isolation of CD25+ regulatory T cells in cynomolgus macaques

CD4+ regulatory T cells were identified by flow cytometry and defined as CD25+FoxP3+. FoxP3 were typically expressed within the CD4+CD25high population in PBMC, as well as in LNMC (see Figure, Supplemental Digital Content 3A, https://links.lww.com/QAD/A112). The proportion of CD25+FoxP3+ cells within CD4+ T cells were higher in LNMC compared with PBMC with an average proportion of 7.1 ± 2.3% and 2.0 ± 0.5%, respectively (see Figure, Supplemental Digital Content 3A, https://links.lww.com/QAD/A112 and Tables 2 and 3). FoxP3 is an intracellular marker and, therefore, cannot be used as marker for selection of live cells; instead, CD25 was targeted for depletion and enrichment of regulatory T cells. CD25+-positive cells were sorted out by positive magnetic bead selection giving rise to more than 95% pure CD25− fractions (see Figure, Supplemental Digital Content 3B and 3C, https://links.lww.com/QAD/A112). The resulting positively enriched fraction, thereafter called CD25+, was less pure with an average of 57 ± 6% CD25+ cells (see Figure, Supplemental Digital Content 3B and 3C, https://links.lww.com/QAD/A112).

In-vitro suppressive activity of CD25+ cells from peripheral blood and peripheral lymph nodes

During chronic infection, the SIV-specific proliferation in PBMC (day 273 postinfection) and LNMC (day 245 postinfection) was low with few responding macaques [31]. Consequently, we used an antigen-nonspecific stimulation, stimulation with anti-CD3 with anti-CD28 antibodies, to study the in-vitro suppressive capacity of CD25+ regulatory T cells during chronic infection. Macaques were considered suppressors when stimulation index of the CD25− (depleted fraction) was at least 10% higher than stimulation index of the total cell population, being significantly different (P = 0.0156). In peripheral blood, seven of 17 macaques displayed suppressive activity of CD25+ cells (Fig. 1a and Table 1). In one macaque, not enough PBMC could be isolated to perform the CD25 depletion. Similarly, in peripheral lymph node cells, seven of 18 macaques exhibited suppressive activity of CD25+ cells (Fig. 1b and Table 2), being significantly different (P = 0.0156). However, suppressive activity in peripheral blood did not necessarily correspond to suppressive activity in peripheral lymph nodes with only three macaques showing evidence of suppressive activity of CD25+ cells in both compartments. When there was a higher proliferation in the CD25−-negative fraction than in total mononuclear cells, the suppressive capacity of CD25+ cells was confirmed by readding these cells to the CD25− fraction in a 1: 1 ratio (Fig. 2).

F1-6
Fig. 1:
The effect of CD25 depletion on proliferative capacity of peripheral blood mononuclear cell and lymph node mononuclear cell. Proliferative response to anti-CD3 with anti-CD28 stimulation of the total cell fraction and CD25-depleted fraction in (a) PBMC and (b) LNMC. Proliferation is expressed as stimulation indexes (SIs) and suppressors are defined as at least 10% higher SI in the CD25-negative fraction compared with the undepleted fraction. Underlined macaques (15 596, 20 565 and 20 695) showed suppressive activity in both compartments. In suppressors, the SIs are significantly higher in the CD25− peripheral blood mononuclear cell (PBMC) and CD25− lymph node mononuclear cell (LNMC), P = 0.0156 and P = 0.0156, respectively.
T1-6
Table 1:
Biological markers of progression and functional characteristics of peripheral blood of 17 cynomolgus macaques infected with SIVmac251.
T2-6
Table 2:
Clinical and functional characteristics of peripheral lymph nodes of 18 cynomolgus macaques infected with SIVmac251.
F2-6
Fig. 2:
The suppressive activity is restored when readding the CD25+ cells. Proliferative responses to anti-CD3 with anti-CD28 stimulation in undepleted fractions [peripheral blood mononuclear cell (PBMC)/lymph node mononuclear cell (LNMC)], CD25-depleted fraction (CD25−), CD25-purified fraction (CD25+) and CD25-depleted fraction with CD25-purified fraction in a 1: 1 ratio in PBMC (a) and LNMC (b). Showing three representative macaques: 20 695, 20 613 and 15 729. Macaque 20 695 displayed suppressive activity in PBMC as well as LNMC, macaque 20 613 in LNMC but not in PBMC and macaque 15 729 in PBMC but not in LNMC. Proliferation is expressed as stimulation indexes (SIs).

In-vitro suppressive activity associated with high CD4+ T-cell counts

On the basis of the antigen-nonspecific in-vitro-suppressive capacity of CD25+ cells in PBMC and LNMC, the macaques were divided into suppressors and nonsuppressors (Tables 1 and 2). PBMCs of suppressors had significantly higher (P = 0.0006) number of CD4+ T cells in peripheral blood, compared with nonsuppressors. The seven macaques with suppressive CD25+ cells in PBMC were the seven macaques with the highest CD4+ T-cell counts in peripheral blood (>700 cell/μl). Moreover, PBMC of suppressors displayed a significantly lower (P = 0.0047) loss of CD4+ T cells compared with baseline. Similarly, LNMC suppressors had significantly higher (P = 0.0145) number of CD4+ T cells in peripheral lymph nodes. Six of seven macaques with suppressive CD25+ cells in LNMC were within the eight macaques with the highest number of CD4+ T cells in peripheral lymph nodes (>70 × 106 cells/g of tissue). As in peripheral blood, the LNMC nonsuppressors appeared to have a lower loss of CD4+ T cells in peripheral lymph nodes compared with baseline (P = 0.0572). Although there were very good associations between suppressive activity and preserved CD4+ T cells, there was no significant association between the suppressive activity and the level of plasma viral load (Tables 1 and 2), suggesting that CD4+ T-cell count was more relevant to regulatory T-cell activity in this assay.

In-vitro suppressive activity of CD25+ cells was not associated with a higher proportion of CD25+FoxP3+ cells within CD4+ T cells (Tables 1 and 2). Even though there was a higher level of CD25+FoxP3+ in LNMC than in PBMC, there was not a higher suppressive activity in LNMC.

In-vitro suppressive activity associated with low T-cell activation

When comparing the expression of the activation markers Ki67, HLA-DR and CD25 on peripheral T cells in suppressors and nonsuppressors (on the basis of the in-vitro suppressive capacity of CD25+ cells in PBMC, Table 1), we identified a significantly lower expression of Ki67 (P = 0.0373) and HLA-DR (P = 0.0297) on CD4+ T cells from suppressors (Table 3). The same comparison also indicated a tendency to lower expression of Ki67 (P = 0.1473) and HLA-DR (P = 0.0701) on CD8+ T cells from suppressors. As CD25 is not only a marker for regulatory T cells but also for activated T cells, we also included this marker in this analysis. However, CD25 expression by T cells was not significantly different between the two groups. Moreover, high expression of the activation markers Ki67 and HLA-DR on CD4+ T cells negatively correlated with CD4+ T-cell counts (P = 0.0160 and P = 0.0077, respectively) (Table 3).

T3-6
Table 3:
Activation status of T cells in peripheral blood of 18 cynomolgus macaques infected with SIVmac251.

Discussion

Recent studies on HIV/SIV pathogenesis have suggested that regulatory T cells play a role in determining the outcome of disease [12–17,19–21]. Exhaustion of the immune system, with the aberrant expression of inflammatory cytokines, upregulation of inhibitory T-cell surface markers and decrease in T-cell proliferative capacity have been identified as making a major contribution to disease progression during chronic HIV infection [32–34]. Also, reduced low level of T-cell activation has been suggested to have a protective role in nonpathogenic SIV infection in African green monkeys [35] and sooty mangabeys [36]. T-cell activation is a better predictor of disease progression than plasma viral load [37]. Immune suppression by regulatory T cells during chronic HIV infection may be beneficial for the host by attenuating chronic immune activation and exhaustion [38]. In this study, we demonstrate that the in-vitro suppressive capacity of CD25+ regulatory T cells isolated from peripheral blood and peripheral lymphoid tissue is associated with preserved CD4+ T-cell counts in these compartments and lower T-cell activation in peripheral blood.

In a previous report, we described a decrease of CD4+CD25+FoxP3+ regulatory T cells during the primary and chronic SIV infection of cynomolgus macaques in peripheral blood [26], consistent with studies in HIV-infected patients [13–16], and SIV-infected rhesus macaques [17,27]. Low numbers of CD4+CD25+FoxP3+ regulatory T cells were associated with high plasma viral load during early and asymptomatic chronic infection [26]. Direct infection and killing have been proposed as possible causes of the decrease in number of CD4+CD25+FoxP3+ regulatory T cells [13], as the expression of CCR5, the main coreceptor for SIV [39], makes these cells susceptible to infection. The loss of regulatory T cells during chronic HIV/SIV infection may favor disease progression.

Lymphoid tissue, the sites at which primary immune responses to most antigens are generated, is the primary site of HIV replication [40]. There are opposing reports on the changes of regulatory T cells in lymphoid tissue during HIV/SIV infection. We did not observe any significant increase in the proportion of CD4+CD25+FoxP3+ T cells in peripheral lymph nodes in SIV-infected cynomolgus macaques, a model displaying various rates of progression, similarly to HIV infection in humans [41,42]. Likewise, Pereira et al.[17] and Chase et al.[27] did not observe any increases in the CD4+CD25+FoxP3 T cells in SIV-infected rhesus macaques and pigtailed macaques, respectively. In contrast, others have described an increase in the numbers of regulatory T cells in the lymphoid tissue of HIV-infected patients displaying progression [16,24] and during early SIV infection in rhesus macaques [25]. Discordance in the changes in regulatory T-cell populations in lymphoid tissues during HIV/SIV infection may be due to differences in the quantification methods and models used and/or differences in disease stage.

To explore further the role of regulatory T cells during HIV/SIV infection, we here evaluated the in-vitro suppressive activity of CD25+ T cells from chronically infected cynomolgus macaques. There was a strong correlation between the suppressive activity of CD25+ cells and preserved number of CD4+ T cells. This was true when comparing the in-vitro suppressive capacity of CD25+ T cells isolated from both peripheral blood and peripheral lymph nodes to the number of CD4+ T cells in the respective compartment. Moreover, we were able to see a strong association between in-vitro suppressive activity of CD25+ T cells and low T-cell activation. Although T-cell activation and regulatory T cells have been considered in pathogenic HIV/SIV infection [14,17,18,38,43] and nonpathogenic SIV infection [35,36], this is the first study in which a correlation between regulatory function and T-cell activation is shown.

Plasma viral load and CD4+ T-cell counts are used as markers of HIV/SIV disease progression [44–46]. Although we previously showed that plasma viral load negatively correlated with CD4+ T-cell counts and absolute FoxP3+CD25+CD4+ T-cell counts negatively correlated with plasma viral load [17,31], in-vitro suppressive activity of CD25+ cells was negatively correlated with CD4+ T-cell counts, but was not correlated with plasma viral load. A correlation between in-vitro suppressive activity of peripheral blood CD25+ cells and lower levels of plasma viral load has previously been described in HIV-infected patients by Kinter et al. [21]. However, the same group later suggested that CD4+ T-cell counts may be more relevant to regulatory T-cell activity [23], in accordance with our results in cynomolgus macaques. Although it cannot be excluded that preserved T-cell counts and low T-cell activation preserves CD25+ regulatory T-cell activity, we suggest that CD25+ regulatory T-cell activity has a positive impact on disease.

Suppressive activity in peripheral blood did not necessarily correspond to suppressive activity in peripheral lymph nodes with only three macaques showing evidence of suppressive activity of CD25+ cells in both compartments. Noteworthy, these macaques displayed the highest CD4 T-cell counts and lowest viral loads, indicative of the positive influence of regulatory T cells. The number of animals in this study is too limited to draw any parallels with regulatory T-cell activity in different compartments and different profiles of disease. We can only speculate that this is a result from different turnover rate and trafficking between compartments depending on wherein immune activation needs to be controlled at different time points.

Although we could detect a higher proportion of CD25 and FoxP3-expressing cells in CD4+ T cells from peripheral lymphoid tissue compared with peripheral blood, we could not demonstrate a higher in-vitro suppressive capacity by CD25+ cells from lymphoid tissue compared with peripheral blood. Equally, there were not higher proportions of FoxP3+CD25+CD4+ regulatory T cells in animals displaying functional in-vitro suppressive/regulatory T-cell activity compared with animals not displaying such activity. This is in agreement with previous studies in human HIV-infected patients [21] not detecting a correlation between in-vitro suppressive activity and the proportion of CD25+ regulatory T cells, and suggests that phenotypic identification does not necessarily correspond to in-vitro suppressive function. Moreover, a recent study by Wang et al.[47] suggests that in humans mRNA expression of a transmembrane protein called glycoprotein A repetitions predominant (GARP) more accurately defines regulatory T cells with suppressive activity than FoxP3.

In conclusion, the in-vitro suppressive activity of CD25+ regulatory T cells isolated from peripheral blood and peripheral lymphoid tissue during chronic SIV infection in cynomolgus macaques is associated with preserved CD4+ T-cell counts in both compartments and lower T-cell activation in peripheral blood. This suggests that suppression of immune activation in chronic HIV/SIV infection by CD25+ regulatory T cells may be lost during disease progression and, thereby, contributes to exhaustion of the immune system.

Acknowledgements

This study was supported by the French national AIDS agency, Agence Nationale de Recherche sur le SIDA et les Hépatites Virales (ANRS). B.M. and I.K. were both supported by grants from the ANRS. The authors would also like to thank the Lennanders Foundation, the Swedish Royal Academy of Sciences and the Faculty of Medicine of Lund University, Sweden.

B.V. and R.L.G. conceived and designed the experiments. I.K., B.M., P.B., B.D. and J.C. performed the experiments. I.K. analyzed the data. I.K. and B.V. wrote the manuscript. In addition, all the authors have read and approved the text as submitted to AIDS.

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Keywords:

macaque; pathogenesis; progression; regulatory T cell; simian immunodeficiency virus

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