Inadequate local cell-mediated immunity appears crucial for the establishment of chronic HIV infection. Accumulation of regulatory T cells (Treg) at the site of HIV replication, the lymphoid organs, may influence the outcome of HIV infection. Our data provide the first evidence that chronic HIV infection changes Treg tissue distribution. Several molecules characteristics of Treg (FoxP3, CTLA-4, glucocorticoid-induced TNFR family-related receptor, and CD25) were expressed more in tonsils of untreated patients compared with antiretroviral-treated patients. Importantly, most FoxP3+ cells expressed CTLA-4, but not CD69. Furthermore, a direct correlation between FoxP3 levels and viral load was evident. In contrast, FoxP3 expression was decreased in circulating T cells from untreated patients, but normalized after initiation of treatment. Functional markers of Treg activity (indoleamine 2,3-dioxygenase, TGF-β, and CD80) were markedly increased in the tonsils of untreated patients. Our data could provide a new basis for immune-based therapies that counteract in vivo Treg and thereby reinforce appropriate antiviral immunity.

Persistent infection with HIV is associated with inefficient anti-HIV immune responses. In most infected patients, HIV-specific CD4+ T cell proliferation is undetectable soon after primary infection (1). HIV-specific CD8+ T cell responses play an important role in limiting acute viral replication. However, decreased cytolytic function, inappropriate maturation, and limited proliferation have been found to associate with the inability of CD8+ T cells to control viral replication in chronic state (2). The molecular basis for such impaired responses is most likely multifactorial. In the present study, we have studied the potential role of endogenous regulatory T cells (Treg). 4 Treg have recently been described as a unique population of CD4+ T cells that can prevent the activation and expansion of self-reactive lymphocytes. Treg constitutively express CD25, CTLA-4, and FoxP3, a transcription factor of the forkhead family (3, 4, 5). Another marker of Treg activity is the glucocorticoid-induced TNFR family-related receptor (GITR), which appears to define this subset more accurately than CD25 (3).

The major hallmark of this T cell subset is its ability to suppress the activation of effector T cells (3, 4, 5). The mechanisms that underlie Treg function have not yet been elucidated, but ligation of CTLA-4 and secretion of anti-inflammatory cytokines have been implicated (5). Treg may also affect the properties of APC, as well as the interactions between APC and T cells. In particular, CTLA-4-expressing Treg induce APC to express the tryptophan-catabolyzing enzyme indoleamine 2,3-dioxygenase (IDO). These interactions result in APC that exert suppressive activity both in vitro and in vivo (6, 7).

The establishment and maintenance of chronic infections have recently been shown to depend on restraint of the vigor of the antimicrobial immune response by Treg (4, 8). In particular, a role for Treg in chronic HIV infection has been proposed, based on the fact that the depletion of CD4+CD25+ T cells from PBMC from HIV-infected donors resulted in increased anti-HIV T cell responses (9, 10, 11). In the present study, we show a direct correlation between FoxP3 expression in lymphoid tissue, the major site of HIV replication in vivo (12), and plasma HIV viral load. Conversely, FoxP3 expression was suppressed in circulating T cells from untreated individuals, with normalization after highly active antiretroviral therapy (HAART)-mediated control of viral replication. Moreover, functional markers of Treg activity (IDO, TGF-β, and CD80) were markedly increased in the tonsils of untreated HIV-infected patients. These data provide evidence that chronic HIV infection influences Treg tissue distribution, and provides a rationale for targeting of Treg as a strategy for therapeutic amplification of anti-HIV immune responses.

Heparinized blood samples were obtained from 20 HIV-1-infected adults (University of Cincinnati), with CD4 counts ranging from 13 to 488/mm3 and detectable viral loads (range, 4,118–1,221,413 copies/ml; Ultrasensitive HIV RT-PCR; Roche). They had no associated opportunistic infections or cancer. Such individuals started HAART within a few days of the initial sample. Blood samples were obtained in 17 of 20 patients after HAART initiation (median, 9.6 wk; range, 5–27 wk). Blood samples from 15 adult HIV-uninfected donors were obtained. All samples were processed within few hours of collection.

Lymphoid tonsilar biopsies were obtained surgically from six untreated and four HAART-treated adults chronically infected with HIV-1 (Karolinska Institutet). The biopsies were immediately snap frozen and kept cryopreserved, embedded in OCT (Sakura) (13). Informed consent was obtained from all the subjects and protocols were approved by the Institutional Review Boards of all participating institutions.

Total RNA extracted from purified T cells (14) and cryopreserved tissues (RNAeasy kits; Qiagen) was reverse-transcribed using SuperScript reverse transcriptase (Invitrogen Life Technologies) and random primers (Roche). Reverse transcriptase products were amplified by real-time PCR (Light-Cycler; Roche), using SYBR green (Roche) and specific primers. Melting curves and electrophoresis established the purity of the amplified bands. A threshold was set in the linear part of the amplification curve. Relative units (RU) were calculated by normalization to CD4 or GAPDH mRNA expression (14).

Biopsy samples were cut 8 μm thick, fixed in 2% formaldehyde, and blocked for endogenous biotin (Vector Laboratories) (13). Anti-human GITR (R&D Systems), CTLA-4 and CD4 (BD Pharmingen), and irrelevant isotype-matched Ab (DakoCytomation) were used. The staining reactions were developed using diaminobenzidine tetrahydrochloride and hematoxylin, and analyzed using a DMR-X microscope (Leica) and the image analysis system Quantimet Q550IW (Leica Imaging Systems). The mean size of the scanned area per section was 6.5 × 105 μm2. Expression of the markers of interest was determined as the percent positive area of the total relevant cell area (mucosal surface of the tonsil sections was excluded). Whole-section scans were performed and each biopsy was assessed twice, with result variations always <10%.

For dual staining, tissues were stained with anti-human CD3 (DakoCytomation), CD4 (RPAT4; BD Pharmingen), CD8 (289-13804; BD Pharmingen), CD25 (CLB-IL2R/TB-30; Pelicluster), CD69 (FN50; DakoCytomation), and FoxP3 (Ab2481; Novus Biologicals) followed by the appropriate Alexa Fluor-conjugated secondary Ab (Molecular Probes). Positive cells were quantified in 10 high-power fields using the Qwin 550 software and a filter-free spectral confocal microsope (Leica TCS SP2 AOBS).

The difference between donors was assessed using Student’s t test with the Satterthwaite correction for unequal variance. Intrasubject difference was compared with a paired t test. Gene expression in biopsies was compared with the Mann-Whitney U test. Proportion of expressing cells was assessed by ordinary least-squares regression after logit transformation of the data. Correlations were determined with Spearman’s rank correlation. A two-tailed p < 0.05 was considered to be significant.

To analyze Treg in HIV infection, we quantified the expression of FoxP3, one of the most specific markers of human Treg (15, 16). FoxP3 mRNA levels were significantly decreased in peripheral T cells from untreated HIV-infected donors compared with uninfected donors (Fig. 1,A). These results are in agreement with recently reported low FoxP3 expression by T cells from a subset of HIV-infected donors (16). HAART was begun in 17 patients shortly after initial sampling, and induced significant decreases in viral load after several weeks (median decrease of 30,000 copies/ml, undetectable viral loads in 11 patients; viral loads remained undetectable in 12 of 13 of these patients after 40 wk of HAART). Strikingly, FoxP3 levels significantly increased in treated patients compared with their pre-HAART levels, reaching levels similar to those measured in uninfected donors’ cells (Fig. 1 A).

FIGURE 1.

FoxP3 mRNA expression in T cells from untreated HIV-infected donors was decreased in blood but increased in lymphoid organs. A, FoxP3 expression in peripheral blood T cells. T cells were purified in uninfected (HIV) donors (n = 15), and HIV-infected (HIV+) donors before (n = 20) and after HAART (n = 17). Results (mean values ± SE) are expressed in RU after normalization for CD4 mRNA expression. The p values indicate the difference between the groups (by unpaired or paired t test). Similar results were obtained when FoxP3 expression was normalized for GAPDH expression, with significantly decreased expression in untreated HIV+ subjects compared with either HIV subjects (0.22 vs 0.44; p = 0.012) or post-HAART samples (0.22 vs 0.42; p = 0.04). B, FoxP3 expression in tonsil biopsies of HIV-infected donors. FoxP3 expression was normalized for GAPDH expression. Horizontal bars within boxes correspond to the median; box limits correspond to the 25th and 75th percentiles; vertical lines extend to the 10th and 90th percentiles. The p value indicates the difference between the two groups (Mann-Whitney U test). C, Correlation between FoxP3 expression in tonsil biopsies of HIV-infected donors and viral load. Untreated donors and HAART-treated donors are represented by closed (•) and open symbols (○), respectively. The p value indicates the level of significance (Spearman correlation coefficient, ρ = 0.812).

FIGURE 1.

FoxP3 mRNA expression in T cells from untreated HIV-infected donors was decreased in blood but increased in lymphoid organs. A, FoxP3 expression in peripheral blood T cells. T cells were purified in uninfected (HIV) donors (n = 15), and HIV-infected (HIV+) donors before (n = 20) and after HAART (n = 17). Results (mean values ± SE) are expressed in RU after normalization for CD4 mRNA expression. The p values indicate the difference between the groups (by unpaired or paired t test). Similar results were obtained when FoxP3 expression was normalized for GAPDH expression, with significantly decreased expression in untreated HIV+ subjects compared with either HIV subjects (0.22 vs 0.44; p = 0.012) or post-HAART samples (0.22 vs 0.42; p = 0.04). B, FoxP3 expression in tonsil biopsies of HIV-infected donors. FoxP3 expression was normalized for GAPDH expression. Horizontal bars within boxes correspond to the median; box limits correspond to the 25th and 75th percentiles; vertical lines extend to the 10th and 90th percentiles. The p value indicates the difference between the two groups (Mann-Whitney U test). C, Correlation between FoxP3 expression in tonsil biopsies of HIV-infected donors and viral load. Untreated donors and HAART-treated donors are represented by closed (•) and open symbols (○), respectively. The p value indicates the level of significance (Spearman correlation coefficient, ρ = 0.812).

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One possible explanation for these data is that Treg are preferentially sequestered in lymphoid tissue during HIV replication. To examine this possibility, we analyzed FoxP3 expression in tonsil biopsies from chronically HIV-infected donors (Table I). FoxP3 mRNA levels were 5-fold higher in samples from untreated donors than in those of treated patients (Fig. 1,B). Strikingly, FoxP3 mRNA levels in the tonsils were highly correlated with the patients’ plasma viral loads (Fig. 1,C). We then analyzed these biopsies for markers expressed by Treg. Both CTLA-4 and GITR expression were significantly higher in tonsils from untreated patients compared with treated patients’ samples (Fig. 2). CTLA-4- and GITR-expressing cells were predominantly localized to the parafollicular T cell-enriched areas (Fig. 2).

Table I.

Clinical characteristics of the patients in whom a biopsy sample was obtained

PatientsViral LoadaCD4bStagec
U1d 1,800 470 A1 
U2 241,861 464 A1 
U3 >100,000 282 A1 
U4 107,204 303 A1 
U5 405,700 136 
U6 78,599 391 A2 
T1d <50 700 B2 
T2 <50 340 A3 
T3 <50 1,220 A2 
T4 <50 740 A2 
PatientsViral LoadaCD4bStagec
U1d 1,800 470 A1 
U2 241,861 464 A1 
U3 >100,000 282 A1 
U4 107,204 303 A1 
U5 405,700 136 
U6 78,599 391 A2 
T1d <50 700 B2 
T2 <50 340 A3 
T3 <50 1,220 A2 
T4 <50 740 A2 
a

Viral loads are expressed in copies per milliliter. For statistical analysis, donor T1–T4’s viral loads were assigned an arbitrary value of 49; donor U3′s viral load was assigned an arbitrary value of 100,001.

b

CD4 are expressed in cells per cubic millimeter.

c

HIV stage disease is indicated according to the CDC classification.

d

U, Untreated patients; T, treated patients, who had received HAART for at least 2 years at the time the biopsy was taken.

FIGURE 2.

Increased expression of CTLA-4 and GITR in tonsils from untreated HIV-infected donors. Expression of CTLA-4 (left column) and GITR (right column) was analyzed in the biopsy from one untreated (top row) and one HAART-treated (middle row) representative HIV-infected individual. Magnification is ×340. Results obtained in six untreated and four HAART-treated patients are summarized in the lowest row. The p values indicate the difference between the two groups (ordinary least-squares). CD4 expression was also increased in untreated donors (median of 11.9 vs 8.9%; results not shown).

FIGURE 2.

Increased expression of CTLA-4 and GITR in tonsils from untreated HIV-infected donors. Expression of CTLA-4 (left column) and GITR (right column) was analyzed in the biopsy from one untreated (top row) and one HAART-treated (middle row) representative HIV-infected individual. Magnification is ×340. Results obtained in six untreated and four HAART-treated patients are summarized in the lowest row. The p values indicate the difference between the two groups (ordinary least-squares). CD4 expression was also increased in untreated donors (median of 11.9 vs 8.9%; results not shown).

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These data demonstrate that expression of three Treg markers was significantly increased in lymphoid tissues from patients with active HIV replication. However, because GITR and CTLA-4 can be up-regulated briefly during the activation of non-Treg, these data could derive from the presence of activated non-Treg. Our previous results do not favor this hypothesis because FoxP3 mRNA expression was decreased in untreated donors’ peripheral T cells despite concomitant up-regulation of the activation markers CD69 and CD134 (14). To determine more precisely the role of immune activation in our findings, we determined the expression of FoxP3 protein by immunohistochemistry. More than 90% of FoxP3+ cells were CD4+ T cells, and >80% of FoxP3+ cells also expressed CTLA-4 (Fig. 3 and Table II). However, despite the fact that prevalence of CD69+ cells in untreated donors was significantly increased (median of 2.85 vs 0.71% in treated patients; p = 0.003), only a minority of FoxP3+ T cells expressed CD69 (∼10%) (Fig. 3 and Table II). Our findings thus suggest that Treg distribution is influenced by local persistent Ag stimulation. Of note, a similar redistribution pattern has been reported in human cancer, because Treg frequency was higher in metastatic lymph nodes than in tumor-free lymph nodes and PBMC (15). A role for functional Treg in peripheral lymphoid tissues of untreated HIV-infected individuals seems paradoxical, as the dominant phenotype is one of activation. However, a similar phenotype has been described in rheumatoid arthritis patients, in whom significant accumulation of functional Treg at the site of inflammation was nevertheless incapable of completely controlling inflammation (17). The underlying mechanisms are not known, but this apparent paradox suggests a complex balance between effector T cells and Treg, with negative feedback mechanisms likely to control such balance (17).

FIGURE 3.

FoxP3-expressing cells also expressed CTLA-4 but not CD69. Coexpression of FoxP3 and CTLA-4 (A) and CD69 (B) was analyzed by confocal microscopy in the tonsil biopsy of a representative untreated HIV-infected donor. Expression of FoxP3 is in green, and the other marker is in red. Magnification is ×640.

FIGURE 3.

FoxP3-expressing cells also expressed CTLA-4 but not CD69. Coexpression of FoxP3 and CTLA-4 (A) and CD69 (B) was analyzed by confocal microscopy in the tonsil biopsy of a representative untreated HIV-infected donor. Expression of FoxP3 is in green, and the other marker is in red. Magnification is ×640.

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Table II.

Characterization of FoxP3+ cells in tonsils from HIV-infected donors

UntreatedaTreateda
CD4 91.2 (90.6–96.5) 92.7 (92.0–93.9) 
CD8 7.6 (6.9–9.2) 6.8 (6.2–8.3) 
CTLA-4 86.1 (85.6–90.0) 82.4 (77.7–87.8) 
CD69 12.2 (9.6–13.4) 9.2 (8.5–9.4) 
CD25 19.4 (14.9–20.4) 15.9 (11.8–17.6) 
UntreatedaTreateda
CD4 91.2 (90.6–96.5) 92.7 (92.0–93.9) 
CD8 7.6 (6.9–9.2) 6.8 (6.2–8.3) 
CTLA-4 86.1 (85.6–90.0) 82.4 (77.7–87.8) 
CD69 12.2 (9.6–13.4) 9.2 (8.5–9.4) 
CD25 19.4 (14.9–20.4) 15.9 (11.8–17.6) 
a

Median (range) percentage of FoxP3+ cells that also expressed the specific marker.

We also studied CD25 expression in the FoxP3+ population in tonsil tissues (Table II). Surprisingly, only ∼20% of FoxP3+ cells expressed CD25 despite the fact that CD25 expression tended to be higher in untreated donors (2.81 vs 1.91% in treated donors; p = 0.3). Expression of CD25 was very diffuse, suggesting intense shedding, as described earlier (18). Our results showing that FoxP3 expression strongly associates with CTLA-4 expression, but not with CD25, are reminiscent from the reported loss of CD25 expression in Treg that have undergone in vivo expansion (19). It is thus likely that Treg in HIV-infected patients will have a different phenotype in lymphoid compartments from the one described in peripheral blood.

Our results suggest that HIV infection results in either the local expansion of Treg and/or their selective recruitment to lymphoid organs. TGF-β1 promotes local Treg expansion (20). Therefore, we analyzed TGF-β1 mRNA expression in the same samples. TGF-β1 expression showed a 2-fold increase in untreated compared with HAART-treated patients (Table III). HIV-induced production of TGF-β by lymphocytes (21) may thus participate in the local expansion of Treg in HIV-infected donors. Little is known about the mechanisms of recruitment of Treg into lymphoid organs. Some circulating human Treg express the α4β1 integrin (22), a homing receptor for T lymphocytes into inflamed tissues that express high levels of its ligand, VCAM-1. It is therefore possible that α4β1-expressing Treg are preferentially recruited into lymphoid organs in which HIV replication is occurring, as suggested by the enhanced adhesion to VCAM-1 of lymphocytes purified from HIV-infected lymph nodes (23).

Table III.

Increased mRNA expression of TGF-β, IDO, and CD80 in tonsils from untreated HIV-infected donors

UntreatedaTreateda
TGF-β 11.2 (7.1–12.4) 6.8 (2.0–9.4) 
IL-10 1.4 (0.7–2.6) 1.0 (0.8–1.2) 
IDO 4.7 (2.7–7.1)b 1.5 (0.9–1.7) 
IFN-γ 0.4 (0.1–0.9) 0.1 (0.1–0.2) 
CD80 10.6 (4.4–16.8) 4.7 (3.0–7.3) 
CD86 0.6 (0.4–1.2) 0.6 (0.4–0.7) 
UntreatedaTreateda
TGF-β 11.2 (7.1–12.4) 6.8 (2.0–9.4) 
IL-10 1.4 (0.7–2.6) 1.0 (0.8–1.2) 
IDO 4.7 (2.7–7.1)b 1.5 (0.9–1.7) 
IFN-γ 0.4 (0.1–0.9) 0.1 (0.1–0.2) 
CD80 10.6 (4.4–16.8) 4.7 (3.0–7.3) 
CD86 0.6 (0.4–1.2) 0.6 (0.4–0.7) 
a

Median (range) gene expression, expressed in RU after normalization for GAPDH expression.

b

Values in bold indicate a significant difference between the two groups (Mann-Whitney test).

Secretion of IL-10, as well as CTLA-4 ligation, have been suggested as mechanisms mediating Treg activity (5). CTLA-4+ Treg up-regulate IDO in APC, through an IFN-γ-dependent pathway. We therefore analyzed IL-10, IDO, and IFN-γ mRNA expression in tonsil tissues. IL-10 levels were similar in both groups (Table III). Of note, Treg purified from HIV-infected donors’ blood acted through IL-10-independent suppressor mechanisms (10, 11), suggesting that IL-10 may not play a major role in Treg activity in HIV infection. Conversely, HAART significantly decreased the expression of IDO and IFN-γ mRNA (Table III). The accumulation of CTLA-4+ cells in the lymphoid organs during chronic HIV infection may thus induce local IDO expression. In vitro HIV infection of APC also up-regulates IDO; however, this property is not shared by all HIV strains (24). It is therefore unlikely that direct infection of APC represents the sole mechanism underlying our results. Because localized control of tryptophan catabolism in tissues constitutes a potent mechanism for peripheral tolerance (6), IDO induction could play a major role in Treg-mediated suppression in HIV infection.

CD80 appears to be the functional ligand of CTLA-4 and is thus involved in the negative control of immune responses (25). Therefore, we analyzed CD80 and CD86 mRNA expression in the tonsil samples from HIV-infected patients, as a marker of functional interactions with CTLA-4+ cells. CD80 expression was >2-fold higher in untreated compared with HAART-treated donors. In contrast, CD86 expression was similar in the two groups (Table III). These results reinforce our hypothesis of increased Treg activity in the lymphoid organs during HIV replication, because CD80 ligation promotes regulatory function by interacting with CTLA-4 (25).

This study provides the first evidence that chronic HIV infection influences the tissue distribution of Treg in humans. Moreover, our data demonstrate a marked increase of several functional markers associated with Treg activity in the lymphoid tissues of untreated HIV-infected patients, compared with patients in whom HAART had controlled viral replication. This accumulation of Treg in lymphoid tissue could play a major role in the institution/maintenance of an environment that would favor the virus maintenance, by hampering protective immune mechanisms. Importantly, strong in vitro HIV-specific Treg function mediated by peripheral Treg was reported to correlate with favorable clinical markers (11). Taken together, our findings and the above report suggest a model in which selective recruitment and/or expansion of Treg at the lymphoid site of massive HIV replication have a detrimental effect on protection against HIV disease progression. Moreover, increased Treg numbers in lymphoid organs may explain the poor success of immune-based therapies in untreated SIV-infected macaques as well as in HIV-infected patients (26, 27). Thus, our findings could provide a new basis for designing therapies that counteract Treg in vivo and thereby reinforce antiviral immunity.

The authors have no financial conflict of interest.

We thank all of the patients involved in this study. We are also grateful to Dr. C. Fichtenbaum and L. Hinds (University of Cincinnati) for their involvement in the follow-up of treated patients. We also thank Lena Radler for expert help, as well as Dr. Y. Belkaid, D. Jankovic, and C. Karp for their critical reading of this manuscript.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1

C.A.C. is partially supported by a grant from the Trustee Board of the Cincinnati Children’s Hospital Medical Center; J.A. has grants from The Swedish Research Foundation, The Swedish Strategic Research Foundation, and The Swedish Cancer Foundation; and G.M.S. is supported by the intramural research program, Center for Cancer Research, National Cancer Institute, National Institutes of Health.

4

Abbreviations used in this paper: Treg, regulatory T cell; GITR, glucocorticoid-induced TNFR family-related receptor; IDO, indoleamine 2,3-dioxygenase; HAART, highly active antiretroviral therapy; RU, relative unit.

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