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Journal of Virology, November 2007, p. 11593-11603, Vol. 81, No. 21
0022-538X/07/$08.00+0     doi:10.1128/JVI.00760-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Regulatory T-Cell Markers, Indoleamine 2,3-Dioxygenase, and Virus Levels in Spleen and Gut during Progressive Simian Immunodeficiency Virus Infection

Adriano Boasso,^1* Monica Vaccari,² Anna Hryniewicz,^2,3 Dietmar Fuchs,⁴ Janos Nacsa,² Valentina Cecchinato,² Jan Andersson,⁵ Genoveffa Franchini,² Gene M. Shearer,¹ and Claire Chougnet⁶

Experimental Immunology Branch, CCR, National Cancer Institute, National Institutes of Health, Bethesda, Maryland,¹ Animal Models and Retroviral Vaccines Section, National Cancer Institute, National Institutes of Health, Bethesda, Maryland,² Department of General and Experimental Pathology, Medical University of Bialystok Mickiewicza 2c, 15-222 Bialystok, Poland,³ Division of Biological Chemistry Biocentre, Innsbruck Medical University, Ludwig Boltzmann Institute of AIDS-Research, Innsbruck, Austria,⁴ Center for Infectious Medicine, Division of Infectious Diseases, Karolinska Institute, Karolinska University Hospital, 141 86 Stockholm, Sweden,⁵ Division of Molecular Immunology, Cincinnati Children's Hospital Research Foundation and Department of Pediatrics, University of Cincinnati College of Medicine, Cincinnati, Ohio⁶

Received 9 April 2007/ Accepted 8 August 2007

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
High levels of viral replication occur in gut-associated lymphoid tissue (GALT) and other lymphoid tissues (LT) since the early phase of human/simian immunodeficiency virus (HIV/SIV) infection. Regulatory T cells (T_reg), a subset of immunosuppressive T cells expressing CTLA-4 and the FoxP3 transcription factor, accumulate in LT during HIV/SIV infection. Here we show that FoxP3 and CTLA-4 mRNA are increased in leukocytes from the spleens, lymph nodes (LN), and mucosal sites of chronically SIV-infected macaques with high viremia (SIV_HI) compared to animals with low viremia (SIV_LO). FoxP3 and CTLA-4 correlated with SIV RNA levels in tissues; SIV virus levels in the spleen, inguinal LN, mesenteric LN, colon, and jejunum directly correlated with the plasma virus level. Importantly, CTLA-4 and FoxP3 mRNA were predominantly increased in the CD25^– subpopulation of leukocytes from SIV_HI, further challenging the classical definition of T_reg as CD4⁺ CD25⁺ T cells. Similar to CTLA-4 and FoxP3, expression of indoleamine 2,3-dioxygenase (IDO), an immunosuppressive enzyme induced by T_reg in antigen-presenting cells, was increased in the spleens, mesenteric LN, colons, and jejuna from SIV_HI compared to SIV_LO and directly correlated to SIV RNA in the same tissues. Accordingly, plasma kynurenine/tryptophan, a marker for IDO enzymatic activity, was significantly higher in SIV_HI compared to SIV_LO and correlated with plasma viral levels. Increased T_reg and IDO in LT of SIV-infected macaques may be the consequence of increased tissue inflammation and/or may favor virus replication during the chronic phase of SIV infection.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Lymphoid tissues, and the gut-associated lymphoid tissue (GALT) in particular, are important sites for viral replication and CD4⁺-T-cell depletion during human/simian immunodeficiency virus (HIV/SIV) type 1 infection(20, 27). Because these tissues are key sites for regulation of immune responses, the characterization of positive and negative immune regulators in this locale is of utmost importance to understand HIV pathogenesis and develop immune-based strategies to contain viral replication.

GALT is the largest compartment of the immune system. Constant exposure to intestinal bacterial flora, food antigens, and insulting pathogens constitutes a chronic immune stimulus within the GALT (48). Thus, even under physiological conditions, the gut mucosa requires fine-tuning of immune responses, with a tight balance between activating and suppressing signals (48). SIV and HIV replicate abundantly in GALT, and severe CD4⁺-T-cell depletion is observed at this site during acute infection(9, 19, 26, 27, 45), likely due to the fact that activated memory CCR5-expressing CD4⁺ T cells, the main target for HIV/SIV infection, are preferentially found in the GALT (26, 27). The levels of activated CD4⁺ T cells are decreased in the GALT throughout the chronic phase of infection (9).

Although viral replication has been studied extensively in lymph nodes (LN) and GALT, less is known about viral replication and T-cell dynamics in the spleen. Viral reservoirs have been described in resting CD4⁺ T cells, which carry integrated viral DNA, from the spleens of SIV-infected macaques when viral replication was suppressed by antiretroviral therapy (39). The potential importance of virus-cell interactions occurring in the spleen is confirmed by the beneficial effect reported for splenectomy on the course of HIV disease progression (3).

Despite increasing interest in the dynamics of CD4⁺-T-cell depletion in lymphoid tissues and gut mucosa, studies on the effects of viral replication on other CD4⁺ T cells at these sites remain limited. We focus here on regulatory T cells (T_reg), a specialized set of CD4⁺ T cells with immunosuppressive activity that behave differently from CD4⁺ T helper cells and exhibit increased survival when exposed to HIV (31).

T_reg constitutively express high levels of CD25, of the negative regulator of T-cell activation, cytotoxic-T-lymphocyte antigen 4 (CTLA-4), and of the transcription factor from the forkhead family, FoxP3(4, 33, 35, 37). FoxP3 is currently considered the most accurate marker for T_reg (14). Its expression is directly associated with regulatory function (23) and is required for CTLA-4 expression (43). One mechanism by which T_reg could affect immune responses is through the binding of Treg-associated CTLA-4 to B7 molecules on antigen-presenting cells (APC), leading to the expression of the tryptophan (Trp)-catabolizing enzyme indoleamine 2,3-dioxygenase (IDO) (6, 13, 18, 30) by APC, which results in powerful immunosuppressive activity (28).

Several recent studies suggest that T_reg may limit the ability of adaptive T-cell responses to control HIV and SIV replication (7, 24). In particular, anti-HIV/SIV responses were increased after in vitro removal of T_reg from peripheral leukocytes of HIV-infected patients (1, 25, 46) and from lymph nodes of SIV-infected rhesus macaques (24). Accumulation of T_reg in lymphoid tissues was associated with ineffective anti-SIV immune responses during primary infection (12), and favorable disease prognosis may depend on the activation of cell-mediated immunity in the absence of accumulating T_reg within the lymphoid tissues (31). A role for IDO in suppressing anti-HIV responses has also been proposed in a mouse model of HIV-induced encephalopathy (34). We have recently shown that inhibition of IDO in vitro increased the proliferation of CD4 T cells from HIV-infected patients (5).

Despite growing evidence that T_reg may be implicated in HIV pathogenesis (7), little is known about their repartition at sites for viral replication, such as GALT and spleen, and how viral replication affects T_reg dynamics. In the present study, we investigated such repartition in SIV-infected macaques, which is a suitable model for HIV infection of humans (10, 15). We investigated the relation between plasma and tissue virus level, a reliable clinical marker for HIV/SIV disease progression, and markers associated with T_reg function (CTLA-4 and FoxP3) and IDO in the GALT, LN, and spleens of chronically SIV-infected animals. T_reg markers were increased in colons, spleens, and LN, including mesenteric LN, of chronically SIV-infected macaques with high viremia and correlate with SIV virus levels in the same tissues. We also show that CTLA-4 and FoxP3 mRNA expression was predominantly increased in the CD25^– subpopulation of leukocytes from spleen and LN in highly viremic animals, further challenging the classical definition of T_reg as CD4⁺ CD25⁺ T cells. Increased T_reg numbers and IDO expression in lymphoid tissues, particularly the spleen and GALT, of SIV-infected macaques may favor viral replication by impairing host immune responses.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Animals. All animals were colony-bred rhesus macaques (Macaca mulatta) obtained from Covance Research Products (Alice, TX). Animals were housed and handled in accordance with the standards of the Association for the Assessment and Accreditation of Laboratory Animal Care International, and the study was reviewed and approved by the animal care and use committees at Advanced BioScience Laboratories (Kensington, MD) and Bioqual (Rockville, MD). The care and use of animals were in compliance with institutional (National Institutes of Health) guidelines. At the time of purchase, all animals were in good health, 2 to 4 years of age, and weighed 4 to 9 kg. Before the study, the animals were seronegative for SIV, simian T-cell lymphotropic virus type 1, and herpesvirus B. Twelve macaques that had been infected with SIV_mac251 for at least 2 years were enrolled in the study. Macaques were infected by the intravenous route, as previously described (42). All animals included in the study had not received antiretroviral therapy for at least 1 year (52 weeks) before euthanasia. SIV_mac251 RNA in plasma was quantified by nucleic acid sequence-based amplification (NASBA), which has a detection limit of 2 x 10³ RNA copies/ml (36). Animals were divided into two groups: low viremic (SIV_LO), which showed plasma viral levels consistently lower than 10⁴ copies/ml for at least 6 months (26 weeks) before euthanasia (Fig. 1A), and high viremic (SIV_HI), which showed plasma viral levels consistently higher than 10⁴ copies/ml for at least 6 months (26 weeks) before euthanasia (Fig. 1B). The plasma viral level at the time of euthanasia and the history of previous treatment are shown for each animal in Table 1. In addition, peripheral blood was drawn from four SIV-uninfected animals (SIV_NEG), and tissues were obtained from two SIV_NEG euthanized for non-SIV-related conditions.

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FIG. 1. Longitudinal plasma viral level measurements for the SIV-infected macaques. Plasma viral RNA was measured by NASBA, as described in Materials and Methods, in SIVLO (A) and SIVHI (B) animals. Plasma viral levels for the last 26 weeks before euthanasia are shown. For all animals, antiretroviral therapy was terminated at least 52 weeks before euthanasia. Dotted lines represent the 104 copies/ml cutoff value between SIVLO and SIVHI. ND*, under detection level.

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TABLE 1. Animals included in the study

Cell preparation and CD25 determination. Mononuclear cells from the blood, LN, and spleen were isolated by density-gradient centrifugation and resuspended in RPMI 1640 medium (Gibco-BRL, Gaithersburg, MD) containing 10% fetal calf serum. The frequency of CD4⁺ CD25⁺ T cells was measured on a FACSCalibur flow cytometer (Becton Dickinson, Franklin Lakes, NJ), using CD3-fluorescein isothiocyanate (clone SP34), CD25-phycoerythrin (PE) (clone M-A251), CD4-PerCP (clone L-200), and CD8-allophycocyanin (clone G42-8) (all from BD Pharmingen). In some experiments, mononuclear cells from the blood, LN, and spleen were sorted according to CD25 expression. Cells were incubated first with PE-conjugated anti-CD25 MAb (Becton Dickinson), washed, and incubated with anti-PE magnetic beads (Miltenyi Biotech, Auburn, CA). Labeled cells were passed twice into separation columns (Miltenyi Biotech), allowing for collection of both CD25^– and CD25⁺ cells. The efficiency of CD25 depletion was assessed by flow cytometry, and the values were 88.6% ± 2.4%, 87.5% ± 7.9%, 92.7% ± 5.1%, and 86.9% ± 5.1% for peripheral blood mononuclear cells (PBMC), spleen, and mesenteric and inguinal LN leukocytes, respectively.

Quantification of mRNA in cells and tissues. Total RNA was extracted from isolated cells by the guanidium thiocyanate-phenol-chloroform method, modified for TRIzol (Invitrogen, Carlsbad, CA), and from snap-frozen tissues by using the RNAeasy extraction kit (QIAGEN, Valencia, CA), according to the manufacturer's instructions. RNA (1 µg) was reverse transcribed into first-strand cDNA by using random hexanucleotide primers, oligo(dT), and Moloney murine leukemia virus reverse transcriptase (Promega, Madison, WI). cDNA quantification was performed by real-time PCR, conducted with an ABI Prism 7900HT (Applied Biosystems, Foster City, CA). All reactions were performed by using a SYBR green PCR mix (QIAGEN), according to the following thermal profile: denaturation at 95°C for 15 s, annealing at 60°C for 15 s, and extension at 72°C for 15 s (data collection was performed during the extension step). Primer sequences for macaque mRNA were designed by using the Primer3 software and are presented in Table 2. Primers for SIVgag have previously been described (22). Because CTLA-4 and FoxP3 are mainly expressed by CD4⁺ T cells, the results for these genes were normalized on CD4 mRNA. Normalization on GAPDH (glyceraldehyde-3-phosphate dehydrogenase) gave comparable results.

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TABLE 2. Primers for real-time PCR assays

Flow cytometry. For fluorescence-activated cell sorting (FACS) analysis, cells were washed with FACS buffer and stained with anti-CD25-allophycocyanin (M-A251; BD Pharmingen), anti-CD8b (2ST8.5H7; Beckman Coulter), and anti-CD4, (L-200; BD Pharmingen). Cells were then fixed and permeabilized according to eBiosciences's instructions, incubated with normal rat serum for 15 min, and stained with anti-FOXP3-fluorescein isothiocyanate (PCH101; eBiosciences, San Diego, CA) for 30 min. The appropriate isotype matched control antibodies were used to define positivity. Marker expression was analyzed by using a FACSCalibur apparatus and CellQuest software (BD Pharmingen). A minimum of 10,000 cells/tube was analyzed.

Trp, Kyn, and neopterin concentration measurement by high-performance liquid chromatography. Plasma samples were collected from all animals at the time of euthanasia and frozen at –20°C until analysis. Trp and kynurenine (Kyn) concentrations were determined by high-performance liquid chromatography as previously described (47). To estimate IDO activity, Kyn/Trp ratios were calculated. Neopterin concentrations were measured by enzyme-linked immunosorbent assay (BRAHMS Diagnostica, Berlin, Germany).

Statistical analysis. Differences between SIV_HI and SIV_LO animals were assessed by using a nonparametric Mann-Whitney U test. Correlations were determined with Spearman's rank correlation. Differences with P values lower than 0.05 were considered statistically significant.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Plasma viral level partially reflects viral RNA levels in lymphoid tissues. Plasma viral level is a clinical marker of HIV/SIV disease progression. However, it is not fully understood how the plasma viral level relates to the virus levels in different lymphoid tissues. We performed an extensive analysis of RNA encoding for the viral polypeptide gag (SIVgag) in biopsies from different lymphoid sites and compared them with plasma viral levels. Plasma virus correlated with SIV RNA levels in the spleen (Fig. 2A), inguinal LN (Fig. 2B), mesenteric LN (Fig. 2C), colon (Fig. 2D), and jejunum (Fig. 2E), and statistically significant correlations were observed in four of five of the tissues analyzed (Fig. 2). Interestingly, the distribution of SIV RNA values differed considerably among the tissues analyzed in that they were more tightly clustered in the colon and jejunum compared to the spleen and LN. These differences can be quantified by the slope values for each correlation (Fig. 2), which were 0.32, 0.91, and 0.67 for the spleen, inguinal LN, and mesenteric LN, respectively, compared to 2.02 and 1.78 for the colon and jejunum. These data suggest that viral replication in the lymphoid tissues directly affects the plasma viral level, and that even limited variations of viral activity in the GALT (colon and jejunum) may lead to large changes in the plasma virus level.

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FIG. 2. Correlation between plasma viral level and viral RNA expression in lymphoid tissues. The plasma viral RNA was measured by NASBA in all SIV-infected animals. SIVgag RNA was measured in biopsy samples from the spleen (A), inguinal LN (B), mesenteric LN (C), colon (D), and jejunum (E) by real-time PCR as described in Materials and Methods. Diagonal lines represent the best-fit regression line, to which the slope values refer. Dotted lines represent the 104 copies/ml cutoff value between SIVLO and SIVHI. ND*, under detection level.

CTLA-4 and FoxP3 mRNA expression in the spleens and LN of SIV-infected macaques and their correlation with local viral RNA expression. We investigated the expression of CTLA-4 and FoxP3 mRNA in leukocytes isolated from the peripheral blood, spleens, and mesenteric and inguinal LN of five SIV_HI and seven SIV_LO macaques, as well as from uninfected (SIV_NEG) animals (PBMC, n = 3; tissues, n = 1). Because both CTLA-4 and FoxP3 are primarily expressed by CD4⁺ T cells, the results were normalized on CD4 mRNA expression rather than on GAPDH, as in previous reports(2, 31). SIV_HI exhibited significantly higher CTLA-4 mRNA in inguinal and mesenteric LN than did SIV_LO (Fig. 3A) _. A similar trend was observed for splenic leukocytes (Fig. 3A). FoxP3 mRNA was also significantly increased in the leukocytes from the spleens of SIV_HI animals versus SIV_LO animals, and a similar trend was observed for the mesenteric and inguinal LN (Fig. 3B). In contrast, PBMC from SIV_NEG, SIV_LO, and SIV_HI animals expressed comparable levels of CTLA-4 and FoxP3 mRNA. We assessed the relationship between CTLA-4/FoxP3 mRNA expression in spleen and LN and SIV RNA levels in the same tissues and found that SIV RNA directly correlated with both CTLA-4 (Fig. 3C) and FoxP3 expression (Fig. 3D). These findings suggest that T_reg are expanded in the LN and spleens of SIV-infected animals with high viremia and that the increase in T_reg markers is associated with active SIV transcription.

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FIG. 3. CTLA-4 and FoxP3 mRNA expression in blood and tissues from SIV-infected macaques. CTLA-4 (A) and FoxP3 (B) mRNA in leukocytes isolated from peripheral blood (PBMC), spleens, and LN (Mes., mesenteric; Ing., inguinal) of SIVNEG (U), SIVHI (H), and SIVLO (L). In panels A and B horizontal bars within boxes correspond to the median, the box limits correspond to the 25th and 75th percentiles, and the vertical lines extend to the 10th and 90th percentiles. CTLA-4 (C) and FoxP3 (D) mRNA correlated with SIVgag RNA in the spleens and LN from SIV-infected animals (spleens, mesenteric LN, and inguinal LN were grouped together for statistical purposes).

Differential expression of CTLA-4 and FoxP3 mRNA in CD25⁺ and CD25^– cells from lymphoid tissues of SIV-infected macaques. Expression of CD25, the high-affinity a-chain of interleukin-2 (IL-2) receptor, has been used as a marker to distinguish T_reg (35). Interestingly, the frequency of CD3⁺ CD4⁺ CD25⁺ cells was comparable between SIV_HI and SIV_LO animals (data not shown), suggesting that the increased expression of T_reg markers does not go hand-in-hand with CD4⁺ CD25⁺ cells. We therefore determined CTLA-4 and FoxP3 mRNA expression in isolated CD25⁺ and CD25^– cells from the peripheral blood and lymphoid tissues. Although no significant differences in CTLA-4 (Fig. 4A) and FoxP3 (Fig. 4B) mRNA expression were observed in CD25⁺ cells from spleen and LN of SIV_HI compared to SIV_LO, a significant difference was observed in CD25^– T cells from spleen and LN between SIV_HI and SIV_LO for CTLA-4 and FOXP3 mRNA expression (Fig. 4C, 4D). As expected, by comparing CD25⁺ and CD25^– cells from the same animals, we found that FoxP3 was expressed at generally higher levels in CD25⁺ than CD25^– cells in PBMC and tissues from both SIV_LO and SIV_HI (compare Fig. 4B to 4D). An exception to this trend was observed in the spleen of SIV_HI, in which FoxP3 expression was similar in CD25⁺ and CD25^– cells (compare Fig. 4B to 4D).

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FIG. 4. CTLA-4 and FoxP3 mRNA expression in CD25+ and CD25– cells from blood and tissues of SIV-infected macaques. CD25+ and CD25– cells were isolated from peripheral blood (PBMC), spleens, and LN (Mes., mesenteric; Ing., inguinal) of SIVHI (H) and SIVLO (L) animals. CTLA-4 (A and C) and FoxP3 (B and D) mRNA in CD25+ (A and B) and CD25– (C and D) cells are shown. Horizontal bars within boxes correspond to the median, box limits correspond to the 25th and 75th percentiles, and vertical lines extend to the 10th and 90th percentiles.

We verified these findings by using flow cytometry in a group of SIV_HI, SIV_LO and SIV_NEG. The mean fluorescence intensity (MFI) for FoxP3 staining was higher in CD25⁺ CD4 T cells than for the CD25^– counterpart in all tissues analyzed, independent from SIV infection and plasma virus levels (Fig. 5). FoxP3 MFI was increased in both CD25⁺ and CD25^– CD4 T cells from inguinal lymph nodes of SIV_HI compared to SIV_LO and SIV_NEG (Fig. 5B). Similar results were observed when CD25⁺ and CD25^– CD4 T cells from spleen of SIV_HI and SIV_NEG were analyzed (Fig. 5C). These observations confirm that the population of FoxP3-expressing cells in lymphoid tissues from SIV_HI is not limited to a CD25⁺ phenotype and that CD25^– FoxP3⁺ may be present in lymphoid tissues during active viral replication.

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FIG. 5. Flow cytometry analysis of FoxP3 expression in CD25+ and CD25– CD4+ T cells. (A) Flow cytometry plot of one example inguinal LN showing the gates for CD25+ and CD25– CD4 T cells. (B and C) Flow cytometry histograms showing intracellular staining for FoxP3 in CD25+ (solid histograms) and CD25– (open histograms) CD4 T cells from the inguinal LN of one SIVNEG, one SIVLO, and three SIVHI (B) and from the spleen of one SIVNEG and one SIVHI (C). Numbers beside each histogram indicate the MFI of FoxP3 staining in CD25+ and CD25– CD4 T cells and the plasma virus levels of each animal at the time of tissue collection.

CTLA-4 and FoxP3 mRNA expression are increased in the colon of SIV-infected macaques with high viremia. The intestine is a major site of viral replication (9, 19, 26, 27, 45); however, little information is available on T_reg number and/or function in this tissue. Therefore, we analyzed CTLA-4 and FoxP3 mRNA expression in jejunum and colon of SIV-infected macaques. Colon biopsies from SIV_HI showed increased CTLA-4 (Fig. 6A) and FoxP3 (Fig. 6B) mRNA levels compared to SIV_LO, approaching statistical significance. In contrast, both CTLA-4 and FoxP3 mRNA appeared to be unaltered in the jejuna of SIV_HI animals compared to SIV_LO animals (Fig. 6C and D). Interestingly, CTLA-4 and FoxP3 expression were generally lower in the jejunum than in the colon (P = 0.038 and P = 0.029 for CTLA-4 and FoxP3, respectively, when SIV_HI, SIV_LO and SIV_NEG were grouped together), suggesting that T_reg dynamics may be different between these two sites, independently of SIV replication. Interestingly, CD4 mRNA expression in jejunum and colon (normalized on GAPDH mRNA) was lower in SIV-infected animals than in SIV_NEG animals, although it was not different between SIV_HI and SIV_LO animals (data not shown). Using flow cytometry, we verified that CD4⁺ T cells were depleted in the jejuna of SIV-infected animals and that few of the remaining CD4⁺ T cells expressed FoxP3 (Fig. 6E).

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FIG. 6. CTLA-4 and FoxP3 mRNA expression in gut tissues from SIV-infected macaques. CTLA-4 (A and C) and FoxP3 (B and D) mRNA were determined in snap-frozen gut tissues of SIVNEG (U), SIVHI (H), and SIVLO (L) macaques. One sample from colon (A and B) and one sample from jejunum (C and D) were analyzed for each animal. Horizontal bars within boxes correspond to the median, box limits correspond to the 25th and 75th percentiles, and vertical lines extend to the 10th and 90th percentiles. (E) Flow cytometry analysis of CD4 and CD8 in T lymphocytes (left panel) and of CD25 and FoxP3 in the gated CD4+ T cells (right panel) in leukocytes from the jejunum of one SIVHI macaque. Numbers within plots represent the frequencies of the subpopulations identified by the gates.

IL-2 expression in lymphoid tissues did not differ between SIV_HI and SIV_LO animals. FoxP3 and CTLA-4 not only are expressed by T_reg but can also be transiently expressed by recently activated T cells (37). IL-2 is a proliferation-inducing cytokine that is rapidly produced by T cells upon activation (41). Thus, we quantified mRNA levels for IL-2 in leukocytes from spleens and mesenteric LN, as well as for jejunum and colon biopsies, of SIV-infected and uninfected animals. We found that both SIV_HI and SIV_LO showed a trend toward increased IL-2 mRNA compared to the uninfected animal in all tissues analyzed (Fig. 7). However, no difference in IL-2 mRNA expression was observed between SIV_HI and SIV_LO (Fig. 7). These data indicate that IL-2 mRNA is upregulated in lymphoid tissues from SIV-infected animals, which is symptomatic of a status of chronic immune activation. However, since no difference in IL-2 mRNA was observed between SIV_HI and SIV_LO, the alterations in FoxP3 and CTLA-4 mRNA that we described for SIV_HI are not likely the consequence of increased number of activated T cells at these sites.

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FIG. 7. IL-2 mRNA expression in lymphoid tissues from SIV-infected and uninfected animals. IL-2 mRNA was measured in leukocytes from spleens (A) and mesenteric LN (B), as well as in biopsy samples from the colons (C) and jejuna (D) of SIVNEG (U), SIVHI (H), and SIVLO (L) animals. Horizontal bars within boxes correspond to the median, the box limits correspond to the 25th and 75th percentiles, and the vertical lines extend to the 10th and 90th percentiles.

IDO expression is increased in spleen and gut of highly viremic SIV-infected macaques and correlates with SIV replication. One of the mechanisms by which T_reg exert immunosuppressive activity is through the induction of IDO in dendritic cells and macrophages, following the engagement of B7 by CTLA-4 (13, 30). Therefore, we analyzed IDO expression in spleen and gut tissue samples. Because IDO is primarily expressed by dendritic cells, macrophages, and other non-T cells, the results were normalized on GAPDH mRNA as in previous reports (2, 5, 6, 24, 31). IDO mRNA expression was significantly increased in SIV_HI compared to SIV_LO in all analyzed tissues (Fig. 8A to D). The uninfected control macaque exhibited among the lowest levels of IDO mRNA. We also quantified SIVgag RNA levels in the same tissues, and they directly correlated with IDO mRNA expression (Fig. 8E), indicating that IDO expression is higher in tissues in which SIV is more transcriptionally active.

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FIG. 8. IDO mRNA expression in spleen and gut tissues and IDO enzymatic activity in SIV-infected macaques. IDO mRNA was measured in lymphoid tissues (A, B, C, and D), and the Kyn/Trp ratios were determined in the plasma of SIV NEG (U), SIVHI (H) and SIVLO (L) animals (F). One sample from the spleen (A), one from the mesenteric LN (B), one from the jejunum (C), and one from the colon (D) were analyzed for each animal. (E) IDO mRNA correlated with the SIVgag RNA measured in the corresponding tissues. (G) Plasma Kyn/Trp ratio correlated with plasma viral level. Horizontal bars within boxes correspond to the median, box limits correspond to the 25th and 75th percentiles, and vertical lines extend to the 10th and 90th percentiles.

IDO activity can be regulated at the posttranscriptional level (28). Therefore, to confirm that increased IDO mRNA expression was associated with increased enzymatic activity, we measured the concentration of Trp and Kyn in plasma samples from SIV-infected macaques. SIV_HI showed reduced Trp and augmented Kyn plasma concentration compared to SIV_LO. Consequently, the Kyn/Trp ratio was significantly higher in SIV_HI than in SIV_LO animals (Fig. 8F) and directly correlated with the viral level (Fig. 8G). Because non-IDO-mediated Trp degradation can also occur at hepatic level, we measured plasma concentration of neopterin, which is a reliable marker for cytokine-induced IDO-mediated Trp catabolism (38). The Kyn/Trp ratio correlated with the plasma concentration of neopterin (R² = 0.68; P = 0.006; data not shown), confirming that increased Kyn/Trp ratios are due to increased IDO activity and not to increased hepatic Trp degradation.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
It is believed that an early loss of CD4⁺ T cells occurs in LN and GALT within the first few weeks of HIV/SIV infection and that such depletion results from infection and killing of activated CD4⁺CCR5⁺ memory T cells at these sites (8). However, limited information is available on how the virus influences the immune functions of other cell types at mucosal and lymphoid tissues. We used the experimental model of SIV infection to perform a broad analysis of viral expression and T_reg markers in multiple lymphoid compartments. We found a consistent increase in T_reg markers and of the immunosuppressive enzyme IDO in the spleen, LN, and gut tissues of animals with high levels of viral replication. Importantly, CTLA-4, FoxP3 and IDO mRNA directly correlated with SIVgag RNA in the tissues analyzed, suggesting a relationship between immunoregulatory mechanisms and viral replication at these sites.

We found that the levels of SIV RNA in the colon and jejunum directly correlated with the plasma viral level, similar to what we observed for the spleen and LN, confirming previous findings (29). However, in both the colon and jejunum the distribution of tissue SIV RNA values generated regression curves with a steeper slope than in other tissues. This observation suggests that, on one hand, individuals with low plasma viral levels might have high levels of viral replication at the mucosal level and, on the other hand, that even small increases in virus levels in the GALT may result in large increments in plasma virus.

T_reg survival is promoted by exposure to HIV and progressive HIV disease associates with accumulation of T_reg in tonsils of HIV-infected patients (31). Thus, while CD4⁺ T helper cells are depleted, T_reg may not only be spared but even favored by HIV/SIV replication. Here we found that, despite the similar levels of SIV transcriptional activity in the jejunum and colon, FoxP3 and CTLA-4 expression are diversely affected at these sites. The colons of SIV_HI animals showed increased T_reg markers, similar to a previous report of increased T_reg in the duodenal mucosa of HIV-infected patients (11). In contrast, no significant change in FoxP3 and CTLA-4 expression was observed in the jejunal tract of the intestine. Surprisingly, when the colon and jejunum from the same animals were compared side by side, the latter had constantly lower levels of T_reg markers, in both SIV_HI and SIV_LO, as well as in the uninfected macaque. Thus, it is possible that T_reg distribute differentially in different sites of the GALT, independently of the presence of SIV or its replication, and that SIV accentuates such a trend. In accordance with this hypothesis, the colon is the primary site of microbial colonization (21), whereas the upper two-thirds of the small intestine (duodenum and jejunum) contain only low numbers of microorganisms (21), suggesting that a stronger antigenic stimulation and consequent requirement for immunoregulation occurs in the colon than in the jejunum. An alternative, nonexclusive explanation for the lack of T_reg in the jejunum of SIV_HI is that the severe depletion of CD4⁺ T cells that occurs at this site may also affect T_reg. Indeed, in accordance with previous reports (9, 19, 26, 27, 45), we found that the jejunal tract was severely depleted of CD4⁺ T cells.

Our results describe for the first time a significant accumulation of T_reg in the spleen in SIV_HI animals, which in turn could explain the finding that the spleen is a preferential viral reservoir site (39). Accordingly, splenectomized HIV-infected patients show improved survival, delayed time to AIDS, reduction of plasma viremia, and increased absolute numbers of CD4⁺ and CD8⁺ T cells (3). Importantly, expression of T_reg markers in spleen and LN directly correlated with viral replication within the tissues. On one side, active viral replication may contribute to the activation, survival, and migration of T_reg into lymphoid tissues (31); on the other, the T_reg-mediated suppression of antiviral cell mediated immunity may in turn favor uncontrolled viral replication (24, 31). The coexistence of these events may render the association between T_reg and viral replication a bidirectional cause-and-effect relation. Our findings that IL-2 mRNA expression in lymphoid tissues was comparable between SIV_HI and SIV_LO suggests that increased T-cell activation is not the cause of high CTLA-4 and FoxP3 in SIV_HI, similar to our previous report (31). In addition, our results for FoxP3 and CTLA-4 mRNA were normalized on CD4 mRNA, and we did not observe any difference in CD4 mRNA expression between the jejunum and the colon in SIV_HI and SIV_LO animals, suggesting that the difference in T_reg distribution is not likely due to variations in the absolute numbers of CD4⁺ T cells.

In the present study we report that significant changes in FoxP3 and CTLA-4 expression in spleen and LN of SIV_HI occur in both the CD25^– and the CD25⁺ population, although the expected pattern of higher FoxP3 expression in the CD25⁺ population was maintained in the majority of tissues analyzed. Three possible explanations could account for this observation: (i) T_reg found in the spleen may result from proliferation of T_reg that lost CD25 expression, similar to that reported in a mouse model of T_reg expansion (32); (ii) a different population of CD25^– T_reg (44) may preferentially relocate to the spleen during chronic SIV infection, under conditions of uncontrolled viral replication; or (iii) the CD25^– population may represent a subset of activated T_reg (32) with higher expression of FoxP3 and CTLA-4 on a per-cell basis.

The rate of IDO-mediated Trp catabolism is increased during HIV disease progression (16, 49). We observed higher IDO expression in the mesenteric LN, jejuna, colons, and spleens of SIV_HI animals compared to the same compartments of SIV_LO animals. Similar to CTLA-4 and FoxP3, IDO directly correlated with SIVgag RNA in the tissues, suggesting that SIV virus levels are higher at sites where IDO is highly expressed. Accordingly, we found that IDO enzymatic activity (as reflected by plasma Kyn/Trp ratios) was increased in SIV_HI and directly correlated with the plasma virus level. Again, the jejunum appeared to be unique among the tissues analyzed, in that increased IDO expression was observed in the absence of increased FoxP3 and CTLA-4, suggesting that other mechanisms, independent of T_reg activity, contribute to induce IDO in lymphoid tissues. IDO can be induced in macrophages by direct exposure to certain strains of HIV or to the HIV proteins Tat and Nef (17, 40) and in plasmacytoid dendritic cells by exposure to HIV (5). Thus, APC present in the jejunum may be triggered to express IDO without the intervention of T_reg. Furthermore, secreted cytokines, such as type I and type II interferons, induce IDO expression in different cell types (5, 28) and could also contribute to increased IDO expression in tissues. The increased IDO activity, whether directly induced by HIV/SIV, by secreted cytokines or through the mediation of T_reg, could contribute to the suppression cell-mediated responses (5, 34).

Sustained viral replication is the driving force of HIV/SIV disease, and understanding the causes of the inability of the immune system to control viral activity is of pivotal importance in the development of effective anti-HIV therapy. The accumulation of T_reg and increased IDO expression at sites of massive viral replication, such as the spleen and gut, is likely to play an important role in maintaining a favorable environment for HIV/SIV persistence and consequent disease progression. Manipulation of these immunoregulatory mechanisms, such as blockade of CTLA-4, has given interesting results in reducing virus levels in lymphoid tissues during chronic SIV infection (24). Our findings provide the rationale for designing novel therapeutic strategies aimed at manipulating the number and/or function of T_reg and/or IDO to ameliorate immune control of viral replication in chronically infected patients.

 
This research was supported by the Intramural Program of the Centers for Cancer Research, National Cancer Institute, National Institutes of Health (G.F. and G.M.S.); by the Intramural AIDS Targeted Antiviral Program (G.M.S.); by a grant from the National Institutes of Health (AI068524 to C.C.); by the government of the State of the Austrian Tyrol (D.F.); and by grants from the Swedish Foundation for Strategic Research, Swedish Cancer Foundation, and Swedish Research Council the Swedish (J.A.).

 
* Corresponding author. Mailing address: Experimental Immunology Branch, National Cancer Institute, National Institutes of Health, 10 Center Dr., Bldg. 10, Rm. 4B36, Bethesda, MD 20892. Phone: (301) 435-6420. Fax: (301) 402-3643. E-mail: boassoa{at}mail.nih.gov

Published ahead of print on 22 August 2007.

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Journal of Virology, November 2007, p. 11593-11603, Vol. 81, No. 21
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