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Published ahead of print on September 4, 2003, doi:10.1165/rcmb.2003-0268OC
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American Journal of Respiratory Cell and Molecular Biology. Vol. 30, pp. 311-318, 2004
© 2004 American Thoracic Society
DOI: 10.1165/rcmb.2003-0268OC

CDllc+ Cells Modulate Pulmonary Immune Responses by Production of Indoleamine 2,3-Dioxygenase

Kena A. Swanson, Yan Zheng, Kathleen M. Heidler, Teruaki Mizobuchi and David S. Wilkes

Departments of Microbiology and Immunology and of Medicine, Indiana University School of Medicine, Indianapolis, Indiana; and Department of Thoracic Surgery, Graduate School of Medicine, Chiba University, Chiba, Japan

Address correspondence to: David S. Wilkes, M.D., Division of Pulmonary and Critical Care Medicine, Indiana University School of Medicine, 1001 West Tenth Street, OPW 425, Indianapolis, IN 46202. E-mail: dwilkes{at}iupui.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Interactions between antigen-presenting cells and T cells can result in T cell activation or suppression. With the use of RNA analysis, high-performance liquid chromatography, mixed leukocyte reactions (MLRs), and animal models, the current study reports that lung interstitial antigen-presenting cells (iAPCs, CDllc+) suppress T cell responses in vitro and in vivo by production of indoleamine 2,3-dioxygenase (IDO), an enzyme that catabolizes tryptophan to its byproduct, kynurenine. IDO mRNA expression was unique to lung iAPCs, as cells similarly isolated from the liver and spleen did not express IDO constitutively, or in response to interferon-{gamma}. Lung iAPCs suppressed proliferation of allogeneic T cells, correlating with increased kynurenine levels; and blockade of IDO activity with 1-methyl-DL-tryptohan (1-MT) or addition of exogenous tryptophan recovered T cell proliferation in MLRs. In contrast, liver and splenic iAPCs were potent stimulators of T cells in MLRs, and IDO inhibition had no effect on T cell responses. In vivo studies showed that systemic blockade of IDO resulted in spontaneous proliferation in lung T cells and pulmonary inflammation. Finally, overexpressing IDO in lung transplants abrogated acute allograft rejection, a T cell–mediated disease. Collectively these data show that lung iAPCs contribute to local regulation of cellular immune responses by production of IDO.

Abbreviations: 1-methyl-DL-tryptophan, 1-MT • alveolar macrophage, AM{phi} • counts per minute, CPM • dendritic cell, DC • delayed-type hypersensitivity, DTH • high-performance liquid chromatography, HPLC • interstitial antigen presenting cell, iAPC • indoleamine 2,3-dioxygenase, IDO • interferon-{gamma}, IFN-{gamma} • lipopolysaccharide, LPS • mixed leukocyte reaction, MLR • macrophage, M{phi}


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The lung interstitium is rich in antigen-presenting cells (APCs) such as dendritic cells (DCs), monocytes, and macrophages (M{phi}s). Data showing that these interstitial APCs (iAPCs) are interdigitated with T cells in the perivascular and peribronchiolar tissues suggest that they are primed to induce T cell activation (1–3). Although it has been reported that iAPCs are only able to stimulate cellular immune responses after migrating to regional lymph nodes (4, 5), a very recent study from Constant and coworkers (6) showed that under appropriate conditions iAPCs may prime pulmonary immune responses in situ. The inability of iAPCs to induce T cell activation in lung parenchyma had been attributed to low expression of MHC class II, and costimulatory molecules, as well as alveolar macrophage (AM{phi})-induced suppression of antigen presentation by these cells (7, 8). However, an alternate hypothesis for the inability to induce cellular immunity could be iAPC-induced suppression of interstitial lung T cells.

Monocytes, M{phi}s, and DCs in nonpulmonary tissues suppress T cell activation by the production of indoleamine 2,3-dioxygenase (IDO), an enzyme that catabolizes tryptophan (9–11). Although tryptophan deficiency may result in cell cycle arrest or apoptosis in T cells, more recent studies suggest that by-products of tryptophan catabolism may further suppress immune responses by direct toxicity to T cells and other lymphoid cells (12, 13).

IDO expression has been reported in lung iAPCs (14–16). However, studies examining the functional role of pulmonary IDO were focused on its potential role in the eradication of microbial pathogens (17–19). IDO may have other functions relative to cellular immunity as shown by its key role in abrogating alloreactivity of maternal T cells in the placenta (20), suppression of antitumor cellular immune responses (21), and preventing rejection of pancreatic islet allografts (22). Accordingly, IDO derived from lung iAPCs may contribute to local immune regulation by suppressing spontaneous proliferation in pulmonary T cells. Furthermore, IDO expression could also have a therapeutic role in T cell–mediated lung diseases such as lung allograft rejection.

In the present study, we report that lung iAPCs produce IDO, and that this activity is unique to these cells, as cells isolated similarly from liver or spleen do not produce IDO, constitutively, or in response to interferon (IFN)-{gamma}, the most potent inducer of IDO expression. Only lung iAPCs suppressed proliferation in allogeneic T cells, and blockade of IDO with 1-methyl-DL-tryptophan (1-MT) or addition of exogenous tryptophan recovered lung iAPC-induced T cell proliferation in MLRs. In contrast, liver and splenic iAPCs were potent stimulators of T cells in MLRs, and IDO inhibition had no effect on T cell responses. In vivo studies show that systemic blockade of IDO resulted in spontaneous lung T cell proliferation, and overexpressing IDO in lung transplants abrogated allograft rejection, a T cell–mediated disease.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Female C57BL/6 (I-Ab, H-2b) and BALB/c (I-Ad, H-2d) mice (8–10 wk old) were obtained from Harlan Sprague Dawley, Inc. (Indianapolis, IN) and Jackson Laboratories (Bar Harbor, ME). MHC (RT1)-incompatible male rats were used for the lung transplantation study: Fischer 344 (F344, RT1lv1), Brown Norway (BN, RT1n), and Wistar Kyoto (WKY, RT1l) rats (250–300 g at the time of transplantation) were obtained from Harlan Sprague Dawley, Inc. Animals were killed by injection (intramuscular) of ketamine cocktail (79.3% Ketaject, 17.5% atropine, and 3.2% acepromazine). All mice were housed in pathogen-free facilities in the Laboratory Animal Resource Center at Indiana University School of Medicine in accordance with Institutional Animal Care and Use Committee guidelines.

Reagents
Complete media (cRPMI): RPMI 1640, 10% fetal bovine serum, 400 mM L-glutamine, 100 U penicillin/streptomycin, 1% ß-mercaptoethanol (5 x 10-4 M) (Invitrogen, Carlsbad, CA), DNase I (Sigma, St. Louis, MO), Collagenase D (Roche Diagnostics, Indianapolis, IN). Other reagents used were the following: 1-methyl-DL-tryptrophan (Aldrich Chemical Co., Milwaukee, WI) (23), recombinant murine IFN-{gamma} (R&D Systems, Minneapolis, MN), lipopolysaccharide (LPS), L-tryptophan, and L-kynurenine (Sigma).

Immunohistochemistry
Lung tissue was prepared as previously reported (24). After blocking steps using biotin, avidin (Sigma), and rabbit immune serum (KPL Laboratories, Gaithersburg, MD), slides were incubated with rabbit anti-mouse IDO polyclonal antibody (kindly provided by Dr. Osamu Takikawa) (25), followed by a biotinylated anti-rabbit secondary antibody (Sigma). Streptavidin alkaline phosphatase (KPL Laboratories) was then conjugated to the biotinylated secondary antibody, followed by addition of new fuschin substrate and hematoxylin counterstain (Sigma). IDO staining was visualized by light microscopy. Murine testis served as a positive control (26). Negative controls were testis and normal murine lung stained with either the IDO or secondary antibody, alone.

Cell Isolation
Lung iAPCs were isolated using a modified procedure previously reported (27). Murine lungs were flushed with 0.9% NaCl to remove all blood from the pulmonary circulation, and lungs resected carefully to avoid all regional lymphoid tissues. Lungs were mechanically and enzymatically digested in a DNase/collagenase solution at 35°C, and lung mononuclear cells (LMNC) isolated after passage over a Percoll density gradient (1.03/1.075) (Amersham-Pharmacia, Uppsala, Sweden) (27). Anti-CD11c magnetic microbeads (Miltenyi Biotec, Auburn, CA) were used to isolate iAPCs from LMNC. CDllc+ APCs were isolated from the liver and spleen by techniques similar to that used for lung iAPCs. In no instance were APCs from lung, liver, or spleen adhered to plastic during the isolation procedure. AM{phi}s were isolated as reported (27). Where indicated, freshly isolated lung iAPCS, or AM{phi}'s (106 cells/ml) were cultured for 24 h in 24-well plates with or without IFN-{gamma} (1,000 U/ml), LPS (5 µg/ml), or both. Culture supernatants and cells were harvested and stored at -80°C until use.

CD3+ lung T cells were isolated from LMNCs by sorting on a FACSVantage or FACStar cell sorter (Becton Dickinson, Franklin Lakes, NJ). Anti-CD90 (Thy1.2) magnetic beads (Miltenyi Biotec) were used to isolate splenic T cells. Purity of all cells was > 97%.

Flow Cytometry
The following fluorochrome-conjugated anti-mouse antibodies were used for flow cytometry experiments: FITC CD11c, PE CD80, PE CD86, PE CD40, PE CD8{alpha}, PE I-Ab (Pharmingen, San Diego, CA). Biotinylated anti-mouse OX40L monoclonal antibody (28), a generous gift of Dr. Naoto Ishii (Tohoku University School of Medicine, Sendai, Japan), was followed by a PE-conjugated anti-rat secondary antibody (Pharmingen). The corresponding isotype control antibodies and an Fc blocking antibody were all purchased from Pharmingen.

MLRs and Proliferation Studies
Gamma irradiated (2,000 rads) C57BL/6 DCs were co-cultured with BALB/c T cells in 96-well flat-bottom plates (Becton Dickinson). Eighteen hours before completion of the 72-h incubation, thymidine was added and T cell proliferation reported as mean counts per minute (CPM) thymidine incorporation in triplicate wells (± SD). In some experiments, the IDO inhibitor, 1-methyl-DL-tryptophan (1-MT) (30, 31), or excess L-tryptophan (0.025 mg/ml) were added to MLRs.

Reverse Transcription–Polymerase Chain Reaction
PCR for the detection of IDO was performed using methods described by Mellor and colleagues (31). Total RNA was isolated and reverse transcription performed. IDO transcripts (740 bp) were identified using IDO-specific forward and reverse primers (forward, 5'-GTACATCACCATGGCGTATG-3', reverse, 3'-GCTTTCGTCAAGTCTTCATTG-5'(29)). ß2m transcripts (349 bp) served as an internal control (forward, 5'-TGACCGGCTTGTATGCTATC-3'; reverse, 3'-CAGTGTGAGCCAGGATATAG-5'). Reaction products were run on a 2% agarose gel in TAE. Images were analyzed using ChemiImager 4,400 low light imaging system (Alpha Innotech, San Leandro, CA).

High-Performance Liquid Chromatography
High-performance liquid chromatography (HPLC) was used to detect tryptophan and kynurenine in culture supernatants as reported by Munn and coworkers (30), with modifications, in the Biochemistry Biotechnology Facility at the Indiana University School of Medicine. Samples (25 µl) were injected into a Luna 5 µ C18 (2) column (250 x 4.6 mm; Phenomenex, Torrance, CA) and eluted over a linear gradient of 0–40% acetonitrile (Sigma) over 20 min. Standards for both L-tryptophan and L-kynurenine were assayed before the test samples to establish retention times and standard curves. Concentrations of tryptophan and kynurenine were quantitated by converting the area values under each peak to nmol of each amino acid using the equations generated from the standard curves.

IDO Inhibitor Treatment
BALB/c mice were treated with slow-release polymer pellets containing the IDO inhibitor 1-MT (70 mg/pellet) or empty pellets (Innovative Research, Sarasota, FL) (31). Anti-CD3/PMA (4 µg/ml, 50 ng/ml; Sigma)-induced T cell proliferation post 1-MT or placebo treatment was determined by 3H thymidine incorporation over an 18-h period. In some experiments, the lungs from normal and pellet-treated (placebo or 1-MT, 2.5 d post-treatment) mice were harvested, snap-frozen, and histology examined by light microscopy.

IDO Overexpression and Lung Transplantation
Replication-deficient type 5 adenoviral vectors (107 PFU/200 µl) with enhanced blue fluorescent protein (Ad-EBFP), IDO (AdIDO), or empty vector control (AdNull) (Gene Vector Core laboratory, University of Pittsburgh, Pittsburgh, PA) were instilled (intratracheally) into anesthetized F344 (RT1lv1) rats. Twenty-four hours later rats were killed, and the lungs were harvested, fixed, and frozen with Tissue-Tek O.C.T. Compound (Miles Inc. Diagnostic Division, Elkhart, IN), and sectioned (5 µm) as previously reported (32–34). Lung sections were then examined by confocal microscopy to detect intrapulmonary deposition of Ad-EBFP vectors. These data confirmed vector-induced IDO overexpression in the lung.

Twenty-four hours after AdNull or AdIDO instillation, the left lungs of F344 rats were transplanted into WKY (RT1l) rat recipients as reported (33, 35). The F344 -> WKY model is completely mismatched at the class I locus of the major histocompatibility complex (16). In brief, after the donor rats (F344) were anesthetized with an intramuscular injection of ketamine (40 mg/kg) and xylazine (5 mg/kg), and the heart and lungs were removed en bloc. The left lung was then resected, heparinised, wrapped in sterile gauze, and placed on ice (4°C) in a sterile beaker until transplantation.

The recipient rats (WKY) were anesthetized with a subcutaneous injection of atropine (0.05 mg/kg), followed by an inhalation of 2% halothane. The left lung was resected and the pulmonary vessels of the donor lung were anastomosed to the recipient by a plastic cuff and 7–0 silk sutures (Kono, Chiba, Japan). The donor and recipient bronchi were sutured together using 8–0 Prolene sutures (Ethicon, Sommerville, NJ). All transplantation procedures were performed by T.M. under a surgical microscope (Micro Tech, Colorado Springs, CO) under sterile conditions. No immunosuppressive therapy was given at any time during the experimental period. The F344 -> WKY transplant model is associated with the development of severe acute rejection (grade 4) by the end of the second week after transplantation (32–34). As previously reported (33, 35, 36), lung allografts were graded for rejection pathology using standard criteria in which Grade 0 refers to the absence of pathology and Grade 4 refers to severe rejection (37).

Delayed-Type Hypersensitivity
Delayed-type hypersensitivity (DTH) responses were performed as reported previously (33, 35, 36). Two weeks after transplantation, lung allograft recipients received 107 irradiated (3,000 rad) donor-derived F344 splenocytes in 30 µl of PBS into the right pinnae by subcutaneous injection using a 26-gauge needle. The left pinnae received an equal volume of diluent, and served as the control site. Naive WKY rats were negative controls, and WKY rats that received untreated or AdNull-treated F344 allografts were positive controls. The ear thickness was measured with a micrometer caliper (Mitutoyo, Field Tool Supply, Chicago, IL) in a blinded fashion immediately before and 24 h after injection. Similar to prior reports, antigen-specific DTH response was calculated according to the following formula: specific ear swelling = (right ear thickness @ 24 h – right ear thickness @ 0 h) – (left ear thickness @ 24 h – left ear thickness @ 0 h) x 10-3mm (19). All data reported as the mean ± SD of triplicate measurements.

Statistics
All experimental data sets were compared using Student's t test. P values < 0.05 were considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Constitutive Expression of IDO in Lung iAPCs
Immunohistochemistry revealed IDO in interstitial lung mononuclear cells that are morphologically similar to monocytes, M{phi}s, or DCs (Figure 1). Analysis of lung iAPCs and APCs from the liver and spleen (CDllc+) showed expression of CD80, CD86, I-A, low levels of CD40, OX40L, and B220. However, these cells did not express CD8{alpha}. In general, compared with cells from liver and spleen, lung iAPCs expressed lower levels of all markers, especially I-A, CD86, and OX40L.



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  		Figure 1. Immunohistochemical detection of lung IDO in interstitial mononuclear cells. To localize the cellular source of IDO, an IDO-specific rabbit anti-mouse polyclonal antibody was used to stain lung tissue sections from naive BALB/c mice. A biotinylated mouse anti-rabbit monoclonal antibody was used as a conjugate followed by addition of substrate. Slides were counterstained with hematoxylin and visualized by light microscopy (x40 magnification). Arrows identify positive-stained cells.

 
Examination of IDO mRNA levels by RT-PCR showed that IDO was not expressed constitutively in AM{phi}s, nor in response to IFN-{gamma} and LPS that are known to induce IDO expression (25, 30, 38–40) (Figure 2). In contrast, IDO was expressed constitutively in lung iAPCs and was upregulated by IFN-{gamma}, the most potent inducer of IDO expression (41, 42), but not LPS. Constitutive IDO expression is unique to the lung, as significant levels of IDO were not detected in APCs isolated from liver or spleen.



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  		Figure 2. High levels of IFN-{gamma}–induced IDO mRNA expression by lung iAPCs. Lung, liver, or splenic APCs and alveolar macrophages (M{phi}s) were cultured in the presence or absence of IFN-{gamma} (1,000 U/ml), LPS (5 µg/ml), or both. RT-PCR was then performed on total RNA using murine IDO- or ß2m-specific primers. Products were run on a 2% agarose gel in TAE.

 
APC-induced T cell activation requires contact-dependent bidirectional signaling between the APC and T cell. A recent report showed that this signaling could potentially upregulate IDO expression in DCs (11). Therefore, we next determined if allogeneic T cells induced IDO expression in iAPCs. To conduct these studies, various quantities of iAPCs from C57BL/6 mice were cultured alone, or in the presence of a fixed quantity of lung T cells (BALB/c). Preliminary studies showed that lung T cells did not express IDO. Therefore, data in Figure 3 that show increased expression in iAPCs in response to T cells indicate that allogeneic T cells can induce IDO expression in iAPCs.



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  		Figure 3. Allogeneic T cells induce IDO expression in iAPCs. Varying quantities of iAPCs were cultured (Alone, 0.45, 0.9, and 1.5 x 105) or with 3 x 105 lung T cells (+ T) in complete media. At the end of a 24-h culture, mRNA was isolated and RT-PCR for IDO performed as reported in MATERIALS AND METHODS. Preliminary studies confirmed that T cells did not express IDO. IDO mRNA levels were normalized to ß2m levels by densitometry. Data is representative of two separate experiments.

 
Freshly Isolated Lung iAPCs Suppress T Cell Proliferation
To determine their ability to induce T cell proliferation, CD11c+ APCs from the lung, liver, or spleen of C57BL/6 mice (I-Ab) were cocultured with allogeneic lung T cells (BALB/c, I-Ad) in MLRs. In contrast to liver and splenic APCs that induced dose-dependent proliferation (P < 0.01), lung iAPCs induced minimal T cell proliferation at the lowest DC:T cell ratio (P < 0.02), and suppressed proliferation at increasing DC:T cell ratios (Figure 4A). T cells from different tissues may be either predominantly naive or memory cells and the majority of lung T cells are believed to have a memory phenotype (43). Therefore, data showing that lung iAPCs are unable to stimulate lung T cells may suggest a phenomenon unique to interactions between iAPCs and T cells from the lung. To address this question, MLRs were also performed using splenic T cells, which are primarily a naive phenotype (44). Liver and splenic APCs induced similar levels of proliferation in splenic T cells, albeit at lower levels, compared with lung T cells as shown in Figure 4A. At higher DC:T ratios lung iAPCs suppressed proliferation in splenic T cells (Figure 4B). Collectively, these data show freshly isolated iAPCs from the lung, but not liver or spleen, and suppress T cell proliferation, and this effect is independent of the memory status or tissue origin of the responding T cells.



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  		Figure 4. Lung iAPC-derived IDO actively suppresses allogeneic T cell proliferation. Freshly isolated irradiated C57BL/6 lung iAPCs (circles), or liver (squares) or splenic (triangles) APCs, were co-cultured with allogeneic (A) lung or (B) splenic T cells (3 x 105) at the indicated ratios. Proliferative responses were determined as mean ± counts per minute (CPM) of thymidine incorporated into the T cells in triplicate cultures (#P < 0.01 compared with DC:T cultures from liver and spleen at same ratios; *P < 0.02 and **P < 0.001 comparing DC:T cultures from liver and spleen at the same ratios). (C) APCs and allogeneic lung T cells were cultured at 0.5: 1 (APC:T) in the presence of increasing concentrations of the IDO inhibitor, 1-MT. As a control, 1-MT did not affect spontaneous proliferation in DCs or T cells cultured alone (*P < 0.01comparing lung DC: lung T co-cultures with 250 and 500 mg/ml 1-MT to untreated cultures).

 
Lung iAPC-Derived IDO Suppresses Antigen-Induced T Cell Proliferation
Post-translational modification may inactivate IDO (45). Therefore, detection of IDO message and protein does not confirm the presence of a functional enzyme. To determine if lung iAPCs suppress T cell proliferation via IDO activity, the IDO inhibitor, 1-MT, was added to MLRs. 1-MT recovered lung iAPC-induced lung T cell proliferation in a dose-dependent manner (Figure 4C). As expected, 1-MT had no effect on liver or splenic APC-induced proliferation in T cells or T cells cultured alone. These data correlate with data in Figure 2 showing that only lung iAPCs expressed IDO message. IDO-induced suppression was specific as addition of neutralizing antibodies to IL-10 or TGF-ß, cytokines that suppress T cell function (5, 46), did not affect T cell proliferation (data not shown). Although IDO expression in T cells has been reported in one study (47), T cell–derived IDO did not contribute to immune suppression as IDO was not detected in lung T cells by RT-PCR (data not shown).

IDO catabolizes tryptophan to its byproduct, kynurenine. To confirm IDO activity, we next determined if IDO produced by lung iAPCs catabolized tryptophan by HPLC analysis of iAPC:T cell culture supernatants. Tryptophan levels were ~ 9-fold higher than kynurenine at the initiation of culture. However, when T cells were co-cultured with lung iAPCs, kynurenine levels increased (Figure 5A, *P < 0.048). Thus far, these data suggest that lung T cells should be sensitive to the levels of available tryptophan. Indeed, addition of exogenous tryptophan to lung iAPC:T cell co-cultures resulted in a nearly 2-fold increase in T cell proliferation (Figure 5B, {dagger}P < 0.001). Collectively, these data confirm that lung iAPCs suppress T cell proliferation by expression of functional IDO.



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  		Figure 5. (A) Kynurenine levels are increased in iAPC:T cell co-cultures. Lung 1APCs (1.5 x 105) were cultured with increasing numbers of CD3-sorted lung T cells. Culture supernatants were collected and examined by HPLC analysis examining kynurenine levels (indicated as nanomoles/25 µl sample). Standards for both tryptophan and kynurenine were run to establish a retention time for each amino acid (*P < 0.048). (B) Tryptophan recovers iAPC-induced suppression of proliferation in T cells. Exogenous tryptophan was added in cultures of BALB/c lung T cells alone or cultured with irradiated C57BL/6 lung iAPCs for 24 h as reported in MATERIALS AND METHODS. Proliferation was determined by the mean ± SD counts per minute (CPM) of 3H incorporation in triplicate cultures ({dagger}P < 0.001).

 
IDO Inhibition Enhances Lung T Cell Proliferation IN VIVO and IDO Overexpression Abrogates Lung Allograft Rejection
By inhibiting systemic IDO activity, we next determined if IDO downregulates pulmonary T cell function in vivo. Slow-release polymer pellets, containing either 1-MT or empty pellets (control), were implanted under the dorsal skin of BALB/c mice. Lung T cells (Thy 1.2+) were harvested 2.5 d after 1-MT treatment, and anti-CD3/PMA–induced proliferation was determined. Compared with placebo-treated mice, T cells from 1-MT–treated mice proliferated vigorously in response to anti-CD3 (Figure 6A *P < 0.01).



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  		Figure 6. Systemic IDO inhibition results in spontaneous lung T cell proliferation and bronchiolar damage. Pellets containing 1-MT or placebo were implanted under the dorsal skin of BALB/c mice as reported in MATERIALS AND METHODS. Two-and-one-half days later, lungs were harvested, T cells isolated, and sections stained with hematoxylin. (A) Lung T cells from normal, placebo (empty pellet), or 1-MT–treated mice were cultured in the presence of anti-CD3 (4 µg/ml) and PMA (50 ng/ml) stimulation. Proliferation was determined as the mean ± SEM of 3H incorporation in triplicate wells after a 24-h culture period. Data are representative of five mice in each group (*P < 0.01). (B) Lung histology of normal, placebo (empty pellet), and 1-MT–pellet treated mice. Compared with normal or empty pellet-treated mice, which show normal lung histology (single arrow), inhibition of IDO by 1-MT resulted in bronchiolar pathology showing disruption of airway epithelium (double arrows). Single arrow in normal mice shows normal airway epithelium. Lung pathology was assessed by light microscopy (x20 magnification). Data are representative of five mice in each group.

 
The peribronchiolar tissues are a rich source of iAPCs and these cells are in close proximity to T cells and other lymphoid cells. If IDO is produced by these cells and contributes to local immune suppression of T cells, then blockade of IDO activity may result in spontaneous inflammation and damage of bronchiolar tissues. Indeed, systemic inhibition of IDO by 1-MT resulted in alteration of bronchiolar structures as shown by disruption of airway epithelium and surrounding tissues. Compared with normal mice, placebo-treated mice had only minor histologic abnormalities (Figure 6B).

Data showing that IDO derived from lung iAPCs suppressed T cells suggests that IDO may have therapeutic potential to prevent lung diseases mediated by cellular immunity. Acute lung allograft rejection is believed to be initiated by iAPCs in the donor lung stimulating host lymphocytes, which results in upregulated cellular immune responses and, ultimately, destruction of the allograft. Therefore, we hypothesized that overexpression of IDO in the donor lung before transplantation would abrogate donor antigen-specific cellular immunity and rejection pathology. Deposition of recombinant viral vectors and gene expression were confirmed by observing strong enhanced blue fluorescent protein (EBFP) expression in airway epithelium of F344 donor lungs 24 h after intratracheal administration of AdEBFP (data not shown).

Two weeks after transplantation, the time of severe acute rejection, we determined the DTH response to donor (F344) antigens in recipient WKY rats. The DTH response is a measure of systemic T cell–mediated immunity. We have reported that untreated lung allograft recipients develop strong DTH responses to donor antigens beginning at 1 wk and persisting for at least 10 wk after transplantation (33, 35, 36). Figure 7 shows that instillation of empty vectors into donor lungs had no effect on DTH responses, which were comparable to that observed in untreated allograft recipients (Control). Significantly, overexpression of IDO in donor lungs before transplantation (IDO vector) abrogated DTH responses to donor antigens (Figure 7, *P < 0.02). Data showing that IDO suppressed DTH responses to donor antigens suggested that IDO activity would downregulate rejection responses. Indeed, Figure 8 shows that overexpressing IDO in donor lungs before transplantation downregulated rejection pathology (mild, Grade 1), as compared with severe acute rejection (Grade 4) pathology observed in untreated allograft recipients or rats that received lung allografts treated with empty vectors (Table 1).



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  		Figure 7. Adenoviral-mediated overexpression of IDO abrogates DTH responses to donor antigens. F344 splenocytes (107 irradiated) were injected into the pinnae of the right ear and diluent into the pinnae of the left ear of normal WKY rats or WKY rats that received untreated lung allografts from F344 rats (Control). DTH was also determined in WKY rats that received empty vector-treated or IDO vector-treated lung allografts from F344 rats. The ear thickness was measured with a micrometer caliper in a blinded fashion immediately before and 24 h after injection. The antigen-specific DTH response was calculated as described in MATERIALS AND METHODS, *P < 0.02.

 


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  		Figure 8. Overexpression of IDO before transplantation suppresses allograft rejection. Upper panel: Gross anatomy of transplanted lungs (L), and native lungs (R) in untreated, empty vector-, or IDO vector-treated allograft recipients. Untreated and empty vector-treated allografts were shrunken, hard, and disfigured. In contrast, IDO vector-treated allografts were soft and exhibited nearly normal gross anatomy. Lower panel: Histology of transplant and native lungs from each group. Untreated and empty vector–treated allografts showed severe (Grade 4) rejection with extensive mononuclear cell infiltrates. In contrast, few cellular infiltrates were observed in IDO-vector–treated allografts consistent with mild (Grade 1) rejection (x20 magnification). Data are representative of three individual transplants per group.

 

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  		TABLE 1 Rejection grade of transplanted lungs

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Under normal conditions the lung is exposed constantly to inhaled and hematogenous antigens. Therefore, unless immune responses are tightly regulated, inflammation would ensue, resulting in severe lung dysfunction. The lung is rich in iAPCs, but despite cognate interactions with T cells in the interstitium they do not stimulate cellular immunity in situ unless perturbed by appropriate stimuli (48). The current study reports that the inability of these iAPCs to stimulate T cells is the result of active suppression via production of IDO. Importantly, these studies demonstrate that, unlike AM{phi}s, which are poor APCs and function mainly to suppress pulmonary immunity (7, 8), iAPCs can either strongly stimulate or suppress cellular immunity.

Hayaishi's group was the first to report that IDO was produced in the lung, and that its expression was upregulated in response to bacterial and viral infections (15). Similar to that report, IDO was not detected in AM{phi}s in the current study, but was detected in interstitial mononuclear cells that are morphologically similar to macrophages or dendritic cells (15). Many studies have reported IDO expression in APCs, specifically DCs and M{phi}s from nonpulmonary tissues (30, 31, 49). However, the current study is the first to report that constitutive expression of IDO is unique to lung iAPCs, as similarly isolated cells from liver or spleen did not express IDO constitutively, or in response to IFN-{gamma}, a potent inducer of IDO expression.

A key question that remains unanswered is the specific type of iAPC in the lung that produces IDO. Although AM{phi}s appear to be terminally differentiated, iAPCs, which are primarily M{phi}s and DCs, may represent cells capable of greater plasticity with the ability to differentiate further. Indeed, interstitial lung DCs in situ are considered "immature"; i.e., they express low levels of MHC class II and costimulatory molecules, and are highly phagocytic (50). Interstitial M{phi}s in situ are remarkably similar to these DCs. Only when the appropriate stimuli are encountered in vivo or adherence to a solid phase in vitro will iAPCs differentiate into a cell with the phenotype and function of a "DC." Indeed, adherence steps have been used in studies examining pure DCs isolated from the lung and other tissues (reviewed in Ref. 50). CDllc expression has been used to identify these DCs. However, this marker is expressed on M{phi}s and DCs (50). Because these cells may be highly plastic, then it is not known if the pure DCs reported in other studies were derived from interstitial M{phi}s. By using freshly isolated cells without adherence steps, the current study focused on iAPCs that most closely represented their phenotype and function in situ. M{phi}s and DCs are believed to develop from monocytes, and there are no cell surface markers that specifically differentiate immature DCs from M{phi}s. Although we speculate that the CD11c+ cells that produced IDO in the current study are immature DCs, it is difficult to determine the specific cellular source of IDO.

Data in the current study does not exclude the possibility that IDO may be produced by other subsets of APCs. For example, Munn and coworkers (51) recently reported that human plasmacytoid DCs are a rich source of IDO. Murine plasmacytoid DCs, which may be CDllc-, have not been reported in the lung. However, similar to human studies, there are multiple subsets of DCs in the mouse (52). Accordingly, there may be murine lung DCs (CD11c+ and CD11c-) that are each capable of producing IDO. These questions will be answered in future studies.

Although IDO has been reported to suppress T cell responses by depletion of tryptophan, recent reports suggest that toxicity of tryptophan byproducts could explain IDO-induced T cell suppression (12, 13). However, data in the current study and others showing that repletion of tryptophan recovers T cell proliferation in cultures that contain tryptophan metabolites suggest that not all T cells are susceptible to the toxic effects of tryptophan metabolites, or that there are other mechanisms of IDO-induced suppression of T cell responses.

These data showing that iAPC-derived IDO suppresses T cell proliferation extends our knowledge of IDO function, and has implications for IDO in the regulation of T cell–mediated pulmonary immunity. For example, diseases such as transplant rejection and cancer may be modulated by IDO activity. A recent study by Miki and colleagues (53) showed that liver allograft rejection, a T cell–dependent process, is downregulated by IDO production. Also, IDO expression in pancreatic islets prolongs their survival after transplantation (22). Furthermore, recent work from Mellor and associates (49) showed that IDO overexpression downregulated murine T cell function in vivo. Similarly, Munn and colleagues reported that IDO derived from a subset of human DCs regulated cellular immune responses (51). Data in the current study showed that overexpression of IDO in lung allografts abrogated antidonor T cell responses as determined by the DTH response to donor antigens (Figure 7). More importantly, IDO abrogated lung allograft rejection, which is a disease process mediated primarily by T cells. These data highlight a potential therapeutic role for IDO in downregulation of other T cell–mediated pulmonary diseases such as asthma and autoimmune pulmonary disorders.

Although there are many reports of mechanisms that upregulate IDO expression, little is known about the molecular mechanisms that suppress IDO activity. Recent data has suggested that ligation of B7 molcules (CD80 and CD86) on the surface of CD11c+ splenic DCs by soluble CTLA-4 induces IDO expression and thus suppresses clonal T cell expansion (11, 54). In the context of lung allograft rejection, allograft tolerance may be partially attributed to this pathway of IDO upregulation; however, this possibility is still unknown. Our group and others have described the development of a regulatory T cell population (Treg) following lung transplantation. Knowing that all Tregs express CTLA-4, this pathway (B7/CTLA-4) of T cell–induced DC-derived IDO expression seems a likely mechanism for IDO regulation. However, because pulmonary immune responses are dependent on the ability of APCs to stimulate T cells, then IDO activity must be downregulated locally so that cellular immune responses can occur in the lung. The processes involved in down regulating IDO expression in lung iAPCs are being examined.


   Acknowledgments
 
The authors thank Dr. John Hawes and his staff in the Indiana University School of Medicine Biochemistry and Biotechnology Facility for technical guidance in HPLC analysis, Allison Thiele for technical assistance, and Dr. Janice Blum for helpful discussions. This work was supported by grants from the National Institutes of Health HL60797, and HL/AI67177 to D.S.W.

Received in original form July 17, 2003

Received in final form August 15, 2003


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Schon-Hegard, M. A., J. Oliver, P. G. McMenamin, and P. G. Holt. 1991. Studies on the density, distribution, and surface phenotype of intraepithelial class II major histocompatibility complex antigen (Ia)-bearing dendritic cells (DC) in the conducting airways. J. Exp. Med. 173:1345–1356.[Abstract/Free Full Text]
  2. Nicod, L. P., L. Cochand, and D. Dreher. 2000. Antigen presentation in the lung: dendritic cells and macrophages. Sarcoidosis Vasc. Diffuse Lung Dis. 17:246–255.[Medline]
  3. Cochand, L., P. Isler, F. Songeon, and L. P. Nicod. 1999. Human lung dendritic cells have an immature phenotype with efficient mannose receptors. Am. J. Respir. Cell Mol. Biol. 21:547–554.[Abstract/Free Full Text]
  4. Banchereau, J., F. Briere, C. Caux, J. Davoust, S. Lebecque, Y. J. Liu, B. Pulendran, and K. Palucka. 2000. Immunobiology of dendritic cells. Annu. Rev. Immunol. 18:767–811.[CrossRef][Medline]
  5. Akbari, O., R. H. DeKruyff, and D. T. Umetsu. 2001. Pulmonary dendritic cells producing IL-10 mediate tolerance induced by respiratory exposure to antigen. Nat. Immunol. 2:725–731.[CrossRef][Medline]
  6. Constant, S. L., J. L. Brogdon, D. A. Piggott, C. A. Herrick, I. Visintin, N. H. Ruddle, and K. Bottomly. 2002. Resident lung antigen-presenting cells have the capacity to promote Th2 T cell differentiation in situ. J. Clin. Invest. 110:1441–1448.[CrossRef][Medline]
  7. Holt, P. G., J. Oliver, N. Bilyk, C. McMenamin, P. G. McMenamin, G. Kraal, and T. Thepen. 1993. Downregulation of the antigen presenting cell function(s) of pulmonary dendritic cells in vivo by resident alveolar macrophages. J. Exp. Med. 177:397–407.[Abstract/Free Full Text]
  8. Holt, P. G., M. A. Schon-Hegrad, and J. Oliver. 1988. MHC class II antigen-bearing dendritic cells in pulmonary tissues of the rat: regulation of antigen presentation activity by endogenous macrophage populations. J. Exp. Med. 167:262–274.[Abstract/Free Full Text]
  9. Shimizu, T., S. Nomiyama, F. Hirata, and O. Hayaishi. 1978. Indoleamine 2,3-dioxygenase: purification and some properties. J. Biol. Chem. 253:4700–4706.[Abstract/Free Full Text]
  10. Frumento, G., R. Rotondo, M. Tonetti, and G. B. Ferrara. 2001. T cell proliferation is blocked by indoleamine 2,3-dioxygenase. Transplant. Proc. 33:428–430.[CrossRef][Medline]
  11. Grohmann, U., C. Orabona, F. Fallarino, C. Vacca, F. Calcinaro, A. Falorni, P. Candeloro, M. L. Belladonna, R. Bianchi, M. C. Fioretti, and P. Puccetti. 2002. CTLA-4-Ig regulates tryptophan catabolism in vivo. Nat. Immunol. 3:1097–1101.[CrossRef][Medline]
  12. Frumento, G., R. Rotondo, M. Tonetti, G. Damonte, U. Benatti, and G. B. Ferrara. 2002. Tryptophan-derived catabolites are responsible for inhibition of T and natural killer cell proliferation induced by indoleamine 2,3-dioxygenase. J. Exp. Med. 196:459–468.[Abstract/Free Full Text]
  13. Terness, P., T. M. Bauer, L. Rose, C. Dufter, A. Watzlik, H. Simon, and G. Opelz. 2002. Inhibition of allogeneic T cell proliferation by indoleamine 2,3-dioxygenase-expressing dendritic cells: mediation of suppression by tryptophan metabolites. J. Exp. Med. 196:447–457.[Abstract/Free Full Text]
  14. Meyer, K. C., R. A. Arend, M. V. Kalayoglu, N. S. Rosenthal, G. I. Byrne, and R. R. Brown. 1995. Tryptophan metabolism in chronic inflammatory lung disease. J. Lab. Clin. Med. 126:530–540.[Medline]
  15. Urade, Y., R. Yoshida, H. Kitamura, and O. Hayaishi. 1983. Induction of indoleamine 2,3-dioxygenase in alveolar interstitial cells of mouse lung by bacterial lipopolysaccharide. J. Biol. Chem. 258:6621–6627.[Abstract/Free Full Text]
  16. Yoshida, R., Y. Urade, M. Tokuda, and O. Hayaishi. 1979. Induction of indoleamine 2,3-dioxygenase in mouse lung during virus infection. Proc. Natl. Acad. Sci. USA 76:4084–4086.[Abstract/Free Full Text]
  17. Choi, A. M., K. Knobil, S. L. Otterbein, D. A. Eastman, and D. B. Jacoby. 1996. Oxidant stress responses in influenza virus pneumonia: gene expression and transcription factor activation. Am. J. Physiol. 271:L383–L391.
  18. Jacoby, D. B., and A. M. Choi. 1994. Influenza virus induces expression of antioxidant genes in human epithelial cells. Free Radic. Biol. Med. 16:821–824.[CrossRef][Medline]
  19. Rottenberg, M. E., A. Gigliotti Rothfuchs, D. Gigliotti, M. Ceausu, C. Une, V. Levitsky, and H. Wigzell. 2000. Regulation and role of IFN-gamma in the innate resistance to infection with Chlamydia pneumoniae. J. Immunol. 164:4812–4818.[Abstract/Free Full Text]
  20. Munn, D. H., M. Zhou, J. T. Attwood, I. Bondarev, S. J. Conway, B. Marshall, C. Brown, and A. L. Mellor. 1998. Prevention of allogeneic fetal rejection by tryptophan catabolism. Science 281:1191–1193.[Abstract/Free Full Text]
  21. Friburg, M., R. Jennings, M. Alsarraj, S. Dessureault, A. Cantor, M. Extermann, A. L. Mellor, D. H. Munn, and S. J. Antonia. 2002. Indoleamine 2,3-dioxygenase contributes to tumor cell evasion of T cell-mediated rejection. Int. J. Cancer 101:151–155.[CrossRef][Medline]
  22. Alexander, A. M., M. Crawford, S. Bertera, W. A. Rudert, O. Takikawa, P. D. Robbins, and M. Trucco. 2002. Indoleamine 2,3-dioxygenase expression in transplanted NOD islets prolongs graft survival after adoptive transfer of diabetogenic splenocytes. Diabetes 51:356–365.[Abstract/Free Full Text]
  23. Cady, S. G., and M. Sono. 1991. 1-Methyl-DL-tryptophan, beta-(3-benzofuranyl)-DL-alanine (the oxygen analog of tryptophan), and beta-[3-benzo(b)thienyl]-DL-alanine (the sulfur analog of tryptophan) are competitive inhibitors for indoleamine 2,3-dioxygenase. Arch. Biochem. Biophys. 291:326–333.[CrossRef][Medline]
  24. Wilkes, D. S., K. M. Heidler, L. K. Bowen, W. M. Quinlan, N. A. Doyle, O. W. Cummings, and C. M. Doerschuk. 1995. Allogeneic bronchoalveolar lavage cells induce the histology of acute lung allograft rejection, and deposition of IgG2a in recipient murine lungs. J. Immunol. 155:2775–2783.[Abstract]
  25. Takikawa, O., T. Kuriowa, F. Yamazaki, and R. Kido. 1988. Mechanism of interferon-{gamma} action: characterization of indoleamine 2,3-dioxygenase in cultured human cells induced by interferon-{gamma} and evaluation of the enzyme mediated tryptophan degradation in its anticellular activity. J. Biol. Chem. 263:2041–2048.[Abstract/Free Full Text]
  26. Takikawa, O., R. Yoshida, R. Kido, and O. Hayaishi. 1986. Tryptophan degradation in mice initiated by indoleamine 2,3-dioxygenase. J. Biol. Chem. 261:3648–3653.[Abstract/Free Full Text]
  27. Wilkes, D. S., L. K. Thompson, O. W. Cummings, S. Bragg, and K. M. Heidler. 1998. Instillation of allogeneic lung macrophages and dendritic cells cause differential effects on local IFN-gamma production, lymphocytic bronchitis, and vasculitis in recipient murine lungs. J. Leukoc. Biol. 64:578–586.[Abstract]
  28. Murata, K., N. Ishii, H. Takano, S. Miura, L. C. Ndhlovu, M. Nose, T. Noda, and K. Sugamura. 2000. Impairment of antigen-presenting cell function in mice lacking expression of OX40 ligand. J. Exp. Med. 191:365–374.[Abstract/Free Full Text]
  29. Habara-Ohkubo, A., O. Takikawa, and R. Yoshida. 1991. Cloning and expression of a cDNA encoding mouse indoleamine 2,3-dioxygenase. Gene 105:221–227.[CrossRef][Medline]
  30. Munn, D. H., E. Shafizadeh, J. T. Attwood, I. Bondarev, A. Pashine, and A. L. Mellor. 1999. Inhibition of T cell proliferation by macrophage tryptophan catabolism. J. Exp. Med. 189:1363–1372.[Abstract/Free Full Text]
  31. Mellor, A. L., J. Sivakumar, P. Chandler, K. Smith, H. Molina, D. Mao, and D. H. Munn. 2001. Prevention of T cell-driven complement activation and inflammation by tryptophan catabolism during pregnancy. Nat. Immunol. 2:64–68.[CrossRef][Medline]
  32. Matsumura, Y., A. Marchevsky, X. J. Zuo, R. M. Kass, J. M. Matloff, and S. C. Jordan. 1995. Assessment of pathological changes associated with chronic allograft rejection and tolerance in two experimental models of rat lung transplantation. Transplantation 59:1509–1517.[Medline]
  33. Yasufuku, K., K. M. Heidler, P. W. O'Donnell, G. N. Smith, Jr., O. W. Cummings, B. H. Foresman, T. Fujisawa, and D. S. Wilkes. 2001. Oral tolerance induction by type V collagen downregulates lung allograft rejection. Am. J. Respir. Cell Mol. Biol. 25:26–34.[Abstract/Free Full Text]
  34. Sekine, Y., K. Yasufuku, K. M. Heidler, O. W. Cummings, N. Van Rooijen, T. Fujisawa, J. Brown, and D. S. Wilkes. 2000. Monocyte chemoattractant protein-1 and RANTES are chemotactic for graft infiltrating lymphocytes during acute lung allograft rejection. Am. J. Respir. Cell Mol. Biol. 23:719–726.[Abstract/Free Full Text]
  35. Haque, M. A., T. Mizobuchi, K. Yasufuku, T. Fujisawa, R. R. Brutkiewicz, Y. Zheng, K. Woods, G. N. Smith, O. W. Cummings, K. M. Heidler, J. S. Blum, and D. S. Wilkes. 2002. Evidence for immune responses to a self-antigen in lung transplantation: role of type V collagen-specific T cells in the pathogenesis of lung allograft rejection. J. Immunol. 169:1542–1549.[Abstract/Free Full Text]
  36. Yasufuku, K., K. M. Heidler, K. A. Woods, G. N. Smith, Jr., O. W. Cummings, T. Fujisawa, and D. S. Wilkes. 2002. Prevention of bronchiolitis obliterans in rat lung allografts by type V collagen-induced oral tolerance. Transplantation 73:500–505.[CrossRef][Medline]
  37. Yousem, S. A., G. J. Berry, P. T. Cagle, D. Chamberlain, A. N. Husain, R. H. Hruban, A. Marchevsky, N. P. Ohori, J. Ritter, S. Stewart, and H. D. Tazelaar. 1996. Revision of the 1990 working formulation for the classification of pulmonary allograft rejection: Lung Rejection Study Group. J. Heart Lung Transplant. 15:1–15.[Medline]
  38. Fallarino, F., C. Vacca, C. Orabona, M. L. Belladonna, R. Bianchi, B. Marshall, D. B. Keskin, A. L. Mellor, M. C. Fioretti, U. Grohmann, and P. Puccetti. 2002. Functional expression of indoleamine 2,3-dioxygenase by murine CD8alpha(+) dendritic cells. Int. Immunol. 14:65–68.[Abstract/Free Full Text]
  39. Fujigaki, S., K. Saito, K. Sekikawa, S. Tone, O. Takikawa, H. Fujii, H. Wada, A. Noma, and M. Seishima. 2001. Lipopolysaccharide induction of indoleamine 2,3-dioxygenase is mediated dominantly by an IFN-gamma-independent mechanism. Eur. J. Immunol. 31:2313–2318.[CrossRef][Medline]
  40. Munn, D. H., J. Pressey, A. C. Beall, R. Hudes, and M. R. Alderson. 1996. Selective activation-induced apoptosis of peripheral T cells imposed by macrophages: a potential mechanism of antigen-specific peripheral lymphocyte deletion. J. Immunol. 156:523–532.[Abstract]
  41. Feng, G. S., and M. W. Taylor. 1989. Interferon gamma-resistant mutants are defective in the induction of indoleamine 2,3-dioxygenase. Proc. Natl. Acad. Sci. USA 86:7144–7148.[Abstract/Free Full Text]
  42. Yuan, W., A. Collado-Hidalgo, T. Yufit, M. Taylor, and J. Varga. 1998. Modulation of cellular tryptophan metabolism in human fibroblasts by transforming growth factor-beta: selective inhibition of indoleamine 2,3-dioxygenase and tryptophanyl-tRNA synthetase gene expression. J. Cell. Physiol. 177:174–186.[CrossRef][Medline]
  43. Becker, S., D. T. Harris, and H. S. Koren. 1990. Characterization of normal human lung lymphocytes and interleukin-2-induced lung T cell lines. Am. J. Respir. Cell Mol. Biol. 3:441–448.
  44. Ernst, D. N., W. O. Weigle, D. J. Noonan, D. N. McQuitty, and M. V. Hobbs. 1993. The age-associated increase in IFN-gamma synthesis by mouse CD8+ T cells correlates with shifts in the frequencies of cell subsets defined by membrane CD44, CD45RB, 3G11, and MEL-14 expression. J. Immunol. 151:575–587.[Abstract]
  45. Sono, M., T. Taniguchi, Y. Watanabe, and O. Hayaishi. 1980. Indoleamine 2,3-dioxygenase: equilibrium studies of the tryptophan binding to the ferric, ferrous, and CO-bound enzymes. J. Biol. Chem. 255:1339–1345.[Free Full Text]
  46. Kehrl, J. H., L. M. Wakefield, A. B. Roberts, S. Jakowlew, M. Alvarez-Mon, R. Derynck, M. B. Sporn, and A. S. Fauci. 1986. Production of transforming growth factor beta by human T lymphocytes and its potential role in the regulation of T cell growth. J. Exp. Med. 163:1037–1050.[Abstract/Free Full Text]
  47. Curreli, S., F. Romerio, P. Mirandola, P. Barion, K. Bemis, and D. Zella. 2001. Human primary CD4+ T cells activated in the presence of IFN-alpha 2b express functional indoleamine 2,3-dioxygenase. J. Interferon Cytokine Res. 21:431–437.[CrossRef][Medline]
  48. Lambrecht, B. N., J. B. Prins, and H. C. Hoogsteden. 2001. Lung dendritic cells and host immunity to infection. Eur. Respir. J. 18:692–704.[Abstract/Free Full Text]
  49. Mellor, A. L., D. B. Keskin, T. Johnson, P. Chandler, and D. H. Munn. 2002. Cells expressing indoleamine 2,3-dioxygenase inhibit T cell responses. J. Immunol. 168:3771–3776.[Abstract/Free Full Text]
  50. Lipscomb, M. F., and B. J. Masten. 2002. Dendritic cells: immune regulators in health and disease. Physiol. Rev. 82:97–130.[Abstract/Free Full Text]
  51. Munn, D. H., M. D. Sharma, J. R. Lee, K. G. Jhaver, T. S. Johnson, D. B. Keskin, B. Marshall, P. Chandler, S. J. Antonia, R. Burgess, C. L. Slingluff, Jr., and A. L. Mellor. 2002. Potential regulatory function of human dendritic cells expressing indoleamine 2,3-dioxygenase. Science 297:1867–1870.[Abstract/Free Full Text]
  52. Shortman, K., and Y. J. Liu. 2002. Mouse and human dendritic cell subtypes. Nat. Rev. Immunol. 2:151–161.[CrossRef][Medline]
  53. Miki, T., H. Sun, Y. Lee, A. Tandin, A. M. Kovscek, V. Subbotin, J. J. Fung, and L. A. Valdivia. 2001. Blockade of tryptophan catabolism prevents spontaneous tolerogenicity of liver allografts. Transp. Proc. 33:129–130.
  54. Mellor, A. L., B. Baban, P. Chandler, B. Marshall, K. Jhaver, A. Hansen, P. A. Koni, M. Iwashima, and D. H. Munn. 2003. Cutting edge: induced indoleamine 2,3-dioxygenase expression in dendritic cell subsets suppresses T cell clonal expansion. J. Immunol. 171:1652–1655.[Abstract/Free Full Text]



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