Tryptamine and dimethyltryptamine inhibit indoleamine 2,3 dioxygenase and increase the tumor‐reactive effect of peripheral blood mononuclear cells

Indoleamine 2,3‐dioxygenase (IDO) is an interferon‐γ (IFN‐γ)–induced tryptophan‐degrading enzyme, producing kynurenine (KYN) that participates in the mechanism of tumor immune tolerance. Thus, IDO inhibition has been considered a strategy for anticancer therapy. The aim of this study was to identify whether the metabolites originated from the competitive routes of tryptophan metabolism, such as the serotonergic or N, N‐dimethyltryptamine (DMT) pathways, have inhibitory effects on recombinant human IDO (rhIDO) activity. Serotonin and melatonin had no effect; on the other hand, tryptamine (TRY) and DMT modulated the activity of rhIDO as classical non‐competitive inhibitors, with Ki values of 156 and 506 μM, respectively. This inhibitory effect was also observed on constitutively expressed or IFN‐γ–induced IDO in the A172 human glioma cell line. TRY and DMT increased the cytotoxic activity of peripheral blood mononuclear cells (PBMCs) in co‐culture assays. We conclude that the IDO inhibition by TRY and DMT contributed to a more effective tumor‐reactive response by the PBMCs. Copyright © 2013 John Wiley & Sons, Ltd.     

Reverse signaling through GITR ligand enables dexamethasone to activate IDO in allergy

Glucocorticoid-induced tumor necrosis factor receptor (GITR) on T cells and its natural ligand, GITRL, on accessory cells contribute to the control of immune homeostasis. Here we show that reverse signaling through GITRL after engagement by soluble GITR initiates the immunoregulatory pathway of tryptophan catabolism in mouse plasmacytoid dendritic cells, by means of noncanonical NF-kappaB-dependent induction of indoleamine 2,3-dioxygenase (IDO). The synthetic glucocorticoid dexamethasone administered in vivo activated IDO through the symmetric induction of GITR in CD4(+) T cells and GITRL in plasmacytoid dendritic cells. The drug exerted IDO-dependent protection in a model of allergic airway inflammation. Modulation of tryptophan catabolism via the GITR-GITRL coreceptor system might represent an effective therapeutic target in immune regulation. Induction of IDO could be an important mechanism underlying the anti-inflammatory action of corticosteroids.     

Interferon-γ induces a tryptophan-selective amino acid transporter in human colonic epithelial cells and mouse dendritic cells

IDO1, which encodes the immunosuppressive and tryptophan-catabolizing enzyme indoleamine 2,3-dioxygenase-1 (IDO1), is a target for interferon-γ (IFN-γ). IDO1-mediated tryptophan catabolism in dendritic cells and macrophages arrests T cell proliferation, thereby providing a molecular basis for the immunosuppressive function of IDO1. Whether the entry of tryptophan into IDO1-expressing cells is also regulated by IFN-γ is not known. Here we used a human colonic epithelial cell line (CCD841) and a mouse dendritic cell line (DC2.4) to test the hypothesis that IFN-γ, which induces IDO1, also induces a tryptophan transporter to promote substrate availability to IDO1. Upon treatment with IFN-γ, there was a marked increase in IDO1 mRNA and a concomitant increase in tryptophan uptake in both cell lines. The induced uptake system was selective for tryptophan and saturable with a Michaelis constant of 36 ± 3 μM in CCD841 cells and 0.5 ± 0.1 μM in DC2.4 cells. The induction by IFN-γ and the tryptophan-selectivity of the induced transport system were demonstrable even in the presence of physiologic concentrations of all other amino acids. Since kynurenine, the catabolic end product of IDO1, is a signaling molecule as an agonist for the aryl hydrocarbon receptor (AhR), we examined if AhR signaling induces the tryptophan-selective transporter. Treatment of the cells with kynurenine and other AhR agonists increased tryptophan uptake. The present studies demonstrate that IFN-γ coordinately induces IDO1 and a tryptophan-selective transporter to maximize tryptophan depletion in IDO1-expressing cells and that the process involves a positive feedback mechanism via kynurenine-AhR signaling.     

An Autocrine Cytokine/JAK/STAT-Signaling Induces Kynurenine Synthesis in Multidrug Resistant Human Cancer Cells

BackgroundMultidrug resistant cancer cells are hard to eradicate for the inefficacy of conventional anticancer drugs. Besides escaping the cytotoxic effects of chemotherapy, they also bypass the pro-immunogenic effects induced by anticancer drugs: indeed they are not well recognized by host dendritic cells and do not elicit a durable anti-tumor immunity. It has not yet been investigated whether multidrug resistant cells have a different ability to induce immunosuppression than chemosensitive ones. We addressed this issue in human and murine chemosensitive and multidrug resistant cancer cells.ResultsWe found that the activity and expression of indoleamine 2,3-dioxygenase 1 (IDO1), which catalyzes the conversion of tryptophan into the immunosuppressive metabolite kynurenine, was higher in all the multidrug resistant cells analyzed and that IDO1 inhibition reduced the growth of drug-resistant tumors in immunocompetent animals. In chemoresistant cells the basal activity of JAK1/STAT1 and JAK1/STAT3 signaling was higher, the STAT3 inhibitor PIAS3 was down-regulated, and the autocrine production of STAT3-target and IDO1-inducers cytokines IL-6, IL-4, IL-1β, IL-13, TNF-α and CD40L, was increased. The disruption of the JAK/STAT signaling lowered the IDO1 activity and reversed the kynurenine-induced pro-immunosuppressive effects, as revealed by the restored proliferation of T-lymphocytes in STAT-silenced chemoresistant cells.ConclusionsOur work shows that multidrug resistant cells have a stronger immunosuppressive attitude than chemosensitive cells, due to the constitutive activation of the JAK/STAT/IDO1 axis, thus resulting chemo- and immune-evasive. Disrupting this axis may significantly improve the efficacy of chemo-immunotherapy protocols against resistant tumors.     

Characterization of differences in substrate specificity among CYP1A1, CYP1A2 and CYP1B1: an integrated approach employing molecular docking and molecular dynamics simulations

Recent trends in new drug discovery of anticancer drugs have made oncologists more aware of the fact that the new drug discovery must target the developing mechanism of tumorigenesis to improve the therapeutic efficacy of antineoplastic drugs. The drugs designed are expected to have high affinity towards the novel targets selectively. Current research highlights overexpression of CYP450s, particularly cytochrome P450 1A1 (CYP1A1), in tumour cells, representing a novel target for anticancer therapy. However, the CYP1 family is identified as posing significant problems in selectivity of anticancer molecules towards CYP1A1. Three members have been identified in the human CYP1 family: CYP1A1, CYP1A2 and CYP1B1. Although sequences of the three isoform have high sequence identity, they have distinct substrate specificities. The understanding of macromolecular features that govern substrate specificity is required to understand the interplay between the protein function and dynamics, design novel antitumour compounds that could be specifically metabolized by only CYP1A1 to mediate their antitumour activity and elucidate the reasons for differences in substrate specificity profile among the three proteins. In the present study, we employed a combination of computational methodologies: molecular docking and molecular dynamics simulations. We utilized eight substrates for elucidating the difference in substrate specificity of the three isoforms. Lastly, we conclude that the substrate specificity of a particular substrate depends upon the type of the active site residues, the dynamic motions in the protein structure upon ligand binding and the physico‐chemical characteristics of a particular ligand. Copyright © 2016 John Wiley & Sons, Ltd.     

IDO1 and IDO2 are expressed in human tumors: levo- but not dextro-1-methyl tryptophan inhibits tryptophan catabolism

Objectives  Indoleamine-2,3-Dioxygenase (IDO) is an immunosuppressive molecule inducible in various cells. In addition to classic IDO (IDO1), a new variant, IDO2, has recently been described. When expressed in dendritic cells (DCs) or cancer cells, IDO was thought to suppress the immune response to tumors. A novel therapeutic approach in cancer envisages inhibition of IDO with 1-methyl-tryptophan (1MT). The levo-isoform (l-1MT) blocks IDO1, whereas dextro-1MT (d-1MT), which is used in clinical trials, inhibits IDO2. Here we analyze IDO2 expression in human cancer cells and the impact of both 1-MT isoforms on IDO activity. Methods  Surgically extirpated human primary tumors as well as human cancer cell lines were tested for IDO1 and IDO2 expression by RT-PCR. IDO1 activity of Hela cells was blocked by transfection with IDO1-specific siRNA and analysed for tryptophan degradation by RP-HPLC. The impact of d-1MT and l-1MT on IDO activity of Hela cells and protein isolates of human colon cancer were studied. Results  Human primary gastric, colon and renal cell carcinomas constitutively expressed both, IDO1 and IDO2 mRNA, whereas cancer cells lines had to be induced to by Interferon-gamma (IFN-γ). Treatment of Hela cells with IDO1-specific siRNA resulted in complete abrogation of tryptophan degradation. Only l-1MT, and not d-1MT, was able to block IDO activity in IFN-γ-treated Hela cells as well as in protein isolates of primary human colon cancer. Conclusions  Although IDO2 is expressed in human tumors, tryptophan degradation is entirely provided by IDO1. Importantly, d-1MT does not inhibit the IDO activity of malignant cells. If ongoing clinical studies show a therapeutic effect of d-1MT, this cannot be attributed to inhibition of IDO in tumor cells.     

Non-hematopoietic expression of IDO is integrally required for inflammatory tumor promotion

Indoleamine 2,3-dioxygenase (IDO) is generally considered to be immunosuppressive but recent findings suggest this characterization oversimplifies its role in disease pathogenesis. Recently, we showed that IDO is essential for tumor outgrowth in the classical two-stage model of inflammatory skin carcinogenesis. Here, we report that IDO loss did not exacerbate classical inflammatory responses. Rather, IDO induction could be elicited by environmental signals and tumor promoters as an integral component of the inflammatory tissue microenvironment even in the absence of cancer. IDO loss had limited impact on tumor outgrowth in carcinogenesis models that lacked an explicit inflammatory tumor promoter. In the context of inflammatory carcinogenesis where IDO was critical to tumor development, the most important source of IDO was radiation-resistant non-hematopoietic cells, consistent with evidence that loss of the IDO regulatory tumor suppressor gene Bin1 in transformed skin cells facilitates IDO-mediated immune escape by a cell autonomous mechanism. Taken together, our results identify IDO as an integral component of ‘cancer-associated’ inflammation that tilts the immune system toward tumor support. More generally, they promote the concept that mediators of immune escape and cancer-associated inflammation may be genetically synonymous.     

Molecular docking analysis of lupeol with different cancer targets

Lupeol is one of the secondary metabolite (triterpenoid) present in many medicinally effective plants. It has numerous biological and pharmacological actions. Lupeol is found to have effective herbs and has immense biological activity against several diseases including its cytotoxic effect on cancer cells. In recent drug designing, molecular study of analysis is usually used for understanding the target and the ligand interaction. Therefore, it is of interest to document the molecular docking analysis data of lupeol with different cancer targets such as Caspase- 3, BCL-2, Topoisomerase, PTK, mTOR, H-Ras, PI3K, and AKT. These molecular docking studies were carried out by using AutoDock tools 4.2 version software. Molecular docking analyses of lupeol with target protein were found to have good dock score and minimum inhibition constant. BCL-2, Topoisomerase, PTK, mTOR and PI3Kdocking studies showed the best binding energy inhibition constant and ligand efficiency. The in-silico molecular docking analysis showed that the lupeol having relatively good docking energy, affinity and efficiency towards the active macromolecule, thus it may be considered as good inhibitor of proliferating cancer cells. By this knowledge of docking results, the lupeol can be used as promising drug for anticancer activity.     

Discovery and preliminary structure–activity relationship of 1H-indazoles with promising indoleamine-2,3-dioxygenase 1 (IDO1) inhibition properties

Indoleamine 2,3-dioxygenase 1 (IDO1)-mediated kynurenine pathway of tryptophan degradation is identified as an important immune effector pathway in the tumor cells to escape a potentially effective immune response. IDO1 is an attractive target for anticancer therapy and the discovery of IDO1 inhibitors has been intensely ongoing in both academic research laboratories and pharmaceutical organizations. Our study discovered that 1H-indazole was a novel key pharmacophore with potent IDO1 inhibitory activity. A series of new 1H-indazole derivatives were synthesized and determined the enzyme inhibitory activities, and the compound 2g exhibited the highest activity with an IC₅₀ value of 5.3 μM. The structure–activity relationships (SARs) analysis of the 1H-indazole derivatives as novel IDO1 inhibitors indicated that the 1H-indazole scaffold is necessary for IDO1 inhibition, and the substituent groups at the both 4-position and 6-position largely affect inhibitory activity. The docking model exhibited that the effective interactions of 1H-indazoles with ferrous ion of heme and key residues of hydrophobic Pocket A and B ensured the IDO1 inhibitory activities. The study suggested that the 1H-indazole was a novel interesting scaffold for IDO inhibition for further development.     

Indoleamine 2,3-dioxygenase pathways of pathogenic inflammation and immune escape in cancer

Genetic and pharmacological studies of indoleamine 2,3-dioxygenase (IDO) have established this tryptophan catabolic enzyme as a central driver of malignant development and progression. IDO acts in tumor, stromal and immune cells to support pathogenic inflammatory processes that engender immune tolerance to tumor antigens. The multifaceted effects of IDO activation in cancer include the suppression of T and NK cells, the generation and activation of T regulatory cells and myeloid-derived suppressor cells, and the promotion of tumor angiogenesis. Mechanistic investigations have defined the aryl hydrocarbon receptor, the master metabolic regulator mTORC1 and the stress kinase Gcn2 as key effector signaling elements for IDO, which also exerts a non-catalytic role in TGF-β signaling. Small-molecule inhibitors of IDO exhibit anticancer activity and cooperate with immunotherapy, radiotherapy or chemotherapy to trigger rapid regression of aggressive tumors otherwise resistant to treatment. Notably, the dramatic antitumor activity of certain targeted therapeutics such as imatinib (Gleevec) in gastrointestinal stromal tumors has been traced in part to IDO downregulation. Further, antitumor responses to immune checkpoint inhibitors can be heightened safely by a clinical lead inhibitor of the IDO pathway that relieves IDO-mediated suppression of mTORC1 in T cells. In this personal perspective on IDO as a nodal mediator of pathogenic inflammation and immune escape in cancer, we provide a conceptual foundation for the clinical development of IDO inhibitors as a novel class of immunomodulators with broad application in the treatment of advanced human cancer.     

Inhibition of indoleamine 2,3-dioxygenase-mediated tryptophan catabolism accelerates collagen-induced arthritis in mice

Indoleamine 2,3-dioxygenase (IDO) is one of the initial and rate-limiting enzymes involved in the catabolism of the essential amino acid tryptophan. In cultured cells, the induction of IDO leads to depletion of tryptophan and tryptophan starvation. Recent studies suggest that modulation of tryptophan concentration via IDO plays a fundamental role in innate immune responses. Induction of IDO by interferon-γ in macrophages and dendritic cells results in tryptophan depletion and suppresses the immune-mediated activation of fibroblasts and T, B, and natural killer cells. To assess the role of IDO in collagen-induced arthritis (CIA), a model of rheumatoid arthritis characterized by a primarily Th1-like immune response, activity of IDO was inhibited by 1-methyl-tryptophan (1-MT) in vivo. The results showed significantly increased incidence and severity of CIA in mice treated with 1-MT. Activity of IDO, as determined by measuring the levels of kynurenine/tryptophan ratio in the sera, was increased in the acute phase of arthritis and was higher in collagen-immunized mice that did not develop arthritis. Treatment with 1-MT resulted in an enhanced cellular and humoral immune response and a more dominant polarization to Th1 in mice with arthritis compared with vehicle-treated arthritic mice. The results demonstrated that development of CIA was associated with increased IDO activity and enhanced tryptophan catabolism in mice. Blocking IDO with 1-MT aggravated the severity of arthritis and enhanced the immune responses. These findings suggest that IDO may play an important and novel role in the negative feedback of CIA and possibly in the pathogenesis of rheumatoid arthritis.     

Cutting edge: silencing suppressor of cytokine signaling 3 expression in dendritic cells turns CD28-Ig from immune adjuvant to suppressant

CTLA-4-Ig and CD28-Ig are both agonist ligands of B7 coreceptor molecules on mouse dendritic cells (DCs), yet they bias the downstream response in opposite directions, and CTLA-4-Ig promotes tolerance, whereas CD28-Ig favors the onset of immunity. Although B7 engagement by either ligand leads to a mixed cytokine response, a dominant IL-6 production in response to CD28-Ig prevents the IFN-gamma-driven induction of immunosuppressive tryptophan catabolism mediated by IDO. In the present study, we show that silencing the expression of suppressor of cytokine signaling 3 (SOCS3) in DCs by RNA interference renders CD28-Ig capable of activating IDO, likely as a result of unrestrained IFN-gamma signaling and IFN-gamma-like actions of IL-6. Thus, in the absence of SOCS3, CD28-Ig becomes immunosuppressive and mimics the action of CTLA-4-Ig on tryptophan catabolism.     

Ligand binding characteristics of two molecular forms,one equipped with a hydrophobic glycosyl phosphatidylinositol tail,of the folate binding protein purified from human milk

Two molecular forms of the folate binding protein were isolated and purified from human milk by a combination of cation exchange- and affinity chromatography. One protein (27 kDa) was a cleavage product of the other 100 kDa protein as evidenced by N-terminal amino acid sequence homology and a reduction in the molecular size of the latter protein to 27 kDa after cleavage of its hydrophobic glycosylphosphatidylinositol tail by phosphatidylinositol-specific phospholipase C. High-affinity binding of [³H]folate was characterized by upward convex Scatchard plots and increasing ligand binding affinity with decreasing concentrations of both proteins. Downward convex Scatchard plots and binding affinities showing no dependence on the protein concentration were, however, observed in highly diluted solutions of both proteins. Radioligand binding was inhibited by folate analogs, and dissociation of radioligand was slow at pH 7.4 but rapid and complete at pH 5.0 and 3.5. Ligand binding quenched the tryptophan fluorescence of the 27 kDa protein suggesting that tryptophan is present at the binding site and/or ligand binding induces a conformation change that affects tryptophan environment in the protein. The 27 kDa protein representing soluble folate binding protein exhibited a greater affinity for ligand binding than the 100 kDa protein which possesses a hydrophobic tail identical to the one that anchors the folate receptor to the cell membrane.     

Induction of Indoleamine 2, 3-Dioxygenase in Human Dendritic Cells by a Cholera Toxin B Subunit—Proinsulin Vaccine

Dendritic cells (DC) interact with naïve T cells to regulate the delicate balance between immunity and tolerance required to maintain immunological homeostasis. In this study, immature human dendritic cells (iDC) were inoculated with a chimeric fusion protein vaccine containing the pancreatic β-cell auto-antigen proinsulin linked to a mucosal adjuvant the cholera toxin B subunit (CTB-INS). Proteomic analysis of vaccine inoculated DCs revealed strong up-regulation of the tryptophan catabolic enzyme indoleamine 2, 3-dioxygenase (IDO1). Increased biosynthesis of the immunosuppressive enzyme was detected in DCs inoculated with the CTB-INS fusion protein but not in DCs inoculated with proinsulin, CTB, or an unlinked combination of the two proteins. Immunoblot and PCR analyses of vaccine treated DCs detected IDO1mRNA by 3 hours and IDO1 protein synthesis by 6 hours after vaccine inoculation. Determination of IDO1 activity in vaccinated DCs by measurement of tryptophan degradation products (kynurenines) showed increased tryptophan cleavage into N-formyl kynurenine. Vaccination did not interfere with monocytes differentiation into DC, suggesting the vaccine can function safely in the human immune system. Treatment of vaccinated DCs with pharmacological NF-κB inhibitors ACHP or DHMEQ significantly inhibited IDO1 biosynthesis, suggesting a role for NF-κB signaling in vaccine up-regulation of dendritic cell IDO1. Heat map analysis of the proteomic data revealed an overall down-regulation of vaccinated DC functions, suggesting vaccine suppression of DC maturation. Together, our experimental data indicate that CTB-INS vaccine induction of IDO1 biosynthesis in human DCs may result in the inhibition of DC maturation generating a durable state of immunological tolerance. Understanding how CTB-INS modulates IDO1 activity in human DCs will facilitate vaccine efficacy and safety, moving this immunosuppressive strategy closer to clinical applications for prevention of type 1 diabetes autoimmunity.     

Inhibitors of human indoleamine 2,3-dioxygenase identified with a target-based screen in yeast

Indoleamine 2,3-dioxygenase (IDO) is a tryptophan degradation enzyme that is emerging as an important drug target. IDO is expressed by many human tumors to help them escape immune detection, and it has been implicated in depression and in the formation of senile nuclear cataracts. There is a need for potent and selective IDO inhibitors for use in research and as lead compounds for drug development. We show that expression of human IDO in a Saccharomyces cerevisiae tryptophan auxotroph restricts yeast growth in the presence of low tryptophan concentrations and that inhibition of IDO activity can restore growth. We use this assay to screen for IDO inhibitors in collections of pure chemicals and crude natural extracts. We identify NSC 401366 (imidodicarbonimidic diamide, N-methyl-N'-9-phenanthrenyl-, monohydrochloride) as a potent nonindolic IDO inhibitor (Ki=1.5 +/- 0.2 microM) that is competitive with respect to tryptophan. We also use this assay to identify the active compound caulerpin from a crude algal extract. The yeast growth restoration assay is simple and inexpensive. It combines desirable attributes of cell- and target-based screens and is an attractive tool for chemical biology and drug screening.     

Indoleamine 2,3-dioxygenase inhibitory activity of derivatives of marine alkaloid tsitsikammamine A

Tsitsikammamines are marine alkaloids whose structure is based on the pyrroloiminoquinone scaffold. These and related compounds have attracted attention due to various interesting biological properties, including cytotoxicity, topoisomerase inhibition, antimicrobial, antifungal and antimalarial activity.Indoleamine 2,3-dioxygenase (IDO1) is a well-established therapeutic target as an important factor in the tumor immune evasion mechanism. In this preliminary communication, we report the inhibitory activity of tsitsikammamine derivatives against IDO1. Tsitsikammamine A analogue 11b displays submicromolar potency in an enzymatic assay. A number of derivatives are also active in a cellular assay while showing little or no activity towards tryptophan 2,3-dioxygenase (TDO), a functionally related enzyme. This IDO1 inhibitory activity is rationalized by molecular modeling studies. An interest is thus established in this class of compounds as a potential source of lead compounds for the development of new pharmaceutically useful IDO1 inhibitors.     

Ligand-based virtual screening,molecular docking,QSAR and pharmacophore analysis of quercetin-associated potential novel analogs against epidermal growth factor receptor

The present study was to explore expectation and examination of therapeutic potential quercetin analogs as efficient anticancer agents against human epidermal growth factor receptor (EGFR), which is a consistent hallmark for moderating the non-small-cell lung carcinoma (NSCLC). Here, ligand-based virtual screening, pharmacophore approach and molecular docking were established as rational strategies for recognition of small analogs against the ligand binding domain of EGFR (PDB code: 1XKK). Adverse effects, toxicogenomics and pharmacokinetics reported that 10 candidates showed reliable consequences with less side effects and more efficient for target receptor. Protein–ligand interaction profiles revealed that the probable H-bonds, atomic-π contacts, salt bridges and van der Waals interactions sustain the complexity and stability of receptor structure; thus, they could complicate to generate single alteration acquired for drug resistance. In silico anticancer properties explain the lead scaffolds which are assumed to be flexible and experimentally proved chemicals. The overall consequences indicated that recognized leads could be utilized as reference skeletons for new inhibitors envisaging toward EGFR to ameliorate NSCLC and other malignant disorders.     

Immuno-Regulatory Function of Indoleamine 2,3 Dioxygenase through Modulation of Innate Immune Responses

Successful long-term treatment of type-1 diabetes mainly relies on replacement of β-cells via islet transplantation. Donor shortage is one of the main obstacles preventing transplantation from becoming the treatment of choice. Although animal organs could be an alternative source for transplantation, common immunosuppressive treatments demonstrate low efficacy in preventing xenorejection. Immunoprotective effects of indoleamine 2,3-dioxygenase (IDO) on T-cell mediated allorejection has been extensively studied. Our studies revealed that IDO expression by fibroblasts, induced apoptosis in T-cells while not affecting non-immune cell survival/function. Since macrophages play a pivotal role in xenograft rejection, herein we investigated the effect of IDO-induced tryptophan deficiency/kynurenine accumulation on macrophage function/survival. Moreover, we evaluated the local immunosuppressive effect of IDO on islet-xenograft protection. Our results indicated that IDO expression by bystander fibroblasts significantly reduced the viability of primary macrophages via apoptosis induction. Treatment of peritoneal macrophages by IDO-expressing fibroblast conditioned medium significantly reduced their proinflammatory activity through inhibition of iNOS expression. To determine whether IDO-induced tryptophan starvation or kynurenine accumulation is responsible for macrophage apoptosis and inhibition of their proinflammatory activity, Raw264.7 cell viability and proinflammatory responses were evaluated in tryptophan deficient medium or in the presence of kynurenine. Tryptophan deficiency, but not kynurenine accumulation, reduced Raw264.7 cell viability and suppressed their proinflammatory activity. Next a three-dimensional islet-xenograft was engineered by embedding rat islets within either control or IDO–expressing fibroblast-populated collagen matrix. Islets morphology and immune cell infiltration were then studied in the xenografts transplanted into the C57BL/6 mouse renal sub-capsular space. Local IDO significantly decreased the number of infiltrating macrophages (11±1.47 vs. 70.5±7.57 cells/HPF), T-cells (8.75±1.03 vs. 75.75±5.72 cells/HPF) and iNOS expression in IDO-expressing xenografts versus controls. Islet morphology remained intact in IDO-expressing grafts and islets were strongly stained for insulin/glucagon compared to control. These findings support the immunosuppressive role of IDO on macrophage-mediated xeno-rejection.