High Indoleamine 2,3-dioxygenase Expression Research Articles

Indoleamine 2,3-dioxygenase expression and activity in patients with hepatitis C virus-induced liver cirrhosis.

Indoleamine 2,3-dioxygenase (IDO) is an immunoregulatory enzyme. It plays a key role in various malignancies, infection and autoimmune diseases. IDO induces immunosuppression through the depletion of tryptophan and its downstream metabolites. Hepatitis C virus (HCV) has infected more than 12 million individuals in Pakistan. The aim of the present study was to assess the expression and activity of IDO in HCV-infected patients. The functional enzymatic activity of IDO was measured by colorimetric assay. Serum samples from 100 HCV-infected patients were taken to examine IDO activity and samples from 100 healthy volunteers were used as controls. Liver sections from patients with HCV (n=35) and healthy controls (n=5) were used for immunohistochemical studies. Immunohistochemical analysis revealed that IDO was overexpressed in 28 of 35 (80%) cirrhotic liver samples, whereas 5 of 35 (14.2%) cases presented moderate and 2 of 35 (5.7%) cases presented mild expression of IDO. The enzymatic activity of IDO was significantly higher in the serum samples of HCV-infected patients as compared with those in the control. These data indicate that the expression of IDO correlated with the pathogenesis of disease. In summary, it is suggested that the high expression of IDO in the progressively cirrhotic livers of HCV-infected patients might contribute to the development of hepatocellular carcinoma. IDO may characterize a novel therapeutic target against HCV.

T Cells Expressing CD19-Specific Chimeric Antigen Receptors Are Inhibited By Indoleamine 2,3-Dioxygenase in Tumors

Indoleamine 2,3-dioxygenase (IDO) is an intracellular enzyme that mediates the metabolism of tryptophan to kynurenines, which have an inhibitory activity on immune cells. IDO-positive tumors thus establish a microenvironment in which NK and T cells are inactivated, and high IDO expression by tumor cells and high serum kynurenine levels correlate with poor prognosis in diffuse large B-cell lymphoma patients.CD19-specific chimeric antigen receptor T cell (CART) therapy is a promising new approach against B-cell malignancies but not every tumor responds. To determine whether the presence of IDO in tumors affects CART activity, we first investigated the expression of IDO by the human B-cell lymphoma cell lines Raji, Daudi, BJAB and Jeko-1. We found that only Jeko-1 expresses IDO and produces kynurenine natively. IDO was not expressed by the other cell lines, even after exposure to IFNγ, a known IDO inducer. Based on these results, we chose Raji as a baseline IDO-negative cell line and made an IDO-positive Raji clone by retroviral transduction with human IDO cDNA (Raji-IDO); a clone transduced with an empty vector served as control (Raji-control). We injected SCID mice subcutaneously in opposite flanks with luciferase-transduced Raji-control and Raji-IDO cells. Seven days later, we injected human non-transduced T cells (NTs) or CARTs intravenously. In the NT group, tumors had similar growth on both sides. In contrast, in the CART group, Raji-control tumors had significantly slower growth than Raji-IDO tumors (3.1 ± 1.1×108 and 20 ± 7.3×108 bioluminescence units [BU] at day 7, respectively, P = 0.03). We also found that CARTs significantly inhibited Raji-control tumor growth (NT: 27 ± 6.8×108 vs CART: 3.1 ± 1.1×108 BU at day 7, P = 0.02), but did not affect Raji-IDO tumors (NT: 24 ± 5.4×108 vs CART: 20 ± 7.3×108 BU at day 7, P = 0.35). In another experiment, Raji-IDO cells were injected subcutaneously, and mice were treated with an IDO inhibitor (1-methyl-tryptophan, 1-MT), CARTs, or both. The combination treatment produced significantly better tumor control than either single therapy (1-MT: 45 ± 6.8×108 and CART: 22 ± 4.6×108 vs both: 8.2 ± 3×108 BU at day 7, P = 0.001 and 0.04, respectively). Thus, the IDO inhibitor protects CARTs against the deleterious effects of IDO-positive tumors.To investigate potential mechanisms for CART inhibition by IDO, we assessed the effect of kynurenine and found that even low concentrations (12.5 µM) inhibited CART proliferation in response to IL-2, IL-7, IL-15 or CD19-positive targets, although there was no effect on proliferation of B-cell lymphoma cell lines at this kynurenine concentration. CART apoptosis was also increased by kynurenine (8.6 ± 0.6%, 13.9 ± 2.2%, and 33.5 ± 10.6% annexin V-positive cells with 0, 12.5, or 25 µM kynurenine). In coculture of CARTs with wild-type Raji cells, the latter were eliminated by day 6 in the absence of kynurenine, but increased in numbers (in parallel with a decrease in CARTs) in its presence. Kynurenine also inhibited the release of IFNγ (13,143 ± 848 pg/mL vs 2,663 ± 1,873 pg/mL, P = 0.02) and IL-2 (718 ± 355 pg/mL and 199 ± 165 pg/mL, P = 0.03) by CARTs. Expression of granzyme B, PD-1 and CTLA-4 on CARTs was not significantly affected.Fludarabine has been reported to downregulate IDO expression in tumors and this drug is used in many lymphodepleting regimens that are administered before CART infusion in an effort to augment the efficacy of these therapies. However, the beneficial mechanism of lymphodepleting chemotherapy drugs is not fully understood. Therefore, we also measured the effect of fludarabine and mafosfamide (a cyclophosphamide analog) on IDO expression by Jeko-1 cells. We found that both drugs downregulate IDO expression by Jeko-1, even in the presence of IFNγ.In summary, expression of IDO by tumor cells inhibits CART activity, likely because kynurenine is produced and has negative effects on proliferation and cytokine secretion by CARTs. Fludarabine and cyclophosphamide downregulate IDO expression in tumors and this effect may contribute to the benefits of lymphodepletion before CART therapy. Direct IDO inhibitors may further improve clinical CART activity against IDO-positive tumors. DisclosuresNo relevant conflicts of interest to declare.

Abstract 1633: Novel specific- and dual- tryptophan-2,3-dioxygenase (TDO) and indoleamine-2,3-dioxygenase (IDO) inhibitors for tumor immunotherapy

Abstract Tryptophan catabolism mediated by indoleamine 2,3-dioxygenase is an central immune checkpoint mechanism that controls peripheral immune tolerance and tumor resistance to immune attack. The IDO pathway mediates immunosuppressive effects by expression of the IDO1 gene by tumor cells and/or host antigen presenting cells. Tryptophan depletion and generation of kynurenine generate downstream signals through GCN2, WARS, mTOR and AHR that ultimately lead to anergy of tumor-specific effector CD8+ T cells and activation of Tregs. High expression of IDO correlates negatively with clinical prognosis in patients with a variety of malignancies. Recently, it has been described that tumors can also achieve immunosuppressive effects mediated by tryptophan degradation through expression of TDO, an enzyme functionally and structurally related to IDO1. Therefore, development of small molecules that can prevent tryptophan degradation mediated by IDO1, TDO or both is a prime goal for immunotherapy of cancer. Here we report the discovery of a novel class of imidazoisoindole-based compounds that show potent and selective inhibition of TDO and IDO1, as well as compounds that show potent dual inhibition of both TDO and IDO1. We present structure-activity relationship studies that explore the molecular determinants of potency and specificity for TDO and IDO1. Compounds of these classes were tested for activity in in vitro assays using purified recombinant human and murine TDO and IDO1 as well as in cells expressing IDO1 or TDO. Many of these compounds are 10-200 fold more potent (IC50 < 50 nM) than previously published TDO-specific inhibitors. The pharmacokinetic properties of these compounds are being investigated and lead optimization efforts are in progress. Citation Format: Mario R. Mautino, Richard A. Metz, Firoz Jaipuri, Jesse Waldo, Sanjeev Kumar, Agnieszka Marcinowicz-Flick, Hima Potturi, James T. Adams, Clarissa Van Allen, Nicholas N. Vahanian, Charles J. Link. Novel specific- and dual- tryptophan-2,3-dioxygenase (TDO) and indoleamine-2,3-dioxygenase (IDO) inhibitors for tumor immunotherapy. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1633. doi:10.1158/1538-7445.AM2014-1633

The subsets of dendritic cells and memory T cells correspond to indoleamine 2,3-dioxygenase in stomach tumor microenvironment.

The abnormal distributions of memory T cells (Tm) and dendritic cells (DC) in stomach cancer are not well understood. Indoleamine 2,3-dioxygenase (IDO), produced by DC, may be an important enzyme affecting function and proliferation of Tm. In this study, IDO expression was examined by immunohistochemical staining. The subsets of Tm and DC were counted by flow cytometry. The percentages of CD4 + Tm and CD4 + central Tm (Tcm) were lower in tumor tissues than in normal tissues (P < 0.05), while the CD4 + effector Tm (Tem) and CD8 + Tem percentages were higher in tumor tissues (P < 0.05). The ratio of myeloid DC (DC1)/plasmacytoid DC (DC2) was significantly lower in tumor tissues (P = 0.009). The high expression of IDO was more frequently observed in tumor tissues (P = 0.001). The percentages of CD4 + Tm and CD8 + Tm were positively associated with DC1 percentage and ratio of DC1/DC2 (P < 0.05). The higher CD8 + Tcm percentage was associated with higher DC2 percentage (P = 0.025). The patients with high IDO expression had significantly lower CD4 + Tm (P = 0.012) and CD8 + Tm percentages (P = 0.033), but higher CD8 + Tem percentage (P < 0.01). Concerning on clinicopathologic features, the higher DC2 percentage was associated with larger tumor size (P = 0.019). The CD4 + Tm and CD8 + Tem percentages were significantly associated with clinical stage and lymph node metastasis; the high IDO expressions were significantly associated with deeper tumor invasion (P = 0.016) and lymph node metastasis (P = 0.038). Thus, DC subsets, Tm subsets, and IDO expression were correlated with each other. They were associated with the established clinicopathologic features, such as tumor size, depth of invasion, lymph node metastasis, and clinical stage.

Indoleamine 2,3-dioxygenase (IDO) is frequently expressed in stromal cells of Hodgkin lymphoma and is associated with adverse clinical features: a retrospective cohort study.

BackgroundRegulation of tumor microenvironment is closely involved in the prognosis of Hodgkin lymphoma (HL). Indoleamine 2,3-dioxygenase (IDO) is an enzyme acting as immune modulator through suppression of T-cell immunity. This study aims to investigate role of IDO in the microenvironment of HL.MethodsA total of 121 cases of HL were enrolled to do immunohistochemistry for IDO, CD163, CD68, CD4, CD8, and FoxP3. Positivity was evaluated from area fractions or numbers of positive cells using automated image analyzer. Correlations between IDO expression and various cellular infiltrates and clinicopathologic parameters were examined and survival analyses were performed.ResultsIDO was expressed in histiocytes, dendritic cells and some endothelial cells with variable degrees, but not in tumor cells. IDO positive cells were more frequently found in mixed cellularity type than other histologic types, and in cases with EBV+, high Ann Arbor stages, B symptoms, and high IPS (all p < 0.05). High IDO expression was associated with inferior survival (p < 0.001) and reflects an independent prognostic factor in nodular sclerosis HL.ConclusionsThis is the first study suggesting that IDO is the principle immunomodulator and is involved to adverse clinical outcomes of HL.

A paradoxical pattern of indoleamine 2,3-dioxygenase expression in the colon tissues of patients with acute graft-versus-host disease

Indoleamine 2,3-dioxygenase (IDO) is a rate-limiting enzyme for tryptophan catabolism that plays an important role in the induction of immune tolerance. It is induced in the colon and exerts its effects there, regulating T-cell proliferation and survival. To address the role of IDO in acute graft-versus-host disease (AGVHD) after human allogeneic hematopoietic stem cell transplantation, we analyzed the relationship between IDO expression in colon tissues and clinical outcomes among 41 AGVHD patients who were diagnosed as gut AGVHD by a colon mucosal biopsy within 100 days posttransplantation. By in situ immunohistochemical analyses, IDO expression was measured in colon mucosal mononuclear cells (MNCs) and endothelial cells (ECs) in GVHD areas. High IDO expression in MNCs and low IDO expression in ECs had a trend toward a lower nonrelapse mortality (p = 0.157 and p = 0.062, respectively). Multivariate analysis showed that high MNC combined with low EC IDO expression (p = 0.046), as well as low disease risk (p = 0.012), are associated with lower nonrelapse mortality. Paradoxical upregulation of IDO expression in colon MNCs and ECs may represent a new predictive factor for prognosis in gut AGVHD after human allogeneic hematopoietic stem cell transplantation.

IDO is highly expressed in breast cancer and breast cancer-derived circulating microvesicles and associated to aggressive types of tumors by in silico analysis

Indoleamine-2,3-dioxygenase (IDO) has been established as a normal mechanism of peripheral tolerance and immunosuppression. Besides, malignant tumors release microvesicles (MV) related with tumor dissemination. The aims of this study were to determine the expression of IDO in breast cancer and circulating microvesicles from breast cancer patients and to perform an in silico analysis to find genes co-expressed to IDO. One hundred and twenty-two tissue and serum breast samples (91 malignant, 21 benign, and 10 normal), and MCF7, MDA-MB-231, and T47D breast cancer cell lines were included. Standard immunohistochemistry (IHC), immunocytochemistry (ICC), Western blot (WB), and RT-PCR were employed. Microvesicle isolation from plasma samples was obtained by serial centrifugation and ultracentrifugation. By IHC, 60 % breast cancer, 43 % benign, and 20 % normal samples were positive. Significant differences were found among normal, benign, and malignant samples. Breast cancer stages I, II, and III expressed IDO in 42, 66, and 71 % of samples, respectively, while breast cancer cell lines also reacted; by WB, 9/25 microvesicles fractions showed bands at 42 kD. In silico analysis of IDO 1 gene expression in breast cancer showed its association with several genes related to immune response and apoptosis. Moreover, IDO and co-expressed genes were found predominately in basal and erbB2 subtypes. The cumulative data indicate a high expression of IDO in breast cancer which increased with higher stages. Furthermore, IDO was found in association with circulating breast cancer MV, while experimental and in silico gene expression revealed that IDO was mainly expressed in a triple-negative subgroup.

Mapping the immunosuppressive environment in uterine tumors: implications for immunotherapy

The major hurdle for cancer vaccines to be effective is posed by tumor immune evasion. Several common immune mechanisms and mediators are exploited by tumors to avoid immune destruction. In an attempt to shed more light on the immunosuppressive environment in uterine tumors, we analyzed the presence of PD-L1, PD-L2, B7-H4, indoleamine 2,3-dioxygenase (IDO), galectin-1, galectin-3, arginase-1 activity and myeloid-derived suppressor cell (MDSC) infiltration. IDO, PD-L1, PD-L2 and B7-H4 were analyzed by immunohistochemistry. PD-L2 was mostly expressed at low levels in these tumors. We found high IDO expression in 21 % of endometrial carcinoma samples and in 14 % of uterine sarcoma samples. For PD-L1 and B7-H4, we found high expression in 92 and 90 % of endometrial cancers, respectively, and in 100 and 92 % of the sarcomas. Galectin-1 and 3 were analyzed in tissue lysates by ELISA, but we did not find an increase in both molecules in tumor lysates compared with benign tissues. We detected expression of galectin-3 by fibroblasts, immune cells and tumor cells in single-cell tumor suspensions. In addition, we noted a highly significant increase in arginase-1 activity in endometrial carcinomas compared with normal endometria, which was not the case for uterine sarcomas. Finally, we could demonstrate MDSC infiltration in fresh tumor suspensions from uterine tumors. These results indicate that the PD-1/PD-L1 interaction and B7-H4 could be possible targets for immune intervention in uterine cancer patients as well as mediation of MDSC function. These observations are another step toward the implementation of inhibitors of immunosuppression in the treatment of uterine cancer patients.Electronic supplementary materialThe online version of this article (doi:10.1007/s00262-014-1537-8) contains supplementary material, which is available to authorized users.

Indoleamine 2,3-Dioxygenase as A Prognostic Factor in Patients with Non-Small Cell Lung Cancer

Background: Indoleamine 2,3-dioxygenase (IDO) is an immunomodulatory enzyme produced by tumor cells and some alternatively activated macrophages and other immunoregulatory cells. The purpose of the present study was to evaluate the prognostic value of the relative expression of the forkhead/winged helix transcription factor 3 (Foxp3) and IDO in non-small cell lung cancer (NSCLC) tissues.Methods: The NSCLC tissues from 141 patients who underwent complete surgical resection were collected at the time of surgery. The relative expression levels of Foxp3 and IDO in the tissues were determined by quantitative RT–PCR.Results: The histological types of cancer seen in these patients included 105 adenocarcinomas, 24 squamous cell carcinomas and 12 other types of carcinoma. The average expression levels of Foxp3 and IDO relative to that of s-actin in the NSCLC tissue were 0.052 ± 0.147% and 0.088 ± 0.157%, respectively. The relative expression of Foxp3 tended to increase with the relative expression of IDO (R=0.451, P=0.001). The five-year survival rates of the patients according to the relative expression of Foxp3 were 78.3% and 71.9% in the lower and higher groups, respectively. According to the relative expression of IDO, the five-year survival rate was 83.2% in the lower expression group, and 67.9% in the higher expression group. There was a significant difference between the lower and higher IDO expression groups (p=0.0389).Conclusions: The expression of IDO tended to have a positive correlation with the expression of Foxp3. The higher expression of IDO was therefore a significantly unfavorable prognostic factor in patients with NSCLC.

The Correlation Between the Subsets of Tumor Infiltrating Memory T Cells and the Expression of Indoleamine 2,3-Dioxygenase in Gastric Cancer

Although many researchers have concentrated on the mechanism of T cell anergy resulting from over-expression of Indoleamine 2,3-dioxygenase (IDO), it remains unclear what alterations will developed in memory T cells (Tm) under over-expression of IDO. Immunohistochemical staining for IDO expression in gastric cancer tissues (n=50) was carried out. Tumor-infiltrating memory Tm cells were counted by flow cytometry. The association between IDO expression and the subsets of tumor infiltrating Tm are discussed. The higher IDO expressions were significantly associated with deeper invasion (P=0.016) and higher frequencies (P=0.038) of lymph node metastasis. The lower tumor-infiltrating CD4+Tm were significantly associated with the advanced clinical stage (P=0.026) and lymph node metastasis (P=0.016). The lower percentages of CD8+Tm were significantly related to undifferentiated histological type (P=0.042) and lymph node metastasis (P=0.037). However, the lower percentage of CD8+Tem was significantly correlated to differentiated histological type (P=0.017), lower frequencies of lymph node metastasis (P=0.014), and earlier clinical stage (P=0.008). The higher IDO expression patients had significantly lower percentages of CD4+Tm (P=0.012) and CD8+Tm (P=0.033). Nevertheless, it was confirmed that the higher level of IDO expression correlated with higher percentages of CD8+Tm cells in univariate and multivariate analysis (P=0.011). IDO over-expression and Tm in tumor microenvironments were correlated with the disease stage and histological type of gastric cancer. Higher IDO expression was related to the lower percentage of CD4+Tm and CD8+Tm, whereas the higher level of IDO expression related with a higher percentage of CD8+Tem.

CD11b+ cells in donor-specific transfusion prolonged allogenic skin graft survival through indoleamine 2,3-dioxygenase

The aim of this study is to show the effect of donor-specific transfusion (DST) in inducing immunological tolerance mediated by regulatory T cells (Treg) and indoleamine 2,3-dioxygenase (IDO). Skin grafts from H2d Balb/c were transplanted into H2k C3H/He 7days after the infusion of donor splenocytes, isolated each immune cell populations. Graft survival prolonged in recipients who received splenocytes, MHC class II+ CD90− cells and CD3−CD19− cells (p<0.001, p<0.05 and p<0.01, respectively). CD11b+ cell infusion resulted in prolongation of graft survival when compared to CD11c+ cell infusion (p<0.01). Foxp3+CD4+CD25+ T cells were increased after the transplant in recipients infused with CD11b+ cells (p<0.05). The mixed lymphocyte reaction showed donor-specificity (p<0.001). High IDO expression was observed in CD11b+ cell infusion group. Graft survival with DST using IDO antagonist (1MT) were not prolonged. In conclusion, DST allows induction of donor-specific tolerance which involves Foxp3+CD4+CD25+ T cells and IDO expression.

Abstract 491: NLG919, a novel indoleamine-2,3-dioxygenase (IDO)-pathway inhibitor drug candidate for cancer therapy.

Abstract The IDO pathway mediates immunosuppressive effects through the metabolization of tryptophan (Trp) to kynurenine (Kyn), triggering downstream signaling through GCN2, mTOR and AHR that can affect differentiation and proliferation of T cells. Expression of the IDO1 gene by tumor cells or host APCs can inhibit tumor-specific effector CD8+ T cells and enhance the suppressor activity of Tregs, and high expression of IDO correlates with worse clinical prognosis in patients with a variety of malignancies. Therefore, targeting the IDO pathway via inhibition of the IDO enzyme or blocking its downstream signaling effects is a prime target for small-molecule immunomodulatory drugs in cancer. Here we describe the pharmacological and biological properties of NLG919, a novel small-molecule IDO-pathway inhibitor. NLG919 potently inhibits this pathway in vitro and in cell based assays (Ki=7 nM; EC50 =75 nM). It is orally bioavailable (F&amp;gt;70%); and has a favorable pharmacokinetic and toxicity profile. In mice, a single oral administration of NLG919 reduces the concentration of plasma and tissue Kyn by ∼ 50%. Using IDO-expressing human monocyte-derived DCs in allogeneic MLR reactions, NLG919 potently blocked IDO-induced T cell suppression and restored robust T cell responses with an ED50=80 nM. Similarly, using IDO-expressing mouse DCs from tumor-draining lymph nodes, NLG919 abrogated IDO-induced suppression of antigen-specific T cells (OT-I) in vitro, with ED50=120 nM. In vivo, in mice bearing large established B16F10 tumors, administration of NLG919 markedly enhanced the anti-tumor responses of naïve, resting pmel-1 cells to vaccination with cognate hgp100 peptide plus CpG-1826 in IFA. In this stringent established-tumor model, NLG919 plus pmel 1/vaccine produced a dramatic collapse of tumor size within 4 days of vaccination (∼95% reduction in tumor volume compared to control animals receiving pmel-1/vaccine alone without NLG919). In conclusion, NLG919 is a potent IDO pathway inhibitor with desirable pharmacological properties, suitable for the treatment of immunosuppression associated with cancer. Citation Format: Mario R. Mautino, Firoz A. Jaipuri, Jesse Waldo, Sanjeev Kumar, James Adams, Clarissa Van Allen, Agnieszka Marcinowicz-Flick, David Munn, Nicholas Vahanian, Charles J. Link. NLG919, a novel indoleamine-2,3-dioxygenase (IDO)-pathway inhibitor drug candidate for cancer therapy. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 491. doi:10.1158/1538-7445.AM2013-491

The Kinetics of Plasmacytoid Dendritic Cell Accumulation in the Pancreas of the NOD Mouse during the Early Phases of Insulitis

In non-obese diabetic (NOD) mice that spontaneously develop autoimmune diabetes, plasmacytoid dendritic cells (pDCs) have a diabetes-promoting role through IFN-α production on one hand, while a diabetes-inhibiting role through indoleamine 2,3-dioxygenase (IDO) production on the other. Little is known about the kinetics and phenotype of pDCs in the NOD pancreas during the development of autoimmune diabetes. While para/peri-insular accumulation of conventional dendritic cells (cDCs) could be observed from 4 weeks of age onwards in NOD mice, pDCs only started to accumulate around the islets of Langerhans from 10 weeks onwards, which is concomitant with the influx of lymphocytes. NOD pancreatic pDCs showed a tolerogenic phenotype as assessed by their high expression of IDO and non-detectable levels of IFN-α and MxA. Furthermore, expression of the pDC-attracting chemokines CXCL10 and CXCL12 was significantly increased in the NOD pancreas at 10 weeks and the circulating pDC numbers were increased at 4 and 10 weeks. Our data suggest that a simultaneous accumulation of IDO+ pDCs and lymphocytes in the pancreas in 10 weeks old NOD mice, which may reflect both an immunogenic influx of T cells as well as a tolerogenic attempt to control these immunogenic T cells.

Analysis of indoleamine 2-3 dioxygenase (IDO1) expression in breast cancer tissue by immunohistochemistry

The immunosuppressive enzyme, indoleamine 2,3 dioxygenase (IDO), is overexpressed in many different tumor types including breast cancer. IDO inhibitors synergize with chemotherapy in breast cancer murine models. Characterizing IDO expression in breast cancer could define which patients receive IDO inhibitors. This study analyzed IDO protein expression in 203 breast cancer cases. The relationship between IDO, overall survival (OS), disease-specific survival (DSS), clinicopathologic, molecular, and immune tumor infiltrate factors was evaluated. Expression of IDO, estrogen receptor (ER), progesterone receptor (PR), human epithelial receptor 2, cytokeratin 5/6, epithelial growth factor receptor, phosphorylated AKT, neoangiogenesis, nitrogen oxide synthetase 2 (NOS2), cyclooxygenase 2 (COX2), FoxP3, CD8, and CD11b on archival breast cancer tissue sections was evaluated by immunohistochemistry. Associations between IDO and these markers were explored by a univariate and multivariate analysis. Survival was analyzed using Kaplan-Meier (OS) and Wilcoxon two-sample (DSS) tests. IDO expression was higher in ER+ tumors compared to ER- tumors. IDO was lower in those with higher neoangiogenesis. OS was better in ER+ patients with high IDO expression. DSS was better in node-positive patients with high IDO expression. IDO activity positively correlates with NOS2. COX2 as positively correlated with IDO on univariate but not multivariate analysis. There was a trend toward greater numbers of CD11b+ cells in IDO-low tumors. IDO protein expression is lower in ER- breast tumors with greater neoangiogenesis. Future clinical trials evaluating the synergy between IDO inhibitors and chemotherapy should take this finding into account and stratify for ER status in the trial design.

Tumoral indoleamine 2,3-dioxygenase expression predicts poor outcome in laryngeal squamous cell carcinoma

The development of laryngeal squamous cell carcinomas (LSCC) is strongly influenced by the host immune system. Indoleamine 2,3-dioxygenase (IDO) can promote and maintain an immunosuppressive microenvironment which can impede the efficacy of anticancer responses. The purpose of the current study is to investigate the prognostic value of intratumoral IDO expression in LSCC. The expression of IDO protein was retrospectively assessed by immunohistochemistry in 187 LSCC patients. The potential association of tumor IDO expression with clinical parameters and tumor-infiltrating lymphocytes (TILs) was analyzed separately. Survival curves were estimated by the Kaplan-Meier method, and differences between groups were determined by log-rank test. Multivariate logistic regression analysis was performed to determine the independent factors associated with survival. Based on the evaluation score, 90 carcinomas (48.1%) were identified with high IDO expression and 97 carcinomas (51.9%) showed low expression. Tumor IDO expression was not associated with clinical stage, presence of metastases, and other clinicopathological parameters. Also, high IDO expression was not correlated with tumor-infiltrating CD3(+) and CD8(+) TILs. Instead it was positively related with the density of FOXP3(+) Tregs. Furthermore, multivariate analysis identified a significant association of overall survival and disease-free survival with tumor IDO status. IDO high expression represents a significant negative prognostic factor in patients with LSCC. Current results provide further support for using IDO as an immunotherapeutic target in LSCC. The precise role of tumoral IDO in human LSCC remains to be elucidated in the future.

Indoleamine 2,3-dioxygenase and forkhead boxP3 on immune tolerance of gallbladder carcinoma cells

Objective To investigate the expressions of indoleamine 2,3-dioxygenase (IDO) and transcription factor forkhead box P3(FOXP3)in gallbladder adenocarcinoma; and to evaluate the relationship between the expressions of IDO and FOXP3 protein,and clinicopathology and lymph node metastasis of gallbladder carcinoma.Methods The expressions of IDO and FOXP3 in 51 cases of primary gallbladder cancer,90 cases of chronic cholecystitis,and 20 cases of normal gallbladder tissues were studied by immunohistochemical staining. Results The positive expression rates of IDO and FOXP3 in gallbladder carcinoma were 72.5％ (37/51) and 60.8％ (31/51),in normal gallbladder mucosa were 5％ (1/20) and 20.0％ (4/20),in cholecystitis and gallstone were 11.1％ (10/90) and 32.2％ (29/90),respectively.There were significant differences among the three groups in IDO and FOXP3 expressions (P＜0.01).The positive expression rates of IDO and FOXP3 were 7.1％ and 14.3％ for simple hyperplasia,17.7％ and 35.3％ for atypical hyperplasia,40％ and 35％ for gallbladder carcinoma (stages Ⅰ-Ⅲ),and 77.4％ and 64.5％ for gallbladder carcinoma (stages Ⅳ-Ⅴ ).There were significant differences among the four groups in IDO and FOXP3 expressions (P＜0.01).There were significant differences among the Nevin stage groups,lymph node metastasis group and gallbladder stone in IDO and FOXP3 expressions.However,there were no significant differences among the sex groups and the age groups in IDO and FOXP3 expressions (P＞0.05).In gallbladder carcinoma,the expression of IDO had a positive correlation with FOXP3 expression in lymphocyte (γ=0.406,P=0.003).Conclusion In gallbladder cancer,a high-expression of IDO and an expression of FOXP3 in lymphocyte are closely related with immune tolerance of gallbladder carcinoma.The results provide a reference for clinical use of immunotherapy in gallbladder cancer. Key words: Primary gallbladder carcinoma; Indoleamine 2,3-dioxygenase; Forkhead box P3

High expression of indoleamine 2,3‐dioxygenase in the tumour is associated with medullary features and favourable outcome in basal‐like breast carcinoma

Medullary breast cancer (MBC) is a basal-like breast carcinoma (BLBC) with a favourable outcome, whereas nonmedullary BLBC has a poor prognosis. Tumour infiltrating lymphocytes (TILs) are present in both MBC and BLBC. We hypothesized that the immunosuppressive enzyme indoleamine 2,3-dioxygenase (IDO) could modulate the TILs effects among these tumours and explain their different outcomes. The amount of TILs and IDO expression were analysed using immunohistochemistry (IHC) in 155 BC cases including MBC (n = 17), atypical MBC (n = 13) and non-MBC (n = 125). Messenger RNA expression of the INDO gene, which encodes IDO, was measured in 262 cases from our institution. INDO mRNA expression and histoclinical data of 1,487 BC cases were collected from public databases. IDO immunostaining was present in both neoplastic and stromal cells in 100% of MBC and was associated with histological medullary features among non-MBC cases. There was a significant correlation between IDO positivity and TIL amounts. In our series including mostly grade-3 BC, IDO immunostaining was the most significant marker (p = 0.02) associated with better survival in multivariate analysis. Among our 262 analysed BC cases, INDO mRNA showed significant overexpression in BLBC as compared to luminal A tumours, and in MBC as compared to basal-like non-MBC. In the pooled series of 1,749 BC cases, INDO mRNA was overexpressed in BLBC and was the most significant predictor of better survival in this subtype using multivariate analysis (p = 0.0024). In conclusion, high IDO expression is associated with morphological medullary features and has an independent favourable prognostic value in BLBC.

Expression of indoleamine 2,3-dioxygenase predicts shorter survival in patients with vulvar squamous cell carcinoma (vSCC) not influencing on the recruitment of FOXP3-expressing regulatory T cells in cancer nests

Objective Regulatory T cells (Tregs), and the enzyme indoleamine 2,3-dioxygenase (IDO), have potential regulatory properties for immune escape in cancer. Inhibitors of IDO are available and could potentially be used in vulvar cancer if IDO was proved to drive progression of the disease. The aim of this study was to evaluate the expression of factor forkhead boxP3 (FOXP3), a marker of Tregs, and IDO in vulvar squamous cell carcinoma (vSCC), and to verify their prognostic significance. Methods 76 primary tumors and 35 lymph node metastases derived from 76 patients with full clinical history were analyzed. The intratumoral infiltration of Tregs and IDO expression within cancer were evaluated by immunohistochemistry. Results The number of Tregs in primary tumor and in corresponding lymph node metastasis was significantly correlated. Intensity of Treg infiltrates in the primary and metastatic sites was not correlated to IDO expression and had no influence on the overall patient survival. High IDO expression was associated with significantly worse overall survival among vSCC patients and was found to be an independent prognostic factor similarly to the tumor grade and patient's age. Conclusions The degree of intratumoral Treg infiltrates is an individual feature and remains stable throughout the course of the disease without impact on the patient's survival. IDO expression predicts shorter survival of vSCC patients. If immunologic tolerance of the tumor is promoted by the overexpression of IDO it will not influence the number of intratumoral Tregs. IDO expression seems to be an independent prognostic factor in patients with vSCC.

568 A permutation-based confidence interval for treatment effect in the identified subset of sensitive patients in biomarker-adaptive threshold design

Indoleamine 2,3-dioxygenase (IDO) is a tryptophan-catabolizing enzyme that induces immune tolerance. The purpose of the present study is to investigate IDO expression and its functional role in ovarian cancer cells in vitro and in vivo.IDO expression was immunohistochemically scored in surgically-resected ovarian cancer tissues (n = 60), and its association with tumor-infiltrating lymphocyte (TIL) count or patient survival was analyzed. Next, IDO cDNA was transfected into the human ovarian carcinoma cell line SKOV3, establishing stable clones of IDO-overexpressing cells (SK-IDO). SK-IDO cells were characterized in vitro as well as in vivo using a nude mouse xenograft model.High IDO expression in tumor cells was found in 34 (56.7%) cases and was correlated with a reduced number of CD8+ TIL. Patients with high IDO expression had significantly impaired overall and progression-free survival compared to patients with no or low IDO expression. There were no significant differences in in vitro cell proliferation, migration, invasion, or chemosensitivity to paclitaxel between the SK-IDO and control vector-transfected (SK-pcDNA) cells. However, tumor peritoneal dissemination was significantly increased in SK-IDO-xenografted mice compared to SK-pcDNA-xenografted mice. This tumor-progressive effect in SK-IDO-xenografted mice was abrogated by oral administration of the IDO inhibitor 1-methyl-tryptophan (1-MT). Finally, treatment with weekly i.p. paclitaxel combined with daily administration of 1-MT significantly prolonged the survival of the SK-IDO-xenografted mice compared to treatment with paclitaxel alone.These results suggest that IDO is involved in ovarian cancer progression in vivo and may be a promising therapeutic target for advanced ovarian cancer.

Association of malignancy of human colorectal carcinoma with IDO gene expression

Objective To investigate the correlation of Indoleamine 2,3-dioxygenase (IDO) gene expression with malignancy of colorectal carcinoma. Methods The expression of IDO in 121 specimens of colorectal carcinoma, adjacent tissue, and normal mucus tissue was detected by using immunohistochemis-try with anti-IDO monoclone antibody, reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting. The correlation of the expression of IDO with various clinicopathologic characteristics of eolorectal carcinoma was analyzed statistically. Results 35.5% (43/121) of colorectal carcinomas ex-pressed IDO, and the expression of IDO was significantly upregulated in carcinoma as compared with adja-cent or normal mucus tissues. The positive rate of IDO in colorectal carcinoma with low-/un-differentiation, lymphnode or liver metastasis was up to 61.1%, 65.3% and 87.5% respectively, in which more than half was intensively stained (+++). Conclusion The upregulation of IDO gene expression in colorectal carcino-ma was correlated to tumor cell differentiation, clinic stage, and metastasis of the lymph node or liver. IDO may be an indicator of colorectal carcinoma prognosis. The high expression of IDO in the metastatic lymph node implies IDO might have a role in tumor immune escape by catalyzing tryptophan metabolism locally. Key words: Colorectal cancer; Gene expression; Indoleamine 2,3-dioxygenase