IDO Activity Research Articles

EXTH-62. EXAMINATION OF THE TUMOR MICROENVIRONMENT OF GLIOMAS TREATED WITH VIROIMMUNOTHERAPY REVEALS THE PRODUCTION OF ONCOMETABOLITES LEADING TO IMPROVED THERAPEUTIC STRATEGIES

Abstract Oncolytic viruses are considered part of immunotherapy and have shown promise in preclinical experiments and clinical trials in a small fraction of patients. To improve the response rate, it is necessary to reshape the tumor microenvironment that shields the tumor from the immune system of the patient. We engineered Delta-24-RGDOX (DNX-2440), an oncolytic adenovirus to express the T-cell activator OX40L, which triggered, in murine glioblastoma models, a dramatic reshaping of the tumor microenvironment dominated by changes in immune processes in an RNA-seq analysis of ingenuity pathways. Paradoxically, network analyses also revealed that Delta-24-RGDOX induced robust activation of the cytokine-driven immunosuppressive IDO-Kynurenine-AhR circuitry, indicating a potential mechanism of resistance of gliomas to oncolytic virotherapy. To reverse this immunosuppression, we combined Delta-24-RGDOX with clinically relevant IDO inhibitors to treat glioma-bearing mice. The combined therapy decreased the activation of the IDO network without affecting viral activity. Flow cytometry assays revealed a decrease in the presence of the immunosuppressive cell populations, MDSCs and Tregs, which presence was higher in Delta-24-RGDOX-treated tumors. These data were further confirmed in gene set enrichment analyses. Functional co-culture studies showed that the combined therapy activated splenocytes against tumor antigens and that this activation was reversed by Kynurenine. Importantly, the combination treatment eradicated the tumors in a CD4-dependent manner and significantly prolonged the survival of glioma-bearing mice. Altogether, these studies indicate that the combination treatment promotes an adaptive immune response while decreasing the immunosuppression caused by the virus-induced IDO activation. Furthermore, our data identified the striking role of immunosuppressive pathways in the resistance of gliomas to oncolytic virotherapy. Specifically, the activity of the tumor microenvironment IDO circuitry was responsible, at least partially, for the remodeling of local immunosuppression after tumor infection. Combining molecular and immune-related therapies may improve outcomes in human gliomas treated with virotherapy.

The immunosuppression pathway of tumor-associated macrophages is controlled by heme oxygenase-1 in glioblastoma patients.

The immunosuppressive tumor microenvironment (TME) in glioblastoma (GBM) is mainly driven by tumor-associated macrophages (TAMs). We explored whether their sustained iron metabolism and immunosuppressive activity were correlated, and whether blocking the central enzyme of the heme catabolism pathway, heme oxygenase-1 (HO-1), could reverse their tolerogenic activity. To this end, we investigated iron metabolism in bone marrow-derived macrophages (BMDMs) isolated from GBM specimens and in in vitro-derived macrophages (Mφ) from healthy donor (HD) blood monocytes. We found that HO-1 inhibition abrogated the immunosuppressive activity of both BMDMs and Mφ, and that immunosuppression requires both cell-to-cell contact and soluble factors, as HO-1 inhibition abolished IL-10 release, and significantly reduced STAT3 activation as well as PD-L1 expression. Interestingly, not only did HO-1 inhibition downregulate IDO1 and ARG-2 gene expression, but also reduced IDO1 enzymatic activity. Moreover, T cell activation status affected PD-L1 expression and IDO1 activity, which were upregulated in the presence of activated, but not resting, T cells. Our results highlight the crucial role of HO-1 in the immunosuppressive activity of macrophages in the GBM TME and demonstrate the feasibility of reprogramming them as an alternative therapeutic strategy for restoring immune surveillance.

Immunologically active phenotype by gene expression profiling is associated with clinical benefit from PD-1/PD-L1 inhibitors in real-world head and neck and lung cancer patients

IntroductionIdentification of tumours harbouring an overall active immune phenotype may help for selecting patients with advanced head and neck squamous cell carcinomas (HNSCC) and non-small cell lung cancer (NSCLC) who may benefit from immunotherapies. Our objective was to develop a reliable and stable scoring system to identify those immunologically active tumours. MethodsUsing gene expression profiles of 421 HNSCC, we developed a score to identify immunologically active tumours. Validation of the ‘HOT’ score was done in 40 HNSCC and 992 NSCLC. Stability of the ‘HOT’ score was tested in paired HNSCC samples from diagnostic biopsies versus surgically resected specimens, untreated versus recurrent samples, and pre-versus post-cetuximab samples in a total of 76 patients. The association between the ‘HOT’ score with overall survival (OS) and progression-free survival (PFS) was tested in 184 patients with HNSCC or NSCLC treated with PD-1/PD-L1 inhibitors. ResultsA 27-gene expression based ‘HOT’ score was correlated with: (i) PD-L1 and IDO1 expression, (ii) TCD8 infiltrate and (iii) activation of the IFN-γ pathway. The HOT score concordance when comparing diagnostic biopsies and surgically resected specimens was higher than in untreated samples versus recurrent or pre-versus post-cetuximab samples. In 102 and 82 patients with HNSCC or NSCLC treated with PD-1/PD-L1 inhibitors, the HOT score was associated with an improved OS and PFS in multivariate analysis. ConclusionThe ‘HOT’ score is a simple and robust approach to identify real-world patients with HNSCC and NSCLC immunologically active tumours who may benefit from PD-1/PD-L1 inhibitors.

IDO1, FAT10, IFI6, and GILT Are Involved in the Antiretroviral Activity of γ-Interferon and IDO1 Restricts Retrovirus Infection by Autophagy Enhancement.

Gamma-interferon (γ-IFN) significantly inhibits infection by replication-defective viral vectors derived from the human immunodeficiency virus type 1 (HIV-1) or murine leukemia virus (MLV) but the underlying mechanism remains unclear. Previously we reported that knockdown of γ-IFN-inducible lysosomal thiolreductase (GILT) abrogates the antiviral activity of γ-IFN in TE671 cells but not in HeLa cells, suggesting that other γ-IFN-inducible host factors are involved in its antiviral activity in HeLa cells. We identified cellular factors, the expression of which are induced by γ-IFN in HeLa cells, using a microarray, and analyzed the effects of 11 γ-IFN-induced factors on retroviral vector infection. Our results showed that the exogenous expression of FAT10, IFI6, or IDO1 significantly inhibits both HIV-1- and MLV-based vector infections. The antiviral activity of γ-IFN was decreased in HeLa cells, in which the function of IDO1, IFI6, FAT10, and GILT were simultaneously inhibited. IDO1 is an enzyme that metabolizes an essential amino acid, tryptophan. However, IDO1 did not restrict retroviral vector infection in Atg3-silencing HeLa cells, in which autophagy did not occur. This study found that IDO1, IFI6, FAT10, and GILT are involved in the antiviral activity of γ-IFN, and IDO1 inhibits retroviral infection by inducing autophagy.

Apo-Form Selective Inhibition of IDO for Tumor Immunotherapy.

The pharmacological inhibition of IDO1 is considered an effective therapeutic approach for cancer treatment. However, the inadequate response of existing holo-IDO1 inhibitors and unclear biomarkers available in clinical practice limit the possibility of developing efficacious IDO1 inhibitors. In the current study, we aimed to elucidate the activity and mechanism of a potent 1H-pyrrole-2-carboxylic acid derivative (B37) targeting apo-IDO1 and to determine its role in tumor therapy. By competing with heme for binding to apo-IDO1, B37 potently inhibited IDO1 activity, with an IC50 of 22 pM assessed using a HeLa cell-based assay. The x-ray cocrystal structure of the inhibitor-enzyme complex showed that the B37-human IDO1 complex has strong hydrophobic interactions, which enhances its binding affinity, determined using isothermal titration calorimetry. Stronger noncovalent interactions, including π stacking and hydrogen bonds formed between B37 and apo-human IDO1, underlay the enthalpy-driven force for B37 for binding to the enzyme. These binding properties endowed B37 with potent antitumor efficacy, which was confirmed in a mouse colon cancer CT26 syngeneic model in BALB/c mice and in an azoxymethane/dextran sulfate sodium-induced colon carcinogenesis model in C57BL/6 mice by activating the host immune system. Moreover, the combination of B37 and anti-PD1 Ab synergistically inhibited tumor growth. These results suggested that B37 may serve as a unique candidate for apo-IDO1 inhibition-mediated tumor immunotherapy.

Shear Stress Enhances the Paracrine-Mediated Immunoregulatory Function of Human Periodontal Ligament Stem Cells via the ERK Signalling Pathway

Relevant immunomodulatory effects have been proposed following allogeneic cell-based therapy with human periodontal ligament stem cells (hPDLSCs). This study aimed to examine the influence of shear stress on the immunosuppressive capacity of hPDLSCs. Cells were subjected to shear stress at different magnitudes (0.5, 5 and 10 dyn/cm). The expression of immunosuppressive markers was evaluated in shear stress-induced hPDLSCs using qRT-PCR, western blot, enzyme activity and enzyme-linked immunosorbent assays. The effects of a shear stress-derived condition medium (SS-CM) on T cell proliferation were examined using a resazurin assay. Treg differentiation was investigated using qRT-PCR and flow cytometry analysis. Our results revealed that shear stress increased mRNA expression of IDO and COX2 but not TGF-β1 and IFN-γ. IDO activity, kynurenine and active TGF-1 increased in SS-CM when compared to the non-shear stress-derived conditioned medium (CTL-CM). The amount of kynurenine in SS-CM was reduced in the presence of cycloheximide and ERK inhibitor. Subsequently, T cell proliferation decreased in SS-CM compared to CTL-CM. Treg differentiation was promoted in SS-CM, indicated by FOXP3, IL-10 expression and CD4CD25CD127 subpopulation. In conclusion, shear stress promotes kynurenine production through ERK signalling in hPDLSC, leading to the inhibition of T cell proliferation and the promotion of Treg cell differentiation.

The immunosuppressive role of indoleamine 2, 3-dioxygenase in glioblastoma: mechanism of action and immunotherapeutic strategies.

Glioblastoma multiforme (GBM) is a fatal brain tumor in adults with a bleak diagnosis. Expansion of immunosuppressive and malignant CD4 + FoxP3 + GITR + regulatory T cells is one of the hallmarks of GBM. Importantly, most of the patients with GBM expresses the tryptophan-degrading enzyme indoleamine 2,3-dioxygenase (IDO). While IDO1 is generally not expressed at appreciable levels in the adult central nervous system, it is rapidly stimulated and highly expressed in response to ongoing immune surveillance in cancer. Increased levels of immune surveillance in cancer are thus related to higher intratumoral IDO expression levels and, as a result, a worse OS in GBM patients. Conversion of the important amino acid tryptophan into downstream catabolite known as kynurenines is the major function of IDO. Decreasing tryptophan and increasing the concentration of immunomodulatory tryptophan metabolites has been shown to induce T-cell apoptosis, increase immunosuppressive programming, and death of tumor antigen-presenting dendritic cells. This observation supported the immunotherapeutic strategy, and the targeted molecular therapy that suppresses IDO1 activity. We review the current understanding of the role of IDO1 in tumor immunological escape in brain tumors, the immunomodulatory effects of its primary catabolites, preclinical research targeting this enzymatic pathway, and various issues that need to be overcome to increase the prospective immunotherapeutic relevance in the treatment of GBM malignancy.

Abstract 4184: RNA-seq analyses reveal remodeling of tumor microenvironment and reversal of glioma resistance to oncolytic viruses by targeting immunometabolism

Abstract Viroimmunotherapy aims to infect cancer cells to elicit anti-tumor immune responses. In clinical trials, glioma treatment with oncolytic viruses induced durable clinical responses in a small fraction of patients. To improve the percentage of responders, it is necessary to reshape the tumor microenvironment that shields the tumor from the immune system of the patient. Thus, we engineered Delta-24-RGDOX (DNX-2440), an oncolytic adenovirus that carries the cDNA of the T-cell activator, OX40L. In this work, we observed that Delta-24-RGDOX triggered a dramatic reshaping of the tumor microenvironment dominated by strong changes in immune processes as indicated by RNA-sequencing via ingenuity pathway analyses in a murine glioblastoma model. Paradoxically, network analyses revealed that Delta-24-RGDOX also induced robust activation of the cytokine-driven immunosuppressive IDO-Kynurenine-AhR circuitry, indicating a potential mechanism of resistance of the cancer cells to oncolytic virotherapy. To reverse this immunosuppression, we combined Delta-24-RGDOX with clinically relevant IDO inhibitors to treat glioma bearing mice. Importantly, addition of the IDO inhibitor to Delta-24-RGDOX decreased the activation of the IDO network. IDO inhibition did not affect virus infection or replication in human or murine glioma cells. Flow cytometry assays revealed that the combination therapy increased the frequency of activated CD8+ T cells and decreased the presence of the immunosuppressive cell populations, MDSCs and Tregs. Gene set enrichment analyses confirmed the decrease of MDSCs and Tregs in the combination treated glioma-bearing mice compared to the virus alone. Functional co-culture studies showed that the combined therapy activated splenocytes against tumor antigens, and that this activation was reversed by kynurenine. Importantly, the combination treatment eradicated the tumors in a CD4-dependent manner and significantly prolonged the survival of glioma-bearing mice. Altogether, these studies indicate that the combination treatment promotes an adaptive immune response while decreasing immunosuppression caused by virus-induced IDO activation. Furthermore, our data identified the striking role of immunosuppressive pathways in the resistance of gliomas to oncolytic virotherapy. Specifically, the activity of the tumor microenvironment IDO circuitry was responsible, at least partially, for the remodeling of local immunosuppression after tumor infection. Combining molecular and immune-related therapies may improve outcomes in human gliomas treated with virotherapy. Citation Format: Teresa T. Nguyen, Dong Ho Shin, Sagar Sohoni, Sanjay K. Singh, Yisel Rivera-Molina, Hong Jiang, Xuejun Fan, Joy Gumin, Frederick F. Lang, Christopher Alvarez-Breckenridge, Marta M. Alonso, Filipa Godoy-Vitorino, Lijie Zhai, Erik Ladomersky, Kristen L. Lauing, Derek A. Wainwright, Juan Fueyo, Candelaria Gomez-Manzano. RNA-seq analyses reveal remodeling of tumor microenvironment and reversal of glioma resistance to oncolytic viruses by targeting immunometabolism [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 4184.

Abstract 5533: YIV-906 could modulate nuclear factor of activated T-cells (NFAT) activity of T cells for immune checkpoint blockade therapy and CAR T-cell therapy

Abstract YIV-906 is a standardized botanical drug inspired by a traditional Chinese herbal formulation, historically used to treat gastrointestinal symptoms including diarrhea, nausea, and vomiting. Results from seven Phase I/II to II clinical studies suggest that YIV-906 has potential to prolong survival and improve quality of life for patients who have liver, pancreatic, colorectal, and rectal cancer and received chemotherapy and/or radiation therapy. Currently, a phase II international clinical trial for using YIV-906 plus sorafenib to target HBV positive hepatocellular carcinoma as first line therapy (NCT04000737) had been initialized. Recently, we demonstrated that YIV-906 could enhance anti-tumor activity of anti-PD1 against hepatoma in mice by enhancing both adaptive immunity and innate immunity thought promoting T cell activation by modulating IDO activity and potentiating IFNg action to polarize macrophages into M1-like type. Here, by using Jurkat cells-staphylococcal superantigen E (SEE)-Raji cells cell culture model, we demonstrated that YIV-906 could promote T cell activation with up-regulation of CD69 by modulating nuclear factor of activated T-cells (NFAT) activity. This could be due to that YIV-906 could trigger protein phosphorylation of T cell receptor signaling cascades including, Lck, Fyn, Zap70, LAT and Pyk2. Importantly, YIV-906 could enhance Anti-PD1 action to recuse the depressed NFAT activity of Jurkat cells-PD1 cells when incubated with Raji-PD-L1 cells. In addition, YIV-906 could also enhance NFAT activity triggered by the interaction of chimeric antigen receptor (CAR-CD19-CD3z) on Jurkat cells and CD19 on Raji cells. Component S (Scutellaria baicalensis Georgi) of YIV-906 was found to play key roles of the above activities. Flavonoids of S, such as baicalein, chrysin and oroxylin A, were identified to have modulation effect on NFAT activity. In conclusion, YIV-906 could modulate both adaptive immunity and innate immunity to facilitate immune checkpoint blockade therapy or CAR T-cell therapy for treatment of cancer in patients. This is worthy of further investigation. (Supported by research fund from Yiviva, inc) Citation Format: Wing Lam, Shwu-Huey Liu, Peikwen Cheng, Yung-Chi Cheng. YIV-906 could modulate nuclear factor of activated T-cells (NFAT) activity of T cells for immune checkpoint blockade therapy and CAR T-cell therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5533.

Self-Delivering Nanodrugs Developed via Small-Molecule-Directed Assembly and Macrophage Cloaking for Sonodynamic-Augmented Immunotherapy.

The self-delivery of sonosensitizers and immunomodulators to tumor areas, which is highly recommended for enhancing sonodynamic immunotherapy, remains a challenge. Herein, a self-delivering nanodrug (HB-NLG8189, drug loading: ≈100 wt%) is developed by the small-molecule self-assembly of "HB" (a new clinical photosensitizer) and NLG8189 (indoleamine-(2,3)-dioxygenase (IDO) pathway inhibitor) for sonodynamic-augmented immunotherapy; this preparation method ensures the absence of excipient-related toxicity and immunogenicity. To evade immune recognition and prolong the circulation time, the HB-NLG8189 nanodrugs are camouflaged using macrophage cell membranes (MPCMs). The constructed HB-NLG8189@MPCM nanodrugs show an ability to preferentially accumulate within tumors. Upon ultrasound triggering, the HB-NLG8189@MPCM is able to generate reactive oxygen species efficiently for robust sonodynamic therapy; it induces immunogenic cell death, initiates an antitumor immune response to activate tumor-specific effector T cells, and promotes the secretion of inflammatory cytokines. The concomitant delivery of NLG8189 reverses the immunosuppressive tumor microenvironment by restraining IDO-1 activation and the intratumoral infiltration of regulatory T cells. Sonodynamic-augmented immunotherapy with HB-NLG8189@MPCM significantly inhibits the growth of both primary and distant tumors with little systemic toxicity. The biomimetic self-delivery nanodrug provides a promising paradigm for improving sonodynamic immunotherapy.

On the determinants of IDO activity in patients with familial Mediterranean fever.

On the determinants of IDO activity in patients with familial Mediterranean fever.

Expression, purification, and kinetic characterization of the human strep-IDO1.

BackgroundTryptophan catabolism leading to T cell suppression mediated by indoleamine 2,3-dioxygenase (IDO1) is an important mechanism of tumor immune escape, and IDO1 inhibitors have attracted increasing attention as anticancer therapeutics. However, the phase III clinical trial (ECHO-301/KEYNOTE-252) of epacadostat (INCB024360) had disappointing outcomes. This revealed that purification of IDO1 with high purity is one of the major constraints that limit the development of its inhibitors.MethodsPan-cancer analysis was used to elucidate the relationship between IDO1 function in tumor immunology. The recombinant pET28a-IDO1-strep plasmid and E. coli Rosetta (DE3) strain were used to express and strep-tactin beads to purify the strep-IDO1 protein. High performance liquid chromatography (HPLC) was used to detect enzymatic activity of IDO1. Ten female C57BL/6 mice was used to prepared polyclonal antibody. Enzyme linked immunosorbent assay (ELISA), Western blot, and immunofluorescence were used to measure polyclonal antibody.ResultsWe described an improved method for the purification of recombinant IDO1 protein based on the strep-tag using an E. coli expression system. We obtained large amount of IDO1 with enhanced purity by employing one-step purification through strep-tactin beads. The polyclonal antibody acquired immunized mice could specifically recognize both recombinant and endogenous IDO1.ConclusionsPurified human strep-IDO1 using the protocol described in our study could be used for further biochemical and structural analyses, which may facilitate functional research and further drug screening study on IDO1.

Is Poor Lithium Response in Individuals with Bipolar Disorder Associated with Increased Degradation of Tryptophan along the Kynurenine Pathway? Results of an Exploratory Study.

Bipolar disorder is associated with an inflammation-triggered elevated catabolism of tryptophan to the kynurenine pathway, which impacts psychiatric symptoms and outcomes. The data indicate that lithium exerts anti-inflammatory effects by inhibiting indoleamine-2,3-dioxygenase (IDO)-1 activity. This exploratory study aimed to investigate the tryptophan catabolism in individuals with bipolar disorder (n = 48) compared to healthy controls (n = 48), and the associations with the response to mood stabilizers such as lithium, valproate, or lamotrigine rated with the Retrospective Assessment of the Lithium Response Phenotype Scale (or the Alda scale). The results demonstrate an association of a poorer response to lithium with higher levels of kynurenine, kynurenine/tryptophan ratio as a proxy for IDO-1 activity, as well as quinolinic acid, which, overall, indicates a pro-inflammatory state with a higher degradation of tryptophan towards the neurotoxic branch. The treatment response to valproate and lamotrigine was not associated with the levels of the tryptophan metabolites. These findings support the anti-inflammatory properties of lithium. Furthermore, since quinolinic acid has neurotoxic features via the glutamatergic pathway, they also strengthen the assumption that the clinical drug response might be associated with biochemical processes. The relationship between the lithium response and the measurements of the tryptophan to the kynurenine pathway is of clinical relevance and may potentially bring advantages towards a personalized medicine approach to bipolar disorder that allows for the selection of the most effective mood-stabilizing drug.

Novel Potential Immune Response Biomarkers to Multidrug-Resistant Tuberculosis in the Last Five Years

Rapid and accurate detection performs an important role in the control of raising MDR-TB. Currently, studies on biomarkers as targets for TB diagnostic tests using immune response products to indicate the presence, mycobacterial load, early markers, and activity, diff erentiation, and progression markers of TB infection are rapidly available. This systematic review aims to summarize the last fi ve years of potential biomarkers studies from the immune response for MDR-TB rapid diagnostic development. The authors performed a literature search on four databases as ProQuest, EBSCO Academic Search, Universitas Gadjah Mada Online Library Journal Database, and Google Scholar, retrieved from January 2016 to December 2021. In total, 18,288 articles were identifi ed and three tudies met the inclusion criteria. Several promising biomarkers were found for MDR-TB diagnosis purposes, such as sCD14, PGLYRP2, FGA, Indoleamine 2, 3- dioxygenase (IDO), and Complement Receptor 2 (CR2). A combination of sCD14, PGLYRP2, and FGA were bringing a diagnostic design with a higher sensitivity (94.7%) and specifi city (80%) than the design of a single protein. Higher IDO activity towards the MDR-TB group than in the DS-TB group with a sensitivity of 87.50 %, specifi city of 72.22 %. CR2 was the main focus due to its association with IL-6. After induction of CR2 peptide in a dose-dependent manner, the expression level of IL-6 was decreased signifi cantly. It might because of CR2 peptide regulating the macrophages proinfl ammatory cytokines secretion to decrease the local infl ammation of the immune response. These biomarkers are strong candidates for MDR-TB diagnosis due to their important role as the pathogenesis marker of MDR-TB. There is a need of further research to investigate those immune response products and their role to eliminate infection of Mycobacterium tuberculosis directly.

Circulating immune profile can predict survival of metastatic uveal melanoma patients: results of an exploratory study

ABSTRACT Metastatic uveal melanoma (UM) is a poor prognosis malignancy. Immunotherapy is commonly employed, despite the low activity, considering the lack of other effective systemic treatments. In this study, the prognostic and predictive role of soluble immune checkpoints and inflammatory cytokines/chemokines in 22 metastatic UM patients was evaluated. Baseline levels of these molecules were assessed, as well as their changes during anti-PD-1 therapy. The correlation between soluble immune checkpoints/cytokines/chemokines and survival was analyzed. A comparison between circulating immune profile of metastatic cutaneous melanoma (CM), for which immunotherapy is a mainstay of treatment, and UM during anti-PD-1 therapy was also performed. Three immune molecules resulted significantly higher in metastatic UM patients with survival <6 months versus patients with survival ≥6 months: IL-8, HVEM and IDO activity. Considering these three molecules, we obtained a baseline score able to predict patients’ survival. The same three molecules, together with soluble(s) CD137, sGITR and sCD27, resulted significantly lower in patients with survival >30 months. We also observed an increase of sCD137, sCD28, sPD-1, sPD-L2 sLAG3, sCD80 and sTim3 during anti-PD-1 treatment, as well as IDO activity, IP-10 and CCL2. Several of these molecules were significantly higher in UM compared to CM patients during anti-PD-1 therapy. The analysis of circulating immune molecules allows to identify patients with poor prognosis despite immunotherapy and patients with long survival treated with an anti-PD-1 agent. The different serum concentration of these molecules during anti-PD-1 therapy between UM and CM reflects the different efficacy of immune checkpoint inhibitors.

In-depth characterization of phenolic profiling of Moraiolo extra-virgin olive oil extract and initial investigation of the inhibitory effect on Indoleamine-2,3-Dioxygenase (IDO1) enzyme

In this research, the phenolic extract from Moraiolo extra-virgin olive oil (EVOO) was thoroughly characterized. A reversed-phase HPLC method with a photodiode detector allowed to measure a total phenol content higher than 500 mg/kg EVOO, with elevated amounts of oleocanthal, oleacein, and oleouropein aglycone (131.2, 213.5, and 158.4 mg/kg EVOO, respectively). Appreciable amounts of (+)-pinoresinol and (+)− 1-acetoxy-pinoresinol, 3.2 and 12.5 mg/kg EVOO respectively, were measured. High-resolution mass spectrometry with Orbitrap mass analyzer technology was used to confirm the identity of the analytes. Afterwards, the extract was tested, for the first time, for its activity on Indoleamine-2,3-Dioxygenase (IDO1). This enzyme appears as a promising target for the modulation of the neuroinflammatory-oxidative processes relying on the pathogenesis of several neurodegenerative diseases. The extract showed an inhibitory effect on the catalytic activity of both human and murine IDO1, with a good safety at the concentrations of 15 and 30 µg/mL.

Lithium inhibits tryptophan catabolism via the inflammation-induced kynurenine pathway in human microglia.

Despite its decades' long therapeutic use in psychiatry, the biological mechanisms underlying lithium's mood-stabilizing effects have remained largely elusive. Here, we investigated the effect of lithium on tryptophan breakdown via the kynurenine pathway using immortalized human microglia cells, primary human microglia isolated from surgical specimens, and microglia-like cells differentiated from human induced pluripotent stem cells. Interferon (IFN)-γ, but not lipopolysaccharide, was able to activate immortalized human microglia, inducing a robust increase in indoleamine-2,3-dioxygenase (IDO1) mRNA transcription, IDO1 protein expression, and activity. Further, chromatin immunoprecipitation verified enriched binding of both STAT1 and STAT3 to the IDO1 promoter. Lithium counteracted these effects, increasing inhibitory GSK3βS9 phosphorylation and reducing STAT1S727 and STAT3Y705 phosphorylation levels in IFN-γ treated cells. Studies in primary human microglia and hiPSC-derived microglia confirmed the anti-inflammatory effects of lithium, highlighting that IDO activity is reduced by GSK3 inhibitor SB-216763 and STAT inhibitor nifuroxazide via downregulation of P-STAT1S727 and P-STAT3Y705 . Primary human microglia differed from immortalized human microglia and hiPSC derived microglia-like cells in their strong sensitivity to LPS, resulting in robust upregulation of IDO1 and anti-inflammatory cytokine IL-10. While lithium again decreased IDO1 activity in primary cells, it further increased release of IL-10 in response to LPS. Taken together, our study demonstrates that lithium inhibits the inflammatory kynurenine pathway in the microglia compartment of the human brain.

Combination of IDO1high and CCL19low expression in the tumor tissue reduces survival in HPV positive cervical cancer

The over expression of Indoleamine 2, 3-Dioxygenase (IDO1), an immune checkpoint inhibitor, is well known in cervical cancer. However, its association with chemokine signals promoting cellular infiltration in the cervical tumor microenvironment, is unknown. In the current study, we evaluated the expression and enzymatic activity of IDO1. We also profiled the expression of chemokine ligand-receptors- CCR4-CCL22, CXCR3-CXCL10, CXCR4-CXCL12, and CCR7-CCL19 using immunohistochemistry (IHC), and studied their association with IDO1, statistically. After getting an informed consent, punch biopsy samples were obtained from 105 patients diagnosed with cervical cancer. HPV typing by Sanger sequencing, realtime PCR for quantifying IDO1 mRNA expression, HPLC for determining the K/T ratio and IHC for all the above chemokine receptor-ligand pairs along with IDO1 were performed. We found a significant increase in the expression of IDO1 and K/T levels in early and locally advanced stages when compared to Stage IV disease. Among the chemokine ligand -receptor pairs profiled, we found that high CCL19 marker expression was a good prognostic indicator of patients’ disease-free (p = 0.013) and overall survival (p = 0.043). Although we could not identify IDO1 as an independent prognostic factor, we found that high levels of IDO1 expression may further reduce survival outcomes in patients with low CCL19 expression. This could be vital for designing immuno therapeutic interventions targeting IDO1.

IMMU-12. TUMOR CELL IDO ENHANCES IMMUNE SUPPRESSION AND DECREASES SURVIVAL INDEPENDENT OF TRYPTOPHAN METABOLISM IN GLIOBLASTOMA

Abstract OBJECTIVE Indoleamine 2,3-dioxygenase 1 (IDO; IDO1) is an immune checkpoint that’s characterized as a potent immunosuppressive mediator through its ability to metabolize tryptophan and wild-type IDH patient-resected glioblastoma (GBM) expresses IDO in ≥ 95% of cases. Recent findings from our group led us to investigate the alternative hypothesis that IDO possesses immunosuppressive effects that are independent of its associated metabolic activity. METHODS Murine GBM cell lines that overexpress either wild-type or enzyme-null IDO were created for in vivo characterization of IDO enzyme-independent immunosuppressive function. Microarray was conducted to identify human IDO expression-correlated genes, which were further investigated in human GBM cell lines, patient GBM tissues and plasma, as well as the TCGA database. Ex vivo cell co-culture assays and syngeneic mouse orthotopic GBM models were employed to study immunosuppressive mechanisms. RESULTS Here, we demonstrate that non-enzymic IDO activity decreases survival in experimental animals and increases the expression of immunosuppressive complement factor H (CFH) in human GBM. CFH mRNA levels positively correlate with those of IDO and many other immunosuppressive genes in patient resected GBM and can be applied as a prognostic marker in both lower grade gliomas and GBM. Similar to IDO, the increased expression of CFH in patient-resected glioma was positively correlated with an increased signature for regulatory T cells (Tregs) and myeloid-derived suppressive cells (MDSCs). High expression of CFH in tumor cells increases intratumoral Tregs levels and decreases overall survival in mice with GBM, while inducing tumor associated macrophage cell differentiation. CONCLUSIONS Here, we demonstrated that glioblastoma (GBM) cell IDO promotes the accumulation of intratumoral FoxP3+ regulatory T cells (Tregs) and tumor progression while decreasing overall survival - independent of IDO enzyme activity. Our study reveals a targetable non-metabolic IDO-dependent mechanism for future therapeutic intervention in patients with GBM.

The Role of Indoleamine 2,3-Dioxygenase in Renal Tubular Epithelial Cells Senescence under Anoxia or Reoxygenation.

Ischemia-reperfusion injury is the commonest form of acute kidney injury (AKI). Tubular epithelial cell senescence contributes to incomplete recovery from AKI and predisposes to subsequent chronic kidney disease. In cultures of primary proximal renal tubular epithelial cells (RPTECs) subjected to anoxia or reoxygenation, we evaluated the role of indoleamine 2,3-dioxygenase 1 (IDO) in cellular senescence. Proteins of interest were assessed with Western blotting or enzyme-linked immunosorbent assay or histochemically. Under anoxia or reoxygenation, IDO expression and activity were increased. Moreover, the two IDO-derived pathways, the general control nonderepressible 2 kinase (GCN2K) pathway and the aryl-hydrocarbon receptor (AhR) pathway, were also activated. A DNA damage response (DDR) took place and led to increased levels of the cell-cycle inhibitors p21 and p16, and senescence-associated β-galactosidase (SA-β-Gal) activity. Cell proliferation was inhibited, and more IL-6 was produced. The IDO inhibitor 1-DL-methyl-tryptophan ameliorated the DDR; decreased p21, p16, and SA-β-Gal activity; restored cell proliferation; and decreased IL-6 production. The AhR inhibitor CH223191 did not affect the above parameters. In conclusion, anoxia and the subsequent reoxygenation upregulate IDO. IDO depletes tryptophan and activates GCN2K. The latter enhances the anoxia- or reoxygenation-induced DDR, resulting in increased p21 and p16 expression and eventually leading to RPTEC senescence. Since cellular senescence affects AKI outcome, the role of IDO in cellular senescence and the possible therapeutic role of IDO inhibitors deserve further investigation.