IDO Expression Research Articles

Guanosine treatment prevents lipopolysaccharide-induced depressive-like behavior in mice

Guanosine is a purinergic nucleoside that has been shown to have neuroprotective effects, mainly through its ability to modulate the glutamatergic system. An increase in pro-inflammatory cytokine levels triggers the activation of the enzyme indoleamine 2,3-dioxygenase 1 (IDO-1), leading to glutamatergic excitotoxicity, which has important roles in the pathophysiology of depression. The aim of this study was to investigate the possible antidepressant-like effects and underlying mechanisms of action of guanosine against lipopolysaccharide (LPS)-induced depression in a mouse model. Mice were orally pre-treated with saline (0.9% NaCl), guanosine (8 or 16 mg/kg), or fluoxetine (30 mg/kg) for 7 days before LPS (0.5 mg/kg, intraperitoneal) injection. One day after LPS injection, mice were subjected to the forced swim test (FST), tail suspension test (TST), and open field test (OFT). After the behavioral tests, mice were euthanized and the levels of tumor necrosis factor-α (TNF-α), IDO-1, glutathione, and malondialdehyde in the hippocampus were measured. Pretreatment with guanosine was able to prevent LPS- induced depressive-like behaviors in the TST and FST. In the OFT, no locomotor changes were observed with any treatment. Both guanosine (8 and 16 mg/kg/day) and fluoxetine treatment prevented the LPS-induced increase in TNF-α and IDO expression and lipid peroxidation as well as decrease of reduced glutathione levels in the hippocampus. Taken together, our findings suggest that guanosine may have neuroprotective effects against LPS-induced depressive-like behavior through preventing oxidative stress and the expression of IDO-1 and TNF-α in the hippocampus.

Design, synthesis and evaluation the bioactivities of novel 1,3-dimethyl-6-amino-1H-indazole derivatives as anticancer agents

Indoleamine 2,3-dioxygenase (IDO1) is a heme-containing enzyme mainly responsible for the metabolism of tryptophan to kynurenine. To date, the IDO1 inhibitors have been developed intensively for the re-activation of the anticancer immune response. In this report, we designed, and synthesized novel 1,3-dimethyl-6-amino indazole derivatives as IDO1 inhibitors based on the structure of IDO1 active site. We further examined their anticancer activity on hypopharyngeal carcinoma cells (FaDu), squamous cell carcinoma of the oral tongue (YD-15), breast cancer cells (MCF7), and human dental pulp stem cells (HDPSC). Of them, compound N-(4-bromobenzyl)-1,3-dimethyl-1H-indazol-6-amine (7) remarkably suppressed IDO1 expression in a concentration - dependent manner. In addition, 7 was the most potential anticancer compound with inducing apoptosis activity as well as selectively activated extracellular signal-regulated kinases (ERK) in mitogen-activated protein kinase (MAPK) pathways on FaDu cells. Finally, compound 7 suppressed cell mobility in wound healing assay with the reduced expression of matrix metalloproteinase MMP9. Taken together, we believe that 7 is the most promising compound, which may be applied to treatment of hypopharyngeal carcinoma.

Indoleamine 2,3-dioxygenase 1 regulates cell permissivity to astrovirus infection

Astroviruses cause a spectrum of diseases spanning asymptomatic infections to severe diarrhea, but little is understood about their pathogenesis. We previously determined that small intestinal goblet cells were the main cell type infected by murine astrovirus-1. Here, we focused on the host immune response to infection and inadvertently discovered a role for indoleamine 2,3-dioxygenase 1 (Ido1), a host tryptophan catabolizing enzyme, in the cellular tropism of murine and human astroviruses. We identified that Ido1 expression was highly enriched among infected goblet cells, and spatially corresponded to the zonation of infection. Because Ido1 can act as a negative regulator of inflammation, we hypothesized it could dampen host antiviral responses. Despite robust interferon signaling in goblet cells, as well as tuft cell and enterocyte bystanders, we observed delayed cytokine induction and suppressed levels of fecal lipocalin-2. Although we found Ido-/- animals were more resistant to infection, this was not associated with fewer goblet cells nor could it be rescued by knocking out interferon responses, suggesting that IDO1 instead regulates cell permissivity. We characterized IDO1-/- Caco-2 cells and observed significantly reduced human astrovirus-1 infection. Together this study highlights a role for Ido1 in astrovirus infection and epithelial cell maturation.

Deletion of the GAS Regulatory Sequence of IFN- γ Induced Gene(ICAM-1) by Using CRISPR Cas9 to Decrease the Jak3/Stat3Dependent IDO Expression to Repress Tumor Cells Development

IDO is a checkpoint molecule that regulates T-cell proliferation. Since IDO contributes to T cell death and creates anenvironment for cancer cells to grow, it is necessary to regulate IDO expression. Previous studies demonstrated thatIFN-gamma-induced genes would indirectly activate IDO expression through the Jak/3 pathway, and ICAM-1 is oneof the IFN-gamma-induced genes. This work investigates the effect of ICAM-1 on IDO expression and the effect of2 GAS segment knockouts on ICAM-1 expression. CRISPR-Cas9, heat shock transformation, PCR, and restrictiondigestion are used to knock out the target sequence (GAS) and insert the successful knocked-out gene into the E. coli.RT-PCR is utilized to measure the expression level of ICAM-1 and IDO after the introduction of successfully knockedout genes. The possible result of this work presents that both GAS gene knockouts are thriving; the new gene withoutGAS – 2787 bps expresses less ICAM-1 protein than the gene without GAS – 115 bps, and ICAM-1 does not affect IDOexpression at all. Results of this work indicate that the GAS sequence that is farther away from the ICAM-1 RBS sitemight decrease the ICAM-1 expression level less than does GAS sequence that is nearer to RBS, and ICAM-1 might nothave any direct impact on IDO expression, which means that tumor cell development cannot be inhibited by regulatingICAM-1 expression. Future studies could research more on functions of IFNgamma-induced genes other than ICAM-1and their relationships with IDO expression.

Molecular characterization of nectin-4 in non-small cell lung cancer subtypes.

e21119 Background: Nectin-4 (Nectin cell adhesion molecule-4) is a tumor associated antigen overexpressed in multiple solid tumor types. Studies show that over 60% of NSCLC are nectin-4 overexpressed. As enfortumab vedotin, an antibody-drug conjugate directed against nectin-4, has been FDA approved for advanced urothelial carcinoma, this holds treatment implications. Here, we investigated nectin-4 expression in NSCLC subtypes. Methods: 27,607 NSCLC tumors were tested for whole transcriptome sequencing (WTS, NovaSeq) and NextGen DNA sequencing (592 gene panel or whole exome sequencing, WES) at Caris Life Sciences (Phoenix, AZ). Tumors were analyzed overall in quartiles by nectin-4 expression (Q4-greatest, Q1-least) and further analyzed in the adenocarcinoma (AD) and squamous carcinoma (SQ) cohorts. PD-L1 expression was tested by IHC (Dako 22C3). Immune cell populations were estimated by QuanTISeq. Statistical significance was determined using Chi-square/Fisher-Exact/Mann-Whitney U tests and adjusted for multiple comparisons (q < 0.05). Results: We saw significantly greater expression of nectin-4 in SQ (median TPM- 15.68) versus AD tumors (median TPM - 9.88) (p < 0.05). We saw a greater prevalence of NFE2L2, STK11, ERBB2 mutations and genomic loss of heterozygosity (LOH) but a lower percentage of PD-L1+ tumors in Q4 vs Q1 tumors (all q-value < 0.05, Table 1). mRNA expression of immune checkpoints PDL1, CTLA4, CD80, CD86, HAVCR2, and IDO1 was increased in Q4 tumors (1.1-1.3-fold, all q < 0.05). Q4 tumors had reduced estimates of M1 macrophages, regulatory T cells (Tregs), and CD8+ T cells and increased estimates of neutrophils and NK cells. The association of nectin-4 expression with the immune landscapes of AD and SQ was similar. Q4 tumors in AD and SQ displayed increased expression of the above-mentioned immune checkpoints (q < 0.05). In AD and SQ, estimates of M1 macrophages, Tregs, and CD8+ T cells were reduced, but neutrophils and NK cells were increased in Q4 tumors (q < 0.05). The pattern of molecular alterations was different in AD and SQ. While KEAP1, STK11 and ERBB2 mutations were significantly enriched in AD Q4 tumors, NFE2L2 mutations were significantly enriched in SQ Q4 tumors (Table 1). Prevalence of LOH was higher and PD-L1+ tumors was lower in Q4 tumors in both AD and SQ (Table 1). Conclusions: NFE2L2 alterations in SQ and STK11, KEAP1 mutations in AD, which are associated with poor immunotherapy (IO) response, were enriched in Q4 tumors. This suggests that the immune landscape associated with high nectin-4 expression in NSCLC may be driven by the dysregulated KEAP1-NFE2L2 pathway. Targeted therapy towards nectin-4 may benefit these NSCLC subtypes that do not respond well to IO. [Table: see text]

Updated molecular analysis of MET exon 14skippingmutations (METex14) in non-small cell lung cancer (NSCLC).

9095 Background: METex14 is a heterogeneous family of mutations (mt) in NSCLC that can be effectively treated with approved targeted agents. Unlike some other drivers in NSCLC, METex14 occurs in both squamous and adenocarcinoma histology and in both smokers and non-smokers. Here, we present updated results from analysis of a large dataset to characterize the mutational landscape within METex14 NSCLC. Methods: NSCLC tissue samples were analyzed with DNA-based next-generation sequencing (NGS; 592 genes, NextSeq) or whole-exome sequencing (NovaSeq), RNA-based whole transcriptome sequencing (WTS, NovaSeq), and PD-L1 immunohistochemistry (Dako 22C3) at Caris Life Sciences (Phoenix, AZ). METex14 was detected by WTS. TMB-high was defined as ≥ 10 mt/Mb. Chi-square, Fisher’s exact or Mann-Whitney U tests were used to determine statistical significance and corrected for multiple hypothesis testing (q<0.05). Immune cell estimates (quanTIseq) and pathway analysis (ssGSEA) were informed by WTS analysis. Results: A total of 711 METex14 cases were detected with 288 distinct METex14 mt. By histology, 79 (11.1%) were squamous (Sq), 478 (67.23%) were nonsquamous (nSq), and 24 (3.23%) were adenosquamous. The most common METex14 mt were D1028H (8.1%), D1028N (7.8%), c.3082+2T>C (5.0%), D1028Y (4.6%), and c.3082+1G>T (4.4%). Co-mutated TP53 was common (43.4%) but varied by specific METex14 mt, observed in 60.0% of MET c.3082+3A>G vs 16.7% of MET G344R . Co-amplified CDK4 was found in 9.3%, with 42.9% in MET c.2924-1G>A vs 6.7% in MET c.3802+1G>T (p<0.05). High TMB was seen in 9%; median TMB ranged from 2 mt/Mb in MET c.3082+2T>A to 6.5 mt/Mb in MET c.3082+2T>G (p<0.05). PD-L1 ≥ 1% was seen in 80.8% compared to 56.2% in METex14-WT(p<0.05), and median PD-L1 tumor proportion score (TPS) ranged from 0% in MET G344R to 75% in MET c.3082+2T>A (p<0.05). Co-mutations varied by histology: in Sq-NSCLC, 18.18% had TP53 mt (q<0.05), 8.97% had POT1 mt (p<0.05), 6.06% had TERT mt (p<0.05), 5.13% CASP8 mt (q<0.05), and 2.53% RNF43 mt (p<0.05), while in nSq-NSCLC, 45.51% had TP53 mt, 3.38% had POT1 mt, 0.87% had TERT mt, and 0% in CASP8 and RNF43 mt. Smoking status was available for 120 cases: 88% were smokers and 12% were nonsmokers. Wnt, Hedgehog, and Notch signaling were enriched in nSq (q<0.05) while upregulation of KRAS signaling, Epithelial-Mesenchymal Transition, and angiogenesis pathways were enriched in smokers with METext14 NSCLC (q<0.2). Higher estimates of neutrophils and lower estimates of M2 macrophages, NK cells, and CD8+ T-cells were observed in Sq-NSCLC. PD-L1, PD-1, HAVCR-2, IDO-1 and IFN-γ expression were higher in nSq than Sq-NSCLC (q<0.05). Conclusions: METex14 NSCLC is highly heterogenous, with variations in co-mutation, TMB, and PD-L1 expression. Although Sq- and nSq-NSCLC harbor METex14, the enrichment of oncogenic pathways and infiltrating immune cells differ between histology and smoking history.

Prognostic significance of pretreatment immune cell infiltration in muscle invasive bladder cancer treated with definitive chemoradiation: Analysis of NRG RTOG 0524 and 0712.

4523 Background: Chemoradiation therapy (CRT) is an organ conserving approach in the treatment of locally advanced bladder cancer. Chemoradiation is thought to potentially result in immunogenic stimulation, and bladder cancer is often a tumor with high immune cell infiltration. Thus, we aimed to profile the tumor immune microenvironment of bladder cancer and identify prognostic immune biomarkers for CRT response by profiling tumor samples from NRG/RTOG 0524 and 0712, two prospective trials of CRT in muscle invasive bladder cancer (MIBC). Methods: Pretreatment tissue samples from both trials were profiled using Cofactor Genomics ImmunoPrism, an RNA sequencing assay that uses gene expression profiles to quantify immune cell populations in the tumor microenvironment (TME). Differential gene expression was estimated for different immune cell type proportions across samples. Kaplan-Meier survival analysis and log rank tests were performed to evaluate differences in overall survival (OS) stratified by genes influenced by immune cell proportions or genes associated with immune response signatures. Results: A total of 70 samples (43 from RTOG 0524 and 27 from RTOG 0712) underwent analysis using the ImmunoPrism assay. Immune cell proportions were as follows: CD8 T cells: median 1.2%, CD4 T cells: median 0.8%, Treg cells: median 9.2%, CD19 B cells: median 5.1%, M2 macrophages: median 0.8%, M1 macrophages: median 0%. Unbiased clustering based on gene expression profiles driven by immune cell proportions demonstrated two groups: cluster 1 with a low percentage of immune cells and shorter OS (median 31 months) and cluster 2 with a high percentage of immune cells and longer OS (median 101 months, p = 0.036). Higher expression of genes associated with T cell infiltration ( CD8A and ICOS) was associated with improved OS (104 vs 35 months, p = 0.028, HR = 0.48 (0.25 – 0.94), p = 0.031) as was higher expression of IDO1, which is associated with the interferon gamma pathway (104 vs 35 months, p = 0.042, HR = 0.49 (0.24 – 0.99), p = 0.046). Conclusions: Bladder tumors have a wide range of immune cell infiltration in the TME. Increased immune cell proportions are prognostic for OS following CRT, as well as a higher expression of genes associated with T cell infiltration interferon gamma signaling. These findings have implications for the integration of immunotherapy in the definitive management of MIBC; and can be explored further in the ongoing NRG/SWOG 1806 trial.

Long-Term Effects of Low-Dose Naltrexone on Immunomodulatory Properties of Human Adipose-Derived Mesenchymal Stem Cells.

Low-dose naltrexone (LDN) is involved in the treatment of inflammatory and immune system diseases and can affect immune cells. Mesenchymal stem cells (MSCs) are known for their immunomodulatory effects and the potential for the treatment of certain types of autoimmune diseases. To investigate the long-term effects of LDN on human adipose-derived mesenchymal stem cells (ASCs) to see how their immunomodulatory properties are affected and also how LDN-treated ASCs interact with other immune cells present in peripheral blood mononuclear cells (PBMCs). After 14 days of treatment, the ability of LDN-treated ASCs to modulate PBMC proliferation in a two-way mixed lymphocyte reaction (MLR) model was assessed using XTT. The relative expression of IDO, PD-L1, COX-2, HGF genes, and the level of IL-6 and TGF-β cytokines were measured in IFN-γ stimulated and unstimulated ASCs (treated and not treated cells) using real-time PCR and ELISA respectively. Unstimulated ASCs treated with 10-8 M Naltrexone (10-8 M NTX) showed higher levels of TGF-β, compared with the controls (P<0.05). Stimulated ASCs treated with 10-6 M NTX showed elevated expression of IDO, PD-L1 genes, and IL-6 level (P<0.05). Our results demonstrated that various LDN concentrations have dissimilar effects on ASCs' immunomodulatory properties. A higher LDN concentration induced an alteration in the immunomodulatory features of ASCs.

Cancer Cells Promote Immune Regulatory Function of Macrophages by Upregulating Scavenger Receptor MARCO Expression.

Expression of macrophage receptor with collagenous structure (MARCO) by tumor-associated macrophages is associated with poor prognosis of multiple types of cancer. In this article, we report that cancer cells (e.g., breast cancer and glioblastoma cell lines) can upregulate surface MARCO expression on human macrophages not only via IL-6-induced STAT3 activation but also via sphingosine-1-phosphate receptor (S1PR)-mediated IL-6 and IL-10 expression followed by STAT3 activation. We further found that MARCO ligation induces activation of the MEK/ERK/p90RSK/CREB signaling cascade, leading to IL-10 expression followed by STAT3-dependent PD-L1 upregulation. Such MARCO-induced macrophage polarization is accompanied by increased expression of PPARG, IRF4, IDO1, CCL17, and CCL22. Ligation of surface MARCO can thus result in decreased T cell responses mainly by reduction of their proliferation. Taken together, cancer cell-induced MARCO expression and its intrinsic regulatory function within macrophages are, to our knowledge, new aspects of cancer immune evasion mechanisms that need to be further studied in the future.

TGF-β antagonizes IFN-γ-mediated adaptive immune evasion via activation of the AKT-Smad3-SHP1 axis in lung adenocarcinoma.

IFN-γ-mediated signaling in tumor cells can induce immunosuppressive responses and cause tumor resistance to immunotherapy. Blocking TGF-β promotes T lymphocyte infiltration and turns immunologically cold tumors into hot tumors, thereby improving the efficacy of immunotherapy. Several studies have shown that TGF-β inhibits IFN-γ signaling in immune cells. We thus sought to determine whether TGF-β affects IFN-γ signaling in tumor cells and plays a role in the development of acquired resistance to immunotherapy. TGF-β stimulation of tumor cells increased SHP1 phosphatase activity in an AKT-Smad3-dependent manner, decreased IFN-γ-mediated tyrosine phosphorylation of JAK1/2 and STAT1, and suppressed the expression of STAT1-dependent immune evasion-related molecules, e.g., PD-L1, IDO1, HVEM, and Gal-9. In a lung cancer mouse model, dual blockade of TGF-β and PD-L1 led to superior antitumor activity and prolonged survival compared to anti-PD-L1 therapy alone. However, prolonged combined treatment resulted in tumor resistance to immunotherapy and increased expression of PD-L1, IDO1, HVEM, and Gal-9. Interestingly, after initial anti-PD-L1 monotherapy, dual TGF-β and PD-L1 blockade promoted both immune evasion gene expression and tumor growth compared to that in tumors treated with continuous PD-L1 monotherapy. Alternatively, treatment with JAK1/2 inhibitor following initial anti-PD-L1 therapy effectively suppressed tumor growth and downregulated immune evasion gene expression in tumors, indicating the involvement of IFN-γ signaling in immunotherapy resistance development. These results demonstrate an unappreciated effect of TGF-β on the development of IFN-γ-mediated tumor resistance to immunotherapy.

Quorum sensing-mediated dynamic regulation of 4-hydroxyisoleucine biosynthesis in Corynebacterium glutamicum

With the development of synthetic biology, some quorum sensing (QS) systems have been studied and applied to coordinate growth and production. Recently, a novel ComQXPA-PsrfA system with different response strengths was constructed in Corynebacterium glutamicum. However, the plasmid-harbored ComQXPA-PsrfA system lacks genetic stability, which restricts the application of this QS system. In this study, the comQXPA expression cassette was integrated into the chromosome of C. glutamicum SN01, resulting in QSc chassis strain. The green fluorescence protein (GFP) was expressed by the natural and mutant PsrfA promoters (PsrfAM) with various strengths in QSc. All the expressions of gfp were activated to the related level in a cell density-dependent manner. Therefore, ComQXPA-PsrfAM circuit was applied for modulating the dynamic biosynthesis of 4-hydroxyisoleucine (4-HIL). First, the expression of ido encoding α-ketoglutarate (α-KG)-dependent isoleucine dioxygenase was dynamically regulated by PsrfAM promoters, resulting in QSc/NI. The 4-HIL titer (125.18 ± 11.26 mM) increased by 45.1% compared to static ido expression strain. Then, to coordinate the α-KG supply between TCA cycle and 4-HIL synthesis, the activity of α-KG dehydrogenase complex (ODHC) was dynamically inhibited by regulating the expression of ODHC inhibitor gene odhI under QS-responsive PsrfAM promoters. The highest 4-HIL titer of QSc-11O/20I (145.20 ± 7.80 mM) increased by 23.2% compared to QSc/20I. This study modulated two critical genes expression in both cell growth and 4-HIL de novo synthesis pathways by the stable ComQXPA-PsrfAM system, and 4-HIL was produced responsively with the cell density. This strategy enhanced the 4-HIL biosynthesis efficiently without additional genetic regulation.

H3K4me3-related lncRNAs signature and comprehensive analysis of H3K4me3 regulating tumor immunity in lung adenocarcinoma

BackgroudThe role of epigenetic modifications in tumorigenesis has been widely reported. However, the role and mechanism of H3K4me3 modification in lung adenocarcinoma (LUAD) are rarely reported systematically. We, therefore, sought to analyze the characteristics of LUAD associated with H3K4me3 modification, build an H3K4me3-lncRNAs score model to predict the prognosis of patients with LUAD and clarify the potential value of H3K4me3 in immunotherapy of LUAD.MethodsWe evaluated H3K4me3-lncRNA patterns and H3K4me3-lncRNA scores of 477 LUAD samples based on 53 lncRNAs closely correlated to H3K4me3 regulators and comprehensive analyzed the role of these patterns in tumorigenesis and tumor immunity. Using Gene set variation analysis (GSVA), we systematically evaluated the H3K4me3 level of every sample and deeply analyzed the effect of H3K4me3 on the prognosis of LUAD. In addition, we included two independent immunotherapy cohorts to study the impact of high H3K4me3 score on the prognosis of patients. We also used an independent cohort with 52 matched paraffin specimens of LUAD to verify the impact of high H3K3me3 expression on the prognosis of patients.ResultsWe identified three H3K4me3-lncRNA patterns that exhibited specific immune characteristics. Characterized by immunosuppressive and increased TGFβ-mediated epithelial-mesenchymal transition (EMT), patients with high H3K4me3-lncRNA score had a poor overall survival and decreased H3K4me3 score. H3K4me3 score was significantly positively correlated with CD4+T-cell and CD8+T-cell activation, programmed cell death and immune checkpoints (ICs) expression, and was negatively correlated with MYC pathway, TP53 pathway, and cell proliferation. Patients with high H3K4me3 score showed elevated expression of ICs, potentiated CD4 T-cell and CD8 T-cell activation, increased programmed cell death, and suppressed cell proliferation and TGFβ-mediated EMT. Patients with high H3K4me3 score and high expression of CTLA4, ICOS, TIGIT, PDCD1LG2, IDO1, CD274, PDCD1, LAG3, or HAVCR2 had the best survival advantage. Two independent immunotherapy cohorts verified that patients with high H3K4me3 score showed an increased inflamed tumor microenvironment (TME) phenotype and enhanced anti-PD-1/L1 immunotherapy response. Immunohistochemistry (IHC) data from 52 matched paraffin specimens of LUAD confirmed that the protein level of H3K4me3 in tumor was significantly lower than that of paracancerous tissues and H3K4me3 brought significant survival benefits to patients with LUAD.ConclusionsWe build an H3K4me3-lncRNAs score model to predict the prognosis of patients with LUAD. More importantly, this study revealed characteristics of H3K4me3 modification in LUAD and clarified the important potential role of H3K4me3 on tumor immunotherapy and patients’ survival.

Chronic LCMV infection regulates the effector T cell response by inducing the generation of less immunogenic dendritic cells

Chronic viral infection impairs systemic immunity in the host; however, the mechanism underlying the dysfunction of immune cells in chronic viral infection is incompletely understood. In this study, we studied the lineage differentiation of hematopoietic stem cells (HSCs) during chronic viral infection to elucidate the changes in dendritic cell (DC) differentiation and subsequent impact on T cell functionality using a chronic lymphocytic choriomeningitis virus (LCMV) infection model. We first investigated the lineage differentiation of HSCs in the bone marrow (BM) to elucidate the modulation of immune cell differentiation and found that the populations highly restrained in their differentiation were common myeloid progenitors (CMPs) and common dendritic cell progenitors (CDPs). Of interest, the main immune cells infected with LCMV Clone 13 (CL13) in the BM were CD11b/c+ myeloid DCs. We next characterized CD11b+ DCs that differentiated during chronic LCMV infection. These DCs displayed a less immunogenic phenotype than DCs in naive or acutely infected mice, showing low expression of CD80 but high expression of PD-L1, B7-H4, IDO, TGF-β, and IL-10. Consequently, these CD11b+ DCs induced less effective CD8+ T cells and more Foxp3+ regulatory T (Treg) cells. Furthermore, CD11b+ DCs generated during CL13 infection could not induce effective CD8+ T cells specific to the antigens of newly invading pathogens. Our findings demonstrate that DCs generated from the BM during chronic viral infection cannot activate fully functional effector CD8+ T cells specific to newly incoming antigens as well as persistent antigens themselves, suggesting a potential cause of the functional alterations in the T cell immune response during chronic viral infection.

IDO1 and inflammatory neovascularization: bringing new blood to tumor-promoting inflammation.

In parallel with the genetic and epigenetic changes that accumulate in tumor cells, chronic tumor-promoting inflammation establishes a local microenvironment that fosters the development of malignancy. While knowledge of the specific factors that distinguish tumor-promoting from non-tumor-promoting inflammation remains inchoate, nevertheless, as highlighted in this series on the 'Hallmarks of Cancer', it is clear that tumor-promoting inflammation is essential to neoplasia and metastatic progression making identification of specific factors critical. Studies of immunometabolism and inflamometabolism have revealed a role for the tryptophan catabolizing enzyme IDO1 as a core element in tumor-promoting inflammation. At one level, IDO1 expression promotes immune tolerance to tumor antigens, thereby helping tumors evade adaptive immune control. Additionally, recent findings indicate that IDO1 also promotes tumor neovascularization by subverting local innate immunity. This newly recognized function for IDO1 is mediated by a unique myeloid cell population termed IDVCs (IDO1-dependent vascularizing cells). Initially identified in metastatic lesions, IDVCs may exert broader effects on pathologic neovascularization in various disease settings. Mechanistically, induction of IDO1 expression in IDVCs by the inflammatory cytokine IFNγ blocks the antagonistic effect of IFNγ on neovascularization by stimulating the expression of IL6, a powerful pro-angiogenic cytokine. By contributing to vascular access, this newly ascribed function for IDO1 aligns with its involvement in other cancer hallmark functionalities, (tumor-promoting inflammation, immune escape, altered cellular metabolism, metastasis), which may stem from an underlying role in normal physiological functions such as wound healing and pregnancy. Understanding the nuances of how IDO1 involvement in these cancer hallmark functionalities varies between different tumor settings will be crucial to the future development of successful IDO1-directed therapies.

Increased tryptophan, but not increased glucose metabolism, predict resistance of pembrolizumab in stage III/IV melanoma

ABSTRACT Clinical trials of combined IDO/PD1 blockade in metastatic melanoma (MM) failed to show additional clinical benefit compared to PD1-alone inhibition. We reasoned that a tryptophan-metabolizing pathway other than the kynurenine one is essential. We immunohistochemically stained tissues along the nevus-to-MM progression pathway for tryptophan-metabolizing enzymes (TMEs; TPH1, TPH2, TDO2, IDO1) and the tryptophan transporter, LAT1. We assessed tryptophan and glucose metabolism by performing baseline C11-labeled α-methyl tryptophan (C11-AMT) and fluorodeoxyglucose (FDG) PET imaging of tumor lesions in a prospective clinical trial of pembrolizumab in MM (clinicaltrials.gov, NCT03089606). We found higher protein expression of all TMEs and LAT1 in melanoma cells than tumor-infiltrating lymphocytes (TILs) within MM tumors (n = 68). Melanoma cell-specific TPH1 and LAT1 expressions were significantly anti-correlated with TIL presence in MM. High melanoma cell-specific LAT1 and low IDO1 expression were associated with worse overall survival (OS) in MM. Exploratory optimal cutpoint survival analysis of pretreatment ‘high’ vs. ‘low’ C11-AMT SUVmax of the hottest tumor lesion per patient revealed that the ‘low’ C11-AMT SUVmax was associated with longer progression-free survival in our clinical trial (n = 26). We saw no such trends with pretreatment FDG PET SUVmax. Treatment of melanoma cell lines with telotristat, a TPH1 inhibitor, increased IDO expression and kynurenine production in addition to suppression of serotonin production. High melanoma tryptophan metabolism is a poor predictor of pembrolizumab response and an adverse prognostic factor. Serotoninergic but not kynurenine pathway activation may be significant. Melanoma cells outcompete adjacent TILs, eventually depriving the latter of an essential amino acid.

Tumor lineage-specific immune response in brain metastatic disease: opportunities for targeted immunotherapy regimen?

Metastases in the brain are the most severe and devastating complication of cancer. The incidence of brain metastasis is increasing. Therefore, the need of finding specific druggable targets for brain metastasis is demanding. The aim of this study was to compare the brain (immune) response to brain metastases of the most common tumor lineages, viz., lung adenocarcinoma and breast cancer. Targeted gene expression profiles of 11 brain metastasis of lung adenocarcinoma (BM-LUAD) were compared to 11 brain metastasis of breast cancer (BCBM) using NanoString nCounter PanCancer IO 360™ Panel. The most promising results were validated spatially using the novel GeoMx™ Digital Spatial Profiler (DSP) Technology. Additionally, Immune cell profiles and expression of drug targets were validated by multiplex immunohistochemistry. We found a more active immune response in BM-LUAD as compared to BCBM. In the BM-LUAD, 138 genes were upregulated as compared to BCBM (adj. p ≤ 0.05). Conversely, in BCBM 28 genes were upregulated (adj. p ≤ 0.05). Additionally, genes related to CD45 + cells, T cells, and cytotoxic T cells showed to be expressed higher in BM-LUAD compared to BCBM (adj. p = 0.01, adj. p = 0.023, adj. p = 0.023, respectively). The spatial quantification of the immune cells using the GeoMx DSP technique revealed the significantly higher quantification of CD14 and CD163 in tumor regions of BM-LUAD as compared to BCBM. Importantly, the immune checkpoint VISTA and IDO1 were identified as highly expressed in the BM-LUAD. Multiplex immunohistochemistry confirmed the finding and showed that VISTA is expressed mainly in BM-LUAD tumor cells, CD3 + cells, and to fewer levels in some microglial cells in BM-LUAD. This is the first report on differences in the brain immune response between metastatic tumors of different lineages. We found a far more extensive infiltration of immune cells in BM-LUAD as compared to BCBM. In addition, we found higher expression of VISTA and IDO1 in BM-LUAD. Taken together, targeted immune therapy should be considered to treat patients with BM-LUAD.

CD86 Is Associated with Immune Infiltration and Immunotherapy Signatures in AML and Promotes Its Progression.

Cluster of differentiation 86 (CD86), also known as B7-2, is a molecule expressed on antigen-presenting cells that provides the costimulatory signals required for T cell activation and survival. CD86 binds to two ligands on the surface of T cells: the antigen CD28 and cytotoxic T lymphocyte-associated protein 4 (CTLA-4). By binding to CD28, CD86-together with CD80-promotes the participation of T cells in the antigen presentation process. However, the interrelationships among CD86, immunotherapy, and immune infiltration in acute myeloid leukemia (AML) are unclear. The immunological effects of CD86 in various cancers (including on chemokines, immunostimulators, MHC, and receptors) were evaluated through a pan-cancer analysis using TCGA and GEO databases. The relationship between CD86 expression and mononucleotide variation, gene copy number variation, methylation, immune checkpoint blockers (ICBs), and T-cell inflammation score in AML was subsequently examined. ESTIMATE and limma packages were used to identify genes at the intersection of CD86 with StromalScore and ImmuneScore. Subsequently, GO/KEGG and PPI network analyses were performed. The immune risk score (IRS) model was constructed, and the validation set was used for verification. The predictive value was compared with the TIDE score. CD86 was overexpressed in many cancers, and its overexpression was associated with a poor prognosis. CD86 expression was positively correlated with the expression of CTLA4, PDCD1LG2, IDO1, HAVCR2, and other genes and negatively correlated with CD86 methylation. The expression of CD86 in AML cell lines was detected by QRT-PCR and Western blot, and the results showed that CD86 was overexpressed in AML cell lines. Immune infiltration assays showed that CD86 expression was positively correlated with CD8 T cell, Dendritic cell, macrophage, NK cell, and Th1_cell and also with immune examination site, immune regulation, immunotherapy response, and TIICs. ssGSEA showed that CD86 was enriched in immune-related pathways, and CD86 expression was correlated with mutations in the genes RB1, ERBB2, and FANCC, which are associated with responses to radiotherapy and chemotherapy. The IRS score performed better than the TIDE website score. CD86 appears to participate in immune invasion in AML and is an important player in the tumor microenvironment in this malignancy. At the same time, the IRS score developed by us has a good effect and may provide some support for the diagnosis of AML. Thus, CD86 may serve as a potential target for AML immunotherapy.

Abstract 2890: Investigating dynamic IDO-Kyn-Ahr pathway induced tumor immunosuppression using imaging modality to optimize therapeutic Intervention

Abstract Despite great clinical responses in several cancers to immune checkpoint blockade (ICB) intervention, there persist many mechanisms of tumor evasion. Tryptophan catabolism by enzymes Indoleamine-2,3-dioxygenase 1 (IDO1) and tryptophan-2,3-dioxygenase 2 (TDO2) into metabolite L-Kynurenine (Kyn) has been shown to drive an immunosuppressive tumor microenvironment (TME). We have shown tumors overexpressing IDO1 recruit and activate the Treg/macrophage suppressive axis and blockade of aryl hydrocarbon receptor (AHR) activation abrogates this. Preclinical models demonstrate inhibition of IDO1 delayed tumor growth and increased T cell function. However, a phase III clinical trial in melanoma patients combining IDO1 inhibition with anti-PD-1 blockade showed no improved efficacy when compared to anti-PD-1 blockade. Kyn can act as a key signaling molecule through activation of AHR. We hypothesize that AHR engagement by kyn within the TME is highly dynamic and elevated AHR engagement must coincide with intervention to maximize therapeutic response. Using an AHR biosensor, we designed a construct with a dioxin response element (DRE) promoter that is engaged upon AHR-ligand complex, translocated to the nucleus, and drives a luciferase gene with a luminescence readout. This allows us to use an image-guided approach to track AHR bioactivity in real-time. In-vitro, we were able to validate our construct using transduced murine B16 melanoma and 4T1 breast cancer models. We treated transduced cells with endogenous AHR ligands, kynurenine, and AHR agonist (FICZ), and observed a significant dose-dependent increase in AHR reporter readout. Additionally, the AHR inhibitor (CH-223191) abrogated AHR activity and subsequent luminescence below control threshold. We implanted both DRE transduced B16 (B16-DRE) and 4T1 (4T1-DRE) intradermally and intramammary fat pad respectively. Tumors were imaged and we observed total luminescence flux corresponding to increased tumor growth. After three weeks, we assessed IDO, AHR, and HIF1a expression which all converge on the IDO-Kyn-AhR pathway using a broad panel of lymphocyte and myeloid markers from tumors and spleen. We identified high expression of IDO, AHR, and HIF1a markers in myeloid cells with an increased frequency of dendritic cells and macrophages. Future experiments will focus on using our reporter construct in the context of ICB treatment and assessing AHR activity overtime as well as sampling peripheral blood to assess the magnitude of tryptophan, kyn, and other polycyclic hydrocarbons including gut microbiome-derived indoles. The characterization of the IDO-Kyn-AhR modulation after therapeutic intervention will inform the rational design of treatment regimens to abrogate immune suppression associated with AHR activity in combination with ICB treatment. Citation Format: Mamadou Alpha Bah, Inna Serganova, Sadna Budhu, Tanya Schild, Jonathan F. Khan, Kayvan R. Keshari, Jedd D. Wolchok, Taha Merghoub. Investigating dynamic IDO-Kyn-Ahr pathway induced tumor immunosuppression using imaging modality to optimize therapeutic Intervention [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2890.

Abstract 3282: Regorafenib enhance PD-L1 expression via ATM activation and evaluate the therapeutic efficacy of combining PD-L1 antibody on bladder cancer

Abstract Bladder cancer is one of the most common cancer in urinary system in Taiwan. According to Ministry of Health and Welfare’s research, bladder cancer plays 15th leading causes of death in Taiwan in 2020. Recently immune checkpoint inhibitor (ICI) therapy show several benefits on preventing T cell exhaustion, accumulating evidences showed ICI therapy still remain some limitation, including unable to regulated immunosuppressor cells and tumor microenvironment (TME) related elements which might lead to ICI therapy dysfunction. Recently, study report that DNA damage may activate the innate immune response and improve ICI efficacy. Thus, regulating DNA damage or illustrate the role of DNA damage related factor may bring the twilight to immunotherapy. To identify the possible combination methods with ICIs, we used MB49 cells and animal model to test the combination property of regorafenib (a multiple receptor tyrosine kinase inhibitor) and 10F.9G2 (a PD-L1 monoclonal antibody). Our research illustrated the mechanism between regorafenib and PD-L1, we proved that regorafenib-induced regulated PD-L1 is associated with ATM activation. TUENL assay showed regorafenib are able to activate ATM by causing DNA break and thus enhance the expression of PD-L1 on tumor surface. This effect could be an important step to open the interaction potential of PD-L1 antibodies and thus enhance its efficacy. Through the interaction analysis of miRNA targeting gene (RNA22), we found that IDO1 mRNA could specifically interact with endogenous miR-124. Here, the propose mechanism in our research is that regorafenib may downregulate IDO1 expression via increasing of endogenous miR-124 expression. In animal model, the combination therapy showed extraordinary therapeutic effect, not only dramatically decreased tumor volume, but also upregulating the positive TME components, such as CD8+T cells and M1 macrophage. Though regorafenib may induce PD-L1 expression, regorafenib still showed several therapeutic benefits, including downregulating immunosuppressive factors, such as IDO1, VEGF, and suppressing the accumulation of regulatory T cells, myeloid-derived suppressor cells and also repolarizing macrophage from M2-to-M1⋯etc. In conclusion, this study points out the combination therapy of regorafenib and anti-PD-L1 has present several benefits to bladder cancer patients and is associated with miR-124/ATM signaling. Citation Format: Kai-Jen Pai, I-Tsang Chiang, Fei-Ting Hsu, Jai-Jen Tsai. Regorafenib enhance PD-L1 expression via ATM activation and evaluate the therapeutic efficacy of combining PD-L1 antibody on bladder cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3282.

Single-cell atlas of the liver myeloid compartment before and after cure of chronic viral hepatitis

Chronic viral infections present serious public health challenges; however, direct-acting antivirals (DAAs) are now able to cure nearly all patients infected with hepatitis C virus (HCV), representing the only cure of a human chronic viral infection to date. DAAs provide a valuable opportunity to study immune pathways in the reversal of chronic immune failures in an invivo human system. To leverage this opportunity, we used plate-based single-cell RNA-seq to deeply profile myeloid cells from liver fine needle aspirates in patients with HCV before and after DAA treatment. We comprehensively characterised liver neutrophils, eosinophils, mast cells, conventional dendritic cells, plasmacytoid dendritic cells, classical monocytes, non-classical monocytes, and macrophages, and defined fine-grained subpopulations of several cell types. We discovered cell type-specific changes post-cure, including an increase in MCM7+STMN1+ proliferating CD1C+ conventional dendritic cells, which may support restoration from chronic exhaustion. We observed an expected downregulation of interferon-stimulated genes (ISGs) post-cure as well as an unexpected inverse relationship between pre-treatment viral load and post-cure ISG expression in each cell type, revealing a link between viral loads and sustained modifications of the host's immune system. We found an upregulation of PD-L1/L2 gene expression in ISG-high neutrophils and IDO1 expression in eosinophils, pinpointing cell subpopulations crucial for immune regulation. We identified three recurring gene programmes shared by multiple cell types, distilling core functions of the myeloid compartment. This comprehensive single-cell RNA-seq atlas of human liver myeloid cells in response to cure of chronic viral infections reveals principles of liver immunity and provides immunotherapeutic insights. This study is registered at ClinicalTrials.gov (NCT02476617). Chronic viral liver infections continue to be a major public health problem. Single-cell characterisation of liver immune cells during hepatitis C and post-cure provides unique insights into the architecture of liver immunity contributing to the resolution of the first curable chronic viral infection of humans. Multiple layers of innate immune regulation during chronic infections and persistent immune modifications after cure are revealed. Researchers and clinicians may leverage these findings to develop methods to optimise the post-cure environment for HCV and develop novel therapeutic approaches for other chronic viral infections.