Immunosuppressive Indoleamine 2,3-dioxygenase Research Articles

Aflatoxin B1-induced redox imbalance in the hippocampus and cerebral cortex of male Wistar rats is accompanied by altered cholinergic, indoleaminergic, and purinergic pathways: Abatement by dietary rutin

The neurotoxic impact of dietary exposure to aflatoxin B1 (AFB1) is well documented in experimental studies. Rutin is a phytochemical with prominent anti-inflammatory and antioxidant activities. There is an information gap on the influence of rutin on AFB1-induced neurotoxicity. This study investigated the influence of rutin on neurobehavioral and biochemical abnormalities in male Wistar rats (six weeks old) orally treated with AFB1 (0.75, and 1.5 mg/kg body weight) or co-administered with rutin (50 mg/kg) for 30 uninterrupted days. Results indicate that AFB1-induced depression-like behavior by Tail Suspension Test (TST) and cognitive impairment by Y-maze was abated following rutin co-administration. Abatement of AFB1-induced decreases in acetylcholinesterase (AChE) activity, and increased antioxidant status, by rutin was accompanied by a marked reduction in oxidative stress markers and increased hydrolysis of the purinergic molecules in the cerebral cortex and hippocampus of rats. Additionally, rutin co-treatment abrogated AFB1-mediated elevation of interleukin-6 (IL-6), nitric oxide (NO) levels, and activity of myeloperoxidase (MPO). Correspondingly, rutin co-treatment lowered the activity and immunocontent of immunosuppressive indoleamine 2, 3-dioxygenase (IDO). Further, rutin co-treatment prevented histological injuries in the cerebral cortex and hippocampus. In conclusion, abatement of AFB1-induced neurobehavioral abnormalities by rutin involves the mechanisms of anti-inflammatory, antioxidant, and regulation of cholinergic, purinergic, and indoleaminergic pathways in rats.

NIR-responsive hollow germanium nanospheres mediate photothermal/photodynamic therapy and restrain immunosuppression to cooperatively eradicate primary and metastatic tumors

Photothermal and photodynamic therapies (PTT and PDT) generating regional hyperpyrexia or reactive oxygen species (ROS) not only kill tumors within irradiated site, but also potentially suppress metastatic tumors far away from irradiated site by activation of antitumor immunity through inducing immunogenic cell death (ICD) (termed as “abscopal effect”). However, mono-PTT or mono-PDT are generally insufficient to produce intensive and long-lasting “abscopal effect”. Moreover, abundant immune suppressors within tumor microenvironment can offset ICD-induced antitumor effects that further cripple phototherapeutic efficacy. Towards effective eradication of both primary and metastatic tumors, it is necessary to combine photothermal and photodynamic antitumor effects as well as reverse immunosuppressive tumor microenvironment. Here, we report a hollow germanium nanosphere (HGNs) with both favorable photothermal and photodynamic effects. HGNs under near-infrared (NIR) irradiation generated hyperpyrexia and intracellular ROS, promoting ICD of tumor cells. Notably, HGNs-mediated photothermal and photodynamic effects reduce the expression of immunosuppressive indoleamine-2,3-dioxygenase (IDO), stemness- and invasion/migration-related genes. In preclinical tumor models, HGNs drives antitumor immunity by promoting effector T-cell infiltration and reducing immunosuppressive regulatory T cells (Tregs) under NIR, thus eradicating both primary and distant tumors along with extension of animal survival. Thus, with good biosafety, HGNs demonstrates a great clinically-translational potential.

Quercetin attenuates cyclophosphamide induced-immunosuppressive indoleamine 2,3-dioxygenase in the hippocampus and cerebral cortex of male Wister rats.

This study investigated the protective effect of quercetin against cyclophosphamide-induced immunosuppressive indoleamine 2,3-dioxygenase (IDO) via the mechanism of oxidative-inflammatory stress and behavioral indices. Cyclophosphamide (CYP) was administered to male Wister rats at a dose of 100 mg/kg with or without quercetin 50 mg/kg every other day for 7 days. Experimental techniques including western blotting, immunohistochemistry analysis, and inflammatory and oxidative stress marker assays were carried out. We also conducted behavioral analyses such as open field, tail suspension, and Y-maze tests for cognitive assessment. The results indicated that quercetin attenuated oxidative-inflammatory stress induced by CYP in the hippocampus and cerebral cortex of male Wister rats by augmenting the activities of antioxidant enzymes and suppressing lipid peroxidationas well as inflammatory mediators such as interleukin-6and interferon-γ. Concomitantly, quercetin partially prevented the alteration in brain tissue histological architecture and mitigated the activities of IDO/tryptophan 2,3-dioxygenase(TDO) and protein expression of IDO1. This was corroborated by the IDO-quercetin model obtained in silico, revealing a favorable inhibitory interaction between quercetin and the enzyme. Finally, the results of behavioral tests suggested that quercetin significantly prevented the depressive-like posture of the CYP-treated rats. Our study for the first time revealed that quercetin ameliorates the effect of CYP-instigated IDO/TDO activities in the cerebral cortex and hippocampus via restoration of antioxidant enzymes and preventing oxidative-inflammatory stress.

Abatement of cyclophosphamide-induced splenic immunosuppressive indoleamine 2, 3-dioxygenase and altered hematological indices in Wister rats by dietary quercetin

The immunotoxicity mediated by cyclophosphamide (CYP) was earlier reported. Quercetin, due to its anti-oxidative/inflammatory properties elicits a plethora of health benefits. However, the influence of quercetin on the splenic/immunotoxicity linked with CYP-induced indoleamine 2,3-dioxygenase (IDO) is unavailable in the literature. We investigated the effects of quercetin on the splenic immunosuppressive IDO and hematological indices of immune response in rats. Animals were treated with CYP (100 mg/kg) alone or co-treated (CYP + quercetin [100 mg/kg + 50 mg/kg respectively]) at days 1, 3, 5, and 7. Results revealed that CYP treatment alone significantly provoked an oxidative-inflammatory response, increased serum kynurenine concentration, and concomitantly increased immunosuppressive IDO and tryptophan 2, 3-dioxygenase (TDO), in the spleen as well as altering hematological indices. Quercetin co-treatment enhanced activities of antioxidant enzymes, inhibited myeloperoxidase (MPO) activity, lowered levels of nitric oxide, interferon-Υ (IFN-Υ), and interleukin-6 (IL-6), and reduced kynurenine concentration as well as IDO/TDO activities. Quercetin co-treatment augmented white blood cell (WBC), CD4-T cells, and other hematological indices of the immune response. In conclusion, quercetin prevented CYP-induced alterations in immune response in rats by lowering the activities of immunosuppressive IDO and TDO, inhibiting oxidative-inflammatory stress, diminution of kynurenine concentrations, and augmenting hematological parameters.

Cyclophosphamide instigated hepatic-renal oxidative/inflammatory stress aggravates immunosuppressive indoleamine 2,3-dioxygenase in male rats: Abatement by quercetin

The hepatic-renal toxicity associated with cyclophosphamide (CYP) treatment in both animals and humans have been reported. Quercetin, a dietary flavonoid, is known to elicit beneficial health effects. However, the influence of quercetin on the hepatic-renal toxicity associated with CYP-instigated indoleamine 2,3-dioxygenase is unavailable in the literature. The current study evaluated the effects of quercetin on the dysfunctional hepatic-renal status triggered by CYP exposure in rats. Experimental animals were exposed to CYP (100 mg/kg) or co-treated with quercetin (50 mg/kg) every other day for 7 days. Results revealed that quercetin treatment significantly assuaged CYP-mediated oxidative-inflammatory response, as well as augmenting serum levels of thyroid hormones. Additionally, quercetin attenuated CYP-induced reduction in antioxidant enzyme activities and enhanced hepatic-renal function markers, namely aspartate aminotransferase (AST), alanine aminotransferase (ALT), Alkaline phosphatase (ALP), and levels of urea and creatinine. Quercetin efficiently mitigated CYP-mediated increase in myeloperoxidase (MPO) activity, levels of nitric oxide and interleukin-6 (IL-6) in liver and kidney of rats. CYP-induced increase in the activities of immunosuppressive indoleamine 2, 3-dioxygenase (IDO) and tryptophan 2, 3-dioxygenase (TDO) in the tissues was abated in quercetin co-treated rats. In conclusion, Quercetin ameliorated deficits in the hepatic-renal function in CYP-exposed rats by lowering the activities/expression of immunosuppressive IDO and TDO via diminution of oxidative-inflammatory stress.

Semiconducting polymer nano-PROTACs for activatable photo-immunometabolic cancer therapy

Immunometabolic intervention has been applied to treat cancer via inhibition of certain enzymes associated with intratumoral metabolism. However, small-molecule inhibitors and genetic modification often suffer from insufficiency and off-target side effects. Proteolysis targeting chimeras (PROTACs) provide an alternative way to modulate protein homeostasis for cancer therapy; however, the always-on bioactivity of existing PROTACs potentially leads to uncontrollable protein degradation at non-target sites, limiting their in vivo therapeutic efficacy. We herein report a semiconducting polymer nano-PROTAC (SPNpro) with phototherapeutic and activatable protein degradation abilities for photo-immunometabolic cancer therapy. SPNpro can remotely generate singlet oxygen (1O2) under NIR photoirradiation to eradicate tumor cells and induce immunogenic cell death (ICD) to enhance tumor immunogenicity. Moreover, the PROTAC function of SPNpro is specifically activated by a cancer biomarker (cathepsin B) to trigger targeted proteolysis of immunosuppressive indoleamine 2,3-dioxygenase (IDO) in the tumor of living mice. The persistent IDO degradation blocks tryptophan (Trp)-catabolism program and promotes the activation of effector T cells. Such a SPNpro-mediated in-situ immunometabolic intervention synergizes immunogenic phototherapy to boost the antitumor T-cell immunity, effectively inhibiting tumor growth and metastasis. Thus, this study provides a polymer platform to advance PROTAC in cancer therapy.

Testing Possible Risk Factors for Idiosyncratic Drug-Induced Liver Injury Using an Amodiaquine Mouse Model and Co-treatment with 1-Methyl-d-Tryptophan or Acetaminophen.

Idiosyncratic drug reactions are unpredictable adverse reactions. Although most such adverse reactions appear to be immune mediated, their exact mechanism(s) remain elusive. The idiosyncratic drug reaction most associated with serious consequences is idiosyncratic drug-induced liver injury (IDILI). We have developed a mouse model of amodiaquine (AQ)-induced liver injury that reflects the clinical characteristics of IDILI in humans. This was accomplished by impairing immune tolerance by using PD-1–/– mice and an antibody against CTLA-4. PD-1 and CTLA-4 are known negative regulators of lymphocyte activation, which promote immune tolerance. Immune checkpoint inhibitors have become important tools for the treatment of cancer. However, as in our model, immune checkpoint inhibitors increase the risk of IDILI with drugs that have an incidence of causing liver injury. Agents such as 1-methyl-d-tryptophan (D-1-MT), an inhibitor of the immunosuppressive indoleamine 2,3-dioxygenase (IDO) enzyme, have also been proposed as anti-cancer treatments. Another possible risk factor for the induction of an immune response is the release of danger-associated molecular patterns (DAMPs). Acetaminophen (APAP) is known to cause acute liver injury, and it is likely to cause the release of DAMPs. Therefore, either of these agents could increase the risk of IDILI, although through different mechanisms. If true, then this would have clinical implications. We found that co-treatment with D-1-MT paradoxically decreased liver injury in our model, and although APAP appeared to slightly increase AQ-induced liver injury, the difference was not significant. Such results highlight the complexity of the immune response, which makes potential interactions difficult to predict.

Rational design of a minimalist nanoplatform to maximize immunotherapeutic efficacy: Four birds with one stone

Chemotherapy sometimes can cause potential tumor-specific T-cell-mediated immune response via stimulating immunogenic cell death (ICD). However, such immune response is usually very weak in chemotherapy because of immunosuppressive tumor microenvironment (ITME), substantially nourished by immunosuppressive indoleamine-2,3-dioxygenase (IDO) and myeloid-derived suppressor cells (MDSCs). It is still a challenge to develop a minimalist drug nanoplatform which can stimulate the inherent immunotherapeutic potential in chemotherapy. Herein, a self-sufficient bi-prodrug nanomedicine strategy was reported to realize a minimalist drug nanoplatform for strengthening immunotherapeutic capability in chemotherapy through its self-owned functions. Gemcitabine (GEM) and 1-methyl-tryptophan (1MT) were designed as a bi-prodrug molecule (GEM-1MT), named for the bioactivity reason of both GEM and 1MT. GEM-1MT bi-prodrug molecules could self-assemble into waste-free nanoparticles (NPs) for cancer therapy. Our GEM-1MT NPs can give full scope to the effect of “kill four birds with one stone”: (I) the released GEM could kill tumor cells for triggering ICD; (II) the selective MDSC depletion could be induced by the released GEM; (III) the released 1MT could result in IDO inhibition in tumor cells; (IV) the released 1MT could also cause IDO inhibition in MDSCs. Therefore, the GEM-1MT NPs exhibited an enhanced immunotherapy, contributing to the overall therapeutic efficacy of self-combining chemo-immunotherapy. This bi-prodrug nanomedicine strategy provides a new concept for rational design of a minimalist drug nanoplatform with a strengthened overall therapeutic efficacy of chemo-immunotherapy.

Decreased expression of ligands of placental immune checkpoint inhibitors in uncomplicated and preeclamptic oocyte donation pregnancies

Oocyte donation (OD) pregnancies are characterized by a complete immunogenetic dissimilarity between mother and fetus, which requires enhanced immunoregulation compared to naturally conceived (NC) pregnancies. The trophoblast expresses co-inhibitory ligands crucial for regulation of the maternal T cell response. Therefore, we studied the role of placental immune checkpoint inhibitors for the establishment of fetal tolerance and their relation to the development of preeclampsia in OD compared to NC pregnancies. Placental tissue from uncomplicated OD (n = 21) and NC (n = 21) pregnancies, and OD (n = 9) and NC (n = 15) pregnancies complicated with preeclampsia were studied. Protein expression of co-inhibitory ligands PD-L1 and CD200 was double blind semi-quantitatively determined by immunohistochemistry. Messenger RNA expression of PD-L1, CD200 and indoleamine 2,3-dioxygenase (IDO) was determined using qPCR. Decreased PD-L1 and CD200 protein expression and increased IDO mRNA expression was observed in uncomplicated OD versus NC pregnancies (all p < 0.05). CD200 protein expression was positively correlated with PD-L1 expression in all groups, with the number of HLA total mismatches and with HLA class I mismatches in uncomplicated OD cases (all p < 0.05). Preeclamptic cases showed lower PD-L1 protein and CD200 protein and mRNA expression in OD compared to NC pregnancies (all p < 0.05). This study shows that signaling by co-inhibitory PD-L1 and CD200 and by immunosuppressive IDO is altered in the placenta of OD pregnancies, suggesting a contribution to the higher risk for preeclampsia. These insights provide future prospects in unraveling the immune paradox of oocyte pregnancy, which are applicable for better risk management and treatment of uncomplicated and preeclamptic pregnancies.

Abstract A30: Combined IDO inhibitor and OX40L-armed-oncolytic-adenovirus therapy improves survival in murine glioblastoma

Abstract Glioblastoma diagnoses associate with upregulation of the immunosuppressive indoleamine-2,3-dioxygenase (IDO) enzyme, which correlates with poor prognoses. IDO inhibitors are immunometabolic adjuvants that can reverse protumor immunosuppression through increasing effector T-cell metabolism. To further stimulate antitumor T-cell activation, the OX40L-armed-oncolytic-adenovirus (Delta-24-RGDOX) can be used, which has shown to induce immune mechanisms that activate antitumor cytotoxic properties of T cells. Thus, we hypothesized that combining IDO inhibitors and Delta-24-RGDOX synergizes to produce a therapeutic effect in glioblastoma. In this study, we used a syngeneic glioblastoma orthotopic immunocompetent model to test the efficacy of combining Delta-24-RGDOX and an IDO inhibitor (1MT or indoximod). Delta-24-RGDOX treatment resulted in a significant survival benefit compared to the control groups (mean survival, 46.5 vs. 38.5 days, P=0.02). Interestingly, the combination of Delta-24-RGDOX and 1MT produced a higher percentage of long-term survivors compared to single therapy with Delta-24-RGDOX (50% vs. 20%, P=0.03). Moreover, when these long-term glioblastoma survivors were rechallenged with the same glioblastoma cells, we observed 100% survival, indicating the establishment of immune memory by the combination therapy. Furthermore, functional studies showed a significant increase in antitumor activity of splenocytes from glioblastoma-bearing combination-treated mice compared to Delta-24-RGDOX-treated mice (P=0.009). Additionally, immunophenotyping by flow cytometry revealed that glioblastoma-bearing combination-treated mice yielded the highest levels of PD-1/TIM-3/CD8+ T cells compared to all other treatment groups and decreased levels of immune-tolerant populations, including regulatory T cells and myeloid-derived suppressor cells, compared to Delta-24-RGDOX-only treated mice, and correlated with complete tumor elimination. Most importantly, these data support the use of immunometabolic adjuvants combined with virotherapy as a treatment for glioblastoma to improve its dismal outcomes. Citation Format: Teresa T. Nguyen, Dong Ho Shin, Hong Jiang, Frederick Lang, Candelaria Gomez-Manzano, Juan Fueyo. Combined IDO inhibitor and OX40L-armed-oncolytic-adenovirus therapy improves survival in murine glioblastoma [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr A30.

Adipose tissue-derived stromal cells retain immunosuppressive and angiogenic activity after coculture with cord blood hematopoietic precursors

Adipose-tissue derived stromal cells (ASCs) are currently considered as a full value alternative source of bone marrow MSCs for prevention of graft-versus-host disease (GVHD) after hematopoietic stem cell transplantation due to their immunosuppressive potential. Besides, ASCs are known to support ex vivo expansion of hematopoietic stem and progenitor cells (HSPCs). Ex vivo expansion enables to amplify significantly the number of HSPCs of different commitment. Mononuclear cells (MNCs) from cord blood (cb) contain HSPCs and are easily assessed. The rarity of those HSPCs is a serious limitation of its application in cell therapy. Here we expanded cbMNCs in stroma-dependent setting to generate heterocellular associates consisting of ASCs and undifferentiated and low committed hematopoietic cbHSPCs. A part of cbHSPCs in associates demonstrated a primitive phenotype confirmed by formation of “cobblestone areas". ASCs associated with cbHSPCs demonstrated up-regulation of immunosuppressive indoleamine 2,3-dioxygenase (IDO), leukemia inhibitory factor (LIF), cyclooxygenase-2 (PTGS2) genes. ASC-cbHSPCs as well as ASCs provoked the suppression of HLA-DR activation and apoptosis of mitogen-stimulated T cells. VEGF transcription and secretion were elevated providing stimulation of blood vessel formation in ovo.Thus, ASCs retain immunosuppressive and proangiogenic capacities evidencing “third party” potential along with the effective support of ex vivo expansion of cbHSPCs. Above functions expand the relevance of ASCs for needs of regenerative medicine.

P094 Strain dependent difference in the progression of dextran sulphate sodium induced colitis is a consequence of the cross-talk between colon-infiltrating dendritic cells and T regulatory cells

Dextran sulphate sodium (DSS) causes colon inflammation in C57BL6 mice (Th1 strain), while BALBc mice (Th2 strain) are relatively resistant to DSS-induced injury. The main aim of this study was to evaluate molecular mechanisms responsible for different progression of colitis in Th1 and Th2-dominant mouse strains. DSS (3%, molecular weight 40 kDa) was given to C57BL/6 and BALBc mice in place of normal drinking water for 5 days, followed by 5 days of recovery period. Disease Activity Index (weight loss, stool consistency, visible blood in faeces) and histology score (sum of “infiltration” and “damage of epithelium” sub-scores) were used to assess the severity of colitis. The phenotype of colon-infiltrated immune cells was determined by flow cytometry. BALBc mice developed less aggressive colitis then C57BL/6 mice. DSS-treated BALBc mice did not develop diarrhoea, gross rectal bleeding, and significant body weight loss. Level of pro-inflammatory cytokines (IL-12 and IL-1β) was significantly lower, while concentration of anti-inflammatory IL-10 was higher in sera of DSS-treated BALBc mice. The total number of IFN-γ and IL-17-producing T cells was lower in colons of DSS-treated BALBc mice. On contrary, there was significantly higher number of IL-4 and IL-10-producing T cells, as well as CD4+FoxP3+ T regulatory cells (Tregs) in colons of BALBc DSS-treated animals. Significantly lower number of IL-12 and IL-1β producing colon-infiltrating CD45+CD11c+ dendritic cells (DCs) and significantly higher number of IL-10 producing DCs was observed in DSS-treated BALBc mice. Lipopolysaccharide stimulated DCs, isolated from BALBc mice, produced lower amounts of IL-12 and expressed lower amounts of CD40, but express significantly higher amount of immunosuppressive Indoleamine 2,3-dioxygenase (IDO) when compared with similarly activated DCs isolated from C57BL/6 animals. Depletion of Tregs significantly aggravates clinical and histological symptoms of DSS-induced colitis in BALBc mice. Interestingly, transfer of DCs in Treg-depleted mice managed to recover Tregs number that resulted with the attenuation of colitis. This phenomenon was completely abrogated in the presence of IDO inhibitor, suggesting the importance of IDO for DC:Treg crosstalk in injured colons. Strain dependent difference in the progression of DSS-induced colitis is a consequence of the cross-talk between colon-infiltrating DCs and T regs.

Nano-enabled pancreas cancer immunotherapy using immunogenic cell death and reversing immunosuppression

While chemotherapy delivery by nanocarriers has modestly improved the survival prospects of pancreatic ductal adenocarcinoma (PDAC), additional engagement of the immune response could be game changing. We demonstrate a nano-enabled approach for accomplishing robust anti-PDAC immunity in syngeneic mice through the induction of immunogenic cell death (ICD) as well as interfering in the immunosuppressive indoleamine 2,3-dioxygenase (IDO) pathway. This is accomplished by conjugating the IDO inhibitor, indoximod (IND), to a phospholipid that allows prodrug self-assembly into nanovesicles or incorporation into a lipid bilayer that encapsulates mesoporous silica nanoparticles (MSNP). The porous MSNP interior allows contemporaneous delivery of the ICD-inducing chemotherapeutic agent, oxaliplatin (OX). The nanovesicles plus free OX or OX/IND-MSNP induce effective innate and adaptive anti-PDAC immunity when used in a vaccination approach, direct tumor injection or intravenous biodistribution to an orthotopic PDAC site. Significant tumor reduction or eradication is accomplishable by recruiting cytotoxic T lymphocytes, concomitant with downregulation of Foxp3+ T cells.

Neem leaf glycoprotein overcomes indoleamine 2,3 dioxygenase mediated tolerance in dendritic cells by attenuating hyperactive regulatory T cells in cervical cancer stage IIIB patients

Tolerogenic dendritic cells (DCs) are a subset of DCs characterized by abundant indoleamine 2,3 dioxygenase (IDO) expressions. IDO may be co-operatively induced in DCs by regulatory T (Tregs) cells and various DC maturation agents. Tregs are markedly amplified in the physiological system of cancer patients, inducing over tolerance in DCs that leads to the hyper accumulation of immunosuppressive IDO in tumor microenvironment, thereby, hampering anti-tumor immunity. Consequently, a major focus of current immunotherapeutic strategies in cancer is to minimize IDO, which is possible by reducing Tregs and using various IDO inhibitors. Neem leaf glycoprotein (NLGP), a natural and nontoxic immunomodulator, demonstrated several unique immunoregulatory activities. Noteworthy activities of NLGP are to mature DCs and to inhibit Tregs. As Tregs are inducer of IDO in DCs and hyperactive Tregs is a hallmark of cancer, we anticipated that NLGP might abrogate IDO induction in DCs by inhibiting Tregs. Evidences are presented here that in a co-culture of DCs and Tregs isolated from cervical cancer stage IIIB (CaCx-IIIB) patients, NLGP does inhibit IDO induction in DCs by curtailing the over expression of Cytotoxic T-Lymphocyte Antigen 4 (CTLA4) on Tregs and concomitantly induces optimal DC maturation. In contrast, in the presence of LPS as maturation agent the DCs displays a tolerogenic profile. This finding suggests the reduction of tolerogenecity of DCs in CaCx-IIIB patients by reducing the IDO pool using NLGP. Accordingly, this study sheds more light on the diverse immunomodulatory repertoire of NLGP.

CTLA4-Ig immunosuppressive activity at the level of dendritic cell/T cell crosstalk

Immunosuppressive cytotoxic T lymphocyte associated antigen-4 immunoglobulin fusion proteins (CTLA4-Ig) block the CD28:CD80/86 costimulatory pathway. On a cellular level, CTLA4-Ig is understood to dampen T cell responses. As a mechanism, CTLA4-Ig has been reported to affect dendritic cell (DC) function via inducing the immunosuppressive indoleamine 2,3 dioxygenase (IDO) pathway and promoting a DC regulatory phenotype. We here probed cellular mechanisms of CTLA4-Ig immunoregulation in an allogeneic setting using C57BL/6 splenic or bone marrow derived DCs (BMDCs) as stimulators of allogeneic Balb/c derived T cells. To address whether CTLA4-Ig immunosuppression affected DCs, we pre-exposed C57BL/6 splenic or BMDCs to CTLA4-Ig and removed unbound CTLA4-Ig before co-culture with allogeneic T cells. CTLA4-Ig disappeared rapidly (within 4h) from the cell membrane by combined internalization and dissociation. These CTLA4-Ig pre-exposed DCs were fully capable of stimulating allogeneic T cell proliferation, suggesting that CTLA4-Ig does not impair the DC stimulatory capacity. Only the presence of CTLA4-Ig during DC/T cell co-culture resulted in the expected inhibition of proliferation. C57BL/6 splenic or BMDCs exposed to CTLA4-Ig did not display IDO activity. We conclude that CTLA4-Ig immunosuppressive activity does not depend on a DC regulatory phenotype but on its presence during DC/T cell interaction.

Ambivalent effects of dendritic cells displaying prostaglandin E2‐induced indoleamine 2,3‐dioxygenase

Prostaglandin E2 (PGE(2)), an abundantly produced lipid messenger in mammalian organisms, has been attributed to possess potent albeit ambivalent immunological functions. Recently, PGE(2) has been reported to stimulate the commonly believed immunosuppressive indoleamine 2,3-dioxygenase (IDO) pathway in human dendritic cells (DCs), but without promoting DC immunosuppressive activity. Here, we report that PGE(2) used as a DC maturation agent apparently has more diverse functions. PGE(2)-matured DCs acquired powerful IDO activity, which was sustained even after removing PGE(2). These IDO-competent DCs were able to stimulate allogeneic T-cell proliferation, but achieved inhibitory activity as their content in DC/T-cell co-cultures increased. The DC inhibitory activity was reversed upon blockade of IDO activity, confirming that the suppressive effect was in fact mediated by IDO and occurred in a dose-dependent fashion. IDO-mediated T-cell suppression was restored upon re-stimulation of T cells in the absence of IDO activity, confirming its reversibility. T cells stimulated by PGE(2)-matured IDO-competent DCs were sensitized to produce multiple cytokines, comprising Th1, Th2, and Th17 phenotypes. Collectively, these data suggest that T cells stimulated by PGE(2)-matured DCs are not terminally differentiated and their ultimate type of response may be formed by microenvironmental conditions.

Abstract 3548: Loss of the type III TGF-β receptor during cancer progression generates an immunotolerant tumor microenvironment: Translational implications for TGF-β inhibition and immunotherapy biomarker development

Abstract We have demonstrated that the type III TGF-β receptor (TβRIII) functions to suppress cancer progression and is downregulated in several malignancies. Previous work has shown TβRIII to undergo ectodomain shedding (sTβRIII), enabling the sequestration of the soluble TGF-β ligand and the inhibition of the downstream TGF-β signaling pathway. The TGF-β cytokine is an immunosuppressive factor expressed by many tumor types to dampen the host immune response and thereby allow for tumor progression. Dendritic cells (DC) are critical antigen presenting cells involved in orchestrating antigen-specific immunity. We therefore propose that the downregulation of TβRIII during cancer development permits enhanced TGF-β signaling within local DC populations of the tumor microenvironment, allowing for evasion of the host immune response. Our data suggests that the tumor suppressor properties of TβRIII in the 4T1 murine breast cancer model are significantly diminished in immunosuppressed hosts. Indeed, loss of TβRIII allows for the progression of more immunogenic Her2/neu-expressing 4T1 tumors in vivo and suppresses Her2/neu-specific T cell responses. Importantly, the local delivery of a TGF-β antibody into 4T1-RIIIlo tumors phenocopies 4T1-RIIIhi tumors suggesting that this phenomenon is mediated by sTβRIII-dependent inhibition of soluble TGF-β in the tumor microenvironment. We have shown that 4T1-RIIIhi tumors as well as their draining lymph nodes (TDLNs) exhibit increased numbers of infiltrating CD8+ T cells, diminished FoxP3+ regulatory T cell infiltration, a TH1-polarized cytokine profile, and a mature DC phenotype relative to 4T1-RIIIlo tumors. In vitro studies have shown 4T1-RIII conditioned media to inhibit TGF-β signaling within DCs and to enhance their maturation. In addition, studies have revealed DC populations within 4T1-RIIIhi tumors to express reduced levels of the immunosuppressive indoleamine 2,3-dioxygenase (IDO) enzyme. Further work has demonstrated that plasmacytoid DCs (pDCs) within TDLNs upregulate IDO expression in response to TGF-β stimulation and that IDO expression is suppressed in the presence of TβRIII-expressing tumors. Indeed, we have found that melanoma patients with elevated sTβRIII plasma levels exhibit superior responses to treatment. We conclude that the loss of the TβRIII tumor suppressor during breast carcinogenesis results in enhanced paracrine TGF-β signaling within the pDC compartment of the tumor microenvironment. This work implies that TβRIII downregulation represents a novel mechanism for evading the host anti-tumor immune response. These findings further support the targeting of TGF-β as a strategy to enhance the efficacy of immunotherapeutic approaches for solid tumors and suggests that sTβRIII may represent a biomarker for immunotherapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3548. doi:1538-7445.AM2012-3548

Molecular analysis of melanoma-induced sentinel lymph node immune dysfunction.

Sentinel lymph nodes (SLNs) of melanoma patients show evidence of tumor-induced immune dysfunction. Our previous works have shown that IL-10 and IFNγ co-regulate indoleamine-2,3-dioxygenase (IDO)-expressing immunosuppressive dendritic cells (DCs) in melanoma SLNs. The goal of this study is to examine the relationship between melanoma SLN tumor burden and the degree of SLN immune dysfunction as a model to study tumor-induced immune dysfunction. We hypothesize that SLN tumor burden correlates with the degree of SLN immune dysfunction. Patients undergoing SLN biopsy for clinical stages I and II melanomas were enrolled in the study under an IRB-approved protocol. During the SLN biopsy, non-hot and non-blue portion of the SLN was harvested, flash-frozen in liquid nitrogen, and mRNA was extracted. By using quantitative real-time PCR, gene expressions of cytokines (IL-4, IL-10, IFNγ, TGFβ, GM-CSF) and the surrogates of immunosuppressive regulatory and effector cells (IDO-expressing DCs and Foxp3-expressing T-regs, respectively) were measured and correlated against the SLN tumor burden (MART1) and against each other. The data were log transformed for normalization. Statistical test used Student's t-test and stepwise multivariate regression analysis. Statistical significance was determined at P<0.05. SLNs of 74 patients were analyzed in this analysis. Ten of seventy-four patients (13.5%) had tumor-positive SLNs. MART1 gene expression showed a significant difference between the SLN (+) and SLN (-) groups (P=0.04). Among the various cytokines, multivariate analysis showed that only IFNγ gene expression correlated independently with MART1 gene expression (P<0.0001, r=0.91). Similar multivariate analyses show that IFNγ (P<0.0001, r=0.78), IL-10 (P=0.0037, r=0.60), and TGFβ (P<0.0001, r=0.95) gene expressions correlated independently with IDO gene expression. IFNγ (P<0.0001, r=0.87) and GM-CSF (P=0.042, r=0.76) gene expressions correlated independently with Foxp3 gene expression. MART1 gene expression showed independent correlation with IDO (P=0.0002, r=0.75) and Foxp3 (P=0.0002, r=0.75) gene expressions. SLN tumor burden correlates with immunosuppressive IDO and Foxp3 expressions within the SLNs of melanoma patients. Our data are consistent with our theory that melanoma induces expressions of specific cytokines, which in turn, stimulate immune suppressors within the SLN. This study also supports our previous finding that IL-10 and IFNγ co-regulate IDO within the SLN. In our data, IFNγ is the sole cytokine that correlates with the SLN tumor burden and seems to play a central role in tumor-induced immunological changes in the SLN immune microenvironment.

Abstract 5321: Molecular analysis of melanoma-induced sentinel lymph node immune dysfunction

Abstract Introduction Sentinel lymph nodes (SLNs) of melanoma patients show evidence of tumor-induced immune dysfunction. Our previous works have shown that IL10 and IFNγ co-regulate indoleamine-2,3-dioxygenase (IDO)-expressing immunosuppressive dendritic cells (DCs) in melanoma SLNs. The goal of this study is to examine the relationship between melanoma SLN tumor burden and the degree of SLN immune dysfunction as a model to study tumor-induced immune dysfunction. We hypothesize that SLN tumor burden correlates with the degree of SLN immune dysfunction. Methods Patients undergoing SLN biopsy for clinical stages I and II melanomas were enrolled in the study under an IRB-approved protocol. During the SLN biopsy, non-hot and non-blue portion of the SLN was harvested, flash-frozen in liquid nitrogen, and mRNA was extracted. By using quantitative real-time PCR, gene-expressions of cytokines (IL4, IL10, IFNγ, TGFβ, GM-CSF), and the surrogates of immunosuppressive regulatory and effector cells (IDO-expressing DCs and Foxp3-expressing Tregs, respectively) were measured and correlated against the SLN tumor burden (MART1) and against each other. The data were log-transformed for normalization. Statistical test used Students’ t-test and stepwise multivariate regression analysis. Statistical significance was determined at p&amp;lt;0.05. Results Of 200 patients enrolled, SLNs of 74 patients were analyzed in this preliminary analysis. Ten of 74 patients (13.5%) had tumor-positive SLNs. MART1 gene-expression showed a significant difference between the SLN(+) and SLN(−) groups (p=0.04). Among the various cytokines, multivariate analysis showed that only IFNγ gene-expression correlated independently with MART1 gene-expression (p&amp;lt;0.0001, r=0.91). Similar multivariate analyses show that IFNγ (p&amp;lt;0.0001, r=0.78), IL10 (p=0.0037, r=0.60), and TGFβ (p&amp;lt;0.0001, r=0.95) gene-expressions correlated independently with IDO gene-expression. IFNγ (p&amp;lt;0.0001, r=0.87) and GM-CSF (p=0.042, r=0.76) gene-expressions correlated independently with Foxp3 gene-expression. MART1 gene-expression showed independent correlation with IDO (p=0.0002, r=0.75) and Foxp3 (p=0.0002, r=0.75) gene-expressions. Conclusion SLN tumor burden correlates with immunosuppressive IDO and Foxp3 expressions within the SLNs of melanoma patients. Our data are consistent with our theory that melanoma induces expressions of specific cytokines, which in turn, stimulate immune-suppressors within the SLN. This study also supports our previous finding that IL10 and IFNγ co-regulate IDO within the SLN. In our data, IFNγ is the sole cytokine that correlates with the SLN tumor burden and seems to play a central role in tumor-induced immunological changes in the SLN immune microenvironment. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5321.

Low dose Zebularine treatment enhances immunogenicity of tumor cells

Strategy: We have investigated how alterations in gene expression induced by the demethylating drug Zebularine affect the immune response tumor cells elicit. The rational has been to treat syngeneic rat colon cancer cells with Zebularine at different concentrations and then use these cells to study gene expression of different genes involved in cancer immunogenicity. Gene expressions were monitored by semi-quantitative PCR and real-time PCR. Results: Intriguingly there was a large increase in the production of indoleamine 2,3-dioxygenase (IDO) after treatment with 100 μM Zebularine as compared with untreated tumor cells, whereas treatment with 20 μM Zebularine caused a significant decrease of the IDO production. After immunization with syngeneic tumor cells, spleen cells were isolated and restimulated in vitro with irradiated tumor cells. Immune reactivity was measured by proliferation, and production of interferon gamma and interleukin10. The immunogenicity of tumor cells treated in vitro with a low dose of Zebularine increased, whereas it decreased after high dose exposure. The inhibition of immunogenicity by 100 μM Zebularine was shown to be counteracted by the IDO inhibitor 1-methyl-tryptophan (1MT), confirming that this effect of Zebularine is mainly caused by IDO induction. Differences using Zebularine-treated or non-treated cells for in vitro restimulation were marginal. Conclusion: Low dose treatment with Zebularine (20 μM) decreases the production of the immunosuppressive IDO from rat colon cancer cells and enhances their immunogenicity, whereas high dose Zebularine treatment (100 μM) enhances the IDO production from the cancer cells and suppresses their immunogenicity. This immunosuppression should be considered when cancer is treated with Zebularine or drugs acting in a similar way.