IMMU-41. IDO1 INCREASES Treg RECRUITMENT INDEPENDENT OF TRYPTOPHAN METABOLISM IN A MODEL OF GLIOBLASTOMA
ABSTRACT: Abstract OBJECTIVE Glioblastoma (GBM) patients with high intratumoral IDO1 mRNA levels have an associated decrease in survival (Zhai et al., 2017; CCR). IDO1 is an immunosuppressive mediator that metabolizes tryptophan (Trp), and through its associated enzyme activity, has long been recognized to increase immunosuppressive CD4+CD25+FoxP3+ Tregs. Accordingly, we previously showed that the genetic knockdown of IDO1 in murine glioma cells, suppresses intratumoral Treg recruitment and increases animal subject survival. Unexpectedly, however, IDO1 knockdown has no effect on intratumoral Trp metabolism (Zhai et al., 2017; BBI). Further surprisingly, we recently demonstrated that, although IDO1 overexpression increases Treg levels in brain tumors, the pharmacological inhibition of IDO1 metabolism has no effect on Treg accumulation (Ladomersky et al., 2018; CCR). These novel data led us to question the requirement for IDO1 enzyme activity to regulate Tregs in malignant glioma. METHODS Lentiviral vectors encoding: (i) vector control-, (ii) wild-type (WT)-, or (iii) enzyme-null-IDO1, were created by site-directed mutagenesis in a plasmid containing WT, murine, IDO1-GFP cDNA. IDO1-deficient glioma cells from transgenic mice [(ERT2)GFAP?Cre;pTENfl/fl;p53fl/fl;Rbfl/fl;IDO1-/-] were transduced with the modified plasmids. RT-PCR, Western blotting and HPLC confirmed IDO1 expression and enzyme activity, in vitro. Syngeneic IDO1-/- mice were intracranially-engrafted with 2 × 105 modified glioma cells and studied for intratumoral Treg levels. RESULTS The substitution of IDO1 histidine 350, to an alanine (H350A), decreases IDO1 enzyme activity by 90%. Syngeneic mice with intracranial, IDO1 enzyme null (H350A) glioma, have similar intratumoral Treg levels as IDO1 WT glioma, at 25% and 26%, respectively, and is significantly increased as compared to the 5.8% Tregs in vector control glioma (P=0.015). CONCLUSIONS Our data challenge current dogma explaining how IDO1 causes Treg accumulation, and are in-line with the recent IDO1 enzyme inhibitor-focused Phase III clinical trial failure [NCT02752074]. We are now focused on revealing the mechanism underlying IDO1-mediated immunosuppression in malignant glioma.
Project description:T-cell activation is characteristic during the development of atherosclerosis. While overall T-cell responses have been implicated in disease acceleration, regulatory T cells (Tregs) exhibit atheroprotective effects. The expression of the enzyme indoleamine 2,3-dioxygenase-1 (IDO1), which catalyzes the degradation of tryptophan (Trp) along the kynurenine pathway, has been implicated in the induction and expansion of Treg populations. Hence, Tregs can reciprocally promote IDO1 expression in dendritic cells (DCs) via reverse signaling mechanisms during antigen presentation. In this study, we hypothesize that triggering the "Treg/IDO axis" in the artery wall is atheroprotective. We show that apolipoprotein B100-pulsed tumor growth factor beta 2-treated tolerogenic DCs promote de novo FoxP3+ Treg expansion in vivo. This local increase in Treg numbers is associated with increased vascular IDO1 expression and a robust reduction in the atherosclerotic burden. Using human primary cell cultures, we show for the first time that IDO1 expression and activity can be regulated by cytotoxic T-lymphocyte associated protein-4, which is a constitutive molecule expressed and secreted by Tregs, in smooth muscle cells, endothelial cells, and macrophages. Altogether, our data suggest that Tregs and IDO1-mediated Trp metabolism can mutually regulate one another in the vessel wall to promote vascular tolerance mechanisms that limit inflammation and atherosclerosis.
Project description:Regulatory T cells (Tregs) are a barrier to anti-tumor immunity. Neuropilin-1 (Nrp1) is required to maintain intratumoral Treg stability and function but is dispensable for peripheral immune tolerance. Treg-restricted Nrp1 deletion results in profound tumor resistance due to Treg functional fragility. Thus, identifying the basis for Nrp1 dependency and the key drivers of Treg fragility could help to improve immunotherapy for human cancer. We show that a high percentage of intratumoral NRP1+ Tregs correlates with poor prognosis in melanoma and head and neck squamous cell carcinoma. Using a mouse model of melanoma where Nrp1-deficient (Nrp1-/-) and wild-type (Nrp1+/+) Tregs can be assessed in a competitive environment, we find that a high proportion of intratumoral Nrp1-/- Tregs produce interferon-? (IFN?), which drives the fragility of surrounding wild-type Tregs, boosts anti-tumor immunity, and facilitates tumor clearance. We also show that IFN?-induced Treg fragility is required for response to anti-PD1, suggesting that cancer therapies promoting Treg fragility may be efficacious.
Project description:Experimental data indicate that FOXP3+ Tregs can markedly curtail host antitumor immune responses, but the properties of human intratumoral Tregs are still largely unknown, in part due to significant methodologic problems. We studied the phenotypic, functional, epigenetic, and transcriptional features of Tregs in 92 patients with non-small-cell lung cancer, comparing the features of Tregs within tumors versus corresponding blood, lung, and lymph node samples. Intratumoral Treg numbers and suppressive function were significantly increased compared with all other sites but did not display a distinctive phenotype by flow cytometry. However, by undertaking simultaneous evaluation of mRNA and protein expression at the single-cell level, we demonstrated that tumor Tregs have a phenotype characterized by upregulated expression of FOXP3 mRNA and protein as well as significantly increased expression of EOS, IRF4, SATB1, and GATA1 transcription factor mRNAs. Expression of these "Treg-locking" transcription factors was positively correlated with levels of FOXP3 mRNA, with highest correlations for EOS and SATB1. EOS had an additional, FOXP3 mRNA-independent, positive correlation with FOXP3 protein in tumor Tregs. Our study identifies distinctive features of intratumoral Tregs and suggests that targeting Treg-locking transcription factors, especially EOS, may be of clinical importance for antitumor Treg-based therapy.
Project description:The development of inhibitory antibodies to factor VIII (FVIII) is a major obstacle in using this clotting factor to treat individuals with hemophilia A. Patients with a congenital absence of FVIII do not develop central tolerance to FVIII, and therefore, any control of their FVIII-reactive lymphocytes relies upon peripheral tolerance mechanisms. Indoleamine 2,3-dioxygenase 1 (IDO1) is a key regulatory enzyme that supports Treg function and peripheral tolerance in adult life. Here, we investigated the association between IDO1 competence and inhibitor status by evaluating hemophilia A patients harboring F8-null mutations that were either inhibitor negative (n = 50) or positive (n = 50). We analyzed IDO1 induction, expression, and function for any relationship with inhibitor occurrence by multivariable logistic regression and determined that defective TLR9-mediated activation of IDO1 induction is associated with an inhibitor-positive status. Evaluation of experimental hemophilic mouse models with or without functional IDO1 revealed that tryptophan metabolites, which result from IDO1 activity, prevent generation of anti-FVIII antibodies. Moreover, treatment of hemophilic animals with a TLR9 agonist suppressed FVIII-specific B cells by a mechanism that involves IDO1-dependent induction of Tregs. Together, these findings indicate that strategies aimed at improving IDO1 function should be further explored for preventing or eradicating inhibitors to therapeutically administered FVIII protein.
Project description:Chemokines and chemokine receptors are implicated in regulatory T cell (Treg) trafficking to sites of inflammation and suppression of excessive immune responses in inflammatory and autoimmune diseases; however, the specific requirements for Treg migration into the inflamed organs and the positioning of these cells within the tissue are incompletely understood. Here, we report that Tregs expressing the TH1-associated chemokine receptor CXCR3 are enriched in the kidneys of patients with ANCA-associated crescentic GN and colocalize with CXCR3(+) effector T cells. To investigate the functional role of CXCR3(+) Tregs, we generated mice that lack CXCR3 in Tregs specifically (Foxp3(eGFP-Cre) × Cxcr3(fl/fl)) and induced experimental crescentic GN. Treg-specific deletion of CXCR3 resulted in reduced Treg recruitment to the kidney and an overwhelming TH1 immune response, with an aggravated course of the nephritis that was reversible on anti-IFN? treatment. Together, these findings show that a subset of Tregs expresses CXCR3 and thereby, acquires trafficking properties of pathogenic CXCR3(+) TH1 cells, allowing Treg localization and control of excessive TH1 responses at sites of inflammation.
Project description:The microenvironment of human follicular lymphoma (FL), an incurable B cell non-Hodgkin's lymphoma, is thought to play a major role in its pathogenesis and course. Microenvironmental cells of likely importance include follicular Th cells (TFH) and regulatory T cells (Tregs), and understanding their interactions with FL tumor cells is necessary to develop novel therapeutic strategies. We found that IL-4 and CD40L are expressed by intratumoral TFH and induce production of CCL17 and CCL22 by FL tumor cells. IL-4 alone induces only CCL17 but enhances stimulation by CD40L of both CCL17 and CCL22. Consistent with our in vitro results, mRNA transcripts of IL-4 correlated with CCL17, but not CCL22, in gene expression profiling studies of FL biopsies, whereas CD40L correlated with both CCL17 and CCL22. Tumor supernatants induced preferential migration of Tregs and IL-4-producing T cells rather than IFN-?-producing T cells, and Abs to CCR4 significantly abrogated the migration of Tregs. Our results suggest that through two distinct mechanisms, intratumoral TFH induce production of CCL17 and CCL22 by FL tumor cells and facilitate active recruitment of Tregs and IL-4-producing T cells, which, in turn, may stimulate more chemokine production in a feed-forward cycle. Thus, TFH appear to play a major role in generating an immunosuppressive tumor microenvironment that promotes immune escape and tumor survival and growth. Our results provide novel insights into the cross talk among TFH, tumor cells, and Tregs in FL, and offer potential targets for development of therapeutic strategies to overcome immune evasion.
Project description:The infusion of donor regulatory T cells (Tregs) has been used to prevent acute graft-versus-host disease (GVHD) in mice and has shown promise in phase 1 clinical trials. Previous work suggested that early Treg migration into lymphoid tissue was important for GVHD prevention. However, it is unclear how and where Tregs function longitudinally to affect GVHD. To better understand their mechanism of action, we studied 2 Treg-associated chemokine receptors in murine stem cell transplant models. CC chemokine receptor (CCR) 4 was dispensable for donor Treg function in the transplant setting. Donor Tregs lacking CCR8 (CCR8(-/-)), however, were severely impaired in their ability to prevent lethal GVHD because of increased cell death. By itself, CCR8 stimulation was unable to rescue Tregs from apoptosis. Instead, CCR8 potentiated Treg survival by promoting critical interactions with dendritic cells. In vivo, donor bone marrow-derived CD11c(+) antigen-presenting cells (APCs) were important for promoting donor Treg maintenance after transplant. In contrast, host CD11c(+) APCs appeared to be dispensable for early activation and expansion of donor Tregs. Collectively, our data indicate that a sustained donor Treg presence is critical for their beneficial properties, and that their survival depends on CCR8 and donor but not host CD11c(+) APCs.
Project description:Glioblastoma multiforme (GBM) is an aggressive adult brain tumor with a poor prognosis. One hallmark of GBM is the accumulation of immunosuppressive and tumor-promoting CD4(+)FoxP3(+)GITR(+) regulatory T cells (Tregs). Here, we investigated the role of indoleamine 2,3 dioxygenase (IDO) in brain tumors and the impact on Treg recruitment.To determine the clinical relevance of IDO expression in brain tumors, we first correlated patient survival to the level of IDO expression from resected glioma specimens. We also used novel orthotopic and transgenic models of glioma to study how IDO affects Tregs. The impact of tumor-derived and peripheral IDO expression on Treg recruitment, GITR expression, and long-term survival was determined.Downregulated IDO expression in glioma predicted a significantly better prognosis in patients. Coincidently, both IDO-competent and deficient mice showed a survival advantage bearing IDO-deficient brain tumors, when compared with IDO-competent brain tumors. Moreover, IDO deficiency was associated with a significant decrease in brain-resident Tregs, both in orthotopic and transgenic mouse glioma models. IDO deficiency was also associated with lower GITR expression levels on Tregs. Interestingly, the long-term survival advantage conferred by IDO deficiency was lost in T-cell-deficient mice.These clinical and preclinical data confirm that IDO expression increases the recruitment of immunosuppressive Tregs that lead to tumor outgrowth. In contrast, IDO deficiency decreases Treg recruitment and enhances T-cell-mediated tumor rejection. Thus, the data suggest a critical role for IDO-mediated immunosuppression in glioma and support the continued investigation of IDO-Treg interactions in the context of brain tumors.
Project description:The immunosuppressive microenvironment is one of the major factors promoting the growth of glioblastoma multiforme (GBM). Infiltration of CD4+CD25+Foxp3+ regulatory T cells (Tregs) into the tumor microenvironment plays a significant role in the suppression of the anti-tumor immunity and portends a dismal prognosis for patients. Glioma-mediated secretion of chemo-attractant C-C motif ligand 2 and 22 (CCL2/22) has previously been shown by our group to promote Treg migration in vitro. In this study, we show that a local implantation of platelet-rich fibrin patch (PRF-P) into the brain of GL261 glioma-bearing mice prolonged the survival of affected animals by 42.85% (p?=?0.0011). Analysis performed on brain tumor tissue harvested from PRF-P-treated mice revealed a specific decrease in intra-tumoral lymphocytes with a preferential depletion of immunosuppressive Tregs. Importantly, co-culture of GL261 or chemo-attractants (CCL2/22) with PRF-P abrogated Treg migration. Pharmacological blockade of the CCL2/22 interaction with their receptors potentiated the inhibitory effect of PRF-P on Tregs recruitment in culture. Moreover, our findings revealed the soluble CD40 ligand (sCD40L) as a major Treg inhibitory player produced by activated platelets entrapped within the fibrin matrix of the PRF-P. Blockade of sCD40L restored the migratory capacity of Tregs, emphasizing the role of PRF-P in preventing the Treg migration to glioma tissue. Our findings highlight autologous PRF-P as a personalized, Treg-selective suppression platform that can potentially supplement and enhance the efficacy of glioma therapies.