654 Analysis of IDO-1 expression on dendritic cells and factors influencing its up- and downregulation in pancreatic cancer

BackgroundPancreatic Ductal Adenocarcinoma (PDAC) is bad. An immunosuppressive tumor microenvironment (TME) with an excess of immunosuppressive immune cells and cytokine/chemokine factors contribute to local and systemic immunosuppression in PDAC.1 Our laboratory has generated single-cell RNA-Sequencing (scRNA-Seq.) data from spleens derived from PDAC patients and healthy counterparts. This data demonstrates the existence of dendritic cell (DC) subsets with a tolerogenic phenotype. These DCs display increased expression of several markers, including Indoleamine 2,3-dioxygenases (IDO-1 and IDO-2), widely accepted as markers for a specific population of DCs: tolerogenic DCs. These cells evoke an immunosuppressive signal leading to activation of regulatory T cells and MDSCs as well as apoptosis of CD8+ and CD4+ effector T cells.2 3MethodsTo validate our scRNA-Seq. data, we performed pilot investigations harvesting DCs from the spleen of PDAC patients and healthy subjects. Besides examining human specimens, we also investigated the IDO-1 expression on splenic DCs from tumor-bearing mice, orthotopically implanted with LSL-KrasG12D; LSL-Trp53R172H/+; Pdx1-Cre (KPC)-derived cell lines. It is known that tumor-derived exosomes can impact DC-differentiation to a tolerogenic phenotype.4 Exosome purification using differential ultracentrifugation is a well-established method in our lab and optimized for our autopsy samples. We analyzed tumor-derived exosomes for their potential in modulating IDO-1 expression on DCs in in vitro assays. Briefly, we incubated DCs with different exosome concentrations and harvested the cells for RNA-sequencing and flow cytometry.ResultsCompared to normal spleens, DCs from PDAC spleens displayed higher expression of IDO-1 (figure 1). Additionally, KPC-tumor-bearing mice showed higher expression of IDO-1 on DCs from the spleen and blood compared to wild-type mice. Further investigating the influence of PDAC-derived exosomes on marker expression on DCs have shown an apparent increase in expression of IDO-1 when culturing splenic-derived DCs with tumor-derived exosomes (figure 2).ConclusionsWhile tolerogenic DCs are essential in regulating the homeostasis between immune response and immune tolerance,5 several studies have shown IDO-1 overexpression in cancer. Investigating tolerogenic DCs is an essential part of our lab's efforts to understand the nature of the immune response in PDAC. Future directions for this project include determining molecular pathways that regulate the expression of IDO-1. Additionally, we will investigate downstream mechanisms through which exosomes modulate the switch to a tolerogenic phenotype. We also plan to further characterize different splenic DC populations by evaluating their interplay with other immune cells in the context of antigen-specificity and other factors influencing these cells' properties.ReferencesMundry CS, Eberle KC, Singh PK, Hollingsworth MA, Mehla K. Local and systemic immunosuppression in pancreatic cancer: targeting the stalwarts in tumor's arsenal. BBA - Reviews on Cancer 2020;1874(1):188387.Liu M, Wang X, Wang L, Ma X, Gong Z, Zhang S, Li Y. Targeting the IDO1 pathway in cancer: from bench to bedside. Journal of Hematology & Oncology 2018;11(1):100.Hornyák L, Dobos N, Koncz G, Karányi Z, Páll D, Szabó Z, Halmos G, Székvölgyi L. The role of Indoleamine-2,3-Dioxygenase in cancer development, diagnostics, and therapy. Frontiers in immunology 2018;9:1.Bronte V, Pittet MJ. The spleen in local and systemic regulation of immunity. Immunity 2013;39(5):806–818.Domogalla MP, Rostan PV, Raker VK, Steinbrink K. Tolerance through education: how tolerogenic dendritic cells shape immunity. Frontiers in Immunology 2017;8:1764.Ethics ApprovalThis study was approved by the University of Nebraska Medical Center Ethics Board; approval numbers IRB#: 440-16-EP and IRB#: 091-01.Abstract 654 Figure 1Expression of IDO-1 and IDO-2 on DCs from PDAC spleen (blue) .and normal spleen (orange)Abstract 654 Figure 2Change in expression of IDO-1 through treatment of DCs with different concentrations of tumor-derived exosomes

657 Characterization and therapeutic targeting of a tumor-associated tolerogenic DC subpopulation driven by SREBP2 and the mevalonate metabolic pathway

BackgroundConventional dendritic cells (DCs) are essential mediators of anti-tumor immunity and the efficacy of anti-PD-1 checkpoint immunotherapies.1 Recent studies suggest that tumor-mediated development of a sub-population of tolerogenic DCs plays an important role in immune evasion.2 3 Metabolic reprogramming regulates tolerogenic DCs in the tumor microenvironment (TME).4 5 Activation of DCs leads to rewiring of cDC metabolism towards glycolysis to support T cell activation while tolerogenic DCs display enhanced fatty acid oxidation.6 Related to DC metabolic alterations, tumor-associated DCs (TADCs) are enriched in lipids and have a reduced capacity to present antigen to T cells. Lipid homeostasis is maintained through a complex network of transcription factors including sterol regulatory element-binding protein-2 (SREBP2) which drives the expression of mevalonate pathway genes.7 The identification of those tumor-controlled pathways that regulate tolerogenic DCs in the TME is expected to lead to the discovery of a novel family of immunotherapeutic targets.MethodsWe use transgenic mouse models of melanoma, sentinel lymph node (LN) tissue specimens derived from melanoma patients, single-cell RNA sequencing (scRNAseq), and flow cytometry-based metabolic assays to identify novel tumor-associated regulatory programs amongst different sub-populations of conventional DCs in the TME.Results scRNAseq of DCs isolated from the tumor-draining LN (TDLN) of a BRAFV600EPTEN-/- transgenic melanoma model revealed critical genetic differences in distinct DC sub-populations. We observed a migratory DC subset enriched in the expression of numerous immunoregulatory genes and identified CD63 as a surface marker to distinguish this DC subset from other conventional cDC1s and cDC2s. Further studies demonstrated CD63+ DCs to suppress T cell activation and promote CD4+FOXP3+ regulatory T cell (Treg) differentiation. Relative to other cDC subsets, CD63+ DCs overexpress genes of the mevalonate pathway leading to increased lipid content. Treatment of melanoma-bearing mice with a pharmacologic inhibitor of SREBP2 leads to a significant reduction in CD63+ DCs in the TDLN and reduced Tregs, resulting in suppressed tumor growth. Importantly, scRNAseq of DCs isolated from sentinel LNs of melanoma patients reveal that this population is conserved in humans.ConclusionsLipid homeostasis in TADCs is a major determinant of their metabolic state, but despite significant advances, the molecular pathways regulating tolerogenic DCs have remained poorly understood. Collectively, this data demonstrates an important role of the mevalonate pathway in driving a tolerogenic DC program and highlights the therapeutic targeting of SREBP2 and DC lipid metabolism as a promising approach to overcoming immune tolerance in the TME and boosting immunotherapy responses.ReferencesGardner A, Ruffell B. Dendritic cells and cancer immunity. Trends Immunol 2016;37:855–865. doi:10.1016/j.it.2016.09.006DeVito NC, Plebanek MP, Theivanthiran B, Hanks BA. Role of tumor-mediated dendritic cell tolerization in immune evasion. Front Immunol 2019;10:2876. doi:10.3389/fimmu.2019.02876Gerhard GM, Bill R, Messemaker M, Klein AM, Pittet MJ. Tumor-infiltrating dendritic cell states are conserved across solid human cancers. J Exp Med 2021;218. doi:10.1084/jem.20200264Plebanek MP, Sturdivant M, DeVito NC, Hanks BA. Role of dendritic cell metabolic reprogramming in tumor immune evasion. Int Immunol 2020;32:485–491. doi:10.1093/intimm/dxaa036Wculek SK, Khouili SC, Priego E, Heras-Murillo I, Sancho D. Metabolic control of dendritic cell functions: digesting information. Front Immunol 2019;10:775. doi:10.3389/fimmu.2019.00775Zhao F. et al. Paracrine Wnt5a-beta-Catenin signaling triggers a metabolic program that drives dendritic cell tolerization. Immunity 2018;48:147-+, doi:10.1016/j.immuni.2017.12.004Xue L. et al. Targeting SREBP-2-Regulated mevalonate metabolism for cancer therapy. Front Oncol 2020;10:1510, doi:10.3389/fonc.2020.01510Ethics ApprovalCollection of human tissue specimens was approved by the Duke Institutional Review Board under the title Immune Markers of Sentinel Nodes in Melanoma and the protocol number Pro00090678. All patients gave informed consent prior to participating in the study. All experiments involving animals were approved by the Duke University Institutional Animal Care and Use Committee (IACUC) under protocol number A174-18-07

Cannabinoids induce functional Tregs by promoting tolerogenic DCs via autophagy and metabolic reprograming

The generation of functional regulatory T cells (Tregs) is essential to keep tissue homeostasis and restore healthy immune responses in many biological and inflammatory contexts. Cannabinoids have been pointed out as potential therapeutic tools for several diseases. Dendritic cells (DCs) express the endocannabinoid system, including the cannabinoid receptors CB1 and CB2. However, how cannabinoids might regulate functional properties of DCs is not completely understood. We uncover that the triggering of cannabinoid receptors promote human tolerogenic DCs that are able to prime functional FOXP3+ Tregs in the context of different inflammatory diseases. Mechanistically, cannabinoids imprint tolerogenicity in human DCs by inhibiting NF-κB, MAPK and mTOR signalling pathways while inducing AMPK and functional autophagy flux via CB1- and PPARα-mediated activation, which drives metabolic rewiring towards increased mitochondrial activity and oxidative phosphorylation. Cannabinoids exhibit in vivo protective and anti-inflammatory effects in LPS-induced sepsis and also promote the generation of FOXP3+ Tregs. In addition, immediate anaphylactic reactions are decreased in peanut allergic mice and the generation of allergen-specific FOXP3+ Tregs are promoted, demonstrating that these immunomodulatory effects take place in both type 1- and type 2-mediated inflammatory diseases. Our findings might open new avenues for novel cannabinoid-based interventions in different inflammatory and immune-mediated diseases.

HIV-1 Induction of Tolerogenic DCs is Mediated by Cellular Interaction with Suppressive T Cells

HIV-1 infection gives rise to a multilayered immune impairment in most infected individuals. The crosstalk between Dendritic cells and T cells plays an important part in the induction of immune responses. The chronic presence of human immunodeficiency virus (HIV)-1 during the dendritic cells (DCs) priming and activation of T cells promotes the expansion of suppressor cells in a contact dependent manner. The mechanism behind the T cell side of this HIV induced impairment is well studied, whereas little is known about the reverse effects exerted on the DCs in this setting.Here we assessed the phenotype and transcriptome profile of mature DCs that have been in contact with suppressive T cells. The DCs in the HIV exposed DC-T cell coculture obtained a more tolerogenic/suppressive phenotype with increased expression of e.g., PDL1, Gal-9, HVEM, and B7H3, mediated by their cellular interaction with T cells. The transcriptomic analysis showed a clear type I IFN response profile as well as an activation of pathways involved in T cell exhaustion.Taken together, our data indicate that the prolonged and strong IFN type I signaling induced by the presence of HIV during DC-T cell cross talk could play an important role in the induction of the tolerogenic DCs and suppressed immune response.

In vivo reprogramming of pathogenic lung TNFR2+ cDC2s by IFNβ inhibits HDM-induced asthma

Asthma is a common inflammatory lung disease with no known cure. Previously, we uncovered a lung TNFR2+ conventional DC2 subset (cDC2s) that induces regulatory T cells (Tregs) maintaining lung tolerance at steady state but promotes TH2 response during house dust mite (HDM)–induced asthma. Lung IFNβ is essential for TNFR2+ cDC2s–mediated lung tolerance. Here, we showed that exogenous IFNβ reprogrammed TH2-promoting pathogenic TNFR2+ cDC2s back to tolerogenic DCs, alleviating eosinophilic asthma and preventing asthma exacerbation. Mechanistically, inhaled IFNβ, not IFNα, activated ERK2 signaling in pathogenic lung TNFR2+ cDC2s, leading to enhanced fatty acid oxidation (FAO) and lung Treg induction. Last, human IFNβ reprogrammed pathogenic human lung TNFR2+ cDC2s from patients with emphysema ex vivo. Thus, we identified an IFNβ-specific ERK2-FAO pathway that might be harnessed for DC therapy.

Therapeutic Liposomal Vaccines for Dendritic Cell Activation or Tolerance

Dendritic cells (DCs) are paramount in initiating and guiding immunity towards a state of activation or tolerance. This bidirectional capacity of DCs sets them at the center stage for treatment of cancer and autoimmune or allergic conditions. Accordingly, many clinical studies use ex vivo DC vaccination as a strategy to boost anti-tumor immunity or to suppress immunity by including vitamin D3, NF-κB inhibitors or retinoic acid to create tolerogenic DCs. As harvesting DCs from patients and differentiating these cells in vitro is a costly and cumbersome process, in vivo targeting of DCs has huge potential as nanoparticulate platforms equipped with activating or tolerogenic adjuvants can modulate DCs in their natural environment. There is a rapid expansion of the choices of nanoparticles and activation- or tolerance-promoting adjuvants for a therapeutic vaccine platform. In this review we highlight the most recent nanomedical approaches aimed at inducing immune activation or tolerance via targeting DCs, together with novel fundamental insights into the mechanisms inherent to fostering anti-tumor or tolerogenic immunity.

Curcumin: a dietary phytochemical for targeting the phenotype and function of dendritic cells

: Dendritic cells (DCs) are the most powerful antigen-presenting cells which link the innate and adaptive immune responses. Depending on the context DCs initiate the immune responses or contribute to immune tolerance. Any disturbance in their phenotypes and functions may initiate inflammatory or autoimmune diseases. Hence, dysregulated DCs are the most attractive pharmacological target for the development of new therapies aiming at reducing their immunogenicity and at enhancing their tolerogenicity. Curcumin is the polyphenolic phytochemical component of the spice turmeric with a wide range of pharmacological activities. It acts in several ways as a modulator of DCs and converts them into tolerogenic DCs. Tolerogenic DCs possess anti-inflammatory and immunomodulatory activities that regulate the immune responses in health and disease. Curcumin by blocking maturation markers, cytokines and chemokines expression, and disrupting the antigen-presenting machinery of DCs render them non- or hypo-responsive to immunostimulants. It also reduces the expression of co-stimulatory and adhesion molecules on DCs and prevents them from both migration and antigen presentation but enhances their endocytosis capacity. Hence, curcumin causes DCs-inducing regulatory T cells and dampens CD4+ T helper 1 (Th1), Th2, and Th17 polarization. Inhibition of transcription factors such as NF-κB, AP-1, MAPKs (p38, JNK, ERK) and other intracellular signaling molecules such as JAK/STAT/SOCS provide a plausible explanation for most of these observations. In this review, we summarize the potential effects of curcumin on the phenotypes and functions of DCs as the key players in orchestration, stimulation, and modulation of the immune responses.