• Combinational immunotherapy of anti-OX40 antibody and IDO inhibitor synergistically enhances anti-tumor immune T cell-mediated response

      Berrong, Zuzana Jirina; Department of Biochemistry and Molecular Biology (2016-05)
      One of the major goals of cancer immunotherapy is to disrupt the immunosuppressive environment that allows tumors to thrive in and to generate potent and enduring antitumor specific-immune responses. Cancer vaccines may elicit antigen-specific immune responses; however, this is, in many tumor models and in human cancer, insufficient for positive outcomes due to existence of multiple immune-inhibitory mechanisms in tumors. A relatively recently introduced strategy to increase the therapeutic efficacy of tumor vaccination is to combine different immunological approaches that target different immunosuppressive pathways and to enhance the efficacy of vaccines by T cell agonists. OX40 is a co-stimulatory receptor expressed on T cells that can lead to proliferation and enhancement ofT cell effector function when bound by its ligand or targeted with agonist antibody. Here, we show that different doses of anti-OX40 antibody (Ab) elicit differential impacts on the T cell immune response resulting in either efficacious or detrimental therapeutic effect in immunized tumor-bearing mice. We demonstrate that treating tumor-bearing mice with an optimal dose of 1 mg/kg anti-OX40 Ab leads to a potent therapeutic and immune anti-tumor effect when combined with vaccine, whereas higher dose at 2.5 mg/kg of anti-OX40 Ab with vaccine increases the accumulation of regulatory T cells in the tumor and diminishes the therapeutic effect. Furthermore, we proposed that OX40 downstream molecular signaling through AKT activation in T cells may elucidate the differential T cell response when stimulated with anti-OX40 Ab. After optimizing the dose of agonist anti-OX40 Ab to stimulate the immune system toward maximal anti-tumor response when combined with vaccine, we strategized to improve the combinational therapy by targeting the so far untouched immunosuppressive environment. One of the immune suppressive molecules correlating with cancer progression is indoleamine-(2,3)-dioxygenase (IDO) enzyme. The catalytic activity of IDO hinders effector T cells from properly eliciting an anti-tumor effect. Here, we further evaluated the therapeutic outcome and immune mechanisms of the vaccineinduced immune response enhancement by agonist anti-OX40 antibody, while inhibiting the immunosuppressive IDO enzyme. We demonstrate that therapeutic efficacy of this combinational treatment leads to a profound inhibition of tumor growth and complete regression of established tumors in 60% of treated mice. We show that the mechanisms responsible for this therapeutic potency are: i) an increase in vaccine-induced tumorinfiltrating effector T cells that is facilitated by anti-OX40 antibody, and ii) a decrease of IDO enzyme activity within the tumor and the enhancement in the functionality of effector T cells that are facilitated by 1-methyl tryptophan (1-MT, IDO inhibitor). Our findings provide a promising and translatable strategy that can enhance the overall efficacy of cancer immunotherapy.
    • Murine CD19+ Plasmacytoid Dendritic Cells Expressing Indoleamine 2,3 Dioxygenase

      Kahler, David J.; Department of Cellular Biology and Anatomy (2008-10)
      Indoleamine 2,3 Dioxygenase (IDO) is a potent immunomodulatory enzyme whose role has been described in diverse physiologic states including pregnancy, cancer, tissue transplants, autoimmune disease, chronic inflammation, and depression. IDO suppresses antigen specific T cell proliferation via mechanisms including tryptophan degradation and the production of toxic metabolites, and the activation of resting regulatory T cells (Tregs). IDO expression is tightly regulated in the murine spleen, as only rare dendritic cell (DC) subsets are competent to express IDO. Therefore, an accurate phenotype by which to identify IDO competent DCs in tissues is important when ascribing the role of IDO competent DCs in disease models. Here we show that IDO competent CD19+ pDCs (CD19+ pDCs) express high levels of costimulatory receptors (CD80 / CD86) under homeostatic conditions indicating a mature or activated phenotype and uniquely express the Class I MHC-like molecule CD1d, and the chemokine receptor CCR6. IDO competent pDCs do not share the same lineage as other murine splenic DCs as they were the only DC subset to express Pax5, and were present in reduced numbers in murine models of B cell development indicating that they develop from B cell precursors. Distinct signaling requirements regulate IDO induction in IDO competent pDCs as MyD88 was required for IDO induction and function in inflamed skin draining lymph nodes following phorbol myristate acetate application but not for IDO transcript expression or STAT1 or STAT2 protein phosphorylation following treatment with recombinant cytokines. CD19+ pDCs from WT mice but not mice genetically deficient for the IDO1 gene formed stress granules (SG) following treatment with IFNγ, which were not prevented by inhibitors of IDO activity indicating that SG formation was not IDO dependent. We hypothesize that IDO competent murine splenic pDCs uniquely expressing CD19 are phenotypically and functionally distinct from other splenic DC subsets and respond to inflammatory signals by expressing IDO. We further hypothesize that activated IDO causes distinct yet undefined biochemical changes within IDO competent pDCs following induction most probably by activating the integrated stress response and the eif2a kinases GCN2, PKR, and PERK.
    • Role of GCN2-dependent metabolic stress in regulating myeloid cell activation and differentiation.

      Liu, Haiyun; Department of Medicine (2014-12)
      Amino acid metabolism is a pivotal regulator of innate and adaptive immunity. During inflammation, myeloid cells expressing enzymes such as indoleamine 2, 3-dioxygenase (IDO) and arginase 1 (ARG1) that degrade the amino acids L-tryptophan (L-Trp) and L-arginine (L-Arg), respectively. This serves a critical role in controlling cellular survival, development, and function. Therefore, it is important to understand the metabolic stress sensing pathways that regulate immune cell behavior, which further control the overall inflammatory environment. General Control Non-depressible 2 (GCN2) is an integrated stress response (ISR) kinase activated by intracellular amino acid limitation. Activated GCN2 phosphorylates eukaryotic initiation factor 2 α (eIF2α), which leads to global translation repression while up-regulating various stress-associated transcription factors. We found that in a murine LPS-induced endotoxemia model, IDO expression in macrophages depleted L-Trp that activated the GCN2. GCN2 signaling promoted macrophage cytokine production (IL-6, IL-12), increased CHOP expression and NF-κB activation. GCN2 knockout (GCN2KO) mice showed significantly lower serum and splenic cytokine levels compared to wild-type (WT) mice, and were protected from septicemia induced mortality. In the murine EG7 tumor model, GCN2 signaling was also activated in myeloid-derived suppressor cells (MDSCs) mediated by ARG1 depletion of L-Arg. We found that GCN2 was required for transcription factor C/EBPβ induction and monocytic bone marrow MDSC (BM-MDSC) development. GCN2KO BM-MDSCs showed significantly reduced ARG1 activity and failed to suppress antigen-specific cytotoxic T lymphocytes (CTLs) in vitro and in vivo. GCN2KO mice also exhibited increased efficacy in eliminating tumor cells after adoptive CTL transfer therapy. These data suggest that myeloid cells actively deplete intracellular amino acids to regulate their own cellular behavior. Different amino acid metabolic stress signals converge on the GCN2 pathway which serves as a secondary messenger to modulate downstream transcription factors. Depending on the type of inflammation, GCN2 can either promote pro-inflammatory responses or the immunosuppressive function of myeloid cells. Thus, targeting the GCN2 pathway in myeloid cells may have great potential in clinical therapy.
    • Role of Splenic Macrophages in the Initiation of Tolerance to Apoptotic Cell Associate Antigens

      Ravishankar, Buvana; Department of Biochemistry and Molecular Biology (2014-03)
      Systemic Autoimmune disease occurs due to the breakdown of tolerance to self-antigens caused in part by impaired clearance of apoptotic cells. The spleen is a primary site for generation of the tolerogenic response to selfantigens in the periphery. The marginal zone (MZ), which contains the specialized macrophage (MΦs) populations: Marginal Zone Macrophages (MZMs) and Metallophilic Marginal zone Macrophages (MMMs), as well as B cells and dendritic cells (DCs) play a requisite role in capture of apoptotic cells and the initiation of tolerance to associated self antigens. Moreover, defective MZ cellular architecture may lead to increased auto-reactivity that exacerbates autoimmune like disease progression. MZMs are specialized to recognize and capture apoptotic cells and promote peripheral tolerance to apoptotic cells and associated antigens. However, the mechanism by which MZMs enforce this tolerance is not known. Thus, the overall goal of our project is to fill the lapse in the scientific knowledge and understanding of the mechanism(s) by which the splenic stromal MΦs drive immune tolerance to apoptotic cell associated antigens.