Theodore S. Johnson

Cells Expressing Indoleamine 2,3-Dioxygenase Inhibit T Cell Responses

Pharmacological inhibition of indoleamine 2,3-dioxygenase (IDO) activity during murine gestation results in fetal allograft rejection and blocks the ability of murine CD8+ dendritic cells to suppress delayed-type hypersensitivity responses to tumor-associated peptide Ags. These observations suggest that cells expressing IDO inhibit T cell responses in vivo. To directly evaluate the hypothesis that cells expressing IDO inhibit T cell responses, we prepared IDO-transfected cell lines and transgenic mice overexpressing IDO and assessed allogeneic T cell responses in vitro and in vivo. T cells cocultured with IDO-transfected cells did not proliferate but expressed activation markers. The potency of allogeneic T cell responses was reduced significantly when mice were preimmunized with IDO-transfected cells. In addition, adoptive transfer of alloreactive donor T cells yielded reduced numbers of donor T cells when injected into IDO-transgenic recipient mice. These outcomes suggest that genetically enhanced IDO activity inhibited T cell proliferation in vitro and in vivo. Genetic manipulation of IDO activity may be of therapeutic utility in suppressing undesirable T cell responses.

TRYPTOPHAN CATABOLISM AND T CELL RESPONSES

Cells expressing indoleamine 2,3 dioxygenase (IDO) play key roles in regulating adaptive immune responses orchestrated by T cells. In this report we discuss our working model, the tryptophan depletion hypothesis, to explain links between IDO expression and inhibition of T cell responses. We posit that IDO+ cells, particularly professional antigen presenting cells (APCs) promote T cell entry but block cell cycle progression due to tryptophan catabolism. We discuss experimental evidence supporting predictions from the tryptophan depletion hypothesis and the implications that this model has for understanding the origin of tolerant states that explain immunological paradoxes, such as fetal survival, tumor persistence and failure to eradicate pathogens like HIV that cause persistent infections.

Indoleamine 2,3-dioxygenase, immunosuppression and pregnancy

Pharmacologic inhibition of indoleamine 2,3-dioxygenase (IDO) activity during murine pregnancy results in maternal T-cell-mediated rejection of allogeneic but not syngeneic conceptuses. Increased risk of allogeneic pregnancy failure induced by exposure to IDO inhibitor is strongly correlated with maternal C3 deposition at the maternal-fetal interface. Here we review evidence that cells expressing IDO contribute to immunosuppression by inhibiting T-cell responses to tumor antigens and tissue allografts, as well as fetal tissues.

The indoleamine 2,3-dioxygenase pathway controls complement-dependent enhancement of chemo-radiation therapy against murine glioblastoma

BackgroundIndoleamine 2,3-dioxygenase (IDO) is an enzyme with immune-suppressive properties that is commonly exploited by tumors to evade immune destruction. Anti-tumor T cell responses can be initiated in solid tumors, but are immediately suppressed by compensatory upregulation of immunological checkpoints, including IDO. In addition to these known effects on the adaptive immune system, we previously showed widespread, T cell-dependent complement deposition during allogeneic fetal rejection upon maternal treatment with IDO-blockade. We hypothesized that IDO protects glioblastoma from the full effects of chemo-radiation therapy by preventing vascular activation and complement-dependent tumor destruction.MethodsTo test this hypothesis, we utilized a syngeneic orthotopic glioblastoma model in which GL261 glioblastoma tumor cells were stereotactically implanted into the right frontal lobes of syngeneic mice. These mice were treated with IDO-blocking drugs in combination with chemotherapy and radiation therapy.ResultsPharmacologic inhibition of IDO synergized with chemo-radiation therapy to prolong survival in mice bearing intracranial glioblastoma tumors. We now show that pharmacologic or genetic inhibition of IDO allowed chemo-radiation to trigger widespread complement deposition at sites of tumor growth. Chemotherapy treatment alone resulted in collections of perivascular leukocytes within tumors, but no complement deposition. Adding IDO-blockade led to upregulation of VCAM-1 on vascular endothelium within the tumor microenvironment, and further adding radiation in the presence of IDO-blockade led to widespread deposition of complement. Mice genetically deficient in complement component C3 lost all of the synergistic effects of IDO-blockade on chemo-radiation-induced survival.ConclusionsTogether these findings identify a novel mechanistic link between IDO and complement, and implicate complement as a major downstream effector mechanism for the beneficial effect of IDO-blockade after chemo-radiation therapy. We speculate that this represents a fundamental pathway by which the tumor regulates intratumoral vascular activation and protects itself from immune-mediated tumor destruction.

Etoposide Selectively Ablates Activated T Cells To Control the Immunoregulatory Disorder Hemophagocytic Lymphohistiocytosis

Hemophagocytic lymphohistiocytosis (HLH) is an inborn disorder of immune regulation caused by mutations affecting perforin-dependent cytotoxicity. Defects in this pathway impair negative feedback between cytotoxic lymphocytes and APCs, leading to prolonged and pathologic activation of T cells. Etoposide, a widely used chemotherapeutic drug that inhibits topoisomerase II, is the mainstay of treatment for HLH, although its therapeutic mechanism remains unknown. We used a murine model of HLH, involving lymphocytic choriomeningitis virus infection of perforin-deficient mice, to study the activity and mechanism of etoposide for treating HLH and found that it substantially alleviated all symptoms of murine HLH and allowed prolonged survival. This therapeutic effect was relatively unique among chemotherapeutic agents tested, suggesting distinctive effects on the immune response. We found that the therapeutic mechanism of etoposide in this model system involved potent deletion of activated T cells and efficient suppression of inflammatory cytokine production. This effect was remarkably selective; etoposide did not exert a direct anti-inflammatory effect on macrophages or dendritic cells, and it did not cause deletion of quiescent naive or memory T cells. Finally, etoposide’s immunomodulatory effects were similar in wild-type and perforin-deficient animals. Thus, etoposide treats HLH by selectively eliminating pathologic, activated T cells and may have usefulness as a novel immune modulator in a broad array of immunopathologic disorders.

Host Indoleamine 2,3-Dioxygenase: Contribution to Systemic Acquired Tumor Tolerance

Indoleamine 2,3-dioxygenase (IDO) is a natural mechanism of creating acquired tolerance in a variety of physiological settings. This endogenous tolerogenic pathway has important functions in regulating the magnitude of immune responses in settings of infection, pregnancy, tissue transplantation, mucosal interfaces and others. Whether for angiogenesis, stromal formation or immunologic tolerance, tumors often rely on recruiting host mechanisms. IDO is one such potent endogenous mechanism that appears to be frequently hijacked by tumors to establish systemic immune tolerance to tumor antigens. IDO can be expressed by tumors themselves, but, in addition, its natural site of expression is the host immune cells recruited by the tumor (particularly dendritic cells and macrophages). Therapeutic strategies that target the IDO pathway have been shown to synergize with standard chemotherapy and experimental immunotherapies to break tumor-induced tolerance. When such strategies target IDO expressed in host cells, they may be able to disrupt tolerance without creating intrinsic tumor cell drug resistance.

Contemporary diagnostic methods for hemophagocytic lymphohistiocytic disorders.

Hemophagocytic lymphohistiocytosis is a life-threatening multi-system hyperinflammatory disorder characterized by dysfunctional cytolytic lymphocyte responses, hypercytokinemia, and widespread lymphohistiocytic tissue infiltration and destruction. Diagnosis and definitive therapy are often delayed as clinical efforts are directed toward treatment of presumed overwhelming infection. Sporadic cases occur in association with underlying immune dysfunction related to autoimmune disease, malignancy, or severe infection. However, familial cases predominate with remarkable associations between underlying genetic defects and dysregulation of immune responses. Here, we review the genetic and immunologic basis of contemporary diagnostic methods for hemophagocytic lymphohistiocytosis.

IDO, PTEN-expressing Tregs and control of antigen-presentation in the murine tumor microenvironment

The tumor microenvironment is profoundly immunosuppressive. This creates a major barrier for attempts to combine immunotherapy with conventional chemotherapy or radiation, because the tumor antigens released by these cytotoxic agents are not cross-presented in an immunogenic fashion. In this Focused Research Review, we focus on mouse preclinical studies exploring the role of immunosuppressive Tregs expressing the PTEN lipid phosphatase, and the links between PTEN+ Tregs and the immunoregulatory enzyme indoleamine 2,3-dioxygenase (IDO). IDO has received attention because it can be expressed by a variety of human tumor types in vivo, but IDO can also be induced in host immune cells of both humans and mice in response to inflammation, infection or dying (apoptotic) cells. Mechanistically, IDO and PTEN+ Tregs are closely connected, with IDO causing activation of the PTEN pathway in Tregs. Genetic ablation or pharmacologic inhibition of PTEN in mouse Tregs destabilizes their suppressive phenotype, and this prevents transplantable and autochthonous tumors from creating their normal immunosuppressive microenvironment. Genetic ablation of either IDO or PTEN+ Tregs in mice results in a fundamental defect in the ability to maintain tolerance to antigens associated with apoptotic cells, including dying tumor cells. Consistent with this, pharmacologic inhibitors of either pathway show synergy when combined with cytotoxic agents such as chemotherapy or radiation. Thus, we propose that IDO and PTEN+ Tregs represent closely linked checkpoints that can influence the choice between immune activation versus tolerance to dying tumor cells.

Modulation of Tumor Tolerance in Primary Central Nervous System Malignancies

Central nervous system tumors take advantage of the unique immunology of the CNS and develop exquisitely complex stromal networks that promote growth despite the presence of antigen-presenting cells and tumor-infiltrating lymphocytes. It is precisely this immunological paradox that is essential to the survival of the tumor. We review the evidence for functional CNS immune privilege and the impact it has on tumor tolerance. In this paper, we place an emphasis on the role of tumor-infiltrating myeloid cells in maintaining stromal and vascular quiescence, and we underscore the importance of indoleamine 2,3-dioxygenase activity as a myeloid-driven tumor tolerance mechanism. Much remains to be discovered regarding the tolerogenic mechanisms by which CNS tumors avoid immune clearance. Thus, it is an open question whether tumor tolerance in the brain is fundamentally different from that of peripheral sites of tumorigenesis or whether it simply stands as a particularly strong example of such tolerance.

Abstract 5023: Synergistic antitumor effects of combinatorial immune checkpoint inhibition with anti-PD-1/PD-L antibodies and the IDO pathway inhibitors NLG-919 and indoximod in the context of active immunotherapy

Combination immunotherapy regimens incorporating agents that target more than a single regulatory pathway or immune inhibitory checkpoint can confer marked enhancement of anti-tumor immune responses when combined with active immunotherapy. We have tested the antitumor effect of the IDO pathway inhibitor NLG-919 (currently in Phase I clinical trial) in combination with indoximod (a different IDO pathway inhibitor currently in Phase II clinical trials), chemotherapy, vaccination and/or PD-1/PD-L1/PD-L2 blockade. Here we show that the highly potent, orally-bioavailable IDO pathway inhibitor NLG-919 is synergistic with blockade of the PD-1/PD-L1/PD-L2 pathway in mouse models of large established tumors. The combination of NLG-919 plus PD-1/PD-L1/PD-L2 blockade showed significantly enhanced anti-tumor effect compared to either agent alone. This synergy was particularly evident in the setting of large established tumors treated with normally ineffective immunotherapy regimens (e.g., a single dose of chemotherapy plus a vaccine against a poorly immunogenic shared-self antigen). In vivo, administration of NLG-919 enhanced anti-tumor vaccine responses against B16F10 melanoma treated with hgp100 vaccine plus resting, non-activated pmel-1 T cells NLG-919 also reduced Treg-mediated suppression in tumor-bearing hosts, and enhanced dendritic cell activation in tumors and TDLNs. In combination with chemotherapy, treatment with NLG-919 allowed effector T cell responses against endogenous tumor antigens released by chemotherapy. By each of the preceding readouts, the in vivo biologic effect of NLG-919 was qualitatively identical to that of indoximod, but the same immunologic effects could be achieved at lower plasma concentrations in vivo. In multiple models, a combination of oral NLG-919 with oral indoximod produced synergistic anti-tumor effects, which was further enhanced by blockade of the PD 1/PD L1/PD-L2 pathway. In a more stringent B16F10 melanoma tumor model that did not involve adoptive transfer of pmel-1 cells, a combination of immune checkpoint inhibition involving NLG-919, indoximod and anti-PD1/PD-L1/PD-L2 antibodies with chemotherapy and hgp100 peptide vaccine was able to achieve a significant antitumor effect. The current preclinical studies suggest a mechanistic rationale for a combining NLG919 with indoximod and with agents targeting the PD 1/PD L1/PD-L2 pathway. Citation Format: Mario R. Mautino, Charles J. Link, Nicholas N. Vahanian, James T. Adams, Clarissa Van Allen, Madhav D. Sharma, Theodore S. Johnson, David Munn. Synergistic antitumor effects of combinatorial immune checkpoint inhibition with anti-PD-1/PD-L antibodies and the IDO pathway inhibitors NLG-919 and indoximod in the context of active immunotherapy. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5023. doi:10.1158/1538-7445.AM2014-5023