Journal Articles

Cancer Immunobiology

Basic research studies that extend knowledge in the field of cancer immunotherapy

Indoleamine 2,3-dioxygenase provides adaptive resistance to immune checkpoint inhibitors in hepatocellular carcinoma

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Hepatocellular carcinoma (HCC) is the second leading cause of cancer-related death worldwide. Immune checkpoint blockade with anti-CTLA-4 and anti-PD-1 antibodies has shown promising results in the treatment of patients with advanced HCC. The anti-PD-1 antibody, nivolumab, is now approved for patients who have had progressive disease on the current standard of care. However, a subset of patients with advanced HCC treated with immune checkpoint inhibitors failed to respond to therapy. Here, we provide evidence of adaptive resistance to immune checkpoint inhibitors through upregulation of indoleamine 2,3-dioxygenase (IDO) in HCC. Anti-CTLA-4 treatment promoted an induction of IDO1 in resistant HCC tumors but not in tumors sensitive to immune checkpoint blockade. Using both subcutaneous and hepatic orthotopic models, we found that the addition of an IDO inhibitor increases the efficacy of treatment in HCC resistant tumors with high IDO induction. Furthermore, in vivo neutralizing studies demonstrated that the IDO induction by immune checkpoint blockade was dependent on IFN-gamma. Similar findings were observed with anti-PD-1 therapy. These results provide evidence that IDO may play a role in adaptive resistance to immune checkpoint inhibitors in patients with HCC. Therefore, inhibiting IDO in combination with immune checkpoint inhibitors may add therapeutic benefit in tumors which overexpress IDO and should be considered for clinical evaluation in HCC.

Author Info: (1) Thoracic and GI Malignancies Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Building 10, Room 3B43, Bethesda, MD, 20892, USA

Author Info: (1) Thoracic and GI Malignancies Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Building 10, Room 3B43, Bethesda, MD, 20892, USA. (2) Thoracic and GI Malignancies Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Building 10, Room 3B43, Bethesda, MD, 20892, USA. Department of Internal Medicine and Liver Research Institute, Seoul National University College of Medicine, Seoul, South Korea. (3) Thoracic and GI Malignancies Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Building 10, Room 3B43, Bethesda, MD, 20892, USA. (4) Thoracic and GI Malignancies Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Building 10, Room 3B43, Bethesda, MD, 20892, USA. (5) Thoracic and GI Malignancies Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Building 10, Room 3B43, Bethesda, MD, 20892, USA. (6) Thoracic and GI Malignancies Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Building 10, Room 3B43, Bethesda, MD, 20892, USA. (7) Thoracic and GI Malignancies Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Building 10, Room 3B43, Bethesda, MD, 20892, USA. (8) Thoracic and GI Malignancies Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Building 10, Room 3B43, Bethesda, MD, 20892, USA. (9) Thoracic and GI Malignancies Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Building 10, Room 3B43, Bethesda, MD, 20892, USA. (10) Thoracic and GI Malignancies Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Building 10, Room 3B43, Bethesda, MD, 20892, USA. tim.greten@nih.gov. National Cancer Institute, Center for Cancer Research, Liver Cancer Program, Bethesda, USA. tim.greten@nih.gov.

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Tumor Cell-Intrinsic Factors Underlie Heterogeneity of Immune Cell Infiltration and Response to Immunotherapy

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The biological and functional heterogeneity between tumors-both across and within cancer types-poses a challenge for immunotherapy. To understand the factors underlying tumor immune heterogeneity and immunotherapy sensitivity, we established a library of congenic tumor cell clones from an autochthonous mouse model of pancreatic adenocarcinoma. These clones generated tumors that recapitulated T cell-inflamed and non-T-cell-inflamed tumor microenvironments upon implantation in immunocompetent mice, with distinct patterns of infiltration by immune cell subsets. Co-injecting tumor cell clones revealed the non-T-cell-inflamed phenotype is dominant and that both quantitative and qualitative features of intratumoral CD8(+) T cells determine response to therapy. Transcriptomic and epigenetic analyses revealed tumor-cell-intrinsic production of the chemokine CXCL1 as a determinant of the non-T-cell-inflamed microenvironment, and ablation of CXCL1 promoted T cell infiltration and sensitivity to a combination immunotherapy regimen. Thus, tumor cell-intrinsic factors shape the tumor immune microenvironment and influence the outcome of immunotherapy.

Author Info: (1) Abramson Family Cancer Research Institute, University of Pennsylvania, 3400 Civic Center Blvd., Philadelphia, PA 19104, USA. (2) Department of Medicine, University of Pennsylvania, 340

Author Info: (1) Abramson Family Cancer Research Institute, University of Pennsylvania, 3400 Civic Center Blvd., Philadelphia, PA 19104, USA. (2) Department of Medicine, University of Pennsylvania, 3400 Civic Center Blvd., Philadelphia, PA 19104, USA; Parker Institute for Cancer Immunotherapy, University of Pennsylvania, 3400 Civic Center Blvd., Philadelphia, PA 19104, USA. Electronic address: byrnek@upenn.edu. (3) Abramson Family Cancer Research Institute, University of Pennsylvania, 3400 Civic Center Blvd., Philadelphia, PA 19104, USA. (4) Abramson Family Cancer Research Institute, University of Pennsylvania, 3400 Civic Center Blvd., Philadelphia, PA 19104, USA. (5) Institute for Immunology, University of Pennsylvania, 3400 Civic Center Blvd., Philadelphia, PA 19104, USA. (6) Cancer Biology and Genetics Program, Sloan-Kettering Institute, NY 10065, USA. (7) Abramson Family Cancer Research Institute, University of Pennsylvania, 3400 Civic Center Blvd., Philadelphia, PA 19104, USA. (8) Abramson Family Cancer Research Institute, University of Pennsylvania, 3400 Civic Center Blvd., Philadelphia, PA 19104, USA. (9) Center for RNA Biology, Department of Biochemistry and Biophysics, Department of Urology, University of Rochester Medical Center, Rochester, NY 14642, USA. (10) Abramson Family Cancer Research Institute, University of Pennsylvania, 3400 Civic Center Blvd., Philadelphia, PA 19104, USA; Parker Institute for Cancer Immunotherapy, University of Pennsylvania, 3400 Civic Center Blvd., Philadelphia, PA 19104, USA. (11) Abramson Family Cancer Research Institute, University of Pennsylvania, 3400 Civic Center Blvd., Philadelphia, PA 19104, USA. (12) Department of Medicine, University of Pennsylvania, 3400 Civic Center Blvd., Philadelphia, PA 19104, USA. (13) Abramson Family Cancer Research Institute, University of Pennsylvania, 3400 Civic Center Blvd., Philadelphia, PA 19104, USA. (14) Abramson Family Cancer Research Institute, University of Pennsylvania, 3400 Civic Center Blvd., Philadelphia, PA 19104, USA. (15) Department of Cell, Developmental and Cancer Biology, Oregon Health & Sciences University, 3181 SW Sam Jackson Park Rd, Portland, OR 97239, USA. (16) Abramson Family Cancer Research Institute, University of Pennsylvania, 3400 Civic Center Blvd., Philadelphia, PA 19104, USA. (17) Abramson Family Cancer Research Institute, University of Pennsylvania, 3400 Civic Center Blvd., Philadelphia, PA 19104, USA. (18) Abramson Family Cancer Research Institute, University of Pennsylvania, 3400 Civic Center Blvd., Philadelphia, PA 19104, USA. (19) Abramson Family Cancer Research Institute, University of Pennsylvania, 3400 Civic Center Blvd., Philadelphia, PA 19104, USA. (20) Abramson Family Cancer Research Institute, University of Pennsylvania, 3400 Civic Center Blvd., Philadelphia, PA 19104, USA. (21) Abramson Family Cancer Research Institute, University of Pennsylvania, 3400 Civic Center Blvd., Philadelphia, PA 19104, USA. (22) Abramson Family Cancer Research Institute, University of Pennsylvania, 3400 Civic Center Blvd., Philadelphia, PA 19104, USA. (23) Abramson Family Cancer Research Institute, University of Pennsylvania, 3400 Civic Center Blvd., Philadelphia, PA 19104, USA; Institute for Immunology, University of Pennsylvania, 3400 Civic Center Blvd., Philadelphia, PA 19104, USA; Department of Pathology and Laboratory Medicine, University of Pennsylvania, 3400 Civic Center Blvd., Philadelphia, PA 19104, USA. (24) Penn Genomic Analysis Core, University of Pennsylvania, 3400 Civic Center Blvd., Philadelphia, PA 19104, USA. (25) Cancer Biology and Genetics Program, Sloan-Kettering Institute, NY 10065, USA; Howard Hughes Medical Institute, Memorial Sloan Kettering Cancer Center, 415 East 68(th) Street New York, NY 10065, USA. (26) Department of Cell, Developmental and Cancer Biology, Oregon Health & Sciences University, 3181 SW Sam Jackson Park Rd, Portland, OR 97239, USA. (27) Abramson Cancer Center, University of Pennsylvania, 3400 Civic Center Blvd., Philadelphia, PA 19104, USA; Parker Institute for Cancer Immunotherapy, University of Pennsylvania, 3400 Civic Center Blvd., Philadelphia, PA 19104, USA; Institute for Immunology, University of Pennsylvania, 3400 Civic Center Blvd., Philadelphia, PA 19104, USA. (28) Abramson Family Cancer Research Institute, University of Pennsylvania, 3400 Civic Center Blvd., Philadelphia, PA 19104, USA; Department of Medicine, University of Pennsylvania, 3400 Civic Center Blvd., Philadelphia, PA 19104, USA; Abramson Cancer Center, University of Pennsylvania, 3400 Civic Center Blvd., Philadelphia, PA 19104, USA; Parker Institute for Cancer Immunotherapy, University of Pennsylvania, 3400 Civic Center Blvd., Philadelphia, PA 19104, USA; Institute for Immunology, University of Pennsylvania, 3400 Civic Center Blvd., Philadelphia, PA 19104, USA. Electronic address: rhv@upenn.edu. (29) Abramson Family Cancer Research Institute, University of Pennsylvania, 3400 Civic Center Blvd., Philadelphia, PA 19104, USA; Department of Medicine, University of Pennsylvania, 3400 Civic Center Blvd., Philadelphia, PA 19104, USA; Department of Cell and Developmental Biology, University of Pennsylvania, 3400 Civic Center Blvd., Philadelphia, PA 19104, USA; Abramson Cancer Center, University of Pennsylvania, 3400 Civic Center Blvd., Philadelphia, PA 19104, USA; Parker Institute for Cancer Immunotherapy, University of Pennsylvania, 3400 Civic Center Blvd., Philadelphia, PA 19104, USA. Electronic address: bstanger@upenn.edu.

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IKZF1 Enhances Immune Infiltrate Recruitment in Solid Tumors and Susceptibility to Immunotherapy

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Immunotherapies are some of the most promising emergent treatments for several cancers, yet there remains a majority of patients who do not benefit from them due to immune-resistant tumors. One avenue for enhancing treatment for these patients is by converting these tumors to an immunoreactive state, thereby restoring treatment efficacy. By leveraging regulatory networks we previously characterized in autoimmunity, here we show that overexpression of the master regulator IKZF1 leads to enhanced immune infiltrate recruitment and tumor sensitivity to PD1 and CTLA4 inhibitors in several tumors that normally lack IKZF1 expression. This work provides proof of concept that tumors can be rendered susceptible by hijacking immune cell recruitment signals through molecular master regulators. On a broader scale, this work also demonstrates the feasibility of using computational approaches to drive the discovery of novel molecular mechanisms toward treatment.

Author Info: (1) Department of Dermatology, Columbia University Medical Center, New York, NY, USA; Department of Systems Biology, Columbia University Medical Center, New York, NY, USA. (2)

Author Info: (1) Department of Dermatology, Columbia University Medical Center, New York, NY, USA; Department of Systems Biology, Columbia University Medical Center, New York, NY, USA. (2) Department of Dermatology, Columbia University Medical Center, New York, NY, USA. (3) Department of Medicine, Herbert Irving Comprehensive Cancer Center, Columbia University Medical Center, New York, NY, USA. (4) Department of Medicine, Herbert Irving Comprehensive Cancer Center, Columbia University Medical Center, New York, NY, USA. (5) Department of Dermatology, Columbia University Medical Center, New York, NY, USA; Department of Genetics and Development, Columbia University Medical Center, New York, NY, USA. Electronic address: amc65@columbia.edu.

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Stereotactic radiosurgery and immunotherapy in melanoma brain metastases: Patterns of care and treatment outcomes

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PURPOSE: Preclinical studies have suggested that radiation therapy (RT) enhances antitumor immune response and can act synergistically when administered with immunotherapy. However, this effect in melanoma brain metastasis is not well studied. We aim to explore the clinical effect of combining RT and immunotherapy in patients with melanoma brain metastasis (MBM). MATERIALS AND METHODS: Patients with MBM between 2011 and 2013 were obtained from the National Cancer Database. Patients who did not have identifiable sites of metastasis and who did not receive RT for the treatment of their MBM were excluded. Patients were separated into cohorts that received immunotherapy versus patients who did not. Univariable and multivariable analyses were performed using Cox model to determine predictors of OS. Kaplan-Meier method was used to compare OS. Univariable and multivariable analyses using logistic regression model were used to determine the factors predictive for the use of immunotherapy. Propensity score analysis was used to account for differences in baseline patient characteristics between the RT and RT+immunotherapy groups. Significance was defined as a P value

Author Info: (1) Department of Radiation Oncology, Washington University School of Medicine, Saint Louis, United States. (2) Department of Radiation Oncology, Washington University School of Medicine, Saint

Author Info: (1) Department of Radiation Oncology, Washington University School of Medicine, Saint Louis, United States. (2) Department of Radiation Oncology, Washington University School of Medicine, Saint Louis, United States. (3) Division of Oncology, Department of Medicine, Washington University School of Medicine, Saint Louis, United States. (4) Division of Oncology, Department of Medicine, Washington University School of Medicine, Saint Louis, United States. (5) Division of Oncology, Department of Medicine, Washington University School of Medicine, Saint Louis, United States. (6) Department of Neurosurgery, Washington University School of Medicine, Saint Louis, United States. (7) Department of Neurosurgery, Washington University School of Medicine, Saint Louis, United States. (8) Department of Neurosurgery, Washington University School of Medicine, Saint Louis, United States. (9) Department of Radiation Oncology, Washington University School of Medicine, Saint Louis, United States. (10) Department of Neurosurgery, Washington University School of Medicine, Saint Louis, United States. (11) Department of Radiation Oncology, Washington University School of Medicine, Saint Louis, United States. (12) Department of Radiation Oncology, Washington University School of Medicine, Saint Louis, United States. Electronic address: cabraham@wustl.edu.

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It is finally time for adjuvant therapy in melanoma

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Although melanoma is amenable to early detection, there has been no decline in the mortality rate of this disease and the prognosis of patients with high-risk primary melanoma or with macroscopic nodal involvement remains poor. The best option for patients with higher-risk melanoma is to receive effective adjuvant therapy in order to reduce their chances of recurrence. Multiple systemic therapeutic agents have been tested as adjuvant therapy for melanoma with durable benefits seen only with interferon- to date. More recently ipilimumab at the high dose of 10mg/kg has shown a significant improvement in terms of Relapse free survival and Overall survival for stage III melanoma patients but at a significant cost in terms of immune-related toxicities. More recently, novel treatment options have emerged. The results from the latest trials with immunotherapy (PD-1 inhibitors) and molecular targeted therapy (BRAF inhibitor+MEK inhibitor) have revolutionized the management of adjuvant treatment for melanoma. As the results from these trials will mature in the next years, a change in the landscape of adjuvant treatment for melanoma is expected, resulting in new challenges in treatment decisions such as optimizing patients' selection through predictive and prognostic biomarkers, and management of treatment related adverse events, in particular immune related toxicities.

Author Info: (1) Oncologia Medica, Dipartimento di Internistica Clinica e Sperimentale "F. Magrassi", Universita degli Studi della Campania "Luigi Vanvitelli", Via S. Pansini 5, Napoli 80131, Italy

Author Info: (1) Oncologia Medica, Dipartimento di Internistica Clinica e Sperimentale "F. Magrassi", Universita degli Studi della Campania "Luigi Vanvitelli", Via S. Pansini 5, Napoli 80131, Italy. (2) Dermatologia e Venerologia, Dipartimento di salute mentale e fisica e medicina riabilitativa, Universita degli Studi della Campania "Luigi Vanvitelli", Via S. Pansini 5, Napoli 80131, Italy. (3) Dermatologia e Venerologia, Dipartimento di salute mentale e fisica e medicina riabilitativa, Universita degli Studi della Campania "Luigi Vanvitelli", Via S. Pansini 5, Napoli 80131, Italy. (4) Oncologia Medica, Dipartimento di Internistica Clinica e Sperimentale "F. Magrassi", Universita degli Studi della Campania "Luigi Vanvitelli", Via S. Pansini 5, Napoli 80131, Italy. (5) Oncologia Medica, Dipartimento di Internistica Clinica e Sperimentale "F. Magrassi", Universita degli Studi della Campania "Luigi Vanvitelli", Via S. Pansini 5, Napoli 80131, Italy. (6) Oncologia Medica, Dipartimento di Internistica Clinica e Sperimentale "F. Magrassi", Universita degli Studi della Campania "Luigi Vanvitelli", Via S. Pansini 5, Napoli 80131, Italy. (7) Oncologia Medica, Dipartimento di Internistica Clinica e Sperimentale "F. Magrassi", Universita degli Studi della Campania "Luigi Vanvitelli", Via S. Pansini 5, Napoli 80131, Italy. Electronic address: teresa.troiani@unicampania.it.

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Neutrophils as myeloid-derived suppressor cells

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Neutrophils form the first line of defense against invading pathogens, such as bacteria and fungi, as part of the innate immune response. Recently, neutrophils have also been discovered as repressors of adaptive immune responses. Under certain conditions, such as cancer and severe injury, an expansion of immature and mature neutrophils has been observed to induce suppression of T cell proliferation. These suppressing cells are known as so-called myeloid-derived suppressor cells (MDSCs), a heterogeneous population of granulocytic-MDSCs and monocytic-MDSCs. Initially, MDSCs were believed to be a specific immature type of myeloid immune cell released from the bone marrow, but mature neutrophils have also been proposed to have suppressive capacity. However, granulocytic MDSCs show a similar morphology and expression of cell surface markers as mature neutrophils. The only characteristic that discriminates granulocytic (g)-MDSCs from mature neutrophils is their suppressive capacity, raising the question whether human g-MDSCs and neutrophils are actually different cell types or whether they are one plastic cell type that can functionally polarize from microbial killers to immunosuppressor cells, depending on local conditions. In this review, we will focus on the MDSC activity of circulating mature neutrophils. This article is protected by copyright. All rights reserved.

Author Info: (1) Department of Blood Cell Research, Sanquin Research and Landsteiner Laboratory, AMC, University of Amsterdam, Amsterdam, The Netherlands. (2) Department of Blood Cell Research, Sanquin

Author Info: (1) Department of Blood Cell Research, Sanquin Research and Landsteiner Laboratory, AMC, University of Amsterdam, Amsterdam, The Netherlands. (2) Department of Blood Cell Research, Sanquin Research and Landsteiner Laboratory, AMC, University of Amsterdam, Amsterdam, The Netherlands. Department of Pediatric Hematology, Immunology& Infectious Disease, Emma Children's Hospital, Academic Medical Center (AMC), University of Amsterdam, Amsterdam, The Netherlands.

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Intraperitoneal oxaliplatin administration inhibits the tumor immunosuppressive microenvironment in an abdominal implantation model of colon cancer

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Recent studies have demonstrated that some chemotherapeutic drugs can enhance antitumor immunity by eliminating and inactivating immunosuppressive cells. Oxaliplatin (OXP) induces immunogenic cell death by increasing the immunogenicity of cancer cells. However, the effects of OXP on the tumor immunosuppressive microenvironment remain unclear. The aim of the present study was to evaluate the antitumor activity of OXP by intraperitoneal (i.p.) administration in an abdominal implantation model of colon cancer and tested the tumor immune microenvironment to observe whether OXP affects the local immune inhibitory cell populations. Abdominal metastasis models were established by inoculation of CT26 cells. The antitumor efficacy of OXP and the tumor immune microenvironment were evaluated. The tumors and spleens of mice were harvested for flow cytometric analysis. Cluster of differentiation (CD)8+CD69+ T cells, regulatory T cells (Tregs), CD11b+F4/80high macrophages and myeloidderived suppressor cells (MDSCs) were evaluated by flow cytometric analysis. In vivo i.p. administration of OXP inhibited tumor growth in the abdominal metastasis model. Furthermore, OXP was observed to increase tumorinfiltrating activated CD8+ T cells in tumors, decrease CD11b+F4/80high macrophages in tumors and decrease MDSCs in the spleen. These results suggested that i.p. administration of OXP alone may inhibit tumor cell growth and induce the antitumor immunostimulatory microenvironment by eliminating immunosuppressive cells.

Author Info: (1) Department of Abdominal Cancer, Cancer Center, The State Key Laboratory of Biotherapy, West China Hospital, West China Medical School, Sichuan University, Chengdu, Sichuan 610041

Author Info: (1) Department of Abdominal Cancer, Cancer Center, The State Key Laboratory of Biotherapy, West China Hospital, West China Medical School, Sichuan University, Chengdu, Sichuan 610041, P.R. China. (2) Department of Abdominal Cancer, Cancer Center, West China Medical School, Sichuan University, Chengdu, Sichuan 610041, P.R. China. (3) Department of Abdominal Cancer, Cancer Center, West China Medical School, Sichuan University, Chengdu, Sichuan 610041, P.R. China. (4) Department of Hematology, West China Hospital, West China Medical School, Sichuan University, Chengdu, Sichuan 610041, P.R. China.

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Modulation of the tumor microenvironment by intratumoral administration of IMO-2125, a novel TLR9 agonist, for cancer immunotherapy

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The objective of cancer immunotherapy is to prime the host's immune system to recognize and attack malignant tumor cells. IMO2125, a Tolllike receptor 9 (TLR9) agonist, exhibited potent antitumor effects in the murine syngeneic A20 lymphoma and the CT26 colon carcinoma models. IMO2125 exhibited superior A20 antitumor activity when injected intratumorally (i.t.) compared with equivalent subcutaneous doses. In mice bearing dual CT26 grafts, the i.t. injection of right flank tumors elicited infiltration of cluster of differentiation (CD)3+ T lymphocytes into tumors, resulting in the regression of injected and uninjected left flank tumors. Depletion of CD8+, but not CD4+, Tcells abrogated the IMO2125mediated antitumor response, suggesting that CD8+ lymphocytes are required for the antitumor activity. In mice harboring right flank CT26 and left flank betagalactosidase (betagal)expressing CT26.CL25 grafts, the i.t. administration of IMO2125 to the CT26 graft resulted in potent and dosedependent antitumor activity against the two grafts. Splenic Tcells isolated from these mice responded to AH1 antigen (present in the two tumors) and betagal antigen (present only in CT26.CL25) in an interferon gamma enzymelinked immunospot assay, suggesting the clonal expansion of Tcells directed against antigens from the two tumors. Mice with ablated CT26 tumors by previous IMO2125 treatment rejected reimplanted CT26 tumor cells, but not A20 tumor cells, demonstrating that the initial IMO2125 treatment created a longlived tumorspecific immune memory of CT26 antigens. A quantitative increase in CD3+ T lymphocytes in injected A20 tumors and an upregulation of selected checkpoint genes, including indoleamine 2,3dioxygenase (IDO)1, IDO2, programmed cell death protein-1 (PD-1); programmed cell death protein ligand 1 (PD-L1), carcinoembryonic antigenrelated cell adhesion molecule 1, tumor necrosis factor receptor superfamily member 4 (OX40), OX40 ligand, Tcell immunoglobulin and mucindomaincontaining 3 protein, lymphocyteactivation gene 3, cytotoxic Tlymphocyteassociated protein 4, were observed following IMO2125 treatment. IMO2125 also increased immune checkpoint gene expression in injected and uninjected contralateral CT26 tumors, suggesting that the coadministration of antiCTLA4, antiPD1 or antiPDL1 therapies with IMO2125 may provide additional therapeutic efficacy.

Author Info: (1) Idera Pharmaceuticals, Inc., Cambridge, MA 02139, USA. (2) Idera Pharmaceuticals, Inc., Cambridge, MA 02139, USA. (3) Idera Pharmaceuticals, Inc., Cambridge, MA 02139, USA. (4)

Author Info: (1) Idera Pharmaceuticals, Inc., Cambridge, MA 02139, USA. (2) Idera Pharmaceuticals, Inc., Cambridge, MA 02139, USA. (3) Idera Pharmaceuticals, Inc., Cambridge, MA 02139, USA. (4) Idera Pharmaceuticals, Inc., Cambridge, MA 02139, USA. (5) Idera Pharmaceuticals, Inc., Cambridge, MA 02139, USA.

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A Consistent Method to Identify and Isolate Mononuclear Phagocytes from Human Lung and Lymph Nodes

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Mononuclear phagocytes (MP) consist of macrophages, dendritic cells (DCs), and monocytes. In all organs, including the lung, there are multiple subtypes within these categories. The existence of all these cell types suggest that there is a clear division of labor and delicate balance between the MPs under steady state and inflammatory conditions. Although great strides have been made to understand MPs in the mouse lung, and human blood, little is known about the MPs that exist in the human lung and lung-draining lymph nodes (LNs), and even less is known about their functional roles, studies of which will require a large number of sorted cells. We have comprehensively examined cell surface markers previously used in a variety of organs to identify human pulmonary MPs. In the lung, we consistently identify five extravascular pulmonary MPs and three LN MPs. These MPs were present in over 100 lungs regardless of age or gender. Notably, the human blood CD141(+) DCs, as described in the literature, were not observed in non-diseased lungs or their draining LNs. In the lung and draining LNs, expression of CD141 was only observed on HLADR(+) CD11c(+) CD14(+) extravascular monocytes (often confused in the LN as resident DCs based on the level of HLADR expression and mouse LN data). In the human lung and LNs there are at least two DC subtypes expressing HLADR, DEC205 and CD1c, along with circulating monocytes that behave as either antigen-presenting cells or macrophages. Furthermore, we demonstrate how to distinguish between alveolar macrophages and interstitial macrophage subtypes. It still remains unclear how the human pulmonary MPs identified here align with mouse MPs. Clearly, we are now past the stage of cell surface marker characterization, and future studies will need to move toward understanding what these cell types are and how they function. Our hope is that the strategy described here can help the pulmonary community take this next step.

Author Info: (1) Department of Pediatrics, National Jewish Health, Denver, CO, USA. (2) Department of Pediatrics, National Jewish Health, Denver, CO, USA. jakubzickc@njhealth.org. Department of Microbiology and

Author Info: (1) Department of Pediatrics, National Jewish Health, Denver, CO, USA. (2) Department of Pediatrics, National Jewish Health, Denver, CO, USA. jakubzickc@njhealth.org. Department of Microbiology and Immunology, University of Colorado, Denver, CO, USA. jakubzickc@njhealth.org.

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Imaging Precision-Cut Lung Slices to Visualize Leukocyte Localization and Trafficking

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Pulmonary dendritic cells (DCs) are potent antigen-presenting cells that can activate both naive and memory/effector T cells. However, very little is known of how movements and localization of DCs contribute to these events. To study this, we have developed new procedures that combine precision-cut lung slices with cell staining using fluorescently tagged antibodies to detect individual cell types. In this chapter, we describe these methods in detail and show how they can be used to study the localization of not only DCs but also other leukocytes of interest, as well as structural cells within the lung.

Author Info: (1) Immunity, Inflammation and Disease Laboratory, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC, USA. (2) Immunity, Inflammation and

Author Info: (1) Immunity, Inflammation and Disease Laboratory, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC, USA. (2) Immunity, Inflammation and Disease Laboratory, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC, USA. (3) Immunity, Inflammation and Disease Laboratory, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC, USA. (4) Immunity, Inflammation and Disease Laboratory, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC, USA.

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