Edgar Engleman - US grants

The overall goal of this project is to define the mechanism of cell-mediated immunological unresponsiveness to Mycobacterium leprae seen in patients with lepromatous leprosy. Possible explanations for this unresponsiveness include: (1) a quantitative or qualitative defect in presentation of M. leprae specific antigens in these patients, (2) inhibition of M. leprae specific responses by antigen specific suppressor cells or alternatively by circulating anti-M. leprae antibodies, (3) a lack of M. leprae reactive T cells in the circulation, and (4) tolerance caused by binding of excess antigen to T cells. To determine the basis of specific immune unresponsiveness in lepromatous leprosy we will study the functional consequences of exposing precursors of suppressor T cells (Leu 2+, 9.3-cells) from lepromatous patients to M. leprae antigens, explore the effect of these M. leprae - treated Leu 2+, 9.3-cells on the M. leprae-induced proliferative responses of HLA-matched, M. leprae - responsive siblings of leprosy patients and analyze the functional activity and surface phenotype of T cells recovered from lepromatous skin lesions. M. leprae reactive T cell clones and T-T hybrids will be established from cells isolated from lesions or peripheral blood and extracts and culture supernatants from these clones and hybrids will be examined for the presence of soluble immunoregulatory factors. The role of defined mycobacterial antigens in the activation of each functional clone will be examined using our panel of human anti-M. leprae monoclonal antibodies, murine monoclonal antibodies, and chemically defined macromolecules derived from leprosy bacilli. The results of these studies should clarify the basis of M. leprae specific immune unresponsiveness, and thereby lead to new methods of intervention in this and possibly other infectious states associated with specific anergy (e.g. leishmaniasis).

Dissection of mature human T cells into those that mediate predominantly helper/inducer effects (Leu 3, T4) and those that mediate predominantly suppressor/cytotoxic effects (Leu 2, T8) was made possible with monoclonal antibodies. Using the mixed leukocyte reaction (MLR) as a model system, we have further fractionated these two major T lineages into subsets with relatively narrow functional repetoires. For example, immunoglobulin synthesis induced in MLR was shown to be regulated by sequential interactions between subsets of Leu 2[unreadable]-[unreadable],3[unreadable]+[unreadable] suppressor inducer cells, Leu\2[unreadable]+[unreadable],3[unreadable]-[unreadable]DR[unreadable]+[unreadable] suppressor-amplifier cells, and Leu 2[unreadable]+[unreadable],3[unreadable]-[unreadable],DR[unreadable]-[unreadable] suppressor-effector cells. We further showed that Leu 3[unreadable]+[unreadable] cells capable of helping B cell differentiation and Ig synthesis can be distinguished from Leu 3[unreadable]+[unreadable] cells that do not provide help on the basis of their expression of the Leu 8 marker. Finally, precursors of Leu 2 suppressor-effector cells (Ts) were distinguished from precursors of Leu 2[unreadable]+[unreadable] cytotoxic cells on the basis of their differential expression of the 9.3 antigen. In summary, these data indicate that human T lymphocytes consist of a larger number of functionally distinct subsets than heretofore recognized, distinguishable from one another with combinations of monoclonal antibodies directed at cell surface markers. (LB)

Evidence that AIDS may be transmitted by transfusion of blood from asymptomatic members of AIDS high risk groups has prompted a search for a screening test that can be used to identify a carrier state for the disorder. We propose to assess selected alternative assays for their ability to predict the development of AIDS or persistent lymphadenopathy syndrome in a group of homosexual men at increased risk for developing these disorders. We will also study two groups of volunteer blood donors with laboratory findings seen uncommonly in our general donor pool, but known to be significantly more prevalent in patients with AIDS and AIDS related disorder. The populations to be studied serially over 3 years will include 150 apparently healthy homosexual men with multiple anonymous sexual partners, 100 volunteer blood donors with persistent inversions of the ratio of helper/inducer to suppressor/cytotoxic T lymphocytes (Th:Ts), 100 volunteer blood donors with antibodies to the human T cell leukemia/lymphoma viruses (HTLV), and 50 volunteer blood donors with Th:Ts within the normal range. Groups of 5 patients with fully expressed AIDS and 50 with persistent generalized lymphadenopathy will be tested at the beginning of the study only. In addition to clinical follow-up, the following assays will be performed at the initiation of the study and at six month intervals thereafter: analysis of T lymphocyte subsets using a panel of monoclonal antibodies, including several novel reagents; measurement in serum of soluble forms of lympohcyte surface molecules; tests to detect antibodies to the three identified types of human T cell leukemia lymphoma viruses (HTLV); tests of circulating immune complexes; serum levels of beta-2-microglobulin and thymosin alpha-1; and tests for the presence of antibody to hepatitis B core antigen (HBcAb). These assays have been chosen for study on the basis of encouraging preliminary studies and potential ease of methodology. Correlation of the clinical and laboratory findings in the AIDS hight risk and blood donor groups may help to identify the procedure or combination of procedures best able to predict subsequent development of AIDS or related disorders, with potential applications to screening donated blood to detect an AIDS carrier state.

The overall goal of this program is to utilize immunologic, molecular, and synthetic approaches to develop means for treating AIDS and controlling HIV infection. The specific approaches to be employed include: 1) generation of HIV specific cytolytic T lymphocyte clones and investigation of the potential for using such clones in adoptive cellular immunotherapy, 2) detailed studies of the interactions between viral envelope glycoprotein and CD4 determinants critically involved in the pathobiology of HIV infection, using synthetic peptide and recombinant DNA approaches to identify and develop therapeutic agents capable of interrupting these crucial interactions between the viral envelope and CD4, 3) production and functional characterization of mouse/human chimeric antibodies and immunotoxin conjugates for selective targeting and destruction of infected cells expressing HIV antigens, and 4) generation of HIV- derived vectors and a viral packaging line for selective targeting and expression of defined genetic sequences to effect blockade of HIV replication and/or removal of HIV infected cells from the host. This multidisciplinary approach involves scientists at three institutions and takes advantage of the special areas of expertise of the Program Leaders and of technical resources already well established in our laboratories, and is based on extensive ongoing collaborative interactions between the Program Leaders. In addition, the research program outlined in this proposal incorporates approaches directed against several distinct phases of the viral life cycle with the ultimate goal of developing a synergistic, multimodal approach of the type that will probably be ultimately required for effective therapy of HIV infection.

The objective of this contract is to support a variety of projects aimed at identifying and characterizing the correlates or markers of the immune status to infection with HTLV-III/LAV and to the subsequent progression of the infection to AIDS. The observation that some members of risk groups do not become infected (seroconvert) following a very high probability of exposure to the virus, and the finding that most individuals infected with the virus have not progressed to symptomatic disease let alone AIDS lends support to the view that protective immune mechanisms exist. A definitive understanding of the immune mechanisms that either prevent an initial infection or the progression of an infection to the fully developed disease would help provide a firmer scientific basis for vaccine development activities.

Emphasis on the study of HIV infection of T lymphocytes has skewed research on the pathogenesis of AIDS and development of novel therapeutic approaches. Direct and indirect effects of HIV infection of non-T cell targets, notably dendritic cells (DC), Langerhans cells (LC), and macrophages (M-phi) play an important role in the pathogenesis of AIDS and the immunopathobiology of HIV infection. However, there are significant technical challenges involved in working with non-T cell targets of HIV, owing chiefly to the fact that primary cell populations must be used that are extremely difficult to isolate, culture and analyze, and are often available in limiting numbers or purity. We have developed a series of state of the art approaches for the isolation, purification, culture, in vitro HIV infection and analysis of non-T cell targets of HIV infection. We will utilize these methods to study the immunological consequences of HIV infection in these cells. In addition, we will evaluate the effects of approved and experimental anti-HIV therapeutics on the immunological functions of these cells. Finally, we will develop methods to efficiently exploit the unique immunological properties of these cells to develop effective approaches for passive cellular immunological approaches to the treatment of HIV infection and AIDS. Specific objectives to be pursued include: 1. Optimization of methods for the use of DC to generate HIV specific cytotoxic T cells; 2. Development of improved methods for the isolation, culture, and analysis of DC; 3. Evaluation of the immunopharmacology and immunotoxicology of antiviral compounds of interest; 4. Evaluation of the effects of HIV infection and exposure to viral proteins on immunologic functions of non-T cells.

DESCRIPTION: Recent studies indicating that cytotoxic T cells (CTL) play a critical role in protective immunity to Mycobacterium tuberculosis suggest that an effective tuberculosis vaccine should contain epitopes that elicit such CTL. However, the mycobacterial epitopes that sensitize and serve as targets for CTL are largely unknown. We propose to analyze M. tuberculosis specific CTL generated in vitro and in vivo for the purpose of identifying the immunogenic epitopes of this organism. To generate primary CTL we will take advantage of a recently developed in vitro vaccination system, which utilizes peripheral blood dendritic cells (DC) to present antigens to naive CTL precursor cells. This system has been used successfully to generate HLA class I restricted CTL to a variety of antigens, including keyhole limpet hemocyanin and Human Immunodeficiency Virus derived peptides. Using this system in combination with peptide binding analysis, we will identify epitopes of the 19, 38, and 65 kDa proteins of M. tuberculosis that give rise to antigen specific CTL. We will also determine if the epitopes that elicit mycobacterium specific CTL, in vitro, serve as targets for CTL generated in vivo in individuals infected with tubercle bacilli or vaccinated with BCG. Although our initial studies will focus on CD8+, class I MHC restricted CTL from individuals expressing the HLA-A2 antigen (the most common HLA class I allele), our studies will eventually encompass CD4+ and CD8+ CTL from individuals with multiple HLA types. In addition, we will determine whether M. tuberculosis specific CTL affect the survival of tubercle bacilli in infected macrophages, and we will determine the spectrum of cytokines produced by these CTL. The results of these studies should facilitate the design of an effective subunit vaccine for use both in the prevention and treatment of tuberculosis.

Dissection of mature human T cells into those that mediate predominantly helper/inducer effects (Leu 3, T4) and those that mediate predominantly suppressor/cytotoxic effects (Leu 2, T8) was made possible with monoclonal antibodies. Using the mixed leukocyte reaction (MLR) as a model system, we have further fractionated these two major T lineages into subsets with relatively narrow functional repetoires. For example, immunoglobulin synthesis induced in MLR was shown to be regulated by sequential interactions between subsets of Leu 2[unreadable]-[unreadable],3[unreadable]+[unreadable] suppressor inducer cells, Leu\2[unreadable]+[unreadable],3[unreadable]-[unreadable]DR[unreadable]+[unreadable] suppressor-amplifier cells, and Leu 2[unreadable]+[unreadable],3[unreadable]-[unreadable],DR[unreadable]-[unreadable] suppressor-effector cells. We further showed that Leu 3[unreadable]+[unreadable] cells capable of helping B cell differentiation and Ig synthesis can be distinguished from Leu 3[unreadable]+[unreadable] cells that do not provide help on the basis of their expression of the Leu 8 marker. Finally, precursors of Leu 2 suppressor-effector cells (Ts) were distinguished from precursors of Leu 2[unreadable]+[unreadable] cytotoxic cells on the basis of their differential expression of the 9.3 antigen. In summary, these data indicate that human T lymphocytes consist of a larger number of functionally distinct subsets than heretofore recognized, distinguishable from one another with combinations of monoclonal antibodies directed at cell surface markers. (LB)

Emphasis on the study of HIV infection of T lymphocytes has skewed research on the pathogenesis of AIDS and development of novel therapeutic approaches. Direct and indirect effects of HIV infection of non-T cell targets, notably dendritic cells (DC), Langerhans cells (LC), and macrophages (M-phi) play an important role in the pathogenesis of AIDS and the immunopathobiology of HIV infection. However, there are significant technical challenges involved in working with non-T cell targets of HIV, owing chiefly to the fact that primary cell populations must be used that are extremely difficult to isolate, culture and analyze, and are often available in limiting numbers or purity. We have developed a series of state of the art approaches for the isolation, purification, culture, in vitro HIV infection and analysis of non-T cell targets of HIV infection. We will utilize these methods to study the immunological consequences of HIV infection in these cells. In addition, we will evaluate the effects of approved and experimental anti-HIV therapeutics on the immunological functions of these cells. Finally, we will develop methods to efficiently exploit the unique immunological properties of these cells to develop effective approaches for passive cellular immunological approaches to the treatment of HIV infection and AIDS. Specific objectives to be pursued include: 1. Optimization of methods for the use of DC to generate HIV specific cytotoxic T cells; 2. Development of improved methods for the isolation, culture, and analysis of DC; 3. Evaluation of the immunopharmacology and immunotoxicology of antiviral compounds of interest; 4. Evaluation of the effects of HIV infection and exposure to viral proteins on immunologic functions of non-T cells.

The prupose of this study is to : 1. Determine the feasibility in patients with multiple myeloma of immunization with dentritic cells loaded with idiotype. 2. Assess the toxicity of immunization. 3. Obtain preliminary data on the clinical efficacy of therapy of serum and/or urine concentration of ideotype protein and B2 microglobulin.

Colorectal carcinoma represents 15% of all malignancies and is the second leading cause of cancer related deaths. In patients with metastatic disease, treatment is palliative and does not prolong survival. In this project we will develop a transfusion based immunotherapeutic approach to colorectal cancer that utilizes dendritic cells (DC) pulsed with carcinoembryonic antigen (CEA), a well characterized membrane glycoprotein overexpressed on the majority of gastrointestinal malignancies, including colorectal cancer. DC are extremely potent antigen presenting cells, capable of sensitizing naive T lymphocytes to protein antigens and eliciting immune responses, in vitro and in vivo. Our group has developed methods for obtaining DC from human peripheral blood and demonstrated that these cells, but not monocytes or B cells, can sensitize naive CD4+ and CD8+ T cells to nominal antigens, in vitro. Recently. in collaboration with Ronald Levy's laboratory we demonstrated in patients with malignant B cell lymphoma that infusion of immunoglobulin idiotype pulsed DC is well tolerated and induces antigen specific T cell responses and tumor regression. In the proposed studies DC expressing CEA determinants as a result of antigen pulsing or gene transfer will be evaluated for their immunogenicity and anti-tumor effects in an animal model of colorectal carcinoma and in patients with disseminated colorectal carcinoma. Four specific aims are proposed: l) development of methods for introducing CEA into DC such that the antigen is processed and presented on the cell surface in association with MHC class I and II determinants, resulting in the induction of CEA-specific T cytolytic and proliferative responses; 2) evaluation and comparison of the immunogenicity of CEA-pulsed DC and DC expressing CEA determinants as a result of gene transfer, in vivo, in a mouse model of colorectal cancer; 3) evaluation of the anti-tumor effects of CEA-pulsed DC in this murine model; and 4) analysis of the immunogenicity, toxicity and anti- tumor effects of CEA pulsed DC in patients with disseminated colorectal carcinoma. The results of these studies should indicate whether transfusion based immunotherapy with DC combined with CEA has a potential role in the treatment of colorectal cancer.

The Flow Cytometry Core, under the supervision of Dr. Engleman, will support all four of the projects by providing cell sorting and immunofluorescence analysis services. Equipment available to this core, which is located at the Stanford Blood Center, include a FACS Vantage flow cytometer capable of four color fluorescence analysis as well as automated cell sorting, a Coulter Epics II analyzer, and a Baker 9000 cell counter. Extensive utilization of this core is anticipated, since identification, isolation and phenotype analysis of leukocyte subpopulations are required for the projects being served.

Identification of functionally distinct leukocyte subpopulations combined with the recognition of the roles played by their surface proteins have created new opportunities for hematopoietic reconstitution and immune modulation, and provide the underlying rationale for this program project application. The proposed program brings together the collective expertise of investigators who have identified cell surface molecules that mediate immunomodulatory and alloimmune effects, developed techniques for isolating and activating rare types of mononuclear leukocytes, and demonstrated their therapeutic effects in model systems. The objective of our program is to utilize these discoveries to develop more effective immune cell based therapies, both autologous and allogeneic, for disorders ranging from malignancies to autoimmune disorders and graft-versus-host disease (GVHD). Four projects are described. Two of these projects seek to exploit the immunotherapeutic potential of dendritic cells pulsed with tumor derived antigens; one focuses on a treatment for malignant lymphoma a tumor of hematopoietic origin. while the other focuses on colorectal carcinoma a solid tumor. Two additional projects address novel approaches to the treatment and prevention of GVHD. One addresses the dual role of a polymorphic cell surface molecule, CD3 1, which appears to play an important pathogenetic role in GVHD; the other focuses on the ontogeny and mechanism of action of immunoregulatory CD3+,CD4-, CD8- T cell's derived from bone marrow. Supporting these projects are two cores, including an administrative and biostatistical core that will provide budgetary oversight, assistance in protocol design and statistical analysis and a flow cytometry core that will provide surface phenotype analysis and cell sorting capabilities. We believe that this interdisciplinary and highly interactive program will ultimately lead to the development of new and more potent forms of cell based immunotherapies for a variety of life-threatening disorders.

Prostatic Acid Phosphatase is a prostate-specific antigen expressed by both normal and malignant prostate tissue. To explore the potential role of xenoantigen immunization in prostate cancer, we initiated a phase I/IIa trial using dendritic cells (DC) pulsed with recombinant mouse PAP as a tumor vaccine. Eligible patients had recurrent prostate cancer, rising serum PSA, and no recent or concurrent changes in therapy.

human therapy evaluation; neoplasm /cancer therapy; lung neoplasms; colon neoplasms; ligands; dendritic cells; growth factor; clinical trial phase I; clinical research; human subject;

DESCRIPTION: (Adapted from applicant's abstract) CTLA-4, a transmembrane protein expressed transiently on activated T cells, downregulates T cell activation and promotes tolerance in experimental animals. However, neither the molecular basis for these effects nor the extent to which CTLA-4 influences the human immune system are known. Our preliminary findings indicate that CTLA-4 triggering blocks cell cycle progression and promotes anergy in human CD4+ T cells, in vitro. The goals of this project are to define the molecular basis for these effects and determine if CTLA-4 plays a role in regulating human T cell responses, in vivo. Four specific aims are proposed: 1) to determine the function of CTLA-4 in the induction and maintenance of anergy in adult human T cells; 2) to define the molecular basis for CTLA-4 signalling by introducing into human T cells selected mutant forms of CTLA-4 and a variety of signalling intermediates; 3) to determine the role of CTLA-4 in regulating the immune response to self and non-self antigens, in vivo, in immunocompetent SCID/Hu mice; and 4) to determine if administration of anti-CTLA-4 antibody can reverse tolerance to a tumor associated antigen, in vivo, under conditions that mimic those in human cancer. The results of these studies should not only allow us to determine if and how CTLA-4 affects tolerance induction and maintenance in humans but potentially provide the basis for an effective immunotherapeutic approach to cancer.

DESCRIPTION (provided by the applicant): Dendritic cells (DC) represent extremely potent antigen presenting cells and are the major cell type responsible for stimulating naive T cells. In this project we propose to use DC pulsed with carcinoembryonic antigen (CEA), alone or in combination with other antigens expressed by colorectal tumors, as a vaccine for the treatment of patients with advanced colorectal carcinoma. Our group in collaboration with Dr. Levy (Project 1) pioneered the use of antigen loaded DC in the treatment of cancer by demonstrating that such cells can induce anti-tumor responses in patients with malignant lymphoma, a relatively immunogenic tumor. However, DC immunotherapy is limited by the number of DC obtainable directly from blood, the inability of DC pulsed with unmodified protein to induce MHC class I restricted cytotoxic T lymphocytes (CTL), which are likely required to attach solid tumors, and the limited availability of well defined tumor antigens. To address the first of these problems we will use Flt3 ligand (FL), a hematopoietic growth factor, to mobilize DC precursors, in vivo. These cells will be harvested from blood, pulsed with CEA and infused as a vaccine. Preliminary results in ten patients treated with FL indicate that circulating DC increased by more than 20-fold and that the expanded cells, once harvested, activated, pulsed with a CEA peptide and infused as a vaccine, induced CTL and in some patients clinical responses. In the proposed project this trial will be expanded to a total of 30 patients. To address the problems of antigen access and processing we will evaluate several methods in model systems including exposure of DC to CEA protein or RNA in combination with immunostimulatory DNA (ISS), and transfer of whole tumor RNA or apoptotic bodies to DC. Exosomes derived from such DC will also be evaluated. Based on the results of these studies a pilot clinical trial will be undertaken that utilizes one or a combination of these strategies. By combining FL expansion of DC precursors with efficient methods of antigen transfer and DC activation, we hope to develop an effective, broadly applicable immunotherapeutic approach to colorectal cancer.

Langerhans cells (LCs) of the epidermis are critical antigen presenting cells required for initiating a protective immune response against pathogens that invade the skin. However, they may also play a detrimental role by triggering graft-versus-host disease (GVHD) in recipients of allogeneic bone marrow transplants (BMT). Whereas host LCs are rapidly replaced by donor derived cells after allogeneic BMT in mice, following transplantation with T cell depleted BM, host LCs are not replaced and GVHD does not occur. We hypothesize that in recipients of BM allografts that contain sufficient T cells, these cells infiltrate the skin where they attack resident LCs and induce the release of cytokines and chemokines that attract donor-derived LCs. In this project we will identify the cells and molecules responsible for these phenomena. In addition, we will directly assess the ability of LCs to stimulate and act as targets for skin GVHD, determine if regulatory T cells can affect LC chimerism and determine if GVHD can be prevented by selectively depleting host LCs or blocking the trafficking of allogeneic T cells to the skin. Finally, in patients who receive allogeneic progenitor cell transplants we will determine if there is a correlation between LC replacement and skin GVHD. Finally, we will determine if GVHD can be prevented or treated in murine models by selectively blocking the trafficking of allogeneic T cells to the skin. The results of these studies should provide fundamental information about the life cycle of LCs and, in keeping with the theme of this program project, possibly lead to safer and more effective ways of using allogeneic stem cells to reconstitute the immune systems of patients with life threatening diseases.

[unreadable] DESCRIPTION (provided by applicant): [unreadable] We have shown that CD 1 reactive NK T cells induce lupus-like disease when injected into irradiated nu/nu BALB/c mice and that administration of anti-CD 1 mAb ameliorates lupus in NZB/NZW mice. The goal of the proposed studies is to test the hypothesis that the interaction between NK T cells and plasmacytoid and myeloid dendritic cells (pDCs and mDCs) via CD 1 is a critical step in the pathogenesis of lupus. In murine as well as human lupus this interaction is theorized to lead to the activation ofpDCs and mDCs, causing them to secrete elevated levels of interferon-alpha and IL-12, respectively, which enhance both innate and cognate immune responses and promote autoimmunity in susceptible hosts. We will use three different murine models of lupus to study the in vitro effects of anti-CD 1 mAb on NK T cell activation and pDC and mDC activation in the presence or absence of otgalacto sylceramide, a glyocolipid presented by CD ld molecules on antigen presenting cells that activates NK T cells. In addition, we will evaluate the in vivo effects of anti-CD 1 mAb on the maturation and activation status of pDCs and mDCs in healthy and lupus-prone mice. Finally, we will determine whether DCs are required for the development of lupus by transplanting lethally irradiated mice lacking functional DCs with syngeneic (DC-/-) or normal histocompatible (DC+/+) bone marrow alone or in combination with disease inducing CD 1 reactive NK T cells. The results of these studies should provide important insights into the role of CD 1 molecules and DCs in the pathogenesis of lupus. [unreadable] [unreadable]

This Core will support all Projects of this program project by providing flow cytometry services and by performing in vitro functional assays to monitor the immune status of subjects participating in clinical trials. For the clinical trials, the Core will be used to document the development of T cell tolerance to donor or host alloantigens, and assess the ability of the reconstituted patients to mount an immune response to newly introduced as well as recall antigens. For all four Projects the Core will provide flow cytometry based cellular phenotype analysis including the determination of adequate host T cell depletion and the monitoring of T cell recovery following hematopoietic progenitor cell transplantation for the patients in the clinical trials.

Langerhans cells (LCs) of the epidermis are critical antigen presenting cells required for initiating a protective immune response against pathogens that invade the skin. However, they may also play a detrimental role by triggering graft-versus-host disease (GVHD) in recipients of allogeneic bone marrow transplants (BMT). Whereas host LCs are rapidly replaced by donor derived cells after allogeneic BMT in mice, following transplantation with T cell depleted BM, host LCs are not replaced and GVHD does not occur. We hypothesize that in recipients of BM allografts that contain sufficient T cells, these cells infiltrate the skin where they attack resident LCs and induce the release of cytokines and chemokines that attract donor-derived LCs. In this project we will identify the cells and molecules responsible for these phenomena. In addition, we will directly assess the ability of LCs to stimulate and act as targets for skin GVHD, determine if regulatory T cells can affect LC chimerism and determine if GVHD can be prevented by selectively depleting host LCs or blocking the trafficking of allogeneic T cells to the skin. Finally, in patients who receive allogeneic progenitor cell transplants we will determine if there is a correlation between LC replacement and skin GVHD. Finally, we will determine if GVHD can be prevented or treated in murine models by selectively blocking the trafficking of allogeneic T cells to the skin. The results of these studies should provide fundamental information about the life cycle of LCs and, in keeping with the theme of this program project, possibly lead to safer and more effective ways of using allogeneic stem cells to reconstitute the immune systems of patients with life threatening diseases.

This Core will support all Projects of this program project by providing flow cytometry services and by performing in vitro functional assays to monitor the immune status of subjects participating in clinical trials. For the clinical trials, the Core will be used to document the development of T cell tolerance to donor or host alloantigens, and assess the ability of the reconstituted patients to mount an immune response to newly introduced as well as recall antigens. For all four Projects the Core will provide flow cytometry based cellular phenotype analysis including the determination of adequate host T cell depletion and the monitoring of T cell recovery following hematopoietic progenitor cell transplantation for the patients in the clinical trials.

The Program in Cancer Immunology and Immunotherapy is focused on understanding the interactions between the host immune system and tumors, and on the discovery and development of clinically effective anti-tumor immunotherapy. The Program is comprised of highly interactive faculty members whose academic interests are in molecular and cellular immunobiology, medicine, surgery and pathology in the Stanford University School of Medicine. Each faculty member has his or her own research program that is supported by investigator initiated grants. In addition, each faculty member is active in teaching graduate and medical students, as well as training postdoctoral fellows and residents. Three complementary approaches are being taken to achieve the three goals of our program. [unreadable] To manipulate cells of the immune system in patients with tumors with the goal of utilizing the exquisite specificity of this system to achieve tumor eradication without significant toxicity. [unreadable] To develop new and more precise ways of measuring and monitoring the immune response to tumors in tumor-bearing hosts. [unreadable] To develop an understanding of the biochemical and signaling pathways used by T cells and other immune effector cells in both their response to tumors and their cellular trafficking patterns. Understanding and manipulating the immune response to tumors, particularly solid tumors, is the major unifying research interest of the participants in this Research Program, and our ability to achieve our objectives is highly dependent on both basic and translational investigators from the other Programs in the Cancer Center and on the Shared Resources.

DESCRIPTION (provided by applicant): Recent evidence suggests that tumors grow in hierarchies driven by distinct cells with the ability to self renew and differentiate, called cancer stem cells (CSCs). The ability to study CSCs in solid tumors has been hampered by a lack of simple mouse models that mimic human disease. We recently discovered mouse pancreatic CSCs that, when introduced into histocompatible recipients with normal immune systems, generate pancreatic tumors that mimic human pancreatic ductal adenocarcinoma (PDA) in terms of histologic appearance and pattern of disease progression. The objective of this project is to study the immune response to murine pancreatic CSCs and identify molecules that distinguish these cells from non-CSC tumor cells. We recently discovered that in the LSL-KrasG12D/+;LSL-p53 R172H/+;Pdx-1-Cre mouse model of pancreatic cancer, cells isolated from liver metastases grow in vitro and display CSC markers. Moreover, as few as 500 cells injected into immunocompetent, histocompatible mice can generate differentiated tumors that are histologically indistinguishable from human pancreatic adenocarcinoma. Primary tumors as well as sites of metastases are rapidly infiltrated with host immune cells. We will 1) generate clonogenic murine pancreatic CSC lines from primary and metastatic tumor cells and characterize these lines with respect to their morphology, chromosomal stability, and cancer stem cell properties;2) characterize the cellular immune response to CSCs and non-CSCs in tumor-bearing and tumor-naive mice;3) use gene profiling to identify genes that are upregulated in CSCs versus non-CSC tumor cells;4) evaluate the ability of short hairpin RNA (shRNA) agents directed at molecules expressed by CSCs to block the growth of these cells in vitro and their generation of tumors in vivo;and 5) evaluate the role of CXCR4 in CSC growth and metastasis and assess the effects of host cells, including immune cells, on CXCR4 expression. The findings from this project should elucidate the biology of pancreatic cancer and lead to the identification of new therapeutic targets for this devastating disease. RELEVANCE: This model, which closely mimics human pancreatic cancer, provides a novel means to study pancreatic CSCs. Since our CSCs have the same genetic mutations (Kras, p53) commonly found in pancreatic cancer and express the same molecular markers as human pancreatic CSCs, knowledge gained about the biology of these cells should prove useful in the design of more effective therapies for human pancreatic cancer.

CORE C. IMMUNE ASSAY, FLOW CYTOMETRY AND GENE PROFILING This Core will support the Projects of this program project by providing flow cytometry and gene expression profiling services and by performing in vitro functional assays to monitor the immune status of subjects participating in clinical trials. For the clinical trial described in Project 1, the Core will be used to document the development of T cell tolerance to donor or host alloantigens, and assess the ability of the reconstituted patients to mount an immune response to newly introduced as well as recall antigens. For all four Projects the Core will provide flow cytometry based cellular phenotype analysis including the determination of adequate host T cell depletion and the monitoring of T cell recovery following hematopoiefic progenitor cell transplantation for the patients in the clinical trials (Project 1) and for cell phenotype analysis and purification (Projects 2-4). The Core will also provide gene expression profiling to idenfify genes associated with clinical immune tolerance in transplant recipients (Project 1), identify specific cell types expressing these genes (Projects 1 and 4) or identify genes that are differentially expressed in functional subsets of dendritic cells (Project 4).

The Program in Cancer Immunology and Immunotherapy is focused on understanding the interactions between the host immune system and tumors, and on the discovery and development of clinically effective anti-tumor immunotherapy. The program comprises faculty members whose academic interests are in molecular and cellular immunobiology, medicine, surgery, psychiatry and pathology in the Stanford University School of Medicine. Each faculty member has his or her own independent research program and also works collaboratively with members of both this program and other Cancer Center programs. In addition, each faculty member is active in teaching graduate and medical students, as well as training postdoctoral fellows and residents. These shared responsibilities, along with regular journal clubs, program project review meetings and an annual immunology retreat, help to foster interactions between faculty members in the program. Three complementary approaches are being taken to achieve the three goals of our program: [unreadable] To develop new and more precise ways of measuring and monitoring the immune response to tumors in tumor-bearing hosts. [unreadable] To develop an understanding of the biochemical and signaling pathways used by T cells and other immune effector cells in both their response to tumors and their cellular trafficking patterns. [unreadable] To manipulate cells of the immune system in patients with solid tumors with the goal of utihzing the exquisite specificity of this system to achieve tumor eradication without significant toxicity. Understanding and manipulating the immune response to tumors, particularly solid tumors, is the major unifying research interest of the participants in this Research Program, and our ability to achieve our objectives is highly dependent on both basic and translational investigators from the other Programs in the Cancer Center and on the Shared Resources.

Project 4. Role of Functionally Distinct Dendritic Cell Subsets in Tolerance and Graft-Versus-Host Disease We recently discovered that effector T cell subsets, including THI, TH2 and Treg cells, can induce monocytes to differentiate into dendritic cells (DCs) that, in turn, selectively induce the formation of THI, TH2 and Treg cells from naive T cells, respectively. We hypothesize that this heretofore unknown immunoregulatory mechanism may play an important role in the control of protective immunity, including proinflammatory responses and tolerance induction, as well as pathologic immunity such as that seen in graft versus host disease (GVHD). We further believe that it may be possible to target and /or manipulate these novel DC subsets to treat or prevent such disorders. The overall goals of this project are to assess the possible role of immunoregulatory DCs (DCrreg) in immune tolerance and GVHD in recipients of hematopoetic stem cell transplants with or without organ transplants, and identify molecules that induce the formation and mediate the functions of these DCs. These goals will be pursued by 1) assessing and comparing the phenotypic, functional and gene expression profiles of T cells and non-T antigen presenting cells in the blood and kidneys of transplant recipients who are immunologically tolerant of their grafts versus those who are not, 2) using neutralizing antibodies and gene expression profiling to identify key factors expressed by Treg cells that are responsible for inducing monocytes to differentiate into DCxreg, as well as the key factors expressed by DCrreg that are responsible for inducing the formation of Treg cells from naive T cells, and 3) evaluating the effects of DCrreg, as well as Treg cells induced by DCrreg, in a murine model of acute GVHD and organ transplant tolerance. This Project is highly dependent on interactions with Projects 1 and 3 as well as Cores B and C, which are the sources of clinical materials, mouse models and immune and gene profiling assays, respectively. Success in this project should ultimately lead to safer and more effective ways to achieve immune tolerance in the setting of allogeneic transplantation.

DESCRIPTION (provided by applicant): Obesity associated Type 2 diabetes (T2D) is a major global cause of morbidity and mortality, and yet current therapies are inadequate. The onset of T2D is often preceded by a condition known as insulin resistance in which tissues become increasingly insensitive to the natural hormone, necessitating the need for more insulin secretion by the pancreas. If this compensatory increase does not occur, blood glucose concentrations rise and T2D occurs. Multiple factors contribute to insulin resistance, but inflammation of visceral adipose tissue (VAT) resulting in chronic release of pro-inflammatory cytokines is known to be a major factor. We recently showed that the adaptive immune system plays a fundamental role in regulating this process. Oligoclonal IFN-¿ producing (Th1) CD4+ T cells in VAT overcome the anti-inflammatory effects of Th2 and Treg cells, and activate M1 macrophages. B-2 cells contribute to the development of insulin resistance by overcoming the anti-inflammatory effects of B-1 cells through their activation of VAT Th1 cells and secretion of pathogenic IgG autoantibodies. Studies of obese non-diabetic humans have revealed that the proportion of Th1 T cells in VAT is highly correlated with body mass index and that insulin resistance is linked to a specific autoantibody signature. These findings strongly support our central hypothesis that the development of insulin resistance in obesity has a significant autoimmune component involving cooperative effects of both B cells and T cells. In this project, we propose to more clearly define the mechanisms contributing to adaptive immune regulation of insulin resistance. We will pursue this objective through the following specific aims: 1) Identiy the mechanisms by which B-1 and B-2 cell subsets exert their opposing effects on insulin resistance in DIO mice. 2) Identify immunologic targets that worsen or protect against obesity related insulin resistance in DIO mice. 3) Assess the clinical significance of the autoantibody signature associated with insulin resistance in obese human subjects. The results of these experiments promise to yield new diagnostic and therapeutic modalities to manage this important disease.

? DESCRIPTION (provided by applicant): Our clinical studies have demonstrated that conditioning patients given combined kidney and hematopoietic cell transplants with total lymphoid irradiation (TLI) and anti-thymocyte globulin reliably induces mixed chimerism and tolerance to the transplanted kidney in HLA matched patients, enabling complete withdrawal of anti-rejection medications without cytopenias, graft versus host disease or other serious complications. Promising clinical studies indicate that this approach also induces persistent mixed chimerism in HLA haplotype matched patients, although withdrawal of anti-rejection medications has not yet been attempted in these chimeric patients. The goal of the proposed research is to determine the cellular and molecular basis for tolerance induction induced by TLI and anti-thymocyte serum (ATS) through studies in well-characterized mouse models. Our previous experiments in mice indicate that stable mixed chimerism and tolerance induced by TLI/ATS is dependent on the presence of both CD8+ antigen cross-priming myeloid DCs and type I invariant NKT cells, since tolerance cannot be induced in Batf3-/- and Ja18-/- recipients, which specifically lack cross priming DCs and invariant NKT cells, respectively. In addition, the conditioning regimen changes the balance of immune cells to favor CD8+ DCs and NKT cells, and results in their activation. Studies are designed to elucidate the effect of TLI/ATS conditioning on surface markers, cytokine secretion patterns and functions of the myeloid DC subsets and NKT cells, and how these effects impact the interactions between the DCs and NKT cells that promote tolerance. We will also analyze the role of donor bone marrow DCs in TLI/ATS tolerance induction, given the capacity of a subset of plasmacytoid DCs to suppress alloimmune responses. The proposed studies will lead to a detailed understanding of the mechanisms responsible for tolerance induction in TLI/ATS based conditioning, and thereby enable the development of enhanced clinical protocols for achieving tolerance in HLA mismatched organ and hematopoietic cell transplants.

? DESCRIPTION (provided by applicant): Mouse models of cancer have provided much necessary insight regarding the processes of oncogenic transformation and tumor development over the last decades. However, few studies have investigated whether the core tumor-immune interactions that regulate cancer development and spread are conserved in mice, and yet accumulating evidence supports a central role for the immune system in impacting all facets of tumorigenesis and progression. Unfortunately, the complexity of the immune system and its behavioral states has surpassed the technical limitations of fluorescence-based flow cytometry and conventional immunohistochemistry (IHC). The development of mass cytometry and multiplexed ion beam imaging (MIBI) allows investigators to assay the immune system as a whole more thoroughly than ever before. Mass cytometry has expanded the number of parameters that can be simultaneously measured on single cells by combining the throughput of flow cytometry with the precision of mass spectrometry. Similarly, MIBI has dramatically increased the number of parameters that can be imaged to dozens of proteins in a single tissue section. The combination of these methods with new analytical algorithms enables a systems-wide comparison of immune organization between mice and humans. We will apply these novel tools to the setting of breast cancer with the specific intent of discerning similarities and differences between mouse models and primary patient samples of the same histological origin. Our specific aims are to 1) reveal similarities in the phenotype and function of immune infiltrates in metastatic versus non-metastatic breast cancer in humans and mice; 2) elucidate the composition and behavior of the immune system in humans and mice through progression from normal tissue through early neoplasia and metastatic disease; and 3) reveal the effect(s) of standard-of-care therapies on the immune infiltrate in mouse and human breast cancer. These studies will result in guidelines for the investigation of the tumor-immune network in pre-clinical mouse models of breast cancer to determine which findings are likely applicable across the species barrier. Moreover, the proposed studies will lay the foundation for determining the suitability of mouse models in any cancer or for any treatment.

ABSTRACT/SUMMARY - Project 1: Murine Modeling of Tumor-Mediated Immunosuppression Like many tumors, melanoma and head and neck squamous cell carcinoma (HNSCC) often metastasize first to regional lymph nodes, and patients with such metastases typically have poor prognoses. It remains unclear why lymph nodes, with their capacity to recognize distinctive antigens such as those produced by tumors, would be hospitable for metastatic outgrowth of the primary tumor. Previous studies of other cancers have identified mediators of tissue-specific metastases to sites such as the lungs, bone, and brain. However, most of these studies have utilized immunodeficient mice and thus have not queried the role of the adaptive immune response in inhibiting or facilitating spread to those tissues. We hypothesize that lymph node metastasis constitutes an essential first step in the metastatic cascade of melanomas and HNSCC in that such metastases act locally upon the adaptive immune system within the lymph nodes to induce tolerance to the tumor and that leukocytes recirculating from these nodes carry the tolerance to distant sites. Our objectives are to establish whether lymph node metastases induce perturbations in anti-tumor immunity and to identify the mechanisms of these perturbations. Starting with murine models, then validating in human tissue, we will a) characterize differences in local and systemic immune responses to metastatic tumors; b) identify differential regulators of tolerance induction by metastatic cells through the use of genomic profiling; and c) identify the molecular mediators of metastatic tolerance induction. Through the use of serial in vivo passaging in a murine model, we have already developed a panel of syngeneic melanoma cell lines that exhibit enhanced lymph node metastatic potential and will create similar lines in HNSCC. We will compare the activation states of these immune cells, cytokine profiles, T cell polarization, and cytolytic activity toward tumor cells using single cell proteomic methods, in partnership with Project 2. Using cytokine profiling and RNA sequencing on the lines, we will apply computational systems biology approaches, in partnership with Project 3, to identify the molecules relevant for induction of tolerance. Samples collected by the Biospecimen and Data Management Core will be used to corroborate our findings in human tissue. If our hypothesis is proven correct that lymph node metastasis is an obligate step in the generation of systemic disease due to tolerance induction, targeting the molecules responsible for lymph node metastasis induced tolerance could prevent and treat metastatic disease.

Project Summary/Abstract The goal of the proposed research is to determine how subsets of myeloid dendritic cells (DCs) in mice conditioned with total lymphoid irradiation (TLI) and anti-T cell antibodies (anti-thymocyte serum; ATS) interact with natural killer T (NKT) cells and Treg cells to promote tolerance to combined organ and bone marrow transplants . Our previous studies showed that stable mixed chimerism and tolerance in this model system are dependent on the presence of both type I invariant NKT cells and the CD8+ antigen cross priming subset of DCs, since tolerance observed in wild type mice is abrogated in Jalpha18-/- and Batf3-/- recipients. In addition, the conditioning regimen changes the balance of immune cells to favor the CD8+ DCs and NKT cells, and results in their activation. Studies are designed to elucidate the changes in surface markers, cytokine secretion patterns, gene expression and in vitro and in vivo functions of the DC subsets and NKT cells in recipients after conditioning, and how these changes are required for the interactions between the DCs and NKT cells that promote tolerance. We will test the hypothesis that the interactions require DC stress protein recognition by NKG2D receptors on NKT cells that lead to NKT cell stimulation of Treg cells and myeloid derived suppressor cells (MDSCs) in an IL-4 dependent manner. We will also test the mechanism by which myeloid DCs in TLI- conditioned kidney transplant patients mediate suppression of T cell activation, including changes in gene expression and cytokine secretion. Since we hypothesize that the activated immunosuppressive cells allow for the acceptance of donor hematopoietic progenitors, chimerism, and associated clonal deletion, these studies should provide important new information that impacts on current clinical trials of tolerance in HLA mismatched kidney and hematopoietic cell transplant recipients using TLI based conditioning.

Project Summary/Abstract This Core will support the Projects with both standard MLR monitoring and flow cytometry, and advanced technology including immune cell subset assays by CyTOF analysis, and T cell clonal repertoire assays by high throughput sequencing of TCR genes from appropriate T cell populations, and mRNA expression in DC populations by RNAseq. The Core will support both preclinical models in Projects 2-3 by analyzing mouse cells, and the clinical study in Project 1 by analyzing human cells. In the case of CyTOF, staining of cells for up to 40 targets simultaneously using heavy metal conjugated mAbs and analysis by spectroscopy is expected to lead to the identification of new subsets of adaptive and innate immune cells that will provide additional insights into cells that contribute to tolerance in the preclinical and clinical studies. In the case of high throughput sequencing, alloreactive T cell clones will be identified and purified from pretransplant MLR cultures with recipient responder cells and donor stimulator cells. Clonal frequencies will be determined in recipients before and after tolerance induction to search for evidence of clonal deletion. In addition, the breadth of the T cell clonal repertoire will be examined to study long term immune reconstitution.

PROJECT SUMMARY/ABSTRACT Background: Immunotherapy has emerged as one of the most promising approaches for the treatment of cancer. Unfortunately, a large number of patients and common tumor types do not respond to existing immunotherapies. Tumor-associated macrophages (TAMs), which accumulate in many cancers and suppress anti-tumor immunity, likely contribute to this problem. Hypothesis and Objective: Based on encouraging preliminary data, we hypothesize that engagement of Dectin-2, a pattern recognition receptor that is highly expressed by TAMs in certain tumors, can reprogram TAMs into potent antigen-presenting cells capable of inducing immunity against a wide range of cancers. Our objective is to validate this hypothesis by analyzing the immunological and anti-tumor effects of natural and synthetic Dectin-2 agonists in mouse models of cancer. Specific Aims: Aim 1: Analyze the factors that regulate Dectin-2 expression and the mechanisms by which Dectin-2 agonists reprogram TAMs and induce anti-tumor immunity. Aim 2: Assess the anti-tumor effects of natural Dectin-2 agonists, alone and in combination with other agents, on a range of aggressive cancers. Aim 3: Synthesize glycopeptide agonists of Dectin-2 and assess their anti-tumor activity alone and in combination with other agents. Aim 4: Construct tumor-targeted antibody-glycopeptide conjugates and evaluate their functional and therapeutic effects. Study Design and Methods: Since TAMs in some tumors express little or no Dectin-2, we will analyze the effects of natural Dectin-2 agonists on TAMs in the presence of GM-CSF, which induces Dectin-2 expression. The anti-tumor effects of the agonists will be studied in mouse models of pancreas, breast, colon, lung, and skin cancer with a spectrum of Dectin-2 expression, both as monotherapies and in combination with GM-CSF and other agents shown to enhance the efficacy of Dectin-2 agonists in our mouse models. Mass cytometry and recently developed informatics tools will be utilized to analyze the effects of Dectin-2 ligands on the anti- tumor immune response in multiple tissues. To generate more effective and clinically applicable therapies, a novel synthetic approach will be used to produce compositionally defined Dectin-2 ligands that are optimized for TAM activation. The most efficacious of these synthetic Dectin-2 ligands will be conjugated to tumor- targeted antibodies for purposes of enhanced tumor delivery and TAM-mediated tumor cell killing, and their safety and efficacy assessed. Expected Results and Impact: We expect these experiments to demonstrate that engaging Dectin-2 on TAMs induces immunity against a wide range of tumors, including tumors resistant to checkpoint blockade, and that Dectin-2 agonists used alone or in combination with other anti-tumor agents can induce durable tumor regression. The most promising of these agonists will be candidates for clinical development.

Project Summary/Abstract Obesity has reached epidemic proportions in the U.S, and recent studies have demonstrated that the children of obese mothers are far more likely to become obese than the children of non-obese mothers. The explanation for this association is unknown. Since obesity is associated with a widespread macrophage- dependent inflammatory state, we hypothesize that obesity-associated factors in obese mothers cross the placental barrier and adversely affect the fetal immune system. We further hypothesize that monocytes, which constitute a substantial portion of immune cells in the developing fetus, are particularly sensitive to such factors. Our preliminary data support this concept; we show that monocytes in the fetuses of diet-induced obese mice are phenotypically altered relative to the same cells in the fetuses of non-obese mice fed a normal diet. Therefore, in this proposal, in Aim 1 we will decipher the effects of maternal obesity on developing monocytes in the liver and placenta at the phenotypic, functional, epigenetic and transcriptional levels. Additionally, we will use mass cytometry in combination with our novel Scaffolds algorithm to analyze changes of monocytes and other major immune cell populations in multiple tissues and at multiple time points in the offspring of normal versus obese mice. In Aim 2, based on preliminary data showing that high-fat diet fed female mice have increased levels of cholesterol and fatty acids, which can cross the placenta, we will directly test the effects of these factors on fetal monocyte function. Upon completion of the proposed studies, we will have identified maternal factors that affect fetal monocytes and potentially other immune cells, and we will learn if their effects persist after birth.

Project Summary/Abstract Background: Radiation therapy (RT) is a core treatment modality that benefits patients with many types of cancer and can synergize with immune checkpoint blockade therapy. However, delivery of maximally effective doses of radiation to tumors is limited by collateral damage to normal tissues. We are developing a next- generation clinical RT platform called PHASER that will deliver ultra-rapid and precise radiation (FLASH) to decrease damage to normal tissues dramatically. Using a unique preclinical FLASH irradiator we developed for mice, our preliminary data show enhanced tumor control with FLASH vs. conventional dose rate irradiation as well as increased infiltration of immune cells into the tumor, suggestive of an immune mediated mechanism. Hypothesis and objective: We hypothesize that FLASH will demonstrate a superior therapeutic index by comparison to conventional dose rate RT for multiple cancers, based not only on its precision but also on the induction of more potent anti-tumor immunity. We will test this hypothesis in experimental models of cancer. Specific Aims and Study Design: Aim 1: Compare the anti-tumor potency, safety and immunological effects of FLASH vs conventional dose rate RT in primary tumors: We will evaluate different doses of FLASH and compare its effects with maximally tolerated doses of conventional dose rate RT in both syngeneic and patient derived xenograft mouse models. In addition to assessing tumor growth, we will analyze the effects of FLASH on the immune response, both locally and systemically through the use CyTOF and our SCAFFOLDS algorithms. This approach will reveal where and which immune cell subsets become activated in successfully treated animals. We will also assess the immunologic correlates of reduced toxicity from FLASH. Aim 2: Analyze the therapeutic effects of FLASH alone and in combination with immune checkpoint antibodies in metastatic disease. To assess our hypothesis that FLASH in combination with PD-1 blockade will exhibit synergistic anti-tumor effects due to an enhanced system-wide immune response, we will study the effects of FLASH, alone and in combination with anti-PD-1, on tumors outside the radiation field, and assess the immune response as in Aim 1. Aim 3: Identify the immune cellular and molecular basis of FLASH efficacy. We will test the hypothesis that efficacy is dependent on T cells as well as antigen presenting dendritic cells (DCs) by treating tumors in Rag-2 KO and BATF3 KO mice, respectively, and will elucidate the role for these cells by transferring T cells or DCs from successfully treated mice to naive mice challenged with tumor. The specific subsets required for efficacy are expected to be those shown to expand in multiple tissues in Aim 1. Lastly, we will explore the role of Type I interferon and its receptor on DCs in the efficacy of FLASH. Expected Results and Impact: These experiments are expected to demonstrate that FLASH in combination with checkpoint therapy promotes durable tumor regression of primary and metastatic tumors with little damage to normal tissues, thus setting stage for evaluating FLASH in clinical trials.

ABSTRACT (Project 3) Pancreatic ductal adenocarcinoma (PDAC), an aggressive malignancy that is poorly responsive to treatment, is characterized by a prominent infiltration of immune cells. In particular, macrophages are abundant within the tumor microenvironment (TME) in PDAC and contribute to disease progression and treatment resistance. However, the factors affecting the recruitment and distribution of immune cells, including macrophages, in PDAC remain largely unknown. Recently, our collaborators in Project 1 found that tumors differing in their Trp53 status exhibited different immune profiles. Our collaborators further observed that the cell type of origin (acinar or ductal) influenced the tumor molecular subtype (classical or basal-like), which is also known to be shaped by the immune composition of the tumor. We postulate that somatic alterations in cancer cells, the cell type of origin, and tumor molecular subtype influence the immune landscape within the TME and throughout the host during PDAC progression. To test this hypothesis, we will determine immune cell frequency, distribution, and function in the TME and throughout the host using high-dimensional analytical tools (CODEX and CyTOF) on genetically engineered mouse models (GEMMs) of PDAC that vary in their driver mutations and cell type of origin (developed by Project 1). Additionally, we will use molecular methods to identify immunomodulatory factors regulating immune cell recruitment in the context of different genetic mutations and cell type of origin, and we will use genetic, pharmacological, and neutralizing antibody-based approaches to confirm the functional effects of these factors in driving PDAC progression. Importantly, we will validate findings from these studies in human PDAC specimens and in cell lines derived from human pancreatic ductal cells. Finally, we recently found that the pattern recognition receptor, Dectin-2, is expressed on tumor- associated macrophages (TAMs) in an aggressive, transplantable model of PDAC. Notably, administration of natural Dectin-2 ligands induces sustained PDAC regression in a T-cell dependent manner by reprogramming immunosuppressive TAMs into immunostimulatory antigen-presenting cells (APCs). We hypothesize that PDAC can be effectively treated by reprogramming Dectin-2+ TAMs into immunostimulatory APCs. We will treat autochthonous tumors arising in the GEMMs developed by Project 1 with Dectin-2 ligands to test this hypothesis. Furthermore, to identify mechanisms by which Dectin-2 stimulation is therapeutically efficacious, we will use CODEX and CyTOF to observe changes to the immune landscape occurring within the TME and throughout the host upon treatment. Molecular approaches will be used to identify immunomodulatory factors responsible for mediating therapeutic efficacy. Together, these studies will improve our understanding of how the immune system is altered during PDAC development in the context of variation in genetic mutations, cell type of origin, and molecular subtype, and in response to TAM reprogramming.

Project Summary/Abstract Colorectal cancer (CRC) is the second leading cause of cancer mortality in the United States, and obesity, which affects 40% of the population, not only increases the risk of developing CRC, but also increases CRC mortality through unknown mechanisms. Our preliminary studies demonstrate that CRC grows faster in mice rendered obese through a high fat diet (HFD), and that the tumor associated macrophages (TAMs) in these mice exhibit higher expression of the acid sensing receptor, GPR65, which is known to dampen the immune response. Moreover, in HFD-induced obese mice lacking GRP65, TAMs secrete more TNF-? and tumor growth is retarded. Given that TAMs but not normal tissue macrophages of obese mice exhibit increased GPR65, we examined the pH of the tumors in these mice and found them to be more acidic. On the basis of these findings, we hypothesize that excess lipids in the HFD alter tumor cell metabolism resulting in increased acid production, which potentiates GPR65 expression and signaling in TAMs, causing them to become immunosuppressive and promote tumor growth. To test this hypothesis we will pursue the following specific aims: 1) Determine the contribution of GPR65 signaling to TAM function and CRC growth under conditions of obesity by determining if the cAMP- PKA signaling axis, which functions downstream of GPR65, is activated in TAMs of obese mice and controls TNF-? production. We will also analyze GPR65 expression and the cytokine secretion capacity of macrophages from healthy blood donors and TAMs in CRC samples from non-obese and obese patients; 2) Identify the mechanism by which HFD promotes GPR65 signaling in CRC TAMs by testing the ability of HFD and oleic acid, a dietary triglyceride that is highly enriched in HFD tumors, to alter the oxidative potential, fatty acid oxidation capacity and acid production of human tumor cells via flow cytometry, CyTOF and Seahorse assays. We will also determine if a high-oleic-acid diet is sufficient to modify GPR65 expression and cytokine production by TAMs, and examine if tumor acidity is required for the blunted inflammatory response of TAMs; and verify the role of GPR65 in human macrophages and 3) Assess the effects of targeting GPR65 for tumor immunotherapy in obese and nonobese mice with CRC and other tumor types, namely hepatocellular carcinoma and melanoma, and assess the effects of checkpoint blocking antibodies on tumor growth and anti- tumor immunity in GPR65+ and GPR-/- mice bearing these tumors. The results of these studies are expected to not only reveal a critical mechanism responsible for accelerated tumor growth in the setting of obesity, but also identify a novel target for the treatment of these cancers.