Linda M Bradley - US grants

DESCRIPTION (Adapted from the Investigator's abstract): Fundamental to protection against pathogens is the development of immunologic memory, a dramatically more effective response to previously encountered infections that is largely regulated by helper T lymphocytes (CD4 cells). Little is known about regulation of T-cell memory in part because there is no evidence for distinct precursors of effector and memory T-cells. While many studies have focused on requirements for maintenance of memory, regulation of the induction and re-expression of memory has received little attention. Identifying parameters that control CD4 memory at these stages is essential for exploiting the immune system as a means to protect against disease. Since memory CD4 cells arise from responding naive cells, it is imperative to delineate factors that regulate memory at the initiation of the primary response. The investigator proposes that antigen (Ag) and Ag-presenting cells (APC) play crucial roles in CD4 memory development, and that memory is regulated by selective processes of decreasing Ag concentrations and changing APC requirements during the primary response. Since the dose and form of antigen (Ag) can affect cell mediated vs humoral responses that are associated with Th1 and Th2 cells, respectively, Ag may also play a role in development of subsets of memory CD4 cells. The goal of the proposed studies are: 1) to assess the role of Ag in the development of memory CD4 cells and in the differentiation of Th0, Th1, and Th2 subsets of memory CD4 cells; 2) to evaluate the role of B-cells as APC in the memory CD4 response, both for optimal memory development and for re-eliciting memory responses; and 3) to characterize the memory response in situ in lymphoid tissue to identify anatomical sites where memory responses take place and to determine whether subsets of memory CD4 cells differ in their capacity to promote B-cell expansion, germinal center formation, and isotype switch. The investigator will use both in vitro and in vivo approaches with normal, thymectomized, transgenic, and knockout mice to determine factors that are required for the development and function of memory CD4 cells. The investigator will exploit the response to pigeon cytochrome c (PCC) in H-2K mice which is restricted to the v-alpha-11, v-beta-3 TCR where the investigator can compare responses of normal CD4 cells to those from PCC-specific transgenic mice with the same TCR when the dose and form of Ag (peptide versus naive protein) are varied. The investigator will assess the impact of primary and memory B-cells on CD4 memory using B-cell-deficient mice, hen egg lysozyme Ig transgenic mice, and the B-cell response to the NP hapten which is restricted to the lambda light chain. The proposed studies represents an integrated investigation of factors that will provide essential, basic information about the regulation of CD4 memory.

Core B is designed provide transgenic animals required by the four projects of the program, including mice that express key molecules/receptors and mice that have defective genes. Core B will breed and screen transgenic animals and backcross them as necessary to render them suitable for the experiments detailed in the individual projects of the program. Because this process is very labor intensive, a centralized facility specialized ot perform this task makes a great deal of sense. Core B will also oversee the collection and preparation of reagents required as key components by at least two or more individual projects. Such reagents would include Ab to potential co-stimulatory molecules and cytokines, antibodies to adhesion molecules, APC cell l lines expressing particular co-stimulatory molecules, cell lines for cytokine bioassays. The Core will obtain the various Tg or KO mice needed for the individual projects, quarantine them if necessary, develop standardized procedures for screening for the Tg/KO gene expression in each case, and will breed and backcross the mice to C57BL/6 and B10.BR inbred strains. The goals are to have healthy, genetically homogenous mice for the investigators in the project. Investigators will be able to obtain these mice for experiments but will maintain the animals separately during the course of the sometimes lengthy adoptive transfer experiments. Many of the experiments conducted so far on the Tg and KO mice have been done with original B6 x SJL founder mice or with mice backcrossed only for one or two generations. In such cases background genes from the non B6 parent may influence experimental results. Use of non-bred mice can also lead to complications due to mismatches in major or minor histocompatibility differences and also to the presence of genes other than the gene of interest due to linkage on the same chromosome. Furthermore, the facility will allow us to generate new strains of mice by inbreeding. For instance TCR Tg mice will be generated with the SCID mutation so that contributions from expression of endogenous alpha chains can be ruled out. Also double KO mice or TCR Tg/KO mice can be created which will allow a more definitive analysis. At the moment we have the following mouse strains to contribute to the core; TCR Tg mice (specific for pigeon cytochrome C (PCC) plus I-Ek (designated AND) backcrossed 5 times to B6 and B10 BR (currently being bred onto a RAG-2 KO background); TCR Tg mice specific for HY plus Dd (already on a SCID background); TCR Tg mice specific for influenza hemagglutinin (HA) peptide plus IAd (CD4 selecting) and TCR Tg specific for HA plus Dd (CD8 selecting); beta2M KO/KO mice; CD4 KO/KO mice backcrossed 8X to B6; IL-2 KO/KO backcrossed 3X to B6; IFNgammaKO/KO backcrossed 3x to B6. IL-4 KO/KO backcrossed 4x to B10.BR; and RAG-2 KO/kO. We also have mum KO/KO mice and mice constitutively expressing high levels of bcl2. Dr. Klinman will provide the Sma58 m ice, which express I=-Ek only on germinal center B cells. Dr. Bradley has already made arrangements to obtain L-, E- and P- selectin Ko mice.

Fundamental to long lasting immunity to infections is the process of immunologic surveillance of tissue that is largely mediated by memory T lymphocytes. At basis of this process is the continuous recirculation of lymphocytes from blood to lymph which is achieved by their adhesion to, and transmigration across vascular endothelium of both lymphoid and nonlymphoid tissue. yet little is known regarding the parameters that regulate the migration of memory T cells. It is thought that the route of immunization influences development of functionally distinct subsets of memory T cells which preferentially recirculate to lymphoid tissue draining the site(s) of initial antigen exposure. Such tissue-specific migration is regulated by homing receptors and their ligand that are expressed on vascular endothelium. Identifying parameters that control memory T cell migration into tissue are critical both for understanding regulation of memory and for strategies to potentiate memory and achieve tissue-specific priming in the design of effective vaccines. In order to identify adhesion molecules that contribute to normal memory cell trafficking, the proposed study will analyze the homing of T cell populations that are exclusively comprised of memory CD4 or CD8 cells. We will investigate the roles of several adhesion molecules including selectins (L-, E-, and P-), several integrins (alpha4beta1, alpha4beta7, and LFA-1) and their counter receptors (ICAM-1, VCAM01), as well as CD44 in this process. We will use antibodies to the selectins or the respective Ig chimeras to block homing and function of memory cells in normal mice in vivo and will examine memory T cell homing and function in selectin KO mice. We will perform a similar analysis of integrin function and the adhesion molecule CD44 in memory T cell migration using antibodies to block the homing and function of memory CD4 and CD8 T cell populations, and the homing of memory B cells, both in responses to experimental antigens and in affording protection, and the homing of memory B cells, both in responses to experimental antigens and in affording protection to influenza as a disease model. Because the route of priming may play a critical role in determining migratory patterns of memory cells and the development of CD4 subsets with different cytokine secretion patterns, we will directly compare responses of CD4 cells generated by parenteral vs oral immunization and will examine selectin land integrin function in their homing. Unlike previous studies, we will not rely solely on phenotypic markers to identify memory cells or in vitro adhesion assays, but will in addition evaluate CD4 and CD8 memory in secondary antigen=-specific responses. The proposed study represents a comprehensive analysis of the biologic role of adhesion molecules in memory cell migration, which together with the studies proposed in projects 1, 2, and 4 to define parameters that regulate memory in the CD4, CD8 and B cell populations will provide valuable insights into methodology for achieving optimal memory responses.

Immunological memory refers to the dramatic and effective response to previously encountered antigens (Ag) that is largely regulated by T cells. Upon challenge, memory T cells localize in sites of Ag exposure and develop in effector cells that produce cytokines and chemokines that regulate the memory response. Identifying parameters that control memory T cell entry into tissue is crucial for understanding regulation of memory and alterations in T cell migration during disease processes that will be essential for strategies to potential memory in the design of vaccines and to dampen memory in allergic and autoimmune responses. Lymphocyte entry into tissue from blood is orchestrated by multiple adhesion receptors whose expression is modulated by cytokines and whose avidity is regulated by chemokines which exert selective chemoattractant effects on subsets of cells. However, mechanisms that initiate and regulate recruitment of memory T cells to sites of antigen exposure have not yet been identified. We proposed that memory T cells become activated by Ag presented by APC in tissues through which they recirculate, and then acquire the capacity to enter other sites of Ag exposure. Upon reencounter with Ag, activated memory CD4 cells produce mediators which regulate cellular recruitment. The proposed studies will investigate 4 key parameters in regulation of memory T cell migration: Ag, adhesion molecules, cytokines, and chemokines. We will use a defined adoptive transfer model to study Ag-induced recruitment of memory CD4 cells into lymphoid tissues but will also extend our studies to CD8 memory cells, in collaboration with Project 2, since there may be fundamental differences in their regulation. We will use TCR transgenic mice to generate defined memory populations and analyze the kinetics of their activation, division, and death with different conditions of immunization and determine if the recruitment response depends upon inflammation. To define contributions of adhesion receptors, cytokines, and chemokines, we will use blocking reagents at various times with respect to immunization to distinguish roles in the initiation versus progression of the response. We will also take advantage of cytokine and adhesion molecule knock out mice. In collaboration with Project 1 and 2, we have the potential to link these factors that may also affect the induction, expansion, and survival of memory cells with their subsequent ability to respond to Ag upon challenge. By using different CD4 and CD8 transgenics as sources of memory cells which can be identified by Class II and Class I tetramers, respectively, we can test predictions about normal memory responses of heterogeneous populations. These studies address fundamental questions regarding the mechanisms by which memory T cells mediate immune surveillance and provide long lasting immunity.

DESCRIPTION: (Adapted from the Investigator's abstract): Infiltration of autoreactive CD4 T lymphocytes into the pancreas represents a hallmark event in the development of insulin-dependent autoimmune diabetes. Chemokines regulate adhesion of cells to vascular endothelium and, via chemoattractant effects, govern cellular recruitment into inflammatory lesions. However, their role in diabetes has not yet been studied. Defining functions of chemokines in regulating inflammation in the pancreas will provide a greater understanding of diabetes and may supply essential new information for advances in treatment. Th1 cells which secrete IFN-gamma, TNF-alpha, and LT are associated with pathogenesis leading to destruction of islet beta cells and disease onset whereas Th2 cells which produce IL-4, IL-5 and IL-10 do not cause disease due to protective effects of IL-4. Early events leading to differences in the entry of autoreactive CD4 subsets into the pancreas may thus be crucial in diabetes and a key point for therapeutic intervention. The investigators have developed an adoptive transfer model to study the migratory behavior islet-specific, transgenic CD4 subsets. Th1 cells, which induce disease within 5-6 days, enter the pancreas more rapidly than Th2 cells and express different chemokines in vitro. Moreover, in vivo, several chemokines partition with Th1 but not Th2 infiltrates. From these studies they propose that polarized secretion of chemokines by Th1 vs. Th2 cells in the pancreas regulates selective recruitment of subsets of cells that determine whether beta islet cells are destroyed. This pilot project will employ several novel strategies to bridge in vitro and in vivo analysis of chemokines in diabetes that will form the basis for more detailed mechanistic studies in the future. They will use blocking studies to identify chemokines that regulate disease onset and progression, and intravital microscopy to assess chemotaxis in vivo in the presence or absence of Ag (islets). Since cytokines regulate chemokine synthesis by multiple cell types, cytokine/chemokine cascades are likely to determine the nature and extent of pancreatic inflammation. The investigators will test the hypothesis that T cell-derived cytokines coordinate chemokine expression and responses within the pancreas. In addition to IL-4 and IFN-gamma, they have an unparalleled opportunity to study TNF family cytokines (TNF-alpha, LT, LT alpha1beta2, LIGHT) using Fc-receptor constructs as blocking agents in experiments that are likely to reveal fundamental aspects of the biology of inflammation. They will determine if cytokines or conditions of TCR stimulation and costimulation can modulate a Th1 to a Th2 pattern of chemokine expression/reactivity and alter their capacity to induce diabetes. Identifying the role of chemokines in diabetes will provide new insights into regulation of the disease process.

DESCRIPTION (provided by applicant): Autoimmune diabetes results from the loss of self-tolerance to pancreatic islet Ag that ultimately leads to inflammatory destruction of insulin-producing beta cells. A growing body of data indicate that unique immunoregulatory CD4 subsets that in some instances are associated with cytokine profiles that distinguish them from Th1 or Th2 cells, control CD4 cell homeostasis and are actively involved in maintaining self-tolerance. It is not yet clear whether different subsets of regulatory cells can function at the induction and effector phases of an autoimmune response. Recent studies indicate that such T cells may be key to the prevention of several organ-specific autoimmune diseases and that diabetes onset may be precipitated when regulatory CD4 cells are overwhelmed by autoaggressive Th1 effector populations. Identifying means to induce such regulatory populations and understanding the mechanisms by which they function to either prohibit activation of autoaggressive CD4 cells or control their responses will enhance our understanding of immune regulation and may yield new insights into immunotherapeutic approaches for treatment of IDDM. We have developed an adoptive transfer model to study regulation of IDDM by CD4 subsets. We find that Thl cells but not Th2 cells that are generated from islet-specific BDC2.5 transgenic CD4 cells cause rapid onset of IDDM in both NOD.scid and NOD recipients. Although Th2 cytokines are associated with protection from IDDM, Th2 cells cannot reproducibly inhibit or delay disease onset in our model to any significant extent. However, we have recently found that TGF-beta1 induces a population of BDC CD4 cells with a cytokine and chemokine secretion pattern typical of Th1 cells, but which can function in IDDM as a regulatory population with the capacity to inhibit Th1-mediated disease in NOD.scid mice and the spontaneous development of IDDM in NOD mice. The aims of the proposed studies are to 1) identify the precursor population(s) that give rise to TR cells and parameters that regulate their development; 2) investigate their in vivo localization and response, turnover, and requirements for Ag for in vivo persistence, and 3) to investigate mechanism(s) by which regulatory cells control the effector phase of Th1-mediated IDDM. From these studies we hope to determine if induction and expansion of regulatory CD4 cells in vitro offers potential for clinical treatment of IDDM.

DESCRIPTION (provided by applicant): The lung is the site of infection by influenza (flu) virus which gains access to airways by aerosol transmission. This infection is confined to lung epithelium, and its control depends upon an effective, local immune response that is mediated by CD4 and CD8 cells. Protective immunity is associated with flu-specific CD4 and CD8 cells that persist for extended durations in airways. Thus, optimal vaccine development to organisms such as flu that could be used for bioterrorism will depend upon greater understanding of the requirements for generating effector cells with the capacity to migrate into, as well as respond and persist in the target tissue. CD44, the product of a single gene that is subject to mRNA splicing and post translational modifications, is a key regulator of inflammatory processes in the lung. Expression of high levels of CD44 is the most widely used marker to distinguish effector and memory T cells from naive T cells, yet little is known regarding it's function in Ag-experienced cells. Our preliminary studies of responses to flu using CD44- deficient mice and CD44-blocking antibodies support the hypothesis that CD44 is essential for T cell migration into lung airways, and provide evidence for additional roles in regulating the responses and survival of effector and memory cells. We have developed adoptive transfer models with TCR transgenic CD4 and CD8 cells with which we can induce and track Ag-specific T cell responses to engineered flu viruses that express the appropriate peptides. With Class I and Class 1I multimers and cytokine reporter mice we can also follow responses ofpolyclonal T cells. With these tools, we propose to investigate mechanisms by which CD44 can contribute to T cell immunity to flu. Aim 1 will test the hypothesis that CD44 mediates an essential adhesion mechanism that enables the transmigration of effector T cells into the airways in response to infection and functions in the maintenance of flu-specific T cells in this site. Aim 2 will test the hypothesis that CD44 regulates cytokine production by effectors, and supports their appropriate expansion and responses to flu within the lung. Aim 3 will investigate the role of CD44 in regulation of the homeostasis oft cell subsets. We believe that these studies will provide new insights into mechanisms by which adhesion pathways and interactions with extracellular matrix components can affect the optimal development of T cell responses in target tissues as well as the longevity of memory.

DESCRIPTION (provided by applicant): Type 1 diabetes (T1D) develops as control of autoreactive CD4 cells is lost, which occurs in part because of escalating responses of cells that escape regulation and are highly pathogenic. While many cell types contribute to this disease, ameliorating the autoimmune response of CD4 cells continues to be an important strategy for treatment of T1D. The goal of the proposed studies is to assess changes in development and function of CD4 cell subsets during progression to T1D. It has become increasingly clear that a changing dynamic of CD4 cell responses occurs in this autoimmune disease. The objective is to determine whether checkpoints can be identified where autoreactive CD4 cells can be modulated to prevent or reverse diabetes onset. Using the NOD mouse model of T1D, our studies show that Th1 and Th17 subsets of CD4 cells, which are distinguished by the production of IFN-3 vs IL-17, respectively, are present at T1D onset and that both subsets can induce T1D. Moreover, we find CD4 cells that differentially secrete these cytokines throughout the prediabetic phase. Recent studies suggest that FoxP3-expressing regulatory CD4 cells (TR cells) can lose function and may even have the potential to convert to Th17 cells in a proinflammatory milieu, which could also contribute to the loss of control that precipitates T1D onset. These findings underscore the need for better understanding of the responses of CD4 cells and how they are altered as the autoimmune response progresses. We find that persisting CD4 cells become indefinitely established in NOD mice with adoptive transfer studies when allelically marked CD4 populations from NOD or TCR-transgenic BDC 2.5 mice are injected into 1 wk old recipients. These cells are thus subject to in situ regulation by the environment which promotes the development of T1D. This powerful in vivo model will enable us to follow changes that arise in CD4 cell subsets with progression to disease. We will also make use of IFN-3 and FoxP3 reporter mice to track subsets of CD4 cells. We propose to: 1) test the hypothesis that effector CD4 cells that are generated in T1D prone individuals in the prediabetic phase over time increase in number and differentiate into highly pathogenic Th1 and Th17 cells with capacity to mount a potent cytokine-driven effector response culminating in disease onset;and 2) determine if TR cells lose function or convert to pathogenic Th17 cells with the advancing autoimmune response that leads to T1D. These basic studies are essential to provide new insights into the development of the autoreactive CD4 cell response in T1D, and may distinguish a late stage of effector differentiation where intervention could inhibit the responses of pathogenic CD4 cells and control or reverse the disease process so that mechanisms of tolerance can be restored. PUBLIC HEALTH RELEVANCE: Type 1 diabetes is a lifelong, incurable autoimmune disease which can be treated with insulin. However, treatment does not prevent the eventual and devastating effects of this disease, which may include kidney failure, blindness, nerve damage, amputations, heart attack, and stroke. The best hope for achieving better treatments and even a cure is to better understand the autoimmune response. The goals of the proposed studies are to investigate whether checkpoints can be identified where responses of pathogenic CD4 cells can targeted to prevent complete destruction of insulin-producing islet cells so that tolerance can be established.

Understanding mechanisms that regulate memory in T cells is crucial for developing strategies to protect against epidemic and pandemic influenza viruses. The goal of this project is to determine the contribution of the selectin family of adhesion molecules (E, P, and L) and the selectin ligand, PSGL-1, to T cell memory Recently, we found that glycosylated, biologically active ligands for selectins are acquired by a subset of CD4 and CD8 effectors in the draining lymph nodes after influenza virus infection and represent the majority of responding T cells in the lungs including IFN-g or IL-17 producers, in vivo correlates of Th1 and Th17 cells, respectively. Moreover, we find that a consistent fraction of memory phenotype T cells express selectin-binding activity, suggesting that this is a stable heritable trait that distinguishes a subset of memory cells. In the absence of functional selectin ligands or PSGL-1, CD4 cell expansion and localization in the lungs after influenza virus infection is unaltered. However, CD4 effectors produce elevated levels of IFN-g and IL-17 suggesting a role for selectin ligands in dampening the effector response. Importantly, the development of memory CD4 cells with the capacity for secondary expansion after infection is impaired, possibly because of altered homeostatic regulation. Our data support the hypothesis that mechanisms regulated by selectins can be essential for the delivery of signals that control CD4 effector cell responses as well as the homeostatic maintenance of a subset of memory cells. We will investigate the novel function(s) of selectins in the regulation of effector and memory T cells in following specific Aims: 1) to determine if differences in selectin-binding capacity identify memory T cell subsets with distinct functional properties and homeostatic regulation;2) to investigate selectin-dependent responses of T cells and the role of selectinbinding capacity in the generation of memory cells after influenza virus infection;and 3) to identify mechanisms by which selectins regulate T cell homeostasis. We will take advantage of mice that are deficient in PSGL-1-/-, mice that lack PSGL-1 signaling, and mice that lack functional selectin ligands due to deficiency of the IV/VII fucosyl transferases. We will use IFN-g and IL-17 reporter mice, selectin ligand fusion proteins, and WT and engineered influenza viruses to enable us to assess the responses of polyclonal and TCR transgenic CD4 cells and make comparisons to CDS cells. In each of these Aims we will collaborate with Projects 1 and 2 to examine defined subsets of CD4 and CD8 cells in the influenza model. We will collaborate with project 4, which will determine the relevance of selectins in the tuberculosis model.

DESCRIPTION (provided by applicant): The goal of this project is to identify the role of selectins in the trafficking and responses of Th1, Th17, and CD8 cells to nonpathogenic and pathogenic doses of influenza viruses in the lungs. These viruses are highly contagious, and cause yearly epidemics worldwide, with sporadic, devastating pandemics. This past influenza season in the northern hemisphere saw the emergence of a new triple reassortant H1N1 influenza strain carrying swine, avian, and human segments. This virus is now causing a worldwide pandemic. Amid growing concern that the new H1N1 virus will become more virulent and the possibility human of human transmission highly pathogenic H5N1 avian virus could occur, it is crucial to develop new strategies to control the morbidity and mortality associated with influenza viruses. Although CD4 and CD8 T cells typically control influenza virus infection in the lungs via cytotoxic- and cytokine-mediated responses, highly pathogenic strains can cause potent and aberrant inflammatory responses to which T cells contribute. It is therefore critical to understand how T cell responses to pathogenic vs nonpathogenic influenza viruses differ in the lungs to identify mechanisms that could be targeted to ameliorate pathological responses of T cells to this infection. Surprisingly little is known about the regulation of T cell trafficking into the lungs in the response to influenza viruses or how the magnitude of inflammation could affect their function. Our studies of the murine model of influenza have shown that Th1 and Th17 cells arise in the primary response with doses of virus that produce immunity and limited morbidity as well as with doses that are highly pathogenic. In the model of immunity, these effector cells, as well as responding CD8 cells, acquire the capacity to bind P-selectin, an endothelial selectin which initiates migration into sites of inflammation. In the absence of functional selectin ligands or PSGL-1 (P-selectin glycoprotein ligand-1), a major ligand for selectins on T cells, CD4 T cells have aberrant expansion and elevated production of IFN-3 and IL-17 in the draining LN, as well as greatly impaired accumulation in the lungs. The data suggest the hypothesis that selectin-ligand interactions could have a dual role in regulating T cell trafficking into the lungs as well as in dampening the effector response. In this application, we propose in vivo and in vitro studies to test this hypothesis using mice that are deficient in PSGL-1, or are unable generate functional selectin ligands, IFN-3 and IL-17 reporter mice, selectin fusion proteins, and WT and engineered influenza viruses. These tools will enable us to assess the responses CD4 cells and CD8 cells in the following specific aims: 1) to evaluate the role of selectins in T cell trafficking and localization in the lungs after infection with non-pathogenic and pathogenic doses of influenza virus;and 2) to determine the role of selectins regulating the responses of effector T cells. We hope to gain new insights into the regulation of effector T cells through mechanisms that control adhesion via selectins that could be targeted to help protect the population from the consequences of pathogenic influenza viruses. PUBLIC HEALTH RELEVANCE: If we find previously unidentified roles for selectins T cell recruitment or responses in the lungs after influenza infection, we will have identified a new mechanism of T cell regulation in the pulmonary response to this virus. If targeting of cells via selectins can reduce inflammation, synthetic selectin inhibitors that are already in use in murine models of disease might offer a new clinical approach to control pathogenic responses to influenza viruses.

DESCRIPTION (provided by applicant): Type 1 diabetes (T1D) is currently an incurable disease because the autoimmune response that destroys insulin-producing b islet cells cannot be controlled. Autoreactive CD4 cells are key orchestrators of T1D. Their escalating responses ultimately escape regulation, leading to acute inflammation in the pancreas and diabetes onset. While it is clear that IFN-g producing Th1 cells are essential to disease pathogenesis, whether or when IL-17-producing Th17 cells contribute to T1D is unknown. Th17 cells that secrete IL-17A and to a lesser extent IL-17F have been implicated in several autoimmune diseases. Our studies show that IL-17A- and IFN-g- secreting CD4 T cells are distinct subsets in pancreatic infiltrates during the insulitis phase and at T1D onset in NOD mice, whereas IL-17F is not detected. In addition, elevated serum levels of IL-17A and IFN-g herald the onset of hyperglycemia. We now find that IL-17A+ CD4 T cells greatly accelerate diabetes in normal (WT) NOD mice. On the basis of our findings and the detection of IL-17A+ cells in T1D patients, we hypothesize that Th17 cells are important contributors to the pathogenesis of T1D. However, without additional tools, it will not be possible to directly address mechanisms that regulate the responses of these cells or their effector functions that could be targeted to control the autoimmune response. Our goal is to develop a novel IL-17A reporter mouse on the NOD background that will enable analysis of the development and responses of Th17 cells in situ by lineage tracing during progression to T1D and, in a future R01 grant we plan to pursue studies of the regulation of Th17 cells in T1D. We propose to use bacterial artificial chromosome (BAC)-based gene reporter and gene knockin strategies to create mice that will enable identification and isolation of CD4 cells that acquire Il17a gene expression, as reported by expression of a GFP-Cre fusion protein inserted in the Il17a locus. We will use a double reporter strategy by breeding these animals to Rosa26-YFP NOD mice to indelibly mark cells with a history of IL-17A production. These mice will provide a novel tool to unequivocally study the contributions of Th17 cells to T1D and the potential functional interrelationships between Th1 and Th17 cells that lead to diabetes onset. The ability to identify changes in the development of Th17 cells and their functions will facilitate the design of appropriate strategies to control autoaggressive CD4 cells and provide new insights into the biology of Th17 cells.

DESCRIPTION (provided by applicant): The goal of this project is to identify mechanisms by which the adhesion receptor, PSGL-1 (P-selectin glycoprotein-1) regulates the establishment of a chronic LCMV infection and to assess whether targeting this receptor represents a clinically translational approach to treating chronic viral infections. With more than 500 million people infected with HIV, HCV, and HBV worldwide, chronic viruses represent a major global health problem. The murine model of chronic LCMV infection has been widely demonstrated to have clinical relevance to human chronic infections. Although numerous anti-viral compounds and biologicals can inhibit viral replication, current therapies are associated with significant toxiciy and/or immunopathology. It is therefore critically important to develop new targets for treat these infections. Of significance, our studies suggest that ligation PSGL-1 on T cells may be a pivotal event that limits the effector T cell accumulation and leads to dysfunctional virus-specific T cell responses that underlie chronic viral infections. Our data show that the chronic LCMV virus (Clone 13/Cl 13) is unable to establish chronicity in mice that are genetically deficient in PSGL-1, a major receptor for the selectin family of adhesion molecules (P, E, and L). Instead, there are dramatic increases in the numbers of virus-specific CD8+ T cells, deletion of virus-specific T cells is curtailed, and persisting anti-viral T cells fail to develop the exhausted phenotype characterized by sequential loss of effector functions. Virus-specific T cells from PSGL-1 KO mice express greatly reduced levels of immune inhibitory receptors, resulting in enhanced CD8+ T cell functional responses during chronic infection. In addition, the absence of PSGL-1 also supports enhanced virus-specific CD4+ T cell responses to LCMV Cl 13. Most notably, the preservation T cell functionality is associated with viral clearance and severe immunopathology, highlighting a critical function for PSGL-1 as an immune regulator during chronic infection. The data show that there is a broad impact of PSGL-1 on T cell chronic anti-viral responses and support the hypothesis that PSGL-1 is a previously unknown key to negative regulation of T cell immune function. On the basis of our findings we propose to investigate the mechanisms engaged by PSGL-1 to limit anti-viral immunity and explore therapeutic approaches to target this receptor to enhance anti-viral effector T cell responses.

PROJECT SUMMARY The goal of this project is to determine if targeting the selectin-ligand, PSGL-1 (P-selectin glycoprotein-1), represents a novel strategy to improve immune-mediated destruction of melanoma potentially by relieving multiple aspects of immunosuppression, a key element that must be achieved for tumor eradication. PSGL-1 is expressed exclusively by hematopoietic cells, and is a highly conserved ligand for the selectin family of adhesion molecules, P, E, and L, that regulate cell migration, but also binds other ligands that include chemokines and extracellular matrix components in both humans and mice. Our studies of chronic viral infections indicate that PSGL-1 is a previously unrecognized negative regulator of T cell responses that can also prevent effective anti-tumor responses to melanoma. In a chronic viral infection model with the LCMV-variant, Clone 13, PSGL-1-deficient mice completely prevent the establishment of chronicity. These mice developed CD8+ and CD4+ effector T cells that were multifunctional and failed to develop the hallmarks of exhaustion characterized by expression of multitude of inhibitory receptors including, PD-1, Lag3, CD160, BTLA and Tim3. PSGL-1-deficient T cells had improved survival leading to maintenance of a significantly greater number of functional effector T cells. Mechanistically, ligation of PSGL-1 on exhausted CD8 T cells inhibited TCR signaling and was linked to upregulation of PD-1. In two melanoma cancer models where T cells are also suppressed, we found that PSGL-1 was highly expressed on all hematopoietic cells in the tumor microenvironment (TME). PSGL-1-deficient mice showed dramatic control of melanoma growth that was coupled to greater numbers of tumor infiltrating, multifunctional CD8+ and CD4+ T cells (TILs) that expressed lower levels of inhibitory receptors. Since targeting PSGL-1 leads to downregulation of multiple inhibitory receptors and promotes effective anti-tumor responses, there may be significant advantages to blocking this receptor compared to combinations of inhibitory receptors, particularly if toxicity is less, or not greater than with current checkpoint blockades. Furthermore, blocking PSGL-1 may have the potential to limit immune suppression in patients that are unresponsive or have suboptimal responses to other therapies. For this project we have generated PSGL-1 conditional KO (PSGL-1fl/fl) mice to interrogate the function of this receptor on lymphoid and myeloid cells in the melanoma TME by cellular and transcriptome analyses. We will use pharmacologic approaches with genetic validation to determine if blocking PSGL-1 function can control melanoma growth by modulating TILs. These studies will provide new insights into the regulation of immune responses by PSGL-1 and mechanisms by which this receptor can contribute to T cell dysfunction in cancer. Most importantly, these studies will establish whether PSGL1 is a new target for immunotherapy of cancer.

PROJECT SUMMARY Effective immune responses require a finely tuned balance of positive and negative signals that support T cell activation, effector development, and protective memory generation, and at the same time provide means for timely termination to prevent pathology. Our studies of murine models of chronic virus infection and aggressive melanoma indicate that the adhesion receptor, PSGL-1 (P-selectin glycoprotein ligand-1, Selplg), a ligand for the selectin family of C-type lectins (E, P, and L), unexpectedly acts as a key negative regulator of CD4 and CD8 effector T cells. Our goals are to generate novel tools that will enable us to identify cell intrinsic mechanisms by which PSGL-1 impacts the responses of T cells as well as other hematopoietic cells that participate in immune regulation and to determine if PSGL-1 represents a new immune checkpoint that can be targeted by mAb blocking to improve immunity in the settings of chronic virus infection and cancer. In the murine model of chronic virus infection with Clone 13 LCMV, which has been widely demonstrated to have clinical relevance to human chronic virus infections (e.g. HIV, HCV, and HBV), we discovered that PSGL-1 deficiency unexpectedly supported viral clearance. This striking outcome was attributable to enhanced T cell intrinsic survival in addition to dramatically increased effector functions of both CD4 and CD8 T cells that was associated reduced expression of the immune inhibitory receptors, PD-1, BTLA, and CD160 that in combination contribute an ?exhausted? state. In exhausted CD8 T cells, PSGL-1 inhibits TCR signals, upregulates PD-1, and limits responsiveness to the ?c family cytokines, IL-7 and IL-2. To gain further insights into roles of PSGL-1 in regulating immune responses in which effector T cells become disabled, we analyzed the responses of PSGL-1 KO mice to an aggressive melanoma line derived from a genetic inducible model that recapitulates many aspects of the human cancer. We find high PSGL-1 levels on tumor infiltrating T cells, dendritic cells, macrophages, and myeloid-derived suppressor cells. Tumor growth was delayed with in PSGL- 1 deficient mice, with preservation of cytokine production by CD4 and CD8 T cells that was linked to lower expression of PD-1. We hypothesize that PSGL-1 can act as a ?brake? that cooperates with immune inhibitory mechanisms to limit the numbers and responses of CD4 and CD8 T cells, which in the contexts of chronic virus infection and cancer contributes to the inability to generate a productive effector response. To test this hypothesis, we propose to develop a PSGL-1fl/fl mouse line that can be used to study conditional and inducible genetic deficiency of PSGL-1 in subsets of T cells and innate cells and to develop PSGL-1 blocking mAb to determine if loss of PSGL-1 engagement after chronic infection or melanoma become established can support reversal of T cell exhaustion an inhibit the expression of PDL-1 and other inhibitory receptors. These tools will enable proof-of-concept studies to determine if targeting PSGL-1 can be a clinical approach to augment adaptive immunity and the mechanisms that contribute to its underappreciated roles in immune regulation.

PROJECT SUMMARY New advances in immunotherapy for cancer, based on decades of fundamental research on the regulation of T cells, have yielded tremendous optimism that even aggressive and fatal cancers, including metastatic melanoma, can one day be cured in the vast majority of patients. However, current approaches benefit only a subset of patients, and new insights into immune processes are urgently needed to define the means to improve therapies and achieve clinical benefits for a variety of different cancers. We recently discovered that the adhesion receptor, PSGL-1 (P-selectin glycoprotein ligand-1), is a new checkpoint inhibitor of T responses to chronic viral infection and a potent regulator of anti-tumor responses. The goal of this project is to determine if targeting PSGL-1 (Selplg) in tumor-specific CD8+ and/or CD4+ T cells represents a strategy to mitigate tolerance of melanoma tumors when used for adoptive cell therapy alone and when combined with additional approaches to alleviate immune suppression. Our studies showed that genetic deletion of PSGL-1 led to downregulation of multiple inhibitory receptors that are hallmarks of dysfunctional or ?exhausted? T cells including PD-1, LAG3, CD160, BTLA and TIM3, indicating that PSGL-1 modulates expression of other checkpoint inhibitors. PSGL-1-deficiency did not alter T cell migration into tissues, but rather enabled CD8+ and CD4+ T cells to mount much greater effector responses due to enhanced survival of multifunctional T cells that mediated effective anti-viral and anti-tumor responses. Our results demonstrate that this receptor may be a novel target with the potential to reverse immune suppression in patients whose cancers are unresponsive or have suboptimal responses to other immunotherapies. In mice, PSGL-1- deficiency led to dramatic control of melanoma tumor growth that was coupled with greater numbers of tumor infiltrating, multifunctional CD8+ and CD4+ T cells that expressed lower levels of PD-1. However, when tumor- specific CD8+ T cells were used to treat tumor-bearing mice by adoptive transfer, tumor control was not sustained, indicating loss of T cell anti-tumor response. In this application, we will test the hypothesis that genetic deletion of PSGL-1 in tumor-specific CD4+ and CD8+ T cells used for adoptive cell therapy, particulalry in combination, can dramatically enhance anti-tumor responses (Aim 1), but their functionality and preservation can be optimized when other mechanisms underlying T cell exhaustion are concurrently inhibited (Aim 2). We will also address whether TCR ?engineered? T cells can form memory and whether checkpoint blockade is advantageous or deleterious for memory T cell induction.

Project Summary This application is set to define a novel mechanism underlying the control of gut microbiota-immune checkpoint interactions by the ubiquitin ligase RNF5, and the implications of such regulation to melanoma development and response to therapy. Despite the most exciting and significant advances made in clinical management of melanoma via the immune-checkpoint based clinical trials, mechanisms underlying their control, the susceptibility to distinct therapies (or not), are among the fundamental questions that remain unsolved. Here, we provide data to support a model whereby immune checkpoints are regulated by gut microbiome, which is defined by the ubiquitin ligase RNF5. Our discovery of impaired growth of Braf/Pten mutant melanoma in syngeneic Rnf5?/? mice, compared with Rnfwt littermates, was linked with enhanced infiltration of tumor- infiltrating lymphocytes (TILs) (CD4 and CD8 positive), macrophages and dendritic cells in the tumors that developed significantly slower in the Rnf5?/? mice. Strikingly, co-housing the Rnf5?/? and Rnfwt animals or antibiotic ablation of the gut microbiota resulted in loss of the above phenotypes?tumor growth was no longer attenuated and immune checkpoint-based phenotypes were largely lost. Assessment of the gut microbiome revealed a distinct subset of bacterial species, which distinguish Rnf5?/? mice from their WT littermates (all ?pure? in-house maintained C57BL/6 strain). Notably, common to the distinct RNF5-microbiome are bacterial species that generate select subset of short chain fatty acids. These observations provide the foundation for our hypothesis that RNF5 controls the intestinal microbiota that affects immune checkpoint mechanisms, which in turn impacts melanoma development. Our proposed studies will (i) define RNF5 effect on the gut microbiome?tumor interactions, (ii) identify microbiome-dependent changes in the regulation of tumor immune checkpoint control by RNF5, and (iii) establish the physiological significance and impact of RNF5 control of microbiome and immune checkpoint on melanomagenesis in different mouse strains, age, and under select combination therapies. Our highly integrated studies will define the fundamental mechanisms that underlie the regulation of both gut microbiome and immune checkpoints, thereby providing new insights into therapeutic modalities for melanoma and other cancers.

PROJECT SUMMARY The goal of this project is to identify potential mechanisms by which CD44 acts as an immune checkpoint inhibitor in the setting of chronic virus infection and whether CD44 represents a prospective therapeutic target to reverse T cell dysfunction. Our studies using the LCMV (lymphocytic choriomeningitis virus) Cl13 chronic infection model show that Cd44 deficiency supports viral clearance rather than chronic infection. Virus- specific CD8 and CD4 T cells became multifunctional as measured by cytokine production, and downregulated PD-1 as well as several other inhibitory receptors. The effective anti-viral response was accompanied by 30% mortality and immune pathology, underscoring that a key brake on the immune response had been released. Cd44-deficiency exclusively in T cells or in hematopoietic cells did not prevent chronic Cl13 infection or T cell exhaustion. Notably, however, Cd44-deficiency only in non-hematopoietic cells promoted T cell responses that led to viral clearance. This outcome was not due to differences in the infectivity of Cl13 in WT vs Cd44-/- mice. Thus, our findings reveal an unrecognized, novel immune inhibitory function of CD44 that is independent of its direct roles in regulating T cells. To initiate studies of non hematopoietic cells in CD44- dependent immune regulation of the response to Cl13, we first focused on fibroblastic reticular cells (FRCs) since these cells are potent regulators of T cell immune responses and contribute to the immune suppression in the Cl13 model. We found that Cd44-/- FRCs had lower expression of Nos2 than WT FRCs after infection, and that independent of infection, Cd44-/- FRCs produced less NO than WT FRCs when stimulated by IFN-? and TNF?, indicating an FRC intrinsic regulatory role of CD44. Cd44-deficiency was also linked to dramatic downregulation Trim11, an E3-ubiquitin ligase that can block IFN-? production, which we found to be produced in higher levels by Cd44-/- FRCs. We did not find differences in expression of PD-L1, IL-7, or Ccl19/21 that could impair T cell responses. From these results, we hypothesize that during chronic infection, CD44 expression by FRCs promotes NO production and limits IFN-? production thereby contributing to inhibition of T cell responses, promoting T cell exhaustion, and supporting viral persistence. However, FRCs produce other inhibitory mediators which we have not yet tested for CD44-dependent regulation that could also impact the anti-viral response. Based on the differences WT and Cd44-/- FRCs we have identified to date, our goal is to determine whether CD44 expression by these cells plays a fundamental role or ancillary role in the regulating T cell responses, to identify the ligands of CD44 that regulate FRC suppressive function, and to identify signaling mechanisms that regulate FRC responses.

PROJECT SUMMARY This proposal seeks support to establish an innovative multi-thematic postdoctoral training program in fundamental and translational immunology to address the role of innate and adaptive immune cells in various disease contexts. Emphasis is placed on understanding the cellular and molecular basis of immune cell activation in the appropriate context of an immune response to infectious organisms and toxins, or the dysregulation that occurs in the context of chronic inflammation and autoimmunity. These fundamental insights provide the basis for novel therapeutic approaches in infectious disease, autoimmunity and cancer that will be tested in pre-clinical models. Sanford Burnham Prebys Medical Discovery Institute (SBP) recently implemented its 10-year strategic vision, which focuses on four disease priorities (cancer, autoimmunity, metabolic and neurological disorders) and four cross-cutting scientific themes including epigenetics, metabolism, proteostasis and immunomodulation. The proposed Program is an important element of this vision as we strive to provide the best training environment for postdoctoral scientists interested in immunology research. Scientific research at the Institute is supported by an infrastructure that includes multiple core facilities, thereby providing access to advanced technologies and expert, dedicated personnel. SBP emphasizes a collaborative approach to biomedical research, where teams of scientists with expertise in the biological sciences work together with chemists, structural biologists, engineers, and computational biologists to achieve synergistic accomplishments. This collaborative approach to science is intimately integrated into the culture of the Institute, as demonstrated by the fact that ~30% of all SBP publications are the result of inter- and intra- programmatic collaborations. Notably, 35% of over 500 publications from the preceptors' laboratories are led by postdoctoral trainees who serve as first authors. Postdoctoral trainees in this T32 will join one of 13 research groups focused on aspects of immunology. The trainees will be guided by mentoring committees focused on promoting scientific progress and career development. Seminar programs, workshops, retreats and journal clubs will enrich research training. The trainees will be selected based on scientific and academic accomplishments, suitability for research projects in the thematic areas and commitment to a scientific career that builds upon the postdoc experience. Trainees will be supported for an average of 2 years, but will remain with the program upon achieving independent support. The Program will support three trainees within the network of postdoctoral researchers at SBP. It is expected that matriculants who have completed this Program will have received a well-rounded training experience in cutting-edge patient-focused immunology research and will be well prepared to embark on a variety of independent research and research-related careers in fundamental and translational immunology.

5.4 Abstract - FLOW CYTOMETRY (Core Group A) The Flow Cytometry Shared Resource (Core Group A) offers Cancer Center users multi-parametric evaluation and high-speed enrichment of phenotypically distinct cell populations using flow cytometry. For analytical flow cytometry, investigators have a choice of full-service analysis, or receiving training for independent use of four analytical flow cytometers (BD LSRFortessa, BD FACSCanto, BD FACSCalibur, Millipore Muse). Investigator training and consulting is also provided on analytical software including FACSDiVa, CellQuest, FlowJo, and ModFitLT. For high-speed cell sorting, the Core provides experimental planning and full-service cell sorting by two expert operators, utilizing two BD FACSAria sorters in biosafety cabinets. The Resource is staffed by two full-time cytometrists with a combined 18 years of experience in NCI- designated Cancer Center flow cytometry cores. Resource staff provide investigators with extensive flow cytometry guidance, from experimental set-up, selection of reagents, setting gating or sorting parameters, data interpretation, and development of new methods. The Core is widely used and, as in the past funding period, services were provided for over 40 Cancer Center members, representing all three Programs, and Flow Cytometry supported key experiments described in at least 91 cancer-relevant publications from Cancer Center members. There have been quite dramatic upgrades to the Core in the past funding period. A half-time staff member assisting the Director was replaced by an accomplished full-time cytometrist, and the Resource implemented iLab web-based software to streamline Core scheduling, project management, and billing. Technology upgrades to the core include the addition of two BD FACSAria sorters (11-color and 16-color) housed in custom biosafety cabinets, a 14-color BD LSRFortessa analyzer with a 96 well plate-loader, and a Millipore Muse Cell Analyzer. An Amnis ImageStreamX Mark II imaging cytometer was purchased and will be installed in May 2014. This instrument with three lasers combines flow cytometry with high resolution microscopy to enable spatial and morphological analysis of cells in flow, including rare cells. Plans for future development of the Resource include evaluation of hardware for time-of-flight mass cytometry, microfluidic sorting of circulating tumor cells, and automated magnetic separation.

PROJECT SUMMARY Immunity to infectious disease and cancer is dependent on a robust T cell response to clear the pathogen or compromised cells and establish a memory population to protect against future challenge. Understanding T cell biology including T cell activation, differentiation, and memory formation can thus guide future studies on the development of novel therapies to augment this response and impact immunological memory. Although studies demonstrate the contributions of both CD4+ and CD8+ T cells to the effector response and memory formation with pathogenic infection, many are focused on the CD8+ T cell compartment and fail to appreciate the contribution(s) of CD4+ T cells. Our studies recently identified PSGL-1 as an inhibitory receptor expressed on T cells and showed that deletion of PSGL-1 led to reduced exhaustion in both the CD4+ and CD8+ T cell compartments in the context of chronic virus infection with LCMV clone 13 (Cl13). This is evidenced by the ability of PSGL-1-deficient mice to clear LCMV Cl13 infection much earlier than their wildtype counterparts. However, this ability to clear the chronic infection is lost upon CD4+ T cell depletion, demonstrating the importance of this population in the immune response necessary for the resolution of infection. We also found that CD4+ T cells, like CD8+ T cells, display greatly enhanced effector responses to acute infection with LCMV Armstrong, with greater persistence of functional memory cells. The goal of the studies presented in this proposal is to further elucidate the contribution(s) of CD4+ T cells in both acute and chronic infection and memory generation and to further understand how PSGL-1-deficiency confers the capability to clear infection. To this end, the contribution of PSGL-1 deficient CD4+ T cells will be assessed using adoptive transfer of antigen-specific PSGL-1 deficient CD4+ T cells. As the CD4+ T cells population forms a more heterogenous population that is known to change with the temporal progression of infection, single-cell sequencing will be used to identify changes in CD4+ T cells. PSGL-1-deficient mice are capable of clearing LCMV Cl13 which otherwise establishes a chronic infection in wildtype mice; the mechanism(s) by which this occurs will be interrogated by studying changes in motility with PSLG-1-deficiency and the impact of antigen load. As PSGL-1 has been demonstrated to affect T cell activation and function, it represents a therapeutic target for altering T cell differentiation and memory formation; conditional deletion and antibody mediated modulation of PSGL-1 signaling will be studied in both acute and chronic models of infection to further elucidate the role PSGL-1 plays in the T cell compartment. The studies presented here offer a means to understand how modulation of PSGL-1 signaling could augment T cell function and memory generation and impact on the ability of CD4+T cells to augment CD8+ T cell responses that could be used in context of infection and cancer as well as vaccine design to improve overall immunity.

SUMMARY The goal of this project is to address metabolic regulation of exhausted T cells (TEX) by the immune inhibitory receptor (IR), PSGL-1 (P-selectin glycoprotein 1) and to determine if blocking its engagement on T cells can reverse metabolic constraints on tumor infiltrating T cells (TILs) to support antitumor immunity. Effector CD8+ T cells (TEFF) are vital in eliminating tumor cells, including melanoma, but within tumors, T cells become progressively dysfunctional and unable to destroy tumors. Exhaustion is associated with a loss of metabolic fitness and with increased co-expression of IRs, including PD-1, LAG3, TIM3, and CTLA-4. Although immune checkpoint blockade (ICB) of PD-1 and CTLA-4 can reinvigorate TEX and eradicate tumors in some patients, in melanoma, the majority (60%) of patients are refractory to treatment or fail to achieve durable responses. For many other cancers, response rates to ICB are much lower. It is therefore essential to identify novel mechanisms to more broadly induce effective antitumor responses and provide benefit to a far greater number of patients. Recent studies indicate that alterations of T cell metabolism within the tumor microenvironment underlie the development of TEX and their rescue with ICB. Intratumoral T cells can lose mitochondrial function and biogenesis, and exhibit decreased glycolysis and increased fatty acid oxidation. Changes in tumor cell metabolism are also linked with T cell responses, with oxidative tumor cell metabolism linked to increased TEX, and glycolytic tumor cell metabolism coupled to responses to PD-1 blockade. It is therefore critical to identify checkpoint regulators that support T cell metabolic parameters that underlie effective antitumor responses. We discovered that PSGL-1 (Selplg) is a previously unknown T cell intrinsic IR that promotes expression of multiple IRs on CD4+ and CD8+ T cells in the contexts of chronic viral infection and tumors, underscoring an integral connection to immune inhibitory pathways. Our studies suggest that PSGL-1 is a key inducer of the IR gene module that promotes TEX. Importantly, Selplg-/- mice demonstrate dramatic control of melanoma tumors and transfer of PSGL-1-deficient tumor-specific CD8+ T cells is sufficient to significantly delay tumor growth. Our new data show that Selplg-/- CD8+ T cells have increased glycolysis compared to their Selplg+/+ counterparts, and single-cell RNA-sequencing (scRNA-seq) analysis of Selplg-/- CD8+ TILs identified greatly increased expression of several genes associated metabolic function. Thus, we hypothesize that blocking PSGL-1 will provide a mechanism of metabolic reprograming of antitumor T cells and subvert inhibition by multiple IRs, thereby enhancing antitumor responses. To test this scientific premise, we propose to define the intrinsic contribution(s) of PSGL-1 signaling to the antitumor T cell metabolome and to assess whether PSGL-1-targeted ICB promotes metabolic changes in TILs that support effective T cell antitumor responses to melanoma using Seahorse assays and the novel scRNA-seq technique, CITE-seq, to evaluate the interplay between metabolic changes in Selplg-/- CD8+ T cells and melanoma tumor cells.

ABSTRACT ? TUMOR MICROENVIRONMENT AND CANCER IMMUNOLOGY PROGRAM The overarching goal of the Tumor Microenvironment and Cancer Immunology (TMCI) Program is to define how the dynamic interplay among cancer cells, immune cells, stromal components, and vasculature regulates the growth and dissemination of malignancies, and in so doing, identify therapeutic approaches to modulate the microenvironment and tumor growth. The Program consists of 13 faculty (one a new recruit) and five adjunct members, and integrates members whose expertise includes cell migration/invasion, molecular structures, cell signaling, cell metabolism, and angiogenesis, with members whose work encompasses therapeutic targeting of innate and adaptive immune cells, and the influence of the microbiome on tumor- and immune cell-directed cancer therapies. This complementary expertise is organized around three interacting themes: (1) Activating Invasion and Metastasis; (2) Avoiding Immune Destruction, and (3) Tumor Promoting Inflammation. These themes encompass many of the molecular processes that coordinate the formation of the microenvironment that enables progressive tumor growth and metastasis. Members interact on several of levels, including monthly faculty meetings, program-led seminars (48 in the last funding period), strategic meetings organized around new collaborative opportunities (for example, the interface of the human microbiome and cancer), and collaborative grants. Program funding is strong, with current total annual grant funding of $4.4M (direct costs) ($2.6M from NCI, 58%). Members currently lead 28 grants including 15 R01s (nine from NCI), and lead or participate in three P01s (two from NCI), and multiple other grants. Over the last funding period, members have participated in 34 (32%) collaborative grants. Our productivity is reflected in 224 cancer-relevant publications in the last funding period, of which 31% were collaborative (19% intra- and 12% inter-programmatic). In 2018, we published 42 cancer-relevant publications, of which 21% were intra- and 5% inter-programmatic. TMCI members have pioneered novel approaches to interrogate the fundamental properties of tumor cells and other cell types within tumors, and are developing immune cell- and tumor-targeted therapies. Members are participating in translational initiatives through the Oncology Disease Teams, C3 Cancer Center Council, and the San Diego Center for Precision Immunotherapy, which support collaborations with local oncologists to enable large collaborative grants applications. In recognition of the role of the immune system in combatting cancer, a key goal of the Program is to build and further strengthen expertise in cancer immunology by hiring at least two additional faculty, including one with expertise in vaccinology. Key scientific goals are to extend the use of infection models to inform studies to harness the immune system for immunotherapy and cancer vaccines as well as to integrate single-cell transcriptomics, proteomics, epigenetics, and metabolic profiling and imaging of cancer cells, immune cells, and stromal cells in tumors to achieve new insights into the regulation of tumor growth outcomes and cellular processes that lead to drug resistance and immune evasion by tumors.

PROJECT SUMMARY The goal of this project is to evaluate novel therapeutics that block PSGL-1 (P-selectin glycoprotein-1), which we identified as a key inhibitory receptor (IR) that is expressed on T cells, and address the impact(s) on immune cell functions in the tumor microenvironment (TME). PSGL-1 is a conserved ligand for the selectin family of adhesion molecules, P, E, and L, that is expressed on most hematopoietic cells and regulates leukocyte migration when fully glycosylated. T cells are the only cells that express the non-selectin binding form of PSGL- 1 that serves as a receptor for the lymph node chemokines, CCL19 and CCL21, and the PD-L1 homologue, VISTA (B7-H5). We showed that PSGL-1-deficient (Selplg-/-) CD8 T cells fail to acquire the hallmarks of exhaustion compared to their wild-type (WT) counterparts after infection with chronic lymphocytic choriomeningitis virus and significantly limit tumor growth in two murine models of melanoma, one that is nonresponsive to anti-PD-1 treatment. We identified that TILs from Selplg-/- mice have greater anti-tumor function, including cytotoxicity and cytokine production. Moreover, expression of multiple inhibitory receptors (IRs) that distinguish exhausted T cells (TEX) were downregulated on Selplg-/- T cells, including PD-1, BTLA, CD160, LAG3, and TIM3. Notably, PSGL-1 ligation on naïve T cells (TN) and TEX by an agonist mAb in the context of T cell receptor stimulation induced/augmented expression of multiple IRs, underscoring an integral connection of PSGL-1 to immune inhibitory pathways and suggesting a link to the recently identified inhibitory gene module that regulates the coordinated transcription of IRs. We identified that a monovalent Fab of the same agonist mAb dramatically improved T cell responses as did treatment with a rPSGL-1 fusion protein, indicating a capacity to block PSGL-1 function. Notably, blocking PSGL-1 did not lead to widespread inflammation or toxicity. We hypothesize that PSGL-1 blockade has the potential to augment anti-tumor T cell responses by limiting expression levels of multiple IRs, and alter immunosuppression in the TME by impacting other immune cells. We propose to evaluate the effects of PSGL-1 blockade on mouse and human T cell anti-tumor responses using rPSGL-1 and Fab anti-PSGL-1 as comparators to novel mouse and human anti-PSGL-1 blocking mAbs. With these tools, we will analyze the impacts of blocking PSGL-1 on IR expression and functions of T cells and immune cells in the TME, as well as potential synergy with anti-PD-1. In the context of these studies, we will assess the role of VISTA as the relevant PSGL-1 ligand in the TME. These studies will address the potential of targeting PSGL-1 for reversal of T cell exhaustion, induction of anti-tumor immunity, and translational potential.