Marco Colonna - US grants

DESCRIPTION (provided by applicant): NK cells provide first line surveillance against cells that have been infected by viruses or undergo neoplastic transformation. Among the numerous cell surface receptors mediating NK cell recognition of target cells, NKG2D is unique in that it recognizes a variety of class I-related molecules that serve as "flags" for infected or abnormal cells. In vitro studies indicate that NKG2D depends solely on the transmembrane adapter DAP10 for signaling. DAP10 contains an YxxM motif that recruits Phosphatidyl Inositol-3 Kinase. The same docking motif has been reported in CD28, which mediates a costimulatory signal in naive T cells. Therefore, NKG2D/DAP10 is currently considered a costimulatory complex on all NK cells as well as on activated cytotoxic T cells. This functional parallel between NKG2D/DAP10 and CD28 raises several important issues: Do NK cells require a costimulatory signal to kill their targets and/or proliferate in vivo? Is NKG2D/DAP10 involved in priming and expansion of CD8+ T cells in peripheral lymphoid organs, similar to CD28? Or, does NKG2D/DAP10 mainly enhance CTL effector responses against cells expressing NKG2D ligands in peripheral tissues? To address these crucial questions we have generated DAP10-deficient mice by gene targeting. Our preliminary results indicate that in NK cells, NKG2D has the capacity to associate not only with DAP10 but also with the adapter DAP12, which mediates direct activation via a distinct intracellular signaling pathway. In contrast, in T cells, NKG2D associates only with DAP10 and therefore is limited to the YxxM "costimulatory" pathway. On the basis of these results, we propose to dissect the role of NKG2D/DAP10 "costimulatory" and NKG2D/DAP12 "activating" pathways in NK cell biology by comparing NK cell function in DAP10 -/- mice with that of DAP12 -/- and normal mice in vitro and in vivo. In addition, we propose to clarify the role of the NKG2D/DAP10 "costimulatory" pathway in anti-viral and anti-tumor CD8+ T cell responses in vivo. For this purpose, we will analyse activation, expansion and acquisition of effector function of DAP10 -/- CD8+ T cell in mouse models of viral infection and tumor engraftment. In addition, we will study the requirement of DAP10 for establishment and maintenance of long-term memory CTLs following primary infections and tumor vaccination. While previous studies have indicated that expression of NKG2D ligands on virally infected and tumor cells is important for generating a protective immune response, it is not understood why and where NKG2D signaling is required during the response. Therefore, detailed knowledge of NKG2D/DAP10 function in vivo based on the analysis of DAP10-/- mice will be of great value, particularly in view of the possible exploitation of NKG2D-NKG2D-ligands interactions in tumor therapy and vaccine design.

DESCRIPTION (provided by applicant): Plasmacytoid Dendritic Cells (pDC) specialize in secreting high levels of type I interferons (IFNs) and proinflammatory chemokines in response to certain viruses. They also stimulate T cell proliferation and differentiation in vitro. Consequently, pDC are viewed as critical in protecting the host from incoming pathogens. This proposal focuses on the role of pDC in anti-tumor immune responses. In our preliminary studies, we analyzed a panel of MCA-induced sarcomas including tumors that are rejected by the immune system in wild type mice (immunogenic tumors) and tumors that grow progressively (non immunogenic tumors). Results demonstrate that: (a) effective rejection of immunogenic sarcomas requires the action of type 1 IFN s on host cells; (b) pDC infiltrate immunogenic MCA-induced sarcomas but not non-immunogenic tumors. Therefore, we hypothesize that pDC infiltrating immunogenic tumors may provide a major source of type I IFNs and proinflammatory chemokines that activate host anti-tumor immune responses. We are uniquely positioned to test this hypothesis because we can analyze the growth of sarcomas following elimination or functional paralysis of pDC by antibodies that specifically deplete or block pDC function in vivo. Our preliminary experiments also demonstrate that pDC infiltrate human melanomas, but appear to be functionally inactive, possibly due to immunosuppressive factors in the tumor microenvironment. Can we reactivate pDC within tumors? Because they express toll like receptor (TLR) 9 and TLR7, pDC can be activated in vitro and in vivo by CpG oligonucleotides and imidazoquinolins. Our preliminary findings demonstrate that CpG-stimulated pDC effectively delay growth of murine B16 melanomas. In addition, we have observed that CpG and one TLR7 ligand, resiquimod, differ in their ability to elicit type I IFN responses in pDC, yet trigger similar secretion of proinflammatory cytokines. Given this, we will test and compare efficacy of CpG- and resiquimod-activated pDC for immunotherapy of poorly immunogenic tumors, including: a) B16 metastatic melanoma; b) a mouse model of spontaneous breast carcinoma expressing a defined tumor-associated antigen, which will also allow us to study specific interactions between pDC, T cells and dendritic cells. Our preliminary findings validate the concept that activated pDC at the tumor site initiate and amplify anti-tumor immune responses. These findings also substantiate the physiological relevance of the proposed studies to clinical human cancer situations.

[unreadable] DESCRIPTION (provided by applicant): Nectin and Nectin-like (Nec-1) molecules are cell surface molecules that mediate cell-cell adhesion and are strongly expressed on tumor cells. NK cells and T cells express receptors that bind Nectins and Nectin-like (Necl) molecules. The importance of Nectin and Necl recognition in anti-tumor immune responses in vivo is unclear though in vitro these receptors allow lymphocytes to recognize and kill tumor cells. In this grant proposal, we will examine importance of the interaction between Necl-2, a nectin-like molecule, and its ligand class I-restricted T cell-associated molecule (CRTAM). In our preliminary studies, we have discovered that NK cells and CDS T cells recognize Necl-2, through a receptor known as (CRTAM). This interaction promotes target cell killing and IFN-y secretion in NK cells and CDS T cells in vitro. Furthermore, Necl-2 has been previously shown to suppress tumorigenesis in vivo and its expression is silenced in many epithelial tumors. We hypothesize that the CRTAM/Necl-2 interaction is a novel and important immunosurveillance strategy in vivo. Tumors may suppress Necl-2 expression to evade this immunosurveillance mechanism. In Specific Aim 1, we will evaluate the impact of CRTAM/Necl-2 interaction in short-term and long-term rejection of primary and metastatic tumors expressing Necl-2 in vivo. In Specific Aim 2 we will determine the impact of CRTAM/Necl-2 interaction on proliferation, cytokine secretion and anergy of anti-tumor CDS T cells using a model in which the tumor cells express a well-defined tumor-associated antigen. Moreover, we will assess if CRTAM/Necl-2 interaction enhances CDS T cell responses to tumor vaccines that either express or do not express a defined tumor-associated antigen. Our discovery and initial characterization of the CRTAM/Necl-2 interaction and its potential role in immunosurveillance places our laboratory in a unique position to examine this interaction in detail. We have extensive expertise in NK biology and priming of T cell responses, and we are in a privileged position to investigate whether recognition of Necl-2 by CRTAM allows the immunosurveillance network to distinguish tumor cells from normal cells in vivo. [unreadable] [unreadable]

[unreadable] DESCRIPTION (provided by applicant): The goal of this project is to definitively establish the role of the cell surface receptor TREM-1 in the Dathogenesis of sepsis. We hypothesize that activation of TREM-1 synergizes with ligation of the Toll-Like Receptors (TLRs) and amplifies the inflammatory response during sepsis. We will test this hypothesis with three specific aims. In the first specific aim, we will use a newly generated strain of mice in which we have humanized the TREM locus by deleting the murine TREM-3 gene (which is a pseudogene in humans) in tandem with the TREM-1 gene. This mouse is the ideal reagent to model the role of TREM-1 in human biology. Our preliminary data indicate that TREM-1/3 double deficient mice are more resistant to sepsis than wild type mice. We will rigorously test the response of this strain of mice in clinically relevant models of sepsis including cecal ligation and puncture and both gram-positive and gram-negative septic pneumonia. In the second specific aim, we will define the signaling pathway through which TREM-1 and the TLRs interact with a focus on identifying nodes in signaling cascades that could serve as therapeutic targets. The third specific aim is to identify the endogenous ligand for TREM-1. In our preliminary experiments we have established a system that detects a ligand on the surface of granulocytes of septic patients and have identified a monoclonal antibody that recognizes the TREM-1 ligand or a component of it. The ligand for TREM-1 represents a novel target for sepsis therapy. Our studies on TREM biology and our previous experience in the study of sepsis positions us well to succeed at our specific aims. We originally discovered and cloned the TREM genes and thus have an extensive collection of molecular-biological tools for manipulating the TREM system. We have successfully generated TREM-1/TREM-3 knockout animals to best model the role of TREM-1 in human biology. Importantly, we have well established collaborations with a multi-disciplinary group of investigators including active critical care physicians who can provide access to clinical materials and direction as to the clinical relevance of our data, experienced sepsis researchers who can provide expertise in animal models of sepsis, and an expert biochemist to collaborate in the identification of novel mediators. Taken together, we feel that this proposal will advance the study of sepsis by defining the biology of TREM-1 in sepsis, establishing the rnechanism by which TREM-1 amplifies inflammation and determining the ligand for TREM-1, a potential novel mediator of sepsis. In lay terms, the goal of this project is to define the role of the TREM-1 protein in the biology of sepsis, a leading cause of death in critically i patients. Our proposed experiments will define the function of TREM-1 using mouse studies that model the human disease and biochemical studies to identify the mechanisms by which TREM-1 functions in the cell. We also have detected a binding partner of TREM-1 (the TREM-1 ligand) and we propose to identify this molecule as it may serve as a novel target for the treatment of septic patients, [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): HIV-1 infection and mucosal immunity are intimately interrelated. Since the acquisition of HIV-1 primarily occurs through mucosal routes, local immunity at the site of infection is essential for controlling viral spread within the host and in transmitting virus to other individuals. Additionally, established HIV-1 infection compromises both innate and adaptive mucosal immunity, facilitating secondary infections and progression to acquired immune deficiency syndrome (AIDS). Thus, if we are to develop an effective vaccine for HIV-1 that protects the key target cells in the mucosa, we first need to understand how to induce protective anti-HIV-1 mucosal immune responses. We recently discovered a novel subset of tonsil and gut innate immune cells that express natural killer (NK) cell markers, but, in contrast to conventional NK cells, produce interleukin (IL)-22 and other cytokines that protect the integrity of the mucosal barriers during infectious and inflammatory diseases. We refer to these cells as NK-22 (Cella M, et al, Nature. 2009;457:722). Our preliminary data indicate that NK-22 cells directly counter HIV-1 replication in infected CD4 T cells in vitro. We also demonstrate that NK-22 cells secrete BAFF, a soluble factor that enhances B cell responses. Based on their unique properties, we propose that NK-22 cells may be crucial for innate and adaptive anti-HIV-1 mucosal responses. In our proposal we will determine the mechanisms by which NK-22 cells counter HIV-1 infection (specific aim 1), identify vaccine adjuvants that expand NK-22 cells in the mucosa (specific aim 2), and determine the impact of NK-22 cells on adaptive B cell responses against HIV-1 (specific aim 3). We are uniquely positioned to successfully address these aims because of our original discovery of human NK-22 cells, our broad expertise in NK cell biology, the strength of our preliminary data, the relatively straightforward experiments that we have chosen and the availability of reagents through our collaboration with the Center for HIV/AIDS Vaccine Immunology (CHAVI). Our proposal will significantly advance our knowledge of anti-HIV-1 mucosal immune responses and identify adjuvants that can be used to generate vaccines for HIV-1 that elicit effective mucosal immunity.

PROJECT SUMMARY Mucosal surfaces must allow the exchange of metabolites required for life into the host and excretion/exclusion of wastes and toxins out of the host, while also maintaining a first line of defense against invasive microbes. However, mucosal inflammation, elicited to combat microbial infection, can also damage the integrity of the mucosal barrier, if not controlled. Many mucosal surfaces are in constant or transient contact with microbes, yet the molecular mechanisms governing the extent of the inflammatory response at the mucosal surface, the healing of the surface after insults and the molecular basis of remodeling changes that affect subsequent mucosal function and inflammatory responses are poorly understood. This proposal will investigate these mechanisms by assembling an experienced team of investigators with diverse and complimentary expertise to further our understanding of the mucosal immune defense mechanisms of the urinary bladder against bacterial infection. Analysis of the bladder mucosal responses to the most common cause of urinary tract infection (UTI), E. coli, in distantly related inbred mouse strains has identified patterns of response that correlate with resistance or susceptibility to both initial and secondary infections. The proposed research will probe differences in the immune defense pathways of the bladder mucosa during acute and recurrent infection, providing critical insights in the field of mucosal immunology and the role of epithelial cells in mucosal response. These studies will specifically investigate the role of bladder mucosal remodeling in modulating innate signaling pathways in previously infected mice (Aim 1) and investigate the protective (Aim 2) and damaging (Aim 3) mechanisms of the acute bladder inflammatory response in naïve mice. These responses correlate with outcomes of disease and subsequent remodeling of the bladder tissue, which in turn affects subsequent infections. Understanding immune responses of previously infected vs. naïve mucosal tissues will likely give important insights into clinical disease, in which patients over their life-time have undoubtably suffered from multiple sequential infections. These investigations will reveal new details of the mechanisms of mucosal defense against bacteria, broadening the understanding of the regulation of mucosal inflammation and the signaling between mucosal epithelia and immune cells, and thus advance our understanding of acute and recurrent infection susceptibility and protection, in this age of increasing microbial antibiotic resistance. These insights will contribute to the development of novel vaccines and immunomodulatory therapeutics targeting the mucosa.

DESCRIPTION (provided by applicant): This renewal application for years 31-35 of a structured, basic research-oriented Diabetes Training Program consists of 14 faculty members to train Ph.D. scientists & physicians to investigate problems of Types 1 & 2 diabetes. Faculty number & composition has been trimmed to enhance research directly related to diabetes including: 1) insulin secretion & (-cell proliferation, 2) development of endodermal lineages including (-cells, 3) processing & presentation of antigens by histocompatibility molecules in Type 1 diabetes, 4) immune tolerance & autoimmunity in Type 1 diabetes, 5) KATP channels & neonatal diabetes, 6) Ca¿+-independent phospholipase A2 & (-cells 7) insulin resistance/diabetes in adult HIV disease, 8) diabetic neuropathy, 9) glucose transporters & Type 2 diabetes , 10) lipid synthesis, metabolism & vascular diseases in diabetes, 11) obesity, diabetes & nutrition, 12) diabetes-induced pregnancy loss & malformations. The mainstay of training is an independent research project in a mentor's laboratory & opportunities to collaborate with other mentors & basic scientists. The trainees & faculty actively participate by presenting their ongoing work at biweekly (-cell biology meetings. To broaden & strengthen their independent research projects, trainees also participate in weekly seminars on both basic & clinical issues in diabetes, endocrinology, metabolism and immunology. Trainees will present their research at national meetings, & career development will be enhanced by structured workshops in grant & manuscript writing and reviewing. All trainees will participate in The Program for the Ethical and Responsible Conduct of Science & Scholarship. Trainees are generally recent graduates with PhD and/or MD degrees in areas of molecular biology & basic sciences & a commitment to research careers in diabetes and metabolic diseases. The duration of training is 3 years. Support is requested for 4 postdoctoral trainees. Our basic research-oriented postdoctoral training program complements & interacts with the Diabetes Research and Training Center, & a parallel clinically-oriented diabetes training program in the Department of Medicine. Washington University School of Medicine is an institution known for its excellence in basic & clinical diabetes research. Goals of this program are to provide postdoctoral trainees with research experience & skills necessary to gain extramural funding & career opportunities to develop into independent investigators with a career commitment to research & teaching in diabetes.

DESCRIPTION (provided by applicant): Natural killer cells are generally viewed as a first line of defense in cancer immunosurveillance and are critical players in immune-mediated cancer rejection. We have recently described a new subset of unconventional NK cells that reside in mucosal tissues and are characterized by production of large amounts of IL-22. We refer to this novel cell subset as NK-22. Although the origin of these cells is still unclear and their relationship to conventional NK cells a matter of debate, we and others have shown that these cells can acquire features typical of conventional NK cells when exposed to inflammatory cytokines. Moreover, recent work has suggested that a similar cell type plays a critical role in initiating tumor rejection. Our preliminary data show that NK-22 are highly dependent on the transcription factor aryl hydrocarbon receptor (AHR) for their development. AHR is known to bind dietary compounds such as indol-3-carbinol (I3C) and flavonoids that are associated with anti-tumor activity. We hypothesize that by engaging AHR through dietary supplements, we can manipulate the numbers and or the activity of NK-22. In Specific Aim 1 we will test the effect of I3C and the flavonoid quercetin on NK-22 expansion and function in vivo. In Specific Aim 2 we will determine whether these supplements impact the anti-tumor function of NK-22 in spontaneous and transplantable tumor models. We discovered NK-22, contributed to the initial characterization of their development and function and have generated unique NK-22 specific reagents as well as mouse strains lacking NK-22 or AHR in several immune and non-immune cell types. Therefore, we are in a unique position to directly test the impact of specific dietary supplements allegedly associated with anti-cancer activity on tumor surveillance by innate immune cells. !

DESCRIPTION (provided by applicant): PROJECT SUMMARY Langerhans cells (LCs) are the dendritic cells of the skin epidermis that detect physical, chemical and microbial injuries and promote immune responses. They play crucial roles in the immune response to skin allergens, melanoma and other skin cancers as well as infectious agents. Recent studies have shown that LCs are unique among myeloid cells in their development: they arise from embryonic progenitors of the yolk sac and the fetal liver that populate the developing skin before birth. LCs proliferate immediately before birth and during the first days of post-natal life and then self-renew with a slow turnover rate. However, the cytokine(s) responsible for driving the development of LC precursors in the skin remain unknown. Although mouse models in which LCs can be depleted either constitutively or conditionally were recently developed, contrasting roles for LCs in contact hypersensitivity (CHS) responses have been reported in studies using these mice. Thus, whether LCs induce tolerogenic or immunogenic responses to skin allergens remains an open question. In our preliminary data we show for the first time that LC development depends on IL-34, a recently discovered skin-derived cytokine that binds the CSF-1 receptor. Moreover, we demonstrate that IL-34-deficient mice selectively lack LCs and have attenuated CHS responses, with fewer IFN-¿-secreting T cells infiltrating the skin, in comparison to wildtype mice. In this proposal, we will take advantage of our IL-34 knock-in/knock-out mice that express ¿- galactosidase as an IL-34 reporter as well as mice we have generated that carry floxed IL-34 alleles. In specific aim 1 we will investigate when and where IL-34 promotes LC development. We hypothesize that IL- 34 is selectively expressed in the skin, especially during the perinatal period, driving the differentiation of LC progenitors that hae previously migrated from the yolk sac and the fetal liver into the developing skin. In specific aim 2, we will test the hypothesis that LCs are required to induce both IFN-¿-producing (type 1) and IL-4-, IL-5-, IL-13-producing (type 2) T cells specific for skin allergens, depending on the innate response to the allergen and/or adjuvant and the resulting cytokine microenvironment that instructs LCs. Our discovery that IL-34 is essential for LC development and our generation of a novel mouse model lacking LCs affords a unique window of opportunity to advance our knowledge of skin and LC immune function, which will provide the framework for new strategies to treat skin pathologies that target IL-34 and LCs.

PROJECT SUMMARY Alzheimer's disease (AD) is the most common form of late-onset dementia, affecting more than 5.5 million Americans. Currently, there are no approved therapies that can halt or reverse AD. AD is initiated by amyloid beta (A?) peptides that form extracellular aggregates; these promote intraneuronal tau hyperphosphorylation and aggregation, which subsequently lead to neuronal death. These lesions elicit a secondary response by microglia, which undergo prominent changes manifest as a transcriptional signature known as disease- associated microglia (DAM). Genetic studies of human AD have suggested that microglia modulate disease course. Most notably, heterozygous hypomorphic variants of the microglial receptor TREM2 increase the risk of AD several fold. Supported by this grant, our mechanistic studies in various mouse models demonstrated that microglia require TREM2 to acquire the DAM profile and respond to AD pathology. Given these advances in our understanding of how TREM2 is relevant to microglial response, it is important to understand the TREM2 signaling pathway in detail and determine whether it can be exploited for AD therapy. In our recent snRNA-seq study of brain specimens from AD patients with and without TREM2 risk variants, we found that human microglia upregulated the expression of genes integral to the DAM signature together with ?homeostatic? genes and the transcription factor IRF8. This signature echoed that of microglia in a mouse model of peripheral nerve injury that reflects a response to neuronal death and is driven by IRF8. We validated IRF8 expression in human AD microglia and demonstrated that IRF8 expression is reduced in patients carrying the TREM2 AD risk variant. Furthermore, in epigenetic studies of mouse microglia we have found that TREM2 promotes transcriptional activation of target genes through IRF8. Specific Aim 1 will test the hypothesis that IRF8 plays a key role in driving the microglial response to AD pathology downstream of TREM2 using mouse models that combine A? and tau aggregation with neuronal death and lack or overexpress Irf8 in microglia. In a second recent study, we investigated the effects of an agonistic anti-human TREM2 mAb in mice that accumulate A? and express human TREM2 variants rather than endogenous TREM2. A single injection of anti-human TREM2 mAb was sufficient to expand metabolically active and proliferating microglia. Moreover, prolonged mAb treatment partially reduced the neurotoxicity of A? plaques, although it did not change the A? load and only mildy modified behavior. Specific Aim 2 will test the hypothesis that changes in the dosing or initiation of mAb treatment can moderate the A? load and improve cognitive behavior through appropriate modulation of the TREM2-IRF8 axis. Since TREM2 gene dosage has been proposed to control the beneficial or detrimental effects of TREM2 on tau pathology, Specific Aim 3 will test the hypothesis that a mouse model of tau may benefit from appropriate mAb engagement of TREM2. Overall, this proposal will provide mechanistic insights into AD pathogenesis and validate TREM2 as promising target for AD prevention and/or therapy.

? DESCRIPTION (provided by applicant): The recent outbreak of Ebola virus in West Africa has emphasized the need for understanding the immune response to acutely infectious, deadly viruses and developing immunotherapeutic options for treating or preventing infection. It is thought that inhibitory receptors attenuate the responses of cytolytic lymphocytes, such as Natural Killer (NK) and CD8+ T cells, to virally infected cells. Indeed, this is the case in the context of chronic viral infections. However, in our preliminary studies we have found that Nkg2a, an inhibitory receptor that is expressed on activated CD8+ T cells and on a subset of NK cells, promotes the response to an acute, deadly viral infection caused the mouse orthopoxvirus Ectromelia (ECTV). Remarkably, Nkg2a does not limit the immune response like other inhibitory receptors, but rather sustains CD8+ T cell responses against ECTV by preventing activation-induced cell death (AICD) of virus-specific CD8+ T cells in a cell intrinsic manner. This observation provides a new, provocative point of view for understanding the immunology of acute, lethal viral infections; it suggests that inhibitory receptors, or at least some of them, ma be essential for effective CD8+ T cell responses and hence may lead to new avenues for therapeutic intervention. We will explore this hypothesis using newly generated Nkg2a-/- mice through the following aims. In specific aim 1, we will determine whether Nkg2a-/- mice CD8+ T cell are susceptible to AICD exclusively within the milieu of an acute lethal virus infection that presumably generates a highly inflammatory cytokine micro-environment or during TCR-mediated activation in general. Moreover, we will ascertain whether anti- apoptotic cytokines can rescue Nkg2a-/- CD8+ T cells in vivo and whether cell-extrinsic mechanisms contribute to the Nkg2a-/- CD8+ T cell defect. In specific aim 2, we will test the impact of Nkg2a on the proliferation and function of virus-specific memory CD8+ T cells during acute lethal poxvirus infection. We hypothesize that Nkg2a augments the magnitude of the memory CD8+ T cell response and long term immunity to ECTV. In specific aim 3, we will focus on another inhibitory receptor structurally related to Nkg2a, known as Klrg1, which we have found to contribute to host defense against ECTV in synergy with Nkg2a. Since Klrg1 and Nkg2a share very similar expression patterns, we will ask whether Klrg1 primarily functions to maintain CD8+ T cell and perhaps NK cell numbers during the anti-ECTV response by preventing AICD or, alternatively, whether Klrg1 prevents activated CD8+ T cells and/or NK cells from discharging excessive lytic mediators and inflammatory cytokines that cause immunopathology. Based on our substantial record in the field of inhibitory receptors, our newly generated Nkg2a-/- mouse line and our expert collaborators in poxvirus biology, we are in a strong position to successfully pursue this paradigm-shifting view of inhibitory receptor function during anti-viral responses.

? DESCRIPTION (provided by applicant): An inappropriate immune response towards microbial flora in genetically predisposed individuals results in inflammatory bowel disease (IBD). The pathogenic response in IBD is driven by CD4+TH17. Immunoregulatory mechanisms that prevent or counter IBD typically rely on Tregs and IL-10. However, recent studies have identified the involvement of intraepithelial CD4+CD8+T cells in the regulation of intestinal inflammation. Here, we have identified a novel molecular interaction between CRTAM on intestinal T cells and CADM1 on intestinal DC and demonstrated that this interaction is required for intraepithelial CD4+CD8+T cells in the steady-state and for TH17 responses during T. gondii parasitic infection. These results mandate further investigation of the impact of CRTAM-CADM1 interactions on intestinal immunity and their potential exploitation for IBD therapy. In Aim 1, we propose experiments to distinguish whether CRTAM-CADM1 interactions act: a) exclusively in the gut mucosa to facilitate the retention of CD4+T cells and CD4+CD8+ T cells, or b) in the mesenteric lymph nodes to promote priming of CD4+T cells and the acquisition of gut homing molecules. Aim 2 is based on the observation that CD4+CD8+ T cells are absent in germ-free mice, whereas short chain fatty acids (SCFA) induce their expansion. Thus, we will investigate the mechanisms by which the microbiota induces the differentiation of CD4+CD8+T cells and ask whether these mechanisms involve CRTAM-CADM1 interaction. CD4+CD8+T cells may require presentation of commensal antigens by intestinal CADM1+DCs. Alternatively, commensal microbiota may release metabolites that facilitate CD4+CD8+T cell differentiation, possibly by acting as histone deacetylase (HDAC) inhibitors that impact the epigenetics of CD4+CD8+T cell progenitors or CADM1+DCs. In Aim 3, we will explore the mechanisms for the CRTAM-CADM1 dependency of TH17 responses to T. gondii: we will determine whether TH17 cells detected during T. gondii infection are directed toward translocated commensals or are specific for T. gondii and whether CRTAM-CADM1 interactions impact TH17 response to commensals in general. Given the emerging importance of CD4+CD8+T cells in the regulation of intestinal immunity, the relevance of TH17 responses to autoimmunity and the need to manipulate these cells for therapy, this proposal to study CRTAM-CADM1 interactions embodies a new perspective in intestinal immunity that is highly innovative and relevant to IBD.

? DESCRIPTION (provided by applicant): Productive elimination of pathogens while minimizing damage to neighboring tissues requires facile communication between cells of the innate and adaptive immune systems. Defects in cellular or soluble components of these communication networks can interfere with pathogen clearance or lead to unchecked inflammatory responses and incumbent tissue damage associated with autoimmunity. In many tissues, pathogenic insult triggers antigen-presenting cells to secrete cytokines that are initially translated by innate lymphoid cells (ILC). In turn, activated ILC subsets secrete signature cytokines, which promote the release of antimicrobial peptides, minimize damage to surrounding cells, and influence the balance of T helper lymphocytes. Emerging evidence suggests that efficient communication during an immune response relies on the presence of cellular homologues, which mediate innate versus adaptive arms of the response. For example, ILC3 are innate immune cells that secrete IL-22 in response to IL-23, promoting elimination of extracellular pathogens and fungi. Their counterparts from the adaptive immune system, Th17 and Th22, express IL-22 in response to activation of their antigen receptor (TCR). The ILC3-Th17-Th22 cohort all develop from common progenitors and share requirements for some transcription factors, but are clearly distinct in function (only Th17/22 express IL-17) and mechanisms of activation (cytokine versus TCR). Elucidation of the developmental relationships between these immune subsets and their functional plasticity remains an important goal in immunology. Resolution of these issues requires a deeper understanding of transcriptional and regulatory programs employed by each subset in their basal and activated states. The applicants' laboratories will leverage their complementary sets of expertise to solve this problem, leading them previously to the discovery of ILC subsets and immune function, as well as chromatin-based identification of gene regulatory circuits in lymphoid cells. The current project aims to dissect whether human ILC3 and Th17/22 employ a combination of overlapping and divergent regulatory elements to express shared genes (e.g., IL-22) in response to distinct cues (cytokines versus antigen receptors), while simultaneously activating signature expression programs (e.g., IL-17) that mediate unique functions of innate or adaptive immunity. Experiments are proposed to (i) isolate human ILC3, Th17/22, and naïve counterparts from distinct microenvironments (tonsil and blood), (ii) map transcriptional and regulatory landscapes of these cells in their basal and activated states, (iii) decipher the regulatory logic for each subset by connecting cis-elements to target genes, and (iv) employ circuit diagrams to establish developmental relationships, functional work-scopes, and plasticity between three related cell types that are critical for elimination of extracellular pathogens. These studies will provide regulatory blueprints for the ILC3 gene expression program, which strikes a homeostatic balance during immune responses to pathogens; keeping inflammation, tissue damage, and oncogenesis in check.

PROJECT SUMMARY/ABSTRACT Approximately one-third of the world's population is latently infected with TB (LTBI) and have a 10% lifetime risk of developing clinical pulmonary TB (PTB). Global efforts to combat TB are hampered by the emergence of drug-resistant strains of Mycobacterium tuberculosis (Mtb), and variable efficacy of the currently licensed vaccine, M.bovis BCG (BCG). After pulmonary infection with Mtb, aerosolized bacteria are inhaled and interact with the host, resulting in the recruitment of immune cells to the lung to form the tubercle granuloma. Although the presence of granuloma has long been considered a hallmark of TB, the immunological differences between a protective granuloma and a non-protective granuloma has been elusive. Our recent published data, suggest that the presence of inducible Bronchus Associated Lymphoid Tissue (iBALT) within granulomas is indicative of protective granulomas that mediate Mtb control. In contrast, infiltrating neutrophils are characteristic of granulomatous inflammation in PTB patients. These new findings significantly change the overall consensus that TB granulomas in general are protective, but instead put forth the new paradigm that during TB, protective granulomas contain iBALT, while non-protective granulomas are neutrophilic. Our new preliminary data demonstrate that Group 3 Innate Lymphoid cells (ILC3) are among the first innate cells to rapidly accumulate in the lungs upon Mtb infection, and localize within B cell follicles in iBALT-containing granulomas. ILC3 deficiency in mice also results in increased early Mtb susceptibility, and coincides with reduced macrophage accumulation and poorly formed iBALT structures. Thus, the work proposed in this grant will mechanistically address a functional innate role for ILC3 in Mtb infection. In Specific Aim 1 we will define the host factors that mediate early lung ILC3 accumulation following Mtb infection. In Specific Aim 2, we will determine the mechanism(s) via which ILC3 mediate formation of iBALT-containing granulomas and facilitate Mtb control. In Specific Aim 3, we will determine a role for ILC3 in vaccine-induced immunity against TB, and identify new ways to target ILC3 to improve immunity against Mtb infection. Together these aims will provide novel evidence for a critical role for ILC3 in mediating iBALT formation and inducing protective host immunity to TB. Identifying new ways to target ILC3 cells to improve vaccine-immunity as proposed here, will open up novel avenues that can be harnessed for TB vaccine design.

PROJECT SUMMARY A delicate regulatory balance must be achieved in cells of the innate and adaptive immune systems to effectively eliminate pathogens, while minimizing damage to neighboring tissues. Defects in regulatory mechanisms that govern expression of cellular or soluble mediators can interfere with pathogen clearance or lead to unchecked inflammatory responses associated with autoimmunity. Recent studies have revealed that the innate immune system includes functional counterparts of T helper (Th) cells, which lack antigen-specific receptors and respond with enhanced kinetics and vigor to danger signals induced by pathogenic insults. The Th counterparts, called innate lymphoid cells (ILCs), have also been implicated in the pathogenesis of several autoimmune diseases, including inflammatory bowel disease (IBD). In discovery-driven profiling studies supported by an R21, the Co-PIs have recently defined the regulatory landscapes of Th-ILC counterparts derived from inflamed human mucosae, revealing collection of enhancers and super-enhancers that may control the expression of key immune mediators. Moreover, many enhancers that were active in specific ILC or Th subsets co-localized with autoimmune-associated disease SNPs, suggesting the regulatory elements may be important for controlling expression levels of nearby genes that mediate autoimmune pathogenesis. Despite this progress, the precise role of these potentially important regulatory elements in cell type-, agonist-, and disease-specific gene expression remains unclear. The goal of this project is to address these outstanding issues, focusing on regulation of the IL22-IL23R-IL1R1-STAT3 axis, which is critical for immune function of ILC3-Th17 counterparts and is rich in autoimmune-associated SNPs. The Co-PIs will also define and test key aspects of ILC3 regulomes that control their functional conversion to ILC1, a process implicated in IBD pathogenesis. To achieve this goal, we will leverage the Co-PIs' complementary expertise. Dr. Colonna's lab discovered several ILC subsets and contributed to our understanding of their biology in mice and humans. Dr. Oltz's lab studies cis-regulatory circuits that drive lymphocyte development and transformation. Three specific aims are proposed to test the hypotheses that: (i) unique sets of enhancers are critical for cell type- and agonist-specific expression of IL22 and IFNG, (ii) a subset of disease-associated SNPs disrupts transcription factor binding and enhancer function to alter IL23R, STAT3, or IL1R expression in ILC3 and Th17 cells during autoimmune pathogenesis, and (iii) ILC3?ILC1 conversion requires full activation of ILC1-associated enhancers that remain poised in ILC3 and, conversely, a decommissioning of ILC3-specific enhancers, perhaps converting them to a poised state. Together, our project will identify key features of the ILC-Th regulomes that dominate expression patterns of genes involved in autoimmunity, providing insights into independent roles of cytokine expressing cells in pathogenesis, and ultimately opening new therapeutic avenues.

PROJECT SUMMARY Innate lymphoid cells (ILCs) include conventional natural killer (cNK) cells and a broad spectrum of cells that belong to three groups based on their development and production of signature cytokines. ILC1s secrete IFN-?, ILC2s produce IL-5 and IL-13, and ILC3s secrete IL-22 and IL-17. In this proposal, we identify a novel tissue-resident ILC subset in the salivary gland (SG) that deviates functionally from other ILCs and cNK cells and may have a regulatory function in protecting the SG from inappropriately activated T and B cells that cause inflammation and tissue damage. Thus, this proposal is highly significant for Sjögren's syndrome (SS), an autoimmune disease typified by chronic inflammation and tissue destruction of the SG; the identification of an ILC subset with potential regulatory function in the SG may help identify new therapeutic avenues for SS. Our preliminary data show that SG ILCs express markers typical of tissue residency - CD103, CD49a and CD69 - and do not rely on transcription factors that are necessary for the development of cNKs and other ILCs, including Nfil3, T-bet and Eomes. Importantly, SG ILCs do not produce IFN-?, but rather express the cell death molecule TRAIL, which endows SG ILCs with the ability to kill activated T cells that express the receptor for TRAIL. We demonstrate that these features are due to ILC exposure to TGF?, which is abundant in the SG. In Aim 1 we will establish when and for how long SG ILCs require TGF? to develop. We hypothesize that SG ILCs originate from hematopoietic progenitors that seed the SG, proliferate, and acquire irreversible tissue resident/regulatory features after the SG has completed development. To test this, we will examine reconstitution of SG ILCs after transfer of congenically marked ILC progenitors into lymphopenic mice. We will also track SG ILC progenitors at various stages of hematopoiesis in inducible reporter mice as well as in parabiotic pairs and define the timing of TGF? action in mice in which TGF? signaling can be inducibly ablated. In Aim 2 we will identify the signals required for SG ILC development downstream of TGF?. We will test the hypothesis that TGF? drives progenitor cells towards ILCs rather than cNKs by suppressing Eomes. As TGF? signals via SMAD-dependent and -independent paths, we will ascertain which route suppresses Eomes. In Aim3 we will assess the regulatory capacity of SG ILCs. Our preliminary data indicate that depletion of SG ILCs augments T and B cell infiltration in the mouse model of submandibular duct ligation. We will determine whether SG ILCs regulate immune responses via TRAIL-TRAIL-R interactions. Because SG ILCs express the IL-21 receptor (IL-21R), we will ask whether IL-21R signaling enables additional regulatory functions, such as the release of granzyme B and IL-10. Moreover, we will examine the potential regulatory function of SG ILCs in NOD.H-2h4 mice, which contain SG ILCs and are relevant to SS. We have also identified SG ILCs in human salivary glands and will investigate their possible contribution to the progression of SS using samples from SS patients and healthy controls obtained from the SICCA biorepository.

Project Summary Alzheimer's disease (AD) is the most common form of dementia, affecting more than 5 million people in the US; about one in eight people that are 65 and older have the disease. AD cannot be prevented, cured or slowed. Recently, GWAS studies revealed that people with an arginine-47-histidine (R47H) mutation of the Triggering receptor expressed on myeloid cells 2 (TREM2) gene are 3-5 times more likely to get AD than those without the mutation. TREM2 is a microglia receptor that detects phospholipids and transmits intracellular signals through an associated adapter DAP12. The TREM2?DAP12 complex promotes activation and proliferation of microglia in response to AD lesions, presumably through recognition of phospholipids exposed on damaged neurons or associated with amyloid-? (A?) plaques. However, the R47H mutation prevents TREM2 binding to phospholipids ligands, which impairs its capacity to sustain microglial responses to AD lesions. Notably, heterozygosity for R47H is sufficient to considerably increase AD risk. Moreover, TREM2 deficiency and haploinsufficiency in the 5XFAD mouse model of AD accelerate disease progression in a dose- dependent fashion. These data suggest that AD progression is quite sensitive to changes in TREM2 expression. Thus, modulation of TREM2 expression levels may offer a powerful strategy for harnessing protective functions of TREM2. Early work on TREM2 reported that IL-4 increases TREM2 surface expression, LPS or IFN? cause loss of expression, while ADAM10 and ADAM17 proteases cleave TREM2 from the microglia cell surface. However, beyond these isolated mechanisms, little is known about regulation of TREM2 surface expression. By performing an unbiased CRISPR-Cas9 knockout library screening for TREM2 surface expression in the microglial cell line BV2, we have identified two novel regulators, DOK1 and TMEM131. DOK1 is a negative regulator of the Ras-ERK pathway. Nothing is known about its function in microglia and brain physiopathology. Our preliminary data demonstrate that DOK1 promotes TREM2 surface expression. TMEM131 is a functionally uncharacterized two-pass transmembrane protein. Almost nothing is known about this molecule. Our preliminary data show that TMEM131 inhibits TREM2 surface expression. In in vitro experiments with knockout and overexpressing BV2 lines, we will test the hypothesis that DOK1 and TMEM131 regulate surface expression of TREM2 by forming a complex with TREM2?DAP12 that is efficiently exported from ER and Golgi to the cell surface. By analyzing Dok1?/? and Tmem131?/? mice in the 5XFAD model of AD, we will determine the impact of DOK1 and TMEM131 on microglial responses to A? plaques and A?-related pathology in vivo. Overall, these studies will identify key pathways that regulate TREM2 expression in steady?state and during A? plaque pathology and will allow us and others to pursue novel, better-targeted therapeutics to boost TREM2 expression.

PROJECT SUMMARY Alzheimer?s disease (AD) is the most prevalent cause of late-onset dementia, affecting millions of people worldwide. Currently, there are no therapies that can halt or reverse AD. Hence, there is an urgent need to identify new therapeutic options. AD pathology is characterized by A? plaques and neurofibrillary tangles, which are linked to synapse loss and neuron death. Microgliosis is an additional pathological feature in regions affected by plaques and tangles. Recently, a rare variant in a microglial receptor, TREM2, was found to be associated with ~3-fold increased risk for sporadic AD, further implicating microglia in AD. TREM2 is a receptor for phospholipids that binds apoptotic cells and lipoprotein particles. The arginine-47-histidine variant associated with a high risk for AD impairs binding to phospholipids. Moreover, TREM2 deficiency and haploinsufficiency in mouse models of AD impair microglial clustering around plaques, which facilitates plaque spreading and damage of surrounding neurons. While optimal TREM2 function seems to protect from AD, it remains unclear how TREM2 sustains microglial responses during the progression of AD. In SPECIFIC AIM 1, we demonstrate that a defect in TREM2 in both mice and humans impairs mTOR signaling along with energetic and anabolic metabolism, and triggers compensatory autophagy, which, however, is insufficient to preserve microglial survival. We further show that this metabolic defect can be corrected by a creatine analog, cyclocreatine, which restores microglial ATP levels in vitro and promotes microgliosis around plaques and prevents neurite dystrophy in vivo. We will test whether, by improving metabolic fitness, cyclocreatine enhances microglial functions in vitro and cognitive function in fast and slow mouse model of AD in vivo. Additional TREM2 variants associated with increased risk of AD are found in the stalk region of TREM2, which undergoes protease cleavage by ADAMs proteases, resulting in the release of soluble TREM2 (sTREM2) at the expense of membrane-bound TREM2. In AD patients, sTREM2 levels in the cerebrospinal fluid correlate with microglial activation in response to neurodegeneration. Remarkably, recent in vitro studies showed that addition of sTREM2 to myeloid cells enhanced viability and suppressed apoptosis. In addition, injection of sTREM2 into the hippocampus of the mouse brain augmented microglia numbers. However, the functions of sTREM2 remain elusive. In SPECIFIC AIM 2, we demonstrate that sTREM2 binds to neurons in a human TREM2 transgenic mouse when crossed to mice that develop A? plaques. Therefore, we propose to test the hypotheses that AD spurs the production of sTREM2, and that full-length TREM2 promotes microglia activation, whereas sTREM2 has a separate, beneficial effect on neurons in vivo. To do this, we will monitor AD pathology in two novel transgenic TREM2 mouse lines that express either human sTREM2 or a modified human TREM2 that is not cleavable by proteases. Overall, this proposal will provide proof of principle that sustaining microglial metabolic fitness and production of sTREM2 are promising novel strategies for therapeutic intervention in AD.

PROJECT SUMMARY Functional plasticity of immune cells is essential to fine-tune their responses to disparate stimuli. Although plasticity may cause unwanted side effects, it may also be harnessed to steer immune responses towards desired outcomes. Innate lymphoid cells (ILC) are lymphocytes devoid of antigen-specific receptors that produce cytokines at early stages of immune responses. Based on specific networks of transcription factors and cytokine profiles, ILC are subdivided into three subsets: T-bet+ ILC1 release IFN-?; GATA3+ ILC2 secrete IL-5 and IL-13; ROR?t+ ILC3 produce IL-22 and IL-17. Recent studies showed that ILC are functionally plastic and heavily influenced by changes in the microenvironment, which raises outstanding questions: 1) While human ILC are functionally plastic in vitro, are they equally flexible in vivo and, if so, what mechanisms are involved? 2) In vivo fate mapping studies in mice have documented ILC plasticity in steady state; what is the extent and impact of ILC plasticity in disease models? 3) What epigenetic circuits control ILC responses to fluctuations in the microenviroment? We will address these questions in three aims. Aim 1 presents the first demonstration of transitional ILC subsets in human tonsils with features of both ILC3 and ILC1, which is evidence for ILC3?ILC1 conversion in vivo. We also present data indicating that the IKZF3-encoded transcription factor Aiolos is required for transition. We will test the hypothesis that Aiolos cooperates with other transcription factors to drive human ILC3?ILC1 conversion, using in vitro and in vivo approaches. We will perform chromatin studies of ILC3?ILC1 conversion to further define regulatory circuits and transcription factors that govern ILC3/ILC1 plasticity. Clonal analyses of transitional ILC subsets will be employed to corroborate their homogeneity and developmental trajectory. In Aim 2, we propose to precisely characterize ILC3/ILC1 transitional populations in the human intestine by mass cytometry and scRNAseq, as they are not as clearly defined as those in tonsils. Moreover, we propose in vivo mouse studies to determine whether diseases that alter intestinal microenvironment induce ILC3/1 plasticity and, in turn, whether plasticity impacts immune responses during gastrointestinal infections and IBD. These experiments will be carried out in Ror?t-reporter mice and in ILC3-deficient mice reconstituted with either ILC3 or ex-ILC3. In Aim 3, we will test the hypothesis that in diseases that induce type 1 and type 3 polarizing cytokines, ILC2 in the gut and skin convert into ILC1/3. We will track ILC2 plasticity in vivo using reporter mice and test the impact of plastic ILC2 in models of infections, IBD and skin inflammation in adoptive transfer experiments. We will also define the regulatory elements controlling ILC2 plasticity in chromatin studies. We hope that our expertise and leadership in the ILC field will yield an integrated view of ILC functional adaptation in immunity.

Project Summary The long term objective of this competitive renewal application is to train exceptional diabetes scientists capable of leveraging the latest research tools to decrease suffering from diabetes. The program trains MD, PhD, and MD PhD scientists for 2 to 3 years through mentored research and structured activities that are seamlessly integrated with basic science departments, clinical departments, and centers in the context of considerable institutional support. Continually evolving in response to self-assessment and external reviews, the training program features strengths that include: A) Research facilities and a research environment encompassing the Institute of Clinical and Translational Sciences (the Washington University CTSA), the NIDDK-supported Diabetes Research Center (DRC) and Nutrition Obesity Research Center (NORC), as well as substantial other resources. B) A required core curriculum in diabetes science in addition to required supplemental courses that include training in Rigor and Reproducibility all provided at no cost to trainees. C) Administrative support to facilitate interdisciplinary and multidisciplinary research training. D) Two Program Directors with complementary skill sets, strong records of training scholars, and significant commitment of effort through institutional support. E) 30 preceptors focused on diabetes and related metabolic diseases. Over the past 10 years, this group has trained >270 postdoctorates of whom 86% have continued in research. Each of the mentors participates in one or more components of this training program, and each has external research support (average current year support >$990,000/preceptor). F) Institution of a new mentoring curriculum that has improved mentoring as determined by anonymous surveys. G) Highly competitive pools of PhD and clinical degree trainees. All available postdoctoral positions have been filled over the past 15 years and the program completion rate is 96%. H) Anonymous feedback from current and former trainees indicating increased satisfaction with the training program over the most recent funding period. I) Successful training record in terms of publications (a substantial increase in publications per trainee compared to the previous funding period), competing for grant support, and remaining in research or research-related careers. J) To enhance diversity, institution of a new underrepresented minority (URM) Metabolic Outreach Program allowing URM scientists from the University of Miami to undergo short-term research training at Washington University. One URM MD fellow has completed such training. K) An integrated short-term research program for medical students coordinated through the Washington University DRC. Overall, this training program prepares a diverse group of scientists across the translational spectrum to exert a sustained influence on research in diabetes and related metabolic diseases.

PROJECT SUMMARY The overall goal of this application is to establish avenues through which we can harness the intestinal microbiota to enhance mucosal immune responses against Clostridium difficile. C. difficile infection (CDI) is a common cause of diarrhea in hospitalized patients and represents a major health threat due to frequent treatment failures and high risks of colectomy and mortality. Patients with recurrent CDI do not benefit from conventional antimicrobial therapies; while transplantation of fecal microbiota derived from healthy donors might be a valid option, regulation of fecal transplantation is problematic. In our preliminary data we show for the first time a remarkable impact of microbiota-derived acetate on CDI, which may constitute a potential therapeutic avenue. We show that acetate enhances neutrophil and innate lymphoid cells of type 3 (ILC3) responses through the surface receptor FFAR2. Moreover, we show that lack of a decoy receptor for IL-22, known as IL-22 binding protein (IL-22BP), amplifies the activity of IL-22 and modifies the microbiota, strengthening its resistance to CDI. We propose three specific aims. In Specific Aim 1, we will test the mechanisms through which acetate-FFAR2 signaling activates the neutrophil response to CDI. We will perform in vitro functional and transcriptional experiments to determine whether FFAR2 promotes neutrophil recruitment to chemoattractants, upregulates expression of inflammasome components, alters the neutrophil transcriptional profile, and/or modifies neutrophil metabolism. The impact of acetate on human neutrophils will be examined as well. In Specific Aim 2, using newly generated mice lacking FFAR2 in ILC3s, along with mice lacking FFAR2 in neutrophils, we will determine whether FFAR2 mediated activation of ILC3s is necessary and sufficient to recapitulate the protective effect of acetate in vivo. Furthermore, we will determine whether FFAR2 impacts ILC3-mediated lymphoid organogenesis in the steady state and whether FFAR2 modifies the transcriptome and/or metabolism of mouse and human ILC3s. Finally, we will ascertain whether acetate can be used in a therapeutic mode. In Specific Aim 3, we will test the hypothesis that lack of IL-22BP and the resulting increased basal activity of IL-22 modify the intestinal microbiota in a manner that facilitates the colonization of bacterial cohorts that enhance protection against CDI. This work is significant because it addresses the major health burden of dysbiosis and CDI, is innovative because it evaluates alternative therapeutic approaches based on acetate and/or blockade of IL-22BP rather than live microbiota and has potential to translate into novel treatments with implications for health and productivity. The project will be accomplished through the ongoing collaboration between the Colonna lab in USA and the Vinolo lab in Brazil, each with distinct and complementary sets of expertise on CDI, short chain fatty acids, and mucosal innate immunity that must be combined to successfully complete this project. The synergy between the two labs has already generated results that serve as the basis for this proposal and will produce fundamental research on the impact of the microbiota on mucosal responses to CDI and unveil new potential therapies.

PROJECT SUMMARY Cancer is a leading cause of death and disease. The recent success of immune checkpoint therapy (ICT) has revolutionized tumor therapy, indicating that manipulation of the immune system is an effective strategy to treat cancer. MAbs inhibiting CTLA-4 and PD-1 have been extensively shown to unleash T cell effector functions to control tumors in both mice and some cancer patients. However, ICT is incompletely effective for certain tumors, which escape using multiple mechanisms, one of which is the generation of a tumor microenvironment rich in immunosuppressive myeloid cells. TREM2 is an immune receptor expressed by tissue macrophages that binds phospholipids and lipoproteins and transmits intracellular signals through the ITAM pathway. Recently, TREM2+ macrophages have been reported in many human tumors. In our preliminary data, we demonstrate that TREM2-deficiency or mouse TREM2 blockade with the mAb 178 curbs subcutaneous tumor growth of the 3- methylcholanthrene (MCA) cell line and leads to complete tumor regression when associated with suboptimal PD-1 immunotherapy. Furthermore, high-resolution analysis of the tumor cell infiltrate in the MCA model reveals complex remodeling of the myeloid cell landscape in Trem2?/? and anti-TREM2 treated mice. The overall goal of this application is to advance our understanding of the therapeutic impact of TREM2 blockade in mouse models and human cancer. In Aim 1 we show that TREM2 targeting enhances ICT mediated by anti-PD1; we propose to determine whether TREM2 deficiency or blockade impact other tumor therapies, such as anti-CTLA4 and chemotherapy, which elicit different types of immune responses. The impact of TREM2 will be assessed using injected MCA cell lines and the spontaneous MMTV-PyMT model of breast cancer. In Aim 2 we will define the mechanisms through which anti-TREM2 impacts the tumor microenvironment. Given that a) immunosuppressive macrophages depend on lipid metabolism and accumulate lipid droplets; b) TREM2 promotes foam cell formation by binding lipoproteins; and c) anti-TREM2 mAb blocks lipid binding to TREM2, we will test the hypothesis that TREM2 blockade converts tumor macrophages from immunosuppressive to immunostimulatory by blocking lipid droplet accumulation and foam cell formation. We will also test an alternative mechanism based on the observation that TREM2 is cleaved from the cell surface by ADAM metalloproteases, generating soluble TREM2 (sTREM2), which promotes survival of macrophages in various disease models. We will test the hypothesis that lack of sTREM2 in a transgenic mouse with uncleavable TREM2 prevents survival of immunosuppressive tumor macrophages. In Aim 3, we show unpublished data indicating that anti-human TREM2 mAb 21E10 delays tumor growth of an injected MCA cell line in mice expressing human TREM2 in place of mouse TREM2. Therefore, we will determine whether TREM2 blockade with a specific mAb can be extended to a preclinical model expressing the human TREM2 receptor. Overall, this proposal will advance our knowledge of a novel therapeutic approach based on TREM2 that broadens our armamentarium for targeting immunosuppressive myeloid cells in tumors.