Miriam Merad - US grants

[unreadable] DESCRIPTION (provided by applicant): As the only dendritic cells resident in the epidermis, Langerhans cells (LCs) play a critical role in skin immunity. Through their stimulation of donor T cells, LCs likely play a key role in skin graft-versus-host-disease (GVHD), a serious complication that limits the use of allogeneic bone marrow (BM) transplantation. Despite their importance, little is known about the life cycle of LCs, their precursor cells in the blood and their homeostasis after allogeneic BM transplantation. Furthermore, the mechanisms by which LCs influence GVHD have not been elucidated. We have recently established that LCs are maintained by a radio-resistant precursor that self-renew in quiescent skin throughout life. In contrast, LCs are replaced by circulating precursors after major skin injury such as exposure to UV light or donor allo-reactive T cells infiltration. In addition we have recently characterized the circulating LC precursor that repopulate LCs in UV injured skin. We hypothesize that this unique cycle of LC homeostasis; controls skin allogeneic immune responses after transplantation. To address our hypothesis, we will employ complementary approaches using adoptive transfer of hematopoietic precursors in mice and a clinically relevant model for allogeneic hematopoietic stem cell transplantation. Aim 1, will characterize the direct circulating precursor that repopulate skin resident LCs after allo-BMT. Aim 2, will characterize the mechanisms leading to the replacement of host LCs by donor LCs after allog-BMT. Aim 3 Will explore the immunological mechanisms that lead to the protective effect of LC chimerism on skin GVHD. These studies should provide valuable insights into LC regulation of skin immune responses and may lead to improved prevention or treatment of skin GVHD, a major medical problem in transplantation therapy of patients with malignancies. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Dendritic cells (DCs) are highly specialized antigen presenting cells found in all lymphoid and non-lymphoid tissues. In the traditional view, DCs are terminally differentiated cells constantly replaced by a pool of circulating precursors. In contrast to this view, we recently demonstrated that in mice, epidermal DCs also called Langerhans cells (LCs) are maintained by local radio-resistant proliferative precursors, throughout life in quiescent skin, and are replaced by circulating precursors only during injuries. We have also discovered that host radio-resistant LCs can persist in recipient mice after allogeneic bone marrow transplant (allo-BMT). Graft versus host disease (GVHD) is a major cause of mortality and morbidity after allo-BMT. Importantly, we found that persistence of host LCs after allo-BMT correlates with severe skin GVHD, while replacement of host LCs by donor LCs improves skin GVHD outcome. Therefore, exploring DC homeostasis in a tissue target of GVHD may help develop new strategies for the treatment of this devastating disease. In addition to LCs, we recently discovered that a subset of DCs in the gut can resist lethal doses of radiation, raising the possibility that LCs may not be unique in being radio-resistant;other tissues may harbor radio- resistant populations that contribute to GVHD. Thus, in aim 1, we will examine the turnover of DCs in the gut, a tissue that is particularly affected by GVHD. Gut DCs constantly transport commensal bacteria and pathogens from the luminal intestine to local lymphoid tissues, playing a critical role in innate and adaptive immunity. To face their continuous migration, gut DCs must be replaced with new cells, but the nature of the committed precursor that maintains the gut DC pool remains elusive. Using novel strategies, we have recently characterized the circulating LC precursor as being a specific subset of circulating monocytes. We also established the sequence of events that leads a monocyte to become a LC and discovered that signaling through the receptor for CSF-1 is required for this process (Nature Immunology in press). Therefore in aim 2, we propose to use similar approaches to explore DC homeostasis in the gut in the steady state and identify the circulating precursor that gives rise to intestinal DCs. Finally, our finding that induction of DC chimerism is critical to prevent GVHD leads us, in aim 3, to determine the nature of the circulating precursor that repopulates DCs in a clinically relevant model for allo-BMT.

Program Director/Principal Investigator (Last, First, Middle): Merad, Miriam 1R01 AI080884-01A1 DISSECTING THE ORIGIN AND THE FUNCTION OF THE CUTANEOUS DENDRTITIC CELL NETWORK

DESCRIPTION (provided by applicant): Langerhans Cell Histiocytosis (LCH) is the most common of histiocytosis, a group of rare diseases that involve tissue-resident macrophages and dendritic cells. Patients with single system disease require minimal treatment, but even in these patients there may be permanent, mainly orthopaedic consequences. At the other extreme and most often in young infants, the presentation is multisystemic with organ failure and can be fatal in 25% of the cases. LCH results from the accumulation of Langerhans cell-like cells also called the LCH cells. LCH cells are always associated with a local cytokine storm and a large T cell infiltrate leading to irreversible damage to several organs that include the lungs, liver, central nervous system and the skin. LCH research has been hampered by problems related to rare diseases of childhood. Individual physicians see few cases and fresh material are hard to collect. The two central questions that remain unresolved and prevent adequate therapy are: 1) what is the origin of the LCH cell and 2) is LCH a true neoplastic or a reactive disorder. Answering these questions would change the way we diagnose and treat the disease. The current paradigm suggests that LCH results from an accumulation of epidermal dendritic cells (DCs) also called Langerhans cells (LCs).This concept is based on observations showing that LCH lesions are infiltrated by langerin+ cells and it associated Birbeck granules, features thought to be restricted to epidermal LCs. However, it has been difficult to understand how LCs, which is normally restricted to stratified epithelia, could give rise to such a multi-focal disorder. We recently identified the presence of interstitial langerin+ DCs, independent of LCs, in most non-lymphoid tissue in mice and in human lung and dermis (Fig. 5). In contrast to langerin- DCs that derive from circulating monocytes, we found that langerin+ DCs derive from a circulating DC restricted progenitor in a Flt3 ligand dependent manner. In addition, we found that inhibition of the receptor Flt3 leads to the specific depletion of langerin+ DCs whereas langerin- DCs and LCs remain unaffected. Our preliminary data also suggest that LCH cells have a phenotype that resemble interstitial langerin+ DCs, and express high levels of Flt3. Based on these findings, we hypothesize that LCH is due to an accumulation of interstitial langerin+ DCs, and not LCs, and can be characterized by dysregulated molecular pathways in the former. We also hypothesize that the receptor Flt3 may represent a novel therapeutic target for the treatment of LCH patients. To address this hypothesis we propose in aim 1 to characterize langerin+ DCs that populate in healthy tissues in humans. In aim 2, we propose to identify the precursors and the mechanisms that control the development of human langerin+, while in Aim 3 we propose to characterize the circulating precursors and langerin+ DCs that accumulate in LCH lesions.

DESCRIPTION (provided by applicant): Respiratory epithelial-cell surfaces present a large interface with the external environment and provide the first line of defense against a broad array of microbes. Initially perceived as a passive barrier between the host and the environment, the lung is now known to constitute a robust system of immune surveillance that include epithelial cells as well as highly specialized professional antigen presenting cells (APC) distributed throughout the conducting airways and the lung parenchyma. However, the critical elements required for development of protective immunity and the maintenance of immune homeostasis in the lung are largely unknown, particularly in the steady state in the human. This is important because uncontrolled or skewed immunity in the lung might lead to illness as for example might be the case in the link between RSV infection in childhood and increased susceptibility to asthma in adult life. Dendritic cells (DC) play a pivotal role in initiating the immune response to foreign antigens and in the maintenance of tolerance to self antigens. Drs Merad and Palucka laboratories have been working on the mechanisms that control the development and function of DC for more than ten years. Recent data from our groups revealed that the DC networks in nonlymphoid tissues consist of developmentally distinct and functionally specialized DC subsets in mice and humans. In this application, we propose to establish the functional specialization of the DC network in the lung and identify the mechanisms that control DC functional specialization in the induction of mucosal antiviral immunity. We will do so by studying mouse DC in vivo, human DC in humanized mice models as well as human lung. In our preliminary studies we have used influenza virus to probe the function of DC subsets in the lung. GFP-expressing virus differently interacts with distinct DC subsets. We show that distinct DC subsets preferentially interact with CD4+ or CD8+ T cells. These results support our hypothesis that the lung DC network consists of different subsets that differently control the induction of cellular and humoral immunity to respiratory viruses. Research proposed will be carried out through a collaborative effort between three established investigators with distinct and complementary expertise uniquely suited to address the central hypothesis of this application. The PI of this application, Dr. Miriam Merad (Mount Sinai School of Medicine (MSSM)) is an expert in mouse DC biology and has made several key contributions to our understanding of mucosal DC development and function in mice. Dr. Karolina Palucka (Joint investigator at Baylor Institute for Immunology Research (BUR) and MSSM) is a world expert in human DC biology and in humanized mouse models. Dr. Adolfo Garcia Sastre (MSSM) is a virologist and a world expert in influenza virus. In addition, Dr. Christian Becker (a pulmonologist at MSSM and a member of Merad's laboratory) has developed a strong human lung explants program with access to more than 100 fresh human lung samples per year. PUBLIC HEALTH RELEVANCE: The critical elements required for development of protective immunity and the maintenance of immune homeostasis in the lung are largely unknown, particularly in the steady state in the human. Here we propose to use mouse models of lung viral infections, humanized mice models and human lung explants to examine the role of dendritic cells subsets in the modulation of mucosal immune defense mechanisms.

DESCRIPTION (provided by applicant): Melanoma is an extremely difficult disease to treat. Chemotherapy is notoriously ineffective with a negligible long-term remission rate. Recent studies have established that inhibition of BRAF signaling induces massive melanoma cell death leading to response rates exceeding 50% in patients who test positive for the BRAFV600E mutation. Responses unfortunately are transient, lasting on average 6.8 months and eventually all patients' progress or relapse. In contrast, immunotherapy can produce long-lasting cures in a small minority of patients. Combination of these approaches is therefore extremely attractive. BRAF inhibition induces cell death and the release of melanoma antigens in patients. This is exciting for the melanoma field because chemotherapy does not reliably kill melanoma tumors in patients. Recent studies suggest that dying tumor cells can be excellent sources of antigen for immunotherapy. BRAF inhibition therefore, because it causes massive tumor cell death, represents a unique opportunity to develop novel effective immunotherapies. This proposal aims to identify immune mediated strategies able to potentiate clinical remissions induced by BRAF inhibition. The study proposed herein provides the first comprehensive assessment of the contribution of cutaneous DC and macrophages to the development, or the lack thereof, of therapeutic immunity to BRAF inhibitors using an inducible model of BRAF-driven tumors. We find this to be of great importance as endogenous vaccination due to BRAF-inhibition-induced melanoma cell death could offer a novel approach to melanoma immunotherapy. Such an approach could overcome numerous drawbacks in the vaccination strategies currently in use. In the last ten years, our group of investigators has provided evidence that the expansion of endogenous DCs followed by their activation might be a valid strategy to promote antitumor immunity in several mouse tumor models. Here, we propose advancing a step further and testing whether combining BRAF inhibitors to increase tumor antigen availability, together with vaccination strategies aimed at expanding and activating tumor-associated CD103+ DC in situ while eliminating tumor-associated immunosuppressive APC via CSF-1R Ab (already in cancer Phase I trial), could prolong response to BRAF therapy. Combination therapy with BRAF inhibitors and immunotherapy is urgently needed by melanoma patients. Results of this study are expected to provide a rationale for the design of key clinical trials to treat this devastating disease.

DESCRIPTION (provided by applicant): Proper control of the immune system is critical to human health. A hypersensitive immune system can lead to allergies and autoimmune disease, whereas impairment of the immune system leaves an individual susceptible to infections and even cancers. Dendritic cells (DCs) play an important role in ensuring proper regulation of the immune system. In response to pathogens, DCs rapidly alter their phenotype to limit the spread of infection, and initiate an adaptive immune response. DCs are also involved in preventing autoimmunity by maintaining self tolerance. It is now understood that DCs are not a homogeneous population of cells. They are comprised of functionally distinct subsets with different abilities to process antigens, respond to environmental stimuli, and engage distinct effector lymphocytes. DC subsets include the CD8+ DCs that are specialized in the cross-presentation of cell associated antigens to CD8+ T cells, and plasmacytoid DC (pDC), which help to establish an antiviral state through the rapid secretion of high amounts of interferon alpha. While we now appreciate that DCs are a heterogeneous population, little is known about the mechanisms that drive DC lineage commitment and differentiation, as well as the mechanisms that control functional specialization. Our preliminary profiling data and functional analysis have led us to hypothesize that specific microRNA (miRNA) play a key role in regulating DC identity. Indeed, when we knocked out a single miRNA specifically in DCs we observed a significant reduction in the number of pDCs, but not other subsets in mice, and the pDCs had a major reduction in their functional response to TLR stimulation. Here, we propose studies aimed at better understanding how this miRNA controls pDC development and function, including studies to identify the relevant targets of this miRNA (Aim 1). We will also investigate the role of two other miRNA that we identified through profiling studies to be differentially expressed between DC subsets (Aim 2). To do this, we will use a state-of-the-art miRNA decoy vector system that we generated, which enables stable inhibition of a miRNA in mouse DCs in vivo and in human DCs in culture. Finally, we will address a broader question of how miRNAs themselves are regulated in DCs using a novel, functional assay that we developed, which permits high resolution assessment of each miRNA's cellular activity (Aim 3). We have focused our efforts on eliciting the role of miRNAs in DCs because these small non-coding RNAs are known to play a role in controlling cell identity, and because the discovery of relevant miRNAs can be used to uncover other genes and pathways that are important to DC function through the identification of the miRNA's regulatory targets. Thus, our studies will not only provide new insight into the role of specific miRNAs, but they will also reveal other genes involved in the regulation of DCs. This, in turn, will help to find potential new causes of immune dysfunction, and to supply new strategies to enhance or subdue immune responses for the treatment of diseases, and in the aid of vaccine development.

DESCRIPTION (provided by applicant): Stress disorders such as depression and anxiety are associated with increases in the pro-inflammatory cytokine interleukin-6 (IL-6), however, the source and functional relevance of this elevation remains unknown. Using a repeated social defeat stress model in mice, we find individual differences in the peripheral immune response to stress-measured by increased IL-6 release from leukocytes-that predicts stress susceptibility. Susceptible mice develop social avoidance and anhedonia, which are established measures of depression-like behavior in rodents. To understand whether leukocyte derived IL-6 is necessary and sufficient for the development of social avoidance and anhedonia, we generated bone marrow (BM) chimeras transplanted with stem cells from stress susceptible or IL-6 knockout (IL-6-/-) mice. Stress susceptible BM chimeras exhibit baseline anhedonia and increased stress-induced social avoidance, whereas IL-6-/- BM chimeras were resistant to the effects of stress on these behaviors. In addition, we have preliminary evidence that IL-6 may be acting within key brain reward regions, such as the nucleus accumbens and prefrontal cortex, to mediate these behavioral effects. Together our work shows that pre-existing differences in stress responsive IL-6 release from leukocytes functionally contributes to depression-like behavioral phenotypes. In this application we will define the detailed mechanisms by which susceptible mice produce and release more IL-6. We will further define the functional relevance of such changes to development of depression-like behavior and test novel therapeutic strategies, such as bone marrow re-engineering to reduce stress susceptibility. We believe that this work holds promise for developing predictive diagnostic tests based on hyperactive IL-6 responses, as well as verification of important targets for novel antidepressant development.

The Human Immune Monitoring Core will characterize phenotypic and functional, ex vivo and in vivo signature as well as analyses of innate and adaptive immune responses to DENV infection and challenges at various time points whereas information on humoral immune responses to DENV will be obtained from the longitudinal studies of infected patients described project 1. Together, these validated assays will generate data to be integrated with results from the genomic and proteomic cores, and contribute to a DENV signature with clinical value. We anticipate that advances in mass spectrometry technologies and sample miniturization procedures may increase the sensitivity of global and functional analyses and may reduce the amount of sample required for these analyses alloing to extend innate immuen comptence studies to infected and vaccinated patients further refining the identification of a signature correlated with clinical status upon DENV infection, vaccine outcome or DC pathogenicity. These assays comprise the groundwork for future development of new immune enhancing strategies to promote the development of a protective immune response.

DESCRIPTION (provided by applicant): There is now clear evidence that tumor tissues co-opt immune-checkpoint pathways to impair T cell ability to recognize and eliminate abnormal cancer cells. The clinical grade antibody, ipilimumab targeting the immune checkpoint cytotoxic T-lymphocyte-associated antigen 4 (CTLA4) was the first therapy to improve overall survival in patients with advanced melanoma. However, despite significant and long lasting remissions observed with CTLA4 blockade, responses remain limited to a subset of patients. In addition, although increasing doses of ipilimumab can significantly improve overall tumor responses, the therapeutic index of anti-CTLA4 mAb remains limited by the occurrence of immune-related adverse effects (irAE) that can be life threatening, unless promptly managed. Other irAE affect the skin, liver and the endocrine system. There is therefore an urgent need to develop novel strategies to not only increase, but to also uncouple, anti-tumor responses from unwanted immune related toxicities. Antitumor responses may be curtailed by alteration of T cell effector functions by an immunosuppressive tumor microenvironment despite the of CTLA4 blockade. T cell effector function is elicited in the lymph nodes (LN) and further modulated at local tissue sites by antigen presenting cells that include dendritic cells (DC) and macrophages (mph). Over the past decade, our group has been focusing on the mechanisms that control the homeostasis and function of DC and mph in normal and tumor tissues. Through this effort we discovered the instructive role played by the tissue environment in modulating DC and mph function. Specifically, we discovered that the cytokine GM-CSF, recently renamed Csf2, was produced by innate lymphocyte cells in the steady state gut in response to commensal signals to promote DC and mph production of retinoic acid and IL-10 that are necessary to induce intestinal T regulatory cell differentiation and expansion. These results extend previous studies by the Dranoff's laboratory showing that Csf2 drive DC and mph immunoregulation. Concomitantly, we and others have found that Csf2 controls the survival and function of cross-presenting cutaneous CD103+ DC and promotes vaccine CD8 cytotoxic immunity when combined with local tissue delivery of TLR agonists. Though on the one hand vaccine strategies utilizing Csf2-producing tumor cells have led to coordinated antitumor immune responses affecting substantial tumor destruction in patients, these successes has been tempered by the potential of Csf2 to promote tumor immunosuppressive effects. Our new results shed a new light into the mechanisms of action of Csf2-regulated immune responses and reveal that the dual regulatory and immunogenic role of Csf2 is dependent on both the tissue microenvironment in which it is produced and on the availability of DC activating signals. Our results also suggest that Csf2's compartmentalized role in tissue immunity could be exploited clinically to modulate CTLA4 outcome in cancer patients. Based on these results we hypothesize that By harnessing Csf2 compartmentalized role on tissue phagocyte function we could uncouple anti-tumoral effects from unwanted immune related toxicity induced by immune checkpoint blockade.

? DESCRIPTION (provided by applicant): The human immune monitoring core (HIMC) at the Icahn School of Medicine at Mount Sinai (ISMMS) is a novel research center spearheaded by Dr. Merad and colleagues in 2011 and funded by the Dean of ISMMS to promote the development of novel immune profiling tools to help identify novel biomarkers of diagnosis, prognosis, and response to therapy. One of the main challenges to studies of the immune system has been the limited availability of human samples reducing our ability to perform in-depth comprehensive analysis of immune functions in patients. The goal of this application is to develop novel immune profiling tools and functional assays to identify novel targets and novel biomarker of diseases and response to therapy in two clinical areas: food allergy and IBD. The initial focus on these two disease areas reflects the extraordinary ISMMS clinical recruitment of allergic and IBD patients, yet any technological development obtained in this grant will be applied to other inflammatory disease such as atherosclerosis, and skin inflammatory disease including psoriasis and atopic dermatitis for which ISMMS is also a referral center. Food allergy is a common disease of childhood for which there are no treatment or prevention strategies. The Pediatric Allergy and Immunology division at ISMMS has built one of the most active clinical research programs in food allergy in the country. Mechanistic insights have lagged behind clinical gains due to the difficulty of profiling allergen-specific cells, particularly given the challenge of small blood volumes available from pediatric patients. Thus one of the goals of this proposal is to develop novel immune and genomics tools to identify and profile allergen-specific T cells at the single-cell level. The ISMMS Inflammatory Bowel Disease (IBD) Center is unique in its close, integrated collaborations between world-leaders in clinical, genetic, microbiome and immunology-based IBD research. While efforts to explore immune regulators causative of disease has focused on exploiting GWAS data and exploring systemic dysregulation, little effort has been devoted to comprehend the nature of the immune dysregulation that occurs at the tissue site mainly due to limited availability of gastrointestinal tissue. Thus Aim 2 of this projet proposes to leverage the extraordinary tissue samples obtained by the ISMMS IBD Center to develop novel immunological assays to profile immune cell compartments that infiltrate and contribute to IBD lesions in early diagnosed patients and at different time after initiation of immunodulatory agents.

? DESCRIPTION (provided by applicant): Langerhans Cell Histiocytosis (LCH) is the most common of histiocytosis, that results from the accumulation of pathologically activated DC associated with granuloma like lesions that consist of T cells and macrophages leading to irreversible tissue damage. Clinical approaches to LCH remain empiric due to poor understanding of LCH biology. During the previous budget period, we established the pivotal functional role of the BRAFV600E mutation in LCH pathogenesis and discovered the multiple origin of the LCH cell. In contrast to the dogma that suggests that LCH derive from pathological epidermal Langerhans cells (LCs), we showed that the LCH cell can derive from V600E mutated hematopoietic progenitors or differentiated DC and that occurrence of the V600E mutation along the DC lineage defines clinical risk in LCH patients. In addition, we generated the first mouse model of LCH-like disease allowing us to characterize the molecular mechanisms that drive LCH. These studies revealed the potential key contribution of physiological ERK activation in DC differentiation, and induction or expansion of Tregs, which may explain why Treg accumulate in high numbers in LCH lesions. These studies also established the dual contribution of sustained ERK activation and extracellular cues provided by T cells to LCH pathogenesis. Thus the goal of this application is to characterize the role of physiological ERK activation in DC and Treg homeostasis in vivo and how sustained ERK activation contributes to LCH pathogenesis. We will also dissect the contribution of cell extrinsic signals to the transformation of pathologically activated DC and to LCH growth. Based on the results of the mechanistic studies we will develop novel combination strategies that target cell intrinsic and extrinsic cues shown to modulate LCH pathogenesis. Specific Aim 1: To characterize the physiological role of the ERK pathway on DC differentiation, DC homeostasis and induction or expansion of T regulatory cells in situ. Specific Aim 2: To determine the contribution of sustained ERK-activation as well as cell extrinsic cues to LCH pathogenesis Specific Aim 3: To test combination strategies that target the ERK pathway along with cell extrinsic cues for the treatment of BRAFV600E-driven LCH lesions.

PROJECT 3 - PROJECT SUMMARY Macrophages are the most abundant innate immune cells in tissues where they function as immune sentinels. They are uniquely equipped to sense and respond to microbial injury1-4 playing a key role in tissue responses to host pathogens. Emerging studies from our lab, and others, have started to reveal that, counter to earlier dogma, macrophages from different tissues are distinct in their phenotype, function and molecular architecture, and this has important implications for how different tissues respond to infections. Most of our understanding of human macrophage biology, including how macrophages react and respond to pathogens and adjuvants, is based on monocyte-derived macrophages generated in vitro in pre-defined culture conditions. It is now clear that in vitro generated macrophages are different in phenotype and function from actual tissue-resident macrophages, and thus our comprehension of human macrophages is lacking and even inaccurate in many ways. For example, we do not know which TLRs are expressed by different macrophages, which clouds our understanding of pathogen and adjuvant responses in various tissues. Macrophages are the first targets of CMV infection, and are crucial for CMV persistence and dissemination. Since macrophages are also a key modulator of the immune response, these cells are at the crossroad between protection and viral pathogenesis. However to date, human macrophage?s response to CMV and the macrophage-specific contribution to the immune response against CMV is poorly defined. Our goal is to fill this gap in knowledge and understand how different human tissue-resident macrophages respond to pathogens, and to broadly determine the underlying programs that control human tissue macrophage biology. Based on the emerging findings from mice and preliminary studies with human samples, we hypothesize that human macrophages are defined by their microenvironment, which results in molecularly and functionally distinct macrophage populations in different tissues, which have unique responses to pathogens, including CMV. The limited availability of healthy human tissue-resident macrophages has made it impossible to test this hypothesis in humans. To overcome this limitation, we have teamed up with the Farber lab, who has established a truly unique pipeline for obtaining primary human cells from different tissues of organ donors. As part of the HIPC, we will isolate macrophages from different human organs and (1) define the specific properties of macrophages in lymphoid and mucosal tissues, (2) identify tissue-specific macrophage response to innate stimuli, and (3) determine the influence of CMV infection on tissue-resident macrophage function. The outcome of these studies will provide a deep characterization of human tissue-resident macrophages across and between individuals, which will also serve as an important hypothesis-generating resource for the community, help us understand the innate response of different tissues, which can aid vaccine design, and determine how a chronic CMV infection impacts the biology of tissue-resident human macrophages.

The Immune Cell Atlas (ICA) is a collaborative consortium of scientists and clinicians with distinct and synergistic expertise in Immunology and Genomics (Benoist, Hacohen, Merad) and Computational Immunology (Regev) with the goal of generating a comprehensive map of all immune cell populations that reside in human tissues at baseline and challenged states. The ICA inherits from and amplifies Immgen, a multi-institutional collaboration of immunologists and computational biologists which used rigorously shared SOPs to generate gene expression profiles of essentially all cell-types in the mouse immune system, and implemented user-friendly online data portals for easy public access that have become a valuable reference. The ICA will be carried out in synergy with a parallel effort in the UK (Oxford and the Wellcome Trust Sanger Institute), giving the project a broader tissue sampling bandwidth and far reaching access to diverse tissue types and disease states. This effort is integrated into the broader Human Cell Atlas Project (HCA), which aims to complete the 150-year-old dream to identify all cell types in the human body. This community resource project will harness the power of single-cell RNA sequencing (scRNA-seq) to generate a unified cartography of human immune cells. To achieve this goal, we will implement rigorous sample acquisition and processing and analytical pipelines to allow the coordinate analysis of immune cells from many locations, and to integrate these datasets for pooled and normalized analysis, such that a cell in any location or condition can be related to all others. Immune cells do not solely reside in the blood, so we will explore lymphoid organs, as well as frontline (gut, lung, skin) and internal (liver, brain, kidney) tissues in which immune cells reside and mount immuno/inflammatory responses. Because the immune system only manifests its potential when challenged, samples from infectious, inflammatory and tumoral lesions will be analyzed in addition to healthy tissues, selected to reveal the states into which immune cells can be coaxed. We will use unsupervised analysis, which avoids relying on pre-determined marker-based classifications of cell populations, to identify stable immune cell subsets and functional continua. Conventional profiling of sorted populations will complement the single-cell profiles, and allow a deeper, more comprehensive dive into the transcriptomes of cell types uncovered by the single-cell analysis. Working with specific Advisory Panels and harnessing community input, we will derive from these data comprehensive tree and genomic maps of immune cell-types and -states, which will be made publicly accessible through a cloud-enabled data platform, a user- friendly online data portal and mobile apps

PROJECT SUMMARY Macrophages (MF) are one of the largest immune cell components of tumor lesions, where their numbers can even exceed cancer cells. MF play a key role in shaping the composition of the tumor microenvironment (TME), the modulation of tumor innate and adaptive immunity, and the response to cancer immunotherapy. Because of their critical roles, MF are an important target for cancer treatment. However, modulating tumor- associated MF has proved extremely difficult. This is in large part because we still do not have a complete understanding of the tumor MF compartment. In order to develop ways to modulate tumor MF and promote cancer immunity, it is vital we gain a deeper understanding of the molecular and functional diversity of MF in their tissue context. Using mass cytometry (CyTOF) and single cell RNA-seq (scRNA-seq), we initiated deep characterization of the immune composition of early human non-small cell lung carcinomas (NSCLC). We uncovered evidence of multiple distinct MF populations enriched in human tumors. Notably, related MF clusters were also identified in mice lung cancer lesions. Using fate mapping and scRNA-seq, we discovered these discrete MF populations differ in developmental origin and have a distinct distribution in the TME. When the different subsets of MF were depleted, tumor growth was impaired, but the alterations in TME differed, suggesting distinct mechanisms of activity. Based on our findings, we hypothesize that the unique subsets of MF have differential molecular states and mediate differential contributions to tumor growth, organization, immunity, and response to PD1 blockade. To address our hypothesis, we will: (1) comprehensively map the MF compartment of human lung tumors, at the single cell level and with spatial resolution, at baseline and during treatment with PD1 blockade, in NSCLC patients enrolled in a neoadjuvant immunotherapy clinical trial, (2) assess the functional contribution of distinct MF subsets to tumor tissue remodeling and immune cell dynamics in the TME, and (3) determine the contribution of distinct MF subsets to lung tumor immunity. We will also (4) investigate the function and activity of a specific cell surface receptor, Trem2, which we found to be exclusively expressed on monocyte-derived MF in both human and mouse lung tumors, and whose knockout impaired lung tumor growth similar to MF depletion; suggesting an important and potentially targetable molecule in MF control of tumor growth. The outcome of these studies will (i) uncover the molecular and functional diversity of the MF compartment of human lung tumors, (ii) determine how distinct MF subsets, and MF-specific genes, influence tumor growth, the TME state, and tumor immunity, and (iii) provide insight into to how MF subsets influence, and are influenced by, PD1 blockade in human NSCLC. These studies have the potential to help us understand some of the factors that contribute to tumor response and resistance to immune editing, and aid in the further development and clinical use of cancer immunotherapy strategies.

PROJECT SUMMARY Dendritic cells (DC) have a major influence on tumor immunity. DC depletion abrogates tumor immunity and response to PD1 (?PD1) immunotherapy in many preclinical tumor models and DC infiltration in human tumors is a positive correlate of clinical outcome. Unfortunately, we still lack effective strategies for harnessing DC to stimulate tumor immunity and this is in large part because we do not fully understand the control of tumor DCs. To address this shortcoming, we performed scRNA-seq on human and mouse lung tumor lesions. Notably, we identified a distinct and nearly identical population of DC in both human and mouse lung tumors which upregulated genes associated with both DC maturation such as CD40 & IL12, and immunoregulation, including PD-L1 & CD200. This led us to annotate the cluster ?mature DC enriched in immuno-regulatory molecules? (mregDC)(Maier et al. Nature 2020). Strikingly, mregDC were the DC carrying tumor antigen (Ag); meaning these DC are responsible for tumor Ag presentation. We hypothesize the immunostimulatory potential of tumor DC is dampened by genes upregulated in the mreg module and this thwarts induction of tumor immunity and response to ?PD1. We propose that by targeting specific mreg genes we can decouple regulatory & stimulatory programs and enhance DC activation of tumor-reactive T cells and promote tumor immunity and ?PD1 response. In support of our hypothesis, blocking signaling of IL4R, one of the upregulated genes in the mreg module, enhanced DC activation, expanded tumor-infiltrating T cells, and reduced tumor burden in a mouse model of NSCLC. Additionally, inhibition of Birc2/3, also upregulated in mregDC, led to substantially enhanced DC activation. To test our hypotheses and reach our objective, we will: (1) Determine the role of IL4R on induction of the mregDC state and tumor immunity. We will knockout IL4R in mouse & human DC and determine how this impacts mreg induction, Ag presentation, & tumor immunity. We will also combine anti-IL4R & anti-PD1 to assess synergy in controlling tumor growth in a preclinical model. (2) Evaluate Birc2/3 inhibition on the physiology, molecular state and immunostimulatory activity of intratumoral DC. We will test the hypothesis that pharmacological inhibition of Birc2/3 will enhance DC production of IL-12, as well co-stimulatory molecules, while facilitating cancer cell death and tumor Ag uptake, and result in robust tumor immunity. (3) Deconvolute the intrinsic regulators of DC phenotype and the mreg gene module. We will utilize a first-of-its-kind CRISPR genomics platform we developed to KO each of the 37 transcriptional related factor (TrF) genes upregulated by mregDC and determine how each impacts tumor DC activation and molecular state. The outcome of this project will provide a major advance in our understanding of intratumoral DC biology by determining the role of specific genes and pathways in dampening tumor DC functions, establish in preclinical models the therapeutic potential of compounds targeting two different pathways operating in mregDC, and identify additional molecules that could be targeted to enhance DC activity.