Matthew F. Krummel - US grants

immunogenetics; cell differentiation; T lymphocyte; developmental immunology; laboratory mouse;

DESCRIPTION (provided by applicant): Cell-cell interactions are of critical importance for expanding the range of the immune response in order to control infection. Yet, the mechanisms that control cell-cell contacts and receptor movement in the immune system remain cryptic. Using high-speed video microscopy, we have been able to demonstrate T cell receptor clustering followed by coalescence of these clusters into the central "synapse". Through simultaneous imaging of intracellular calcium levels, it has become apparent that while initial microclusters are associated with the onset of signaling, a program of cellular re-polarization is necessary for the formation of the central synapse structure and for sustained signaling. The overall goal of my research is to define spatial and temporal maps of the initiating events of immune recognition. The hypothesis underlying this project is that cellular myosin motors triggered by initial TCR signals are crucial for polarization of key membrane receptors into signaling complexes. The specific aims of this project are: 1. To define the myosin family members that are uniquely responsible for synapse formation in T cells, using biochemistry, in situ localization techniques and dominant negative mutations. 2. To determine the biochemical roles for myosins in T cell signaling by examining the interaction partners and phosphorylation of myosins upon TCR ligation. 3. To define the overlapping role of myosin-motors with signaling mediated by costimulatory signals and with signaling mediated by chemokines. These studies will not only provide fundamental information about the initial steps in lymphocyte activation, but may also lead to clues about ways to manipulate T cell responses in vitro and in vivo.

DESCRIPTION (provided by applicant): The nature of the signals that are perceived as T cell activating versus tolerogenic is as yet unclear. A recently revisited readout for T cell activation is receptor clustering at the T celI-APC interface followed by coalescence of these clusters into the central "synapse". We and others have demonstrated that while initial micro-clusters are associated with the onset of signaling, a program of cellular re-polarization is necessary for the formation of the central synapse structure and for sustained signaling. All of the known signaling players in T cell activation that have been examined have been shown to coalesce at the synapse making this site a critical hub for signaling and making participation in this structure a 'biosensor' at some level for participation in signaling. In this proposal, we screen a library of T cell expressed gene-products for their participation in the immunological synapse under varying activation conditions. To do this, we will i.) Construct fluorescent-protein fusion libraries in T cells using gene-trapping and/or cDNA fusions. This technology is already partially developed in our laboratory, ii.) The library will be phenotypically screened for localization and differential localization of fusions to the synapse under varying activation conditions. For this, we will develop and utilize novel medium-high throughput imaging-based single-cell assays including instrumentation and analysis algorithms. In addition to identifying these gene products, our study will open up a technology for larger scale analyses of activating and tolerant signaling. Thus, consistent with this R21 PA, our studies will investigate the unique and innovative use of existing methodology to explore a new area and will develop novel techniques that could have a major impact on the field of biomedical research.

DESCRIPTION (provided by applicant): Intravital imaging has become a vital tool to study cells in their native context and yet there are almost no methods or tools suitable for real-time spatially-resolved analysis of signaling in living tissues. Light-based fluorescence has the best resolution for subcellular resolution of signaling and can be used in real-time. Numerous groups have developed protocols for applying multiphoton fluorescence imaging to target tissues and organs in the mouse. These protocols are currently limited to observation of cell morphology, positioning and motility parameters while progress requires development of probe systems that respond to signaling onset. In vitro, the use of GFP-tagged biomarkers, genetically expressed and used to track molecular behaviors within cells has proven to be extremely useful in tracking cellular activation. However, the situation in vivo has been considered difficult, as a result of considerations of probe intensity, difficulty with interpretation in the absence of fiducials, and regional autofluorescence. We show that, with new improved GaAs detection in 2-photon microscopy, excitation and detection of weak fluorescent protein expression is not the primary hindrance to molecular imaging so long as the molecule of interest is expressed at or near a now-defined level. Instead, the limitations are autofluorescence reduction and strategies that provide fiducial markers to characterize biosensing fusion-proteins. We propose that a collection of multiplexed biosensor arrays will permit the imaging of nuclear import of transcription factors, activation of PI3kinase enzymes and T cell receptors. The genes encoding these will be integrated as P2A-based fusion into the tissue-independent ROSA-26 locus with lox-stop- lox control so that they can be conditionally expressed under the many existing tissue-specific- promoter Cre driver-strains. Based on the design, which mimics our previous success with the T cell receptor first in vitro and now in vivo, these will be functional even in an organ with uneven levels of autofluorescence. Our goal is to develop these tools and subsequently apply them to a T cell interacting with a newly improved model for spontaneous tumor outgrowth. We will also make the mice expressing these biosensors available to the community through non-restrictive sharing and/or the placement of mouse strains into a repository.7. Project Narrative: While our science is achieving great success at understanding the behavior of our cells in isolation, it is more difficult to study the way cells work in their native context. This proposal will create new mouse strains that will permit us to look into tissues using microscopy and observe cells being activated to divide and/or remodel the tissue of which they are a part. It is important for us to understand which cells are being activated and deactivated in the context of many diseases of humans, and our results are likely to provide great insight into the nature of many of these.

DESCRIPTION (provided by applicant): The immune system reacts to the evolving tumor, so why does it not eradicate tumors? T cell clones that recognize tumor-specific antigens are expanded in cancer patients, yet tumors are rarely spontaneously eradicated by the immune system. Similarly, immune therapies that boost T cells, though showing some efficacy, are inefficient. It appears that the immune response frequently is stymied in the tumor microenvironment. There, T cells are exposed to inhibitory and stimulatory signals, either in the form of soluble or cell-surface derived stimuli. Much is still unclear about how tumor-reactive immune cells function and behave in the microenvironment of naturally evolving tumors. We have literally been blind to the types of dynamic interactions that occur between T cells and antigen presenting, innate immune cells in tumors. However, the nature of the collaboration of the innate and adaptive arms of the immune system can now be fully addressed in situ, within the tumor microenvironment, using mouse models accessible to imaging. Based on preliminary data, we hypothesize that a population of innate immune cells regulate the cells of the adaptive immune system in the microenvironment, protecting the tumor from immune attack. Capitalizing on our ability to concomitantly image innate and adaptive immune cells in situ in mouse models of cancer, we will undertake an assessment of the types of immune cell interactions that occur in the tumor. We will address how interactions between adaptive T cells and innate antigen presenting cells are influenced by microenvironments and by tumor types and how it evolves with tumor progression. We will further seek to identify pathways involved in the collaboration between the innate and adaptive immune responses. Finally, we will use our models to visualize and define what immune and cytotoxic therapy does to the immune response in real-time. In this latter point, direct imaging will guide the development and optimization of therapies. Throughout, we will also coordinate with clinical researchers to undertake concurrent analyses of human biopsy samples to translate our findings into diagnosis and therapy.

Why don't tumor-reactive T cells eliminate tumors? Although priming in the draining lymph node is often suboptimal, more frequently, the immune response is stymied at the tumor itself. This may be as a result of a protection of the tumor from immune surveillance (tolerance) or inadequate repriming and feedback to the local draining lymph node (an interrupted feedback loop). Based on a panel of preliminary data, we hypothesize that a subpopulation of APCs in the tumor microenvironment protect the tumor from CTLs by direct interaction with the T cells, by their failure to mature in response to the interaction, and by cooperation with regulatory T cells. We believe the microenvironment builds an arsenal of these cells and collects the immune system at 'inactivating foci'that prevent stable and effective surveillance of the tumor by primed CTLs. By simultaneously preventing the I cells from seeing the tumor and suboptimally activating them there, these cells may effectively neutralize the response. We propose to study this in a new model system of human breast cancer, the PyMTCherry-OVA mouse that we have produced and characterized expressly to address this poorly accessible problem. The model allows direct visualization of the tumor AND the tumor-sampling APCs via intrinsic fluorescence. This is significant as we are able to study what is happening to the T cell as it contacts these distinct cell types, specifically via 2-photon microscopy. It is also significant because we are also able to phenotype this very important APC population using multicolor flow cytometry, microarray, and in vitro assays and provide key data about the co-development of APC and I cell population that results in tumor acceptance. It should be noted that the studies proposed are amongst the first of their kind that will be achievable in a non-ectopic tumor model that very closely resemble human breast cancer in many important ways. The spontaneous eteliology of our model is also important, as there are many reasons that ectopic tumors are unlikely to faithfully replicate important aspects of immune-evasion, including the microenvironment, by a primary tumor. Thus, studies in this system are much more likely to recapitulate the features of human disease and the 'normal'establishment of immune evasion. The results of these experiments are going to reveal the dynamics of I cells over time in the developing hyperplasia through to established carcinomas. They will also definitely reveal the interaction of regulatory I cells with cells of the local I cell response. Finally, and not insignificantly, they will shed considerable light upon a previously inaccessible cell population which may protect the tumor from the immune response and thus permit tumor growth and metastasis.

A unifying theme of the Program Project is the focus on cellular and molecular studies of the evolving microenvironment of airway inflammation. All three projects take advantage of genetic mouse models. Mycoplasma pulmonis infected mice as a model of chronic airway inflammation, and cutting-edge 2-photon and confocal imaging of cells and tissues in the ainways and lung. To this end, the mouse tools core, jointly administered by the co-directors, will serve the common needs of the projects by providing three main functions: (i) to standardize M. pulmonis preparation, infection procedures, and handling of infected mice; (ii) to develop, optimize and implement genotyping procedures for identifying mutant mice; and (iii) to develop and provide access to cutting edge methods for the real-time analysis of cells involved in the development of inflammation in the ainways and lung. First, the core staff will produce stocks of virulent M. pulmonis organisms, which will be tested for use in all experiments to ensure a consistent supply with minimal variability. Second, the core will use standardized flow cytometric, DNA preparation and polymerase chain reaction procedures to genotype mice from the mutant colonies that will be used in the three projects. Third, the core will provide access to and assist in novel and newly developed techniques for real-time viewing of living cells in the ainways and lung through a collaboration with the UCSF Biological Imaging Development Center (www.ucsf edu/bidc/). Centralizing and coordinating infection, genotyping procedures, and imaging approaches will avoid duplication of resources, ensure that standardized procedures are used by all groups, facilitate the exchange of information, and foster collaborative interactions.

The lung Is a unique epithelial surface at which inhaled particles, be they inert, bacteria or viral, are trapped in very close proximity to the bloodstream and in a filigree of epithelial surfaces. This presents unique spatial challenges for the T cells of the immune system to properly survey these antigens. Like many epithelial tissues, a selection of Immune macrophage and dendritic cells are poised within and beneath the surface to engulf and either neutralize, or to induce a more profound response to the Insult. During inflammatory insults including those of allergic but also bacterial or viral insults, the lung microenvironment physically changes. This new Project 2 will use live-imaging of viable lung and airways to determine how antigens traffic Into the lung and subsequently activate T cells, focusing the majority of effort upon a mouse model of asthma. The primary Issue being addressed is how inflammatory environments create a feedback loop that promotes accelerated immune responses and thus an enhanced inflammatory environment. We hypothesize that inflammation in the lung airway and aveolae creates a hyperreactive milieu for T lymphocyte priming. This milieu in turn is highly facilltative for synaptic interactions and thus increased production of lung remodeling factors Including cytokines. We propose that there are three distinct components of this. First, that there is a shift in the numbers, localization, antigen uptake and trafficking features between tolerizing phagocytic macrophages and highly immunogenic dendritic cells. The features of inflammatory antigens as well as the features of a remodeled milieu are both proposed to be directly responsible for the shift in the nature of APC which interact with T cells. Second, a shift In local polyclonal and antigen-specific regulatory T cells,modulated in part by the changes in APC population, subsequently modulates the activation of T cells. Finally, that T cells and their APC are modulated by airway remodeling and inflammation induced by agents such as Mycoplasma pulmonis or mast cell depletion. Thus, inflammation creates a fertile stimulatory ground for responses to inhaled antigens.

DESCRIPTION (provided by applicant): It is now well established that chronic infiltration of leukocytes into neoplastic tissue potentiates solid tumor development. Many experimental and clinical studies have revealed that tumor-promoting leukocytes regulate essentially all aspects of cancer development. However, the molecular natures of individual leukocyte subtypes associated with developing breast cancers (BCs) are inadequately understood. Our overarching hypothesis is that leukocyte biomarkers in human BC can provide prognostic and predictive information regarding BC outcomes, and that these can also be utilized as platforms for design of new, individualized diagnostic and therapeutic agents. This TMEN proposal is based on our collective goal of generating a comprehensive understanding of the composition and transcriptome characteristics of leukocytes in normal breast tissue as compared to BC. Based on identification of predictive immune-based biomarkers that correlate with BC stage, outcome or response to therapy, we will generate innovative, clinically applicable diagnostic and possibly therapeutic, or combined "theragnostic" "probes" for non-invasive in vivo molecular image analysis. Development of these novel and unique reagents will advance our understanding of the tumor microenvironment in BC. As such, we predict that the immune microenvironment in BC can be utilized and exploited to not only predict risk of recurrent disease, and response to therapy, but also to monitor response to therapy and together, these advances will improve outcomes for patients with BC. As such Project 1 will identify clinically significant leukocyte biomarkers in breast cancer. Project 2 will reveal correlation of outcomes in human breast cancer with leukocyte transcriptomes and novel leukocyte biomarkers;and Project 3 will drive translation of leukocyte biomarkers into clinically applicable diagnostic and therapeutic probes.

DESCRIPTION (provided by applicant): We are requesting funds to purchase a state-of-the-art commercial multiphoton microscope specifically configured and situated to accommodate a portfolio of translational imaging approaches, largely dedicated to pursuing studies of cell-cell dynamics in human normal and diseased biopsy tissues and organ slices. The instrument will be specifically configured and certified for human tissues/BSL-2 and will be located in the UCSF Parnassus Heights Biological Imaging Development Center's (BIDC) development space, adjacent to the UCSF hospital. This application represents a collaboration of expertise from clinical and basic scientists, as described in the proposal. This will provided much-needed capacity through a facility well-versed in the art and will facilitate the transition of the technology into promising areas that are currently marginalized by instrument availability and configuration. The instrument will be primarily used by a core of 11 NIH funded investigators and their laboratories which facilitate 15 NIH-funded R- P- or U- level projects. The principal type of experiment for which this microscope system will be used include multidimensional timelapse imaging of cells in living tissues, typically revealed with fluorochrome conjugated non-stimulatory antibodies or protein conjugates, directly applied to tissue biopsies or to redundant transplant tissues. It will also accommodate imaging of a wide variety of fluorescent proteins in comparative mouse models. Located in the BIDC and utilized approximately 80% by the investigators on this proposal, it will also be generally accessible by the larger campus community served by this facility. The system we are requesting, a Zeiss 710LM was chosen amongst A-B trials comparing our custom built microscopes, whose design has proven compatible with deep tissue/high sensitivity data collection, with the commercial offerings. The microscope will be equipped with an environmental chamber as well as modules for on-stage organ culture, a motorized stage, and high-efficiency PMTs for high resolution and high sensitivity imaging. Dipping water immersion objectives are requested for the optimal collection of data from live cells or tissues in a 3D environment at the highest achievable optical resolution. A combination of high-sensitivity PMTs and 32-element 'spectral'PMT detection arrays is requested to permit imaging of all commonly used dye combinations and/or fluorescent protein variants but also to extend local success using multichannel autofluorescence background subtraction methods to enhance weaker signals. The public userbase at UCSF has two highly functional custom-built multiphoton instruments and two older commercial adaptations. However, the functional instruments are oversubscribed due to the high volume and long timelapses of the typical experiments being run. In addition, 'translational'imaging requires a microscope which is both specifically certified for human studies and which has sufficient capacity to accommodate the short lead-times of human tissue samples.

The pulmonary bronchiolar and alveolar epithelia are involved prominently in a number of important human diseases associated with loss of alveolar integrity, including emphysema and pulmonary fibrosis. In each of these conditions the capacity to generate new alveolar epithelium, and its associated vascular bed, would be of great potential therapeutic value, but such capacity remains beyond the reach of current technology. This application is predicated on the idea that better understanding of distal ainA/ay and alveolar epithelial cell progenitor biology is a crucial part of any effort to move the field of directed distal lung remodeling or repair toward the clinical arena. Toward that end, this application brings together investigative groups with different expertise but overiapplng interests in epithelial progenitor cell biology to advance the understanding of distal lung development, maintenance, and repair. The major objectives are (1) To define the transcriptional program of heretofore uncharacterized distal airway and alveolar progenitors and test the hypothesis that differential expression of adhesion receptors underiies the capacity of epithelial subtypes to self-organize and promote repair. (2) Define the requirement for neuroendocrine cells (PNECS )and alveolar progenitor cells in maintenance and reconstitution of distal airway and alveolar cells following lung injury. (3) Analyze and further develop a novel, single cell in vivo lung organoid assay in kidney capsules in order to optimize the capacity of adult epithelial progenitor cells to generate functional respiratory units de novo. Important tools and approaches developed to achieve these aims include mice with inducible cre activity knocked into lineage-defining genomic loci, flow cytometry-based techniques to isolate and transcriptionally profile mouse and human embryonic and adult epithelial progenitors, and innovative imaging that allows real time capture of stable images of lung and lung organoids over time. We anticipate that by the completion of these studies we should be able to adapt our in vivo assay toward orthotopic transplantation of cellular units capable of lung development. Overall, these studies should provide crucial conceptual and technological infrastructure for the clinical translation of progenitor cell biology to human lung disease.

DESCRIPTION (provided by applicant): It is believed that the lung is a permissive organ for the seeding of certain metastasizing tumors. Recent studies have supported this, finding that distal primary tumors instigate bone marrow-derived cell (BMDC) migration into the lung, forming a pre-metastatic niche (PreMN), and that these cells are crucial for robust and efficient metastasis. These studies have led to a growing recognition of the importance of the immune system and the pulmonary PreMN in lung metastasis, but many important questions remain and have been hitherto inaccessible. Most notably, there has been no direct way of assessing the behavior and fate of DTCs within the lung PreMN versus normal 'less-permissive' lung tissue. Further, the PreMN contains a diverse set of immune cells and the effect of interaction with these populations on DTC survival is unknown. In order to understand and therapeutically target pulmonary metastasis we must first address these crucial questions, in vivo The basis for this R21 exploratory grant is to apply novel real-time intravital imaging approaches to understand how the lung deals with incoming cells. Of particular relevance are to understand why and how metastatic cells survive in the environment and how host-cells 'receive' them and protect them from normal elimination of cells from the vasculature. We hypothesize that normal removal of cells from microvasculature is a repair process which metastatic cells, helped by host cells, extend in duration to achieve successful colonization. The overall success of this project will be defined by our knowledge of the spatiotemporal landscape that metastatic cells face upon their arrival in the lung. Are tumor cells seeded followed by being joined by 'helper' host cells or are successful mets captured directly by these cells? What are the kinetics of the proliferation of incoming metastatic cells relative to the recruitment of macrophages and bone-marrow derived cells? In what way is this process accelerated by ongoing damage repair? Better understanding of this, along with the development of a method to study it, will permit much more rational approaches toward blocking tumor metastasis. PUBLIC HEALTH RELEVANCE: This proposal will apply novel intravital imaging of the lung to define the first hours following the arrival of metastatic cells into the mouse lung. As we know very little about why metastatic tumor cells survive in this environment, this represents a major undertaking in determining how to decrease their success.

Asthma affects millions of individuals in the US. It is a disease propagated by the immune system in interaction with itself and components of the lung. Despite decades of study, there has previously been little methodology to study the dynamics of the key members of the immune response at the living-tissue level, particularly in subclasses of asthma. Thus, treatment strategies have not profited from an understanding of how very specific subsets of immune infiltrates interact with the tissue, deposit cytokines upon them and propagate the allergic state. This project will apply advanced real-time imaging methodologies to determine how and where the immune response takes place in real-time. We will characterize both IL4/13 and IL-17 producing cell types and will include analyses of adaptive (T cells) and innate (e.g. eosinophils, Ih2). We will determine how and when they interact with one another within the tissue and how and when cytokine exchanges take place. We will then transfer this technology to the analysis of human lungs from the shared Clinical Subject and Biospecimen Core. These studies will in turn allow us to understand the homing and interaction zones for two specific classes of T helper cells, secreting IL13 and IL17. These analyses will take place in living lung biopsies and will develop technologies for visualizing ongoing biology in clinically relevant subclasses of asthma; notably before/after allergen challenge, before/after inhaled corticosteroid and in severe asthmatic lung. Together with critical reagent and method development that will be useful for many other asthma and lung applications, this proposal will provide exquisite insight into how the immune system propagates the allergic state in a variety of clinically-relevant settings.

It is now well established that chronic infiltration of leukocytes into neoplastic tissue potentiates solid tumor development Many experimental and clinical studies have revealed that tumor-promoting leukocytes regulate essentially all aspects of cancer development. However, the molecular natures of individual leukocyte subtypes associated with developing breast cancers (BCs) are inadequately understood. Our overarching hypothesis is that leukocyte biomarkers in human BC can provide prognostic and predictive information regarding BC outcomes, and that these can also be utilized as platforms for design of new, individualized diagnostic and therapeutic agents. The goals of Project 1 are to further define leukocyte subtypes correlating with aggressiveness and/or specific molecular subtypes of BC and recurrent disease (Project 1, Aim 1). Identify leukocyte biomarkers that predict long-term outcome and response to therapy for patients with BC (Project 2, Aim 2). Define molecular/cellular tumor-promoting activities of high risk leukocytes in mouse models of BC to enable biomarker and target validation for anticancer therapy (Project 1, Aim 3), We will leverage human BC samples and existing murine models to define immune-stromal biomarkers in tissues that correspond to tumor-supportive or tumor-rejecting environments. Predictive/prognostic biomarkers, using our access to and expertise in complementary mouse and human BC samples, will be defined in three ways. First we will use the subdivision of macrophage and dendritic cell populations based on surface expression and/or tumor localization to define novel genetic signatures for prognosis that can then be translated into protein reagents (i.e. antibodies). We will further subdivide leukocyte infiltration using protein reagents that detect activation states for leukocytes in the microenvironment. Finally, throughout, we will track prognosis versus marker expression for a variety of BC subtypes to determine whether biomarkers sort by BC subtype.

DESCRIPTION (provided by applicant): T lymphocytes require dynamic movement for all of their critical functions. Motility, synapse formation and signaling are intricately linked through he actions of the cellular cytoskeleton. Data from our lab and others indicates that the T cell cortex is tightly regulated from within, using myosin motors and the associated septin cytoskeleton that control cortical integrity and membrane tension, all of which function via intimate association with a continuously remodeling actin cytoskeleton. This control has profound implications for the process of scanning organs for antigens, for the process of interacting with antigen-presenting cells, and for the process of interacting with targets. It is clear that there remains a dearth of understanding about which individual system controls T cell membrane biology and specifically how these: 1. Control cell-intrinsic scanning behavior such that organs are completely surveyed. 2. Control cortical and membrane tension so that membrane-membrane interactions (synapses) are optimized. 3. Drive the large-scale aggregation of proteins into domains that encourage signaling or lead to its cessation. This project will study these fundamental issues. We have assembled an unrivalled panel of genetic knockouts in the myosin/septin pathway and have revealed critical roles actin depolymerization as a co-factor in synapses and likely cell motility. This project will delve into how the T cell works in its native habitat and will reveal nvel mechanisms that regulate immunity and tolerance.

? DESCRIPTION (provided by applicant): Treating the immune response within tumors is a major focus of new therapeutic development. For emerging therapies, there are clear responders and non-responders and at present, we are blind to knowing in how many ways the immune system is deficient. We are also unable to know which of the current or emerging therapies will work for a given patient. The current best-approach is to put each patient through a sequence of therapies before (hopefully) hitting upon one that works. In the future, we believe we will be able to tap the wealth of information that is contained in the just-excised biopsy of each patient. Each biopsy contains significantly more information than we currently glean in the form of the spatiotemporal cellular interactions that are taking place therein. Many immune activities continue to occur when biopsies are collected, maintained and imaged under appropriate conditions. We hypothesize that it is possible to study the functionality of the human tumor microenvironment `live' by the development of standard practices for `live biopsy'. If we are right, patient samples can be both characterized overall but also used as a test-bed for single or combinations of therapies to determine the most likely one to induce tumor rejection. Rare, small and information-rich human biospecimens will be directly and reproducibly studied and will be critical in next-gen discovery and diagnostic platforms. This study will define a serie of parameters by which biopsy samples can enter this important discovery stream.

? DESCRIPTION (provided by applicant): What actually happens to responding T cells in the lymph node during vaccination? Specifically: What is the nature of the developing physical `niche' in which cells activate? Who are the members of this cohort and what are the critical temporal features that bridge two concurrent responses? We intend to discover the answers to these questions as a route to determining how to achieve solid CD8 T cell immunity to viruses. We will focus efforts both in lymph node and in the lung and the latter will access cutting-edge imaging approaches developed in our lab. We and others have recently used live imaging to demonstrate that T cell priming in the lymph node and reactivation in the lung take place under highly dynamic conditions that would appear to permit considerable mixing of ongoing responses. Our lab has established a series of cutting-edge imaging approaches including multiphoton-based cell-tracking and synapse-analysis, paired with our expertise in `classical' immunological assays, to study this `collective' activation; activation of multiple T cells that occupy the same reactive lymph node. A key finding that is corroborated by others is that there is a `Critical Differentiation Period' that coincides with individual activating T cell clones comig together into a T-T synapse-mediated contact. Based on our work and that of others in the field, we hypothesize that there are both natural and synthetic forms of cell- cell interactions that can be leveraged during elicitation of a CD8 response and that these will alter the outcome of a viral challenge. Through addressing this hypothesis, we aim to finally provide a rationale understanding of how immune cells co-activate and improving vaccination for CD8 responses.

? DESCRIPTION (provided by applicant): We are woefully ignorant of which host cells are educated in the tumor and contribute to distal sites. Further, although it is very likely that specific immune cells (e.g. myeloid) travel to the distal lymph nodes, key features (their identity lifespan, self-renewal, specific functions) of such cells remains unaddressed. Remarkably, we cannot currently know which host cells in a metastatic site first arrived following residence in a primary tumor. In this application we will develop new technologies to lineage-track cells from one organ to all others, over the entire lifespan of the cell and its progeny. We will use this to test the hypothesis that immune cells, particularly those of the myeloid lineage, are 'educated' in the tumor microenvironment and contribute to the composition of distant sites such as lymph node, spleen and possibly metastatic sites; furthermore we will determine the functional consequences of such a contribution. Notably, this will represent the first direct and unambigious demonstration of which cells traffic to and present antigens in draining lymph nodes, a site-to-site mapping of entire lineages, a discovery of novel lineages that traffic out of tumors and a test that metastatic sites contain immune cells that were once within another lesion. This technology solves a major deficit with existing photoswitchable approaches and broadly extends cutting edge live-imaging approaches towards lineage tracing.

Identification of cancer drug targets using high throughput screens of tumor cell lines has led to a number of agents presently in clinical trials. In addition, recent advances in drugs that attack immune cells within tumors, such as ?CTLA4 and ?PD-1, have highlighted the importance of immune modulation as a strategy for cancer therapy. The next phase of cancer drug target discovery will seek to integrate these strategies to identify combinations of drugs that most efficiently target both tumor cells and the immune components in advanced cancers. The goal of this proposal is to identify and validate these combinations using large-scale data mining and mouse pre-clinical cancer models that mimic the major genetic features of human cancer. This proposal addresses both mechanisms of immune escape by a) finding genetic targets that may enhance tumor mutation load, and b) carrying out high throughout screens in T cells or myeloid cells for targets that promote immune cell infiltration. We will exploit unique mouse models that mirror major genetic categories of human cancer ? high vs low mutation load, and strong vs weak immune infiltrate. Applying single-cell RNAseq and mass cytometric proteomic analyses, cutting edge immune composition databases and novel computational network approaches to cancer target discovery using existing large databases, we propose to identify vulnerabilities addressed by combining small molecule drugs with immunotherapy. We will make immunologically ?cold? tumors, that do not engage the immune system, into ?hot? tumors that present more or stronger antigens, or that encourage infiltration by immune effector cells. To achieve this goal, we propose three highly innovative aims centered on perturbation of specific targets: first by a CRISP/Cas9 screen in immune cells of the tumor microenvironment, second through increasing antigen load in tumors to optimize immune recognition and finally through a network-based identification of tumor-expressed targets that may confer susceptibility to existing immune-oncology therapies. This represents a true `network' of our collective expertise as well as a measured collection of candidate and screening approaches. AIM 1 ?We will perform CRISPR screens in monocytes and T-cells to identify genes associated with tumor entry and function in two distinct tumor types. AIM 2? We will use genetic or pharmacological perturbation of newly generated candidate genes involved in metabolic stress and ROS-induced DNA damage to increase mutation load and antigen abundance in a tumor- specific manner, leading to improved responses to immunotherapy. AIM 3 ? We will exploit gene expression networks to identify druggable targets and pathways that augment immune responses. This proposal identifies pathways and perturbants for accelerating immunotherapies.

Project Summary/Abstract Treating the immune response within tumors is a major focus of new therapeutic development. Much of the focus has been place on T cells, in particular via checkpoint therapies such as anti-CTLA4 or anti-PD1. Little is currently known of how individual populations of myeloid cells can be partners for T cells. We have recently isolated rare populations of myeloid cells that appear critical for robust responses but we don't yet fully understand how they work. We hypothesize that rare stimulatory dendritic cells traffic antigens and stimulate T cell according to specialized rules and that harnessing and modulation of this pathway is part of the reason that checkpoint blockades may work. We further hypothesize that specific tissue-based cells are responsible for upregulating the critical cytokine to make stimulatory dendritic cells but that tissue production is dysregulated in cancer and possibly improved with checkpoint therapies. In this proposal we will be vastly extending an approach that my lab has been pursuing over the last few years. Specifically we will be extending our cell-biology based studies of these critical cells (Aim1) to understand how they play a fundamental role in antigen trafficking. Additionally, we will seek to understand how they hand off antigen to other antigen-presenting cells in the lymph node to engage T cells (Aim2) and how both of these processes are affected by checkpoint blockades. Finally, in aim 3, we will seek to understand the normal and intratumoral production of the cytokine Flt3L, a key player in regulating the number of these rare cells. At the end of this work, we will understand how these intratumoral myeloid cells function on their own and in concert with T cell therapies.

Project Summary/Abstract T cells utilize surface-bound T cell receptors (TCR) at the immunological synapse with antigen-presenting cells (APC). Detection of their peptide-MHC ligands results in rapid intracellular signaling, necessary for acquisition of effector functions and for profound adaptive immunity. While we now understand some of the fundamental proteins in this processing, understanding how each works together in the context of a rapidly moving T cell has proven difficult. TCR recognition happens as surface deformations provide initial contact. However, despite various fixed and lower-resolution approaches to understanding this process, it has not been previously possible to study this complete surface in real-time in the full 3-dimensions in which it takes place. Here, we will use novel and advanced imaging approaches to define how cytoskeletally-driven membrane movements provide a backbone for efficient ligand detection and we will describe how a range of widely variant environmental cues as well as novel pathways alter this process and affect immune surveillance. This project will define how T cells effectively `find' their ligands amidst a sea of competing MHC. This efficiency of search and detection has clear implications for the ability of T cells to discovery rare epitopes and initiate a response, for example during the early phases of a viral infection.

Abstract/Summary Core C: Microscopy Core. Diabetes research depends on studies of the spatial relationships of cells within tissues or structures within cells, and the relative positions and amounts of molecular factors at those sites. Microscopy enables the detection of alterations in those differences upon the initiation of disease and/or during the normal developmental and physiologic processes investigated as a component of diabetes research. Purpose: The Microscopy Core consolidates, enhances and disseminates resources and expertise in tissue and cell imaging technologies. The Core provides the following capabilities that, in the past 2 years, were used by 122 researchers in 22 DRC laboratories. DRC use averaged 2,332 hours/year over the last four years. 1. Fluorescence Microscopy. Three optical sectioning (two confocal; one Apotome) and two widefield microscopes with highly complementary capabilities are present in the Core. Up to five different markers may be imaged in a single sample. The inverted microscopes also are retrofitted for live cell imaging studies. 2. High Throughput Fluorescence Microscopy. Up to 80,000 fields per day can be collected on a robotic system for large-scale identification of compounds or factors affecting microscopically measurable processes. 3. Histology. Three microtomes, a cryostat, a tissue processor, a histoembedder and a brightfield microscope are available in the Core. 4. Image Processing. Three computers are available exclusively for post-acquisition image analysis. 5. Information and Training. The Core trains investigators on use of equipment, provides advice on the application of imaging technology, and provides software and reagents for microscopy applications. This includes advice and/or assistance with multiphoton and electron microscopy available in other UCSF Cores. Benefits to DRC Community: The Core accelerates diabetes research by providing DRC investigators access to advanced imaging technology in a practical and economic fashion. 27 NIH-funded and 29 other diabetes- related projects totaling $13,769,481 in annual direct costs currently benefit through these efforts. Technology Development: The Core continues to evolve to meet demand. Over the past four years of operation, existing equipment continues to be maintained under Core recharge mechanisms. New Zeiss Apotome and Leica SP5 confocal microscopes were purchased through shared equipment grants, departmental funds and gifts from donors. Access by DRC investigators to electron microscopy was enabled under an arrangement in which the DRC Core Manager manages microscopes owned by others. For all acquisitions, the DRC Microscopy Core defined equipment needs, acquired the instruments and trained investigators in use of the equipment. Other services under development activities approved by the DRC Executive Committee include three-dimensional imaging on stained, whole-mount tissues. These mechanisms for providing instruments and support will continue to evolve the Core in alignment with DRC needs.

PROJECT SUMMARY This is an administrative supplement to the parent R01 AI052116 ?SPATIOTEMPORAL CONTROL OF T CELL SYNAPSE STABILIZATION AND SIGNALING? which for my entire career has been my central grant for studies of T cell interactions leading to tolerance or activation. Here, we apply our considerable immune and tissue-immune experience towards generating and exploiting a RapidPath platform to find rapid actionable immunotherapeutic targets for COVID-19 patients for limiting damage due to SARS-CoV-2 infections. In aim 1 of this study, we will build a lung plus virus plus immune platform in which the role of specific T cells of different activation status ?alone and through their modulation of myeloids cells?will be assessed in the response of damage to lung epithelium plus/minus endothelium (organoid, with Roose/Gordon and lung slice with Looney). This supplement will interact intensely with parallel studies of those labs and also with ongoing studies that will also leverage RapidPath but are not in this first cohort of applications. This will provide `best in class' model systems in human biology and will leverage our collective expertise. In aim 2 of this study, we will test a panel of immunomodulatory drugs to determine if acute exposure to them can modulate lung damage, likely through modulating myeloid biology. The net result will be validated immunotherapeutic paths in robust pre-clinical human systems that recapitulate key features of COVID-19.