PROJECT ABSTRACT The SARS coronavirus-2 (SARS-CoV-2) has rapidly emerged over the past four months leading to a critical pandemic of coronavirus disease (COVID-19) with over 1.4M cases worldwide (https://coronavirus.jhu.edu/map.html) and roughly 100,000 projected fatalities in the US alone by August 2020 (See https://covid19.healthdata.org/projections). SARS-CoV-2 causes a lethal ARDS. Despite our improved mechanistic understanding of ARDS, intervention clinically is challenging. NOT-AI-20-31 indicated several needs, such as development of reagents and assays for virus characterization, understand critical aspects of viral infection, replication, pathogenesis, and transmission, identification and evaluation of the cellular and humoral immune responses to SARS-CoV-2, which we address in this proposal. Indeed, there is an urgent need to understand the immunopathology of COVID-19 and study the interactions of the lung epithelium and tissue, the immune system and the virus to understand the biology of this multipartite interaction. We need to better understand the immunopathology of COVID-19 to explore novel therapeutic approaches that have the potential to work in COVID-19 patients. Our proposal addresses this need from the perspective of the lung epithelium response to SARS- CoV-2 infection and from a T cell perspective in COVID-19. Simultaneously, or efforts will also provide a sharable research platform of lung airway organoids/SARS-CoV-2/immune cells that will expedite testing of experimental therapeutics. Results from my supplement program will be shared with Drs. Gordon, Looney, and Krummel in our ‘RapidPath’ program (see supporting letter) to promote rapid discovery and progress and will be compared to insights from COVID-19 patient immune systems, being simultaneously profiled in the UCSF IMPACC project. In this this Administrative Supplement we will capitalize on my lab’s established expertise in T cell signaling, T cell activation, antigen recognition, inflammation, and autoimmune diseases. Those are broad topics of the parent P01 (2P01AI091580, Weiss). Uniquely, we will combine our T cell expertise with our expertise in the generation and studies of epithelial cell organoids. We already have an “Airway Organoid Biobank” that we will expand as a resource for the community. We will characterize the epithelial response to six different SARS-CoV-2 strains compared to H1N1pdm virus, using airway organoid-, single cell RNAseq-, and CyTOF- technology (Aim 1). In order to better understand SARS-CoV-2 and adaptive immunity, we will obtain mechanistic insights into T cell activation and T cell signaling in the context of SARS-CoV-2- and H1N1pdm- infection of seven, diverse Airway Organoids and two NSCLC organoids (Aim 2). High-resolution imaging and CyTOF analysis of these “virus-T cell-organoids” will provide much needed immunological insights into SARS-CoV-2 and its T cell biology as indicated in NOT- AI-20-31 and will synergize with other projects in ‘RapidPath’ and in UCSF IMPACC programs.

PUBLIC HEALTH RELEVANCE: In this program project renewal, our overall goal is to capitalize on our current progress and bring together approaches from structural biology, proteomics, immunology, and computational biology to understand TCR signaling. In project #1, we will study the distinct features of the T cell-expressed tyrosine kinases that make these most suitable for antigen receptor (TCR) signaling in T cells. In project #2, we hope to understand how TCR signaling regulates Ras, a critical regulator of cell activation, to establish basal homeostasis and allow for efficient activation of T cell responses. Project-001: Project 1 Project Leader (PL): Weiss, Arthur DESCRIPTION (provided by applicant): This application is a renewal of an ongoing project in which four investigators with different but complementary expertise have worked together to understand how the T cell antigen receptor (TCR) regulates the proximal tyrosine kinases (SFKs, Syk kinases and Tec kinases) that control critical downstream tyrosine phosphorylation. We have made considerable progress in understanding the structural basis for the specificity differences that are encoded in the kinase domains and the autoregulatory constraints that control the activities of these kinases, but a full understanding will require new approaches. We propose to capitalize on our current progress and bring together approaches from structural biology, physical sciences, proteomics, immunology, and computational biology to perform studies that are aimed at understanding the distinct features of the T cell-expressed SFKs, Syk and Tec kinases that make the individual kinases more suitable for antigen receptor signaling in T cells than in B cells. We hypothesize that the characteristics of Lck and Fyn, ZAP-70 and Itk and their signaling regulators have been optimized in T cells to establish signaling circuitry that serves to maintain a basal signaling state that is resistant to perturbations by non- agonist peptides and also establishes a sensitive threshold for optimal recognition and response to agonist pMHC. We will explore this hypothesis in experiments designed to: 1) understand the unique features of the proximal kinases that are advantageous in TCR signaling; 2) define the regulatory mechanisms that constrain the activity of the proximal tyrosine kinases; 3) determine how TCRs maintain basal homeostasis and distinguish biological noise from antigenic stimuli; and, 4) define the key intracellular events needed to initiate downstream signal propagation by the TCR.