PROJECT SUMMARY - Core 3: Imaging Mass Cytometry Core The overall goal of the proposed CCHI U19 grant is to determine the mechanisms of mucosal immune responses to human-restricted enteric pathogens, S. typhi and Shigella, which to date remain significant global health concerns. To determine the limitations of current vaccine approaches and immune protection, we need to better understand the immunologic changes and the mechanisms thereof that are occurring systemically and locally within the gut mucosa. Leveraging our CCHI U19 team’s longstanding expertise in and unparalleled clinical infrastructure for evaluating the immune responses to these enteric pathogens, our proposed center seeks to understand the changes within major immune compartments, including T cells, B cells, and innate cells, upon clinical vaccinations and/or pathogen challenge. To accomplish this, we propose to phenotype the different immune cell subtypes and their functional states in-depth at the molecular level and interrogate how they are epigenetically regulated. Furthermore, to determine how the immune responses are orchestrated in situ within the mucosa, we propose to determine the spatial coordination among the immune cells in human mucosal biopsy samples and integrate this information with other molecular features. To enable synergy across all Research Projects, the Imaging Mass Cytometry Core will operate as a centralized service core, providing an imaging pipeline to be implemented on all biospecimens. The overarching purpose of the Imaging Mass Cytometry Core is, therefore, to generate high- parameter (30-40+ markers) spatial immune profiles with subcellular resolution for all three Research Projects. The Core will support the Research Project investigators with the expertise necessary to most optimally utilize imaging mass cytometry techniques. We established an efficient workflow to receive mucosal biopsy samples from University of Maryland and process them for imaging mass cytometry at Johns Hopkins. This Core will deliver the following: Aim 1, optimized and validated antibody panels suited for each Research Project along with the resulting multicolored quality-controlled images; and Aim 2, segmented single-cell data from the acquired images for compositional and neighbor/distance-based spatial analyses. The Core’s leadership and staff have demonstrated a strong track record of employing imaging mass cytometry to advance immunology studies, ensuring success of the proposed aims. The incorporation of our Core’s spatial analysis expertise will uniquely enhance the efforts of the proposed CCHI U19 to understand the immunologic barriers to effective protection and ultimately motivate new strategies for improved vaccine development.