Successful immunity relies on the rapid orchestration of diverse immune cell types that work in a dynamic and cooperative manner to drive location-dependent effector function. Immune cells do not work in isolation, and cooperation between immune cell types requires the sensing of local position-dependent cues that enable immune cells to aggregate into niches to exchange signals in a series of complex feedback loops. These coordinated interactions are critical throughout the developing immune response, from early activation events in inflamed lymph nodes through to effector function and resident memory in inflamed/infected targeted nonlymphoid tissues. A comprehensive understanding of immune responses thus requires the ability to examine the dynamic spatial and temporal exchange of information, in situ, in changing locations within and between tissues. To gain new insight into the biological complexity of immune activation in tissues, this P01 renewal brings immunologists and engineers together into the Programs multi-disciplinary scientific team, adding new concepts, perspectives, and tools to enable immunological questions to be addressed at ever higher spatial and temporal resolution. We aim to integrate dynamic intravital multiphoton microscopy with spatially defined transcriptomics to address fundamental questions about how topographical organization shapes T cell priming and effector function in response to infection. Shared models of infection examined in the lymph nodes and respective skin and lung tissue will enable integration of information across immune response time and space. The goal of this Program Project is to establish new paradigms in tissue immunity by gaining insight into cooperative events that shape immune cell positioning for exchange of signals that regulate effector function in lymphoid and non-lymphoid tissues. By spatially resolving immune response dynamics and transcriptional programs together, we aim to uncover new actionable strategies, based on the principles of immune position/zonation, to overcome poorly immunogenic environments and diminish overt inflammation