Project Summary The opportunistic fungal pathogen Aspergillus fumigatus presents a major health concern in immunodeficient and critically ill patients, with invasive disease contributing to high mortality rates. Infection with A. fumigatus elicits a diverse adaptive CD4 T cell response. Our preliminary data have demonstrated distinct TH1 and TH17 spatial neighborhoods within mediastinal lymph nodes (LNs) during A. fumigatus airway infection. This spatial organization positions activated CD4 T cells to receive tailored signals (e.g., antigens, cytokines) for optimal effector cell differentiation and function. Lymph node stromal cells (LNSCs) are known to provide guidance cues for immune cell trafficking during monotypic effector T cell responses. However, the mechanisms by which distinct TH1 and TH17 microenvironments are concurrently established within LNs remain unknown. Defining these mechanisms will offer insights into the functional importance of LN microenvironments in establishing human immunity to infection and inflammatory disorders for the identification of novel therapeutic strategies. Based upon our single-cell RNA-sequencing and flow cytometric analyses of FRC and effector T cell subsets during A. fumigatus challenge, we propose that (i) FRC subsets establish spatially distinct TH1 and TH17 neighborhoods by dynamically regulating chemotactic receptor-ligand axes, such as CXCR3:CXCL9/CXCL10 and CCR6:CCL20 and that ii) these neighborhoods can persist after pathogen clearance. We will address these hypotheses in a clinically relevant murine model of invasive pulmonary aspergillosis. In Specific Aim 1, we will characterize distinct spatiotemporal microenvironments formed by CD4 T cells and FRCs in A. fumigatus infection by applying spatial transcriptomic profiling followed by high-content immunofluorescence methods paired with a novel computational approach for image analysis. In Specific Aim 2, we will define the functional role of CD4 T cell spatial organization in A. fumigatus infection by perturbing FRC- dependent chemokine gradients in a cell type-dependent manner using FRC-specific gene targeting. We will also conduct adoptive co-transfers of A. fumigatus-specific CD4 T cells sufficient and deficient in CXCR3 and CCR6. In Specific Aim 3, we will investigate the durability of infection-driven spatial FRC diversity by studying epigenetic modifications in A. fumigatus-experienced FRCs. We will also employ high-content confocal imaging to determine whether prior infections affect FRC formation of T cell neighborhoods in subsequent infections. This study employs and develops novel genetic tools, microscopy methods, and computational approaches to generate a systems level understanding of secondary lymphoid organ immunobiology. Furthermore, this proposal is tailored for a physician-scientist in training as it investigates the mechanisms by which stromal cells induce adaptive immunity to the clinically relevant pathogen A. fumigatus, with ...