Germinal centers (GCs) are the microstructural sites in secondary lymphoid organs, but GC structures are impaired in AIDS patients due to the loss of supporting CD4+ T cells, leading the deficiencies in immune responses and even inefficacies in antiretroviral therapies in HIV-infected individuals. To solve this challenge, rhesus macaques are well-established nonhuman primate models (NHPs) to study the immunopathogenesis of HIV. However, the spatial coordination of Follicular dendritic cells (FDCs), T cells, B cells, extracellular matrix (ECM), and cytokine regulation of simian immunodeficiency virus (SIV)+ infected macaques and SIV- macaques is still not clearly understood. Thus, there is a critical need to decipher the spatial and temporal control of (1) cytokine, (2) ECM, and (3) T-B cell interactions at the single cell level in GCs for identifying the (1) spontaneous activation and (2) SIV-infection-induced responses in the immune system of HIV/SIV disease. To shed light on immune regulation in lymph node tissues of SIV+ and SIV- macaques and human donors, this project will leverage spatial proteomic and transcriptional profiling to map B cell subsets and their interactions with T cells, ECM, and cytokines in the lymph node tissues. The long-term goal is to generate single cell insights into B cell development in GCs of SIV+ and SIV- macaques in response to spontaneous activation and infections. The goal of this project is to define spatially resolved cellular interactions and cytokine/ECM gradients pixel-by-pixel in fixed macaque and human tissues. The hypothesis is that (1) spatial distributions of cell types, cytokines, and ECM of spontaneous GC activation are uniquely controlled by CD4/FDC and B cells in NHPs compared to humans, and (2) B-cell responses are spatiotemporally regulated by CD4/FDC cells in NHPs with and without SIV infections. The rationale for this hypothesis is based on the evidence that (1) IL- 10+ cells were spatially close to viral SIV-DNA+ lymphoid cells in SIV+ tissues and (2) heterogeneous GC activation and spatial GC organization maps in macaque and human tissues. The central hypothesis will be tested by pursuing two specific Aims. Aim 1 will evaluate the effect of multiplexed cytokine, ECM, and CD4/FDC interactions with B cell subsets in SIV- macaque (n=24 each) and HIV- human tissues (n=24). Aim 2 will evaluate cytokines, ECM, CD4/FDC, and B cell maturation in lymph node tissues of SIV- and SIV+ macaques (n=24). To accomplish these Aims, spatial cell phenotyping and cytokine gene expression profiling will be used to analyze B cell development and statistical comparisons of spatial cell neighboring features in macaque and human tissues. This project builds an interdisciplinary team integrating experts from spatial omics, NHP immunology and pathology, and bioinformatics. The proposed application is innovative because it uses cutting-edge technology to study spatial proteomics and transcriptomics of lymph node tissues of macaques and humans. This research is significant because it defines spatial GC organization to understand why SIV+ infections induce GC defects and how it deviates from human immune responses.