PROJECT SUMMARY/ABSTRACT Age-related diseases, such as Alzheimer's disease (AD), are defining public health concerns of the 21st century and are the leading cause of disability worldwide. A growing body of evidence notes that central nervous system immune changes superimposed on ongoing chronic neurodegeneration may have a major impact on disease progression in AD and other neurodegenerative conditions. The role of the peripheral immune system in AD, including cellular elements of both the innate and adaptive immunity are still enigmatic. In this proposal, we investigate immune cell identity and abundance in the periphery and cerebrospinal fluid (CSF) at single cell resolution among preclinical-AD, MCI-AD, and AD-Dementia stages and among normal aging controls. Furthermore, a novel aspect of this study will be to corroborate the severity of blood brain barrier (BBB) changes associated with specific immune cell profiles noted in CSF and the periphery across AD stages and its impact on longitudinal cognitive decline. Our overall goal is to characterize immune cell identity and abundance in the periphery and the CNS at single cell resolution across different AD clinical stages and in tandem, to clarify the peripheral anatomical context of immune cell activation and the degree of BBB changes, with the goal of understanding the temporal course of immune response in relation to initiation of clinical decline and subsequent phenotypic heterogeneity of AD trajectories. Towards this, we will investigate immune cell phenotypes in the CSF and peripheral blood by a powerful single-cell proteomic analysis technique, mass cytometry, and BBB changes will be delineated by dynamic contrast-enhanced MRI techniques in the same subjects. Data integrating peripheral and CSF adaptive and innate immune cell activation along with BBB changes across different stages of AD will help develop a clear rationale for the use of these markers in the AmyloidTau(Neurodegeneration) framework. Additionally, these insights will help future therapeutic targeting of specific immune and BBB changes at the most effective clinical stage of disease. The results from the study will also enable the identification of novel mechanisms that regulate immune cell homeostasis in the periphery and the CNS, and the state of BBB, providing potential means to manipulate these immune cells for therapeutic purposes in the future.