In Alzheimer’s disease (AD), danger signals including dead neurons, dystrophic axons, tau and Ab alter the functional phenotype of microglia from a homeostatic (M0) to a neurodegenerative (MGnD) or DAM (disease associated microglia) phenotype, which in turn drives neuroinflammation and promotes disease. In addition, it is now known that there is a population of disease-associated astrocytes (DAA) in AD which appear at early disease stages and increase in abundance with disease progression. However, the investigation of microglia and astrocytes in AD has been hampered by the lack of understanding their phenotype and function and the lack of therapeutic approaches that can target neuroinflammation associated with these cells. In the mouse model of chronic EAE, we have found that nasally administered anti-CD3 mAb localizes to cervical lymph nodes where it induces IL-10-secreting regulatory T cells (Tregs) that then migrate to the brain and suppress both microglia and astrocyte neuroinflammation. We have recently shown that nasal anti-CD3 improved cognitive deficits in the 3xTg mouse model of AD, an effect independent of amyloid beta deposition, but that was associated with modulation of the microglia phenotype from a MGnD to a M0 gene signature. We have obtained preliminary data in which we found close contact between T cells and microglia in the brains of 3xTg mice treated nasally with anti-CD3 indicating that nasally induced T cells migrate to site of disease in the brain. We hypothesize that induction of Tregs by nasal anti-CD3 will ameliorate disease in the 3xTg AD model by modulating disease associated microglia and astrocytes. Our specific aims are: Aim 1. Investigation of nasal anti-CD3 modulation of microglia and astrocytes in the 3xTg model of AD. We will perform pharmacokinetic (PK) studies to determine the biodistribution as well as optimal dose and regimen of nasal anti-CD3 that modulates microglia and astrocytes. Moreover, mice will be treated with nasal anti-CD3 at different regimens of administration and will undergo behavioral testing. We will 1) quantify Ab and tau levels in the brain; 2) characterize microglia and astrocytes using high throughput RNA sequencing; 3) perform histopathology analysis to measure markers of M0 and MGnD microglia and disease associated astrocytes; 4) investigate microglia function in vivo by measuring phagocytic ability in clearing dead neurons. Aim 2. Investigation of Treg modulation of microglia and astrocytes in the 3xTg model of AD. We will perform fate mapping studies using Foxp3-eGFPCre/ETR2:3xTg-ROSA26Td-TomatoFlox to track Treg cells in the brains of these mice and to determine how long IL-10-secreting Tregs remain active in the brain following nasal anti-CD3 administration. In addition, we will use 3xTg:IL-10Rflox/floxClec7aCreETR2 and 3xTg:IL- 10Rflox/floxGFAPCreETR2 conditional and tamoxifen-induced knockout mice to investigate the role of IL-10 produced by nasal anti-CD3-induced Tregs in mod...