PROJECT SUMMARY Recent largest GWAS identified HAVCR2 (TIM3) genetic risk factor for late-onset Alzheimer’s disease (LOAD). Our laboratory discovered and cloned Tim3 as an inhibitory molecule that induces T cell exhaustion in cancer1. Blocking antibodies to Tim3 are being approved for the treatment of cancer. However, we have now identified that Tim3 is not only expressed on T cells, but also on myeloid cells and dendritic cells, where TIM3 restrains dendritic cell function and regulate anti-tumor immunity2. In the CNS, HAVCR2 was identified as one of the top 100 enriched transcripts and is specifically expressed in both mouse and human microglia3-5, but its role and function in microglia is unknown. Our long-term goal is to define the role of TIM3 in regulation of microglia in neurodegeneration. We made the following observations: 1) Tim3 inhibits microglial activation and phagocytosis: deletion of Tim3 in dendritic cells boosted antigen presentation and we find that TIM3 also regulates microglial activation and phagocytosis; 2) TGFb-Tim3 axis regulates microglia phenotype switch in neurodegeneration: we find that TGFb is the key driver for the induction of Tim3 and once expressed it synergizes with TGFBR to potentiate TGFB signaling, loss of Tim3 switches M0-homeostatic microglia to an MGnD-nondegenerative phenotype; and 3) TIM3 deletion in microglia reduces plaque burden in 5xFAD mice. These data support the genetic linkage studies and show the importance of Tim3 in regulating disease pathology in AD by modulating microglial function. Based on these studies, we hypothesize that TIM3 is a key regulatory molecule in microglia that inhibits their response to neurodegeneration, migratory and phagocytic functions and thereby inhibit plaque clearance resulting in promotion of Ab deposition, development, and progression of AD in aging brain. Based on this hypothesis we have proposed three aims: Aim 1: Define how TIM3 regulates phenotype and functions in 5xFAD and P301S mouse AD models. We propose to study the effect of microglial deletion of Tim3 in neurodegeneration and brain tauopathy using the mouse models of AD. Aim 2: What is the role of TGFb signaling in the regulation of Tim3 expression and function in microglia and development of AD? Since TGFb plays a critical role in maintenance of the homeostatic phenotype in microglia, we propose to study how TGFb signaling induces Tim3 expression and promotes homeostatic behavior of microglia by cooperating with TGFb receptor signaling. Aim 3: Define the role of TIM3 in the regulation of human microglial function in AD. Determine how TIM3 impacts human iPSC-derived microglia activation and functions. We will examine whether genetic or pharmacologic inhibition of TIM3 has a similar effect on iPSCs-derived human microglia expressing the MGnD phenotype by utilizing a humanized chimeric mouse model of AD for treatment with human anti-Tim3 antibody IN SUMMARY, targeting TIM3 in microglia may provide a novel a...