Abstract Interferon gamma (IFNγ) is a pro-inflammatory cytokine that is upregulated during chronic infections and leads to activation, differentiation, and eventual loss of hematopoietic stem cells (HSCs). We previously showed that these effects may contribute to bone marrow (BM) insufficiency and cytopenias during chronic infection.1 Despite its clinical significance, the mechanism by which IFNγ activates and depletes HSCs is unknown. Since HSCs are heavily influenced by their interaction with the BM microenvironment2, we previously studied the impact of IFNγ on HSC-niche interactions. Our preliminary data show that HSCs activated by IFNγ re-localize in the BM away from quiescent-promoting CXCL12-abundant reticular (CAR) cells. We used transcriptomic analysis to identify Bone Marrow Stromal Antigen 2 (BST2) as the only surface protein upregulated upon IFNγ stimulation during the early stages of HSC migration. Our work to date shows that BST2-deficient mice have more HSCs present in the BM, their HSCs are less proliferative than wild type (WT) HSCs, and BST2 KO HSCs do not re- localize from CAR cells during IFNγ induction suggesting that BST2 could play a role in regulating activation. Previous reports have suggested that BST2 could serve as an adhesion protein to facilitate migration in other cell types, and we suspect that it may perform a similar function in HSCs. The overarching goal of this proposal is to elucidate the role of BST2 in HSC activation by determining BST2-protein interactions and its impact on HSC interactions within the niche. We hypothesize that increased expression of BST2 promotes HSC activation during inflammatory stress by facilitating binding to alternative niche cell types. To test the hypothesis, in Aim 1, we will establish BST2-surface protein interactions in HSCs using cross-linking aided affinity purification mass spectrometry. We will further determine whether blocking these interactions will affect HSC activation by measuring HSC numbers, proliferation and cell cycle status. In Aim 2, we will identify the BM niche cells that facilitate BST2-mediated HSC activation by performing intravital imaging using a variety of BM reporter mice to identify the spatial location of HSCs relative to the other niche cells. Direct interaction between BST2-positive HSCs and niche cells will also be tested. Collectively, the experiments proposed here aim to expand our understanding of the mechanism by which BST2 alters HSC-niche interactions. Elucidating how HSCs become activated during IFNγ stimulation will provide insight into the mechanisms by which inflammation drives HSC exhaustion and will potentially lead to novel therapeutic strategies for bone marrow failure syndromes associated with excessive inflammation.