Project Abstract Although the stem cell niche has been extensively studied as a site that regulates stem cell functions, immunological attributes of the niche have remained largely unexplored. Interestingly, the testis and the placenta, organs for residence of stem cells, are known to be immunological sanctuaries for stem cells, termed immune privileged (IP) sites. Rigorous immune suppression in IP sites is evidenced by survival of transplanted allogeneic or xenogeneic grafts without any exogenous immune suppressive therapy. Little is known about whether tissue-committed stem cell niches are IP sites, like the testis and the placenta. The overall hypothesis is that one of the best-characterized stem cell niches, the hematopoietic stem and progenitor cell (HSPC) niche in the bone marrow (BM), is an IP site. IP in the niche may prevent rejection of transplanted allogeneic (allo-) HSPCs, and shield malignant stem cells from immune attack. Therefore, IP may be a novel principle that provides a mechanistic underpinning of several clinical settings. Our previous study indicates that the HSPC niche is an IP site. Highly sensitive in vivo microscopy (IVM) enabled detection of allo-HSPCs persisting in immune competent mice without immune suppressive therapy for more than 60 days. Moreover, FoxP3+ regulatory T cells (Tregs) frequently formed clusters with HSPCs. Systemic Treg depletion led to allo-HSPC rejection. The data suggest that Tregs endow the niche with IP. We further observed that MLL-AF9 leukemia stem cells (LSCs) home to BM Tregs, a hallmark of IP sites, suggesting that IP shields LSCs. To develop a new therapy to manipulate IP to promote allo-HSPC engraftment or eradicate BM malignancies, it is important to obtain further mechanistic insights into IP. In Aim 1, we will investigate the role of BM Tregs, mesenchymal cells, and CD11b+Gr1+ myeloid cells in IP maintenance and allo-HSPC persistence without immune suppression. To achieve this goal, we will employ the following four approaches: a) inhibition of Treg homing to the BM due to conditional deletion of CXCR4 in Tregs; b) conditional deletion of CD39 in Tregs; c) conditional deletion of CXCL12 in lepr+ mesenchymal cells; and d) CD11b+Gr1+ cell depletion by anti-Gr1 antibody treatment. We will determine whether these approaches lead to rejection of allo-HSPCs transplanted without immune suppression, by using in vivo microscopy, flow cytometry, and secondary transplantation assay. In Aim 2, we will test whether IP shields LSCs from immunity, and further elucidate the role of BM Tregs, mesenchymal cells, and CD11b+Gr1+ myeloid cells in allowing LSCs to evade immunity. We will determine whether rejection of MLL-AF9 leukemia is induced by a) lack of BM Tregs due to CXCR4 deletion in Tregs, b) CD39 inhibitor treatment, c) CXCL12 deletion in lepr+ mesenchymal cells, and d) myeloid cell depletion. Successful studies will create a novel paradigm that the HSPC niche is an immunological sanctuary for both allo-HSPCs and malignant stem cells. Furthermore, these mechanistic and systematic analyses of IP will establish novel therapeutic strategies for allo-HSPC transplantation and malignancies.