PROJECT SUMMARY Immune checkpoint inhibitors (ICIs) enable anti-tumor immunity by blocking immunoregulatory receptor-ligand interactions (e.g., programmed cell death protein 1 [PD-1] and its ligand [PD-L1]). Despite their widespread success as an anti-cancer therapy, ICIs function poorly in patients with bone metastases, and bone loss and elevated fracture risk are reported in patients receiving ICI therapy. My preliminary data indicate that PD-1 global knockout or pharmacologic blockade decreases bone mass in both tumor naïve mice and mice with bone-metastatic disease (E0771 tumor cells). Furthermore, PD-1 blockade reduces bone strength compared to IgG control treatment in mice with bone metastases. These data are consistent with reports of bone loss and fractures in patients treated with ICIs. I have also found that osteoclast activity and CD8+ T cell populations capable of secreting pro-osteoclastogenic cytokines are elevated in the bone marrow after PD-1 blockade. I therefore hypothesize that the detrimental effects of PD-1 blockade on bone stem from CD8+ T cell expansion, which promotes osteoclast-induced bone destruction, and that tumor resistance to ICIs in the bone microenvironment stems from T cell dysfunction. I propose to (1) determine how immune cells influence PD-1 blockade-induced bone loss, and (2) investigate the mechanism behind T cell dysfunction and tumor resistance to ICIs in the bone microenvironment. In Aim 1 (F99 phase), I will use T cell depletion and adoptive transfer studies to investigate the role of bone marrow immune cells on PD-1 blockade-induced bone loss in tumor-inoculated mice. I will also treat tumor-inoculated mice with α-PD-1 in combination with bisphosphonates to assess prevention of bone loss. In both of these studies, I will assess the bone phenotype via microCT and biomechanical strength testing, and the tumor and immune phenotypes via flow cytometry and histology. In Aim 2 (K00 phase), I will utilize digital spatial profiling and scRNAseq technology to characterize T cell infiltration into bone metastases and identify key signaling pathways between tumor cells, immunoregulatory cells (myeloid derived suppressor cells, tumor associated macrophages T regulatory cells) and cytotoxic T cells, which are important in escape of bone metastatic tumor cells from the T cell anti-tumor response. I will use de- identified human breast cancer samples to validate my findings and elucidate the mechanism(s) by which bone metastases escape T cell surveillance (e.g., T cell suppression, T cell exhaustion, lack of T cell infiltration into the tumor microenvironment, etc.). With the current lack of understanding of the mechanism behind bone loss in patients treated with ICIs and tumor cell resistance to ICIs after bone dissemination, this proposal will help manage bone health in the millions of patients receiving ICIs each year and confirm mechanistically whether anti-resorptives reverse ICI-induced bone loss. Furthermore, I...