The presence of CD8 T cells (TCD8) in tumors is a positive prognostic indicator of patient survival, but representation of TCD8 in many tumors is poor. While this could be a consequence of poor intratumoral proliferation, it is also the case that only a very small fraction of adoptively transferred TCD8 or CAR-T enter tumors. Our published and preliminary data indicates that homing receptor ligand (HRL) expression on tumor vasculature is suboptimal and that entry of TCD8 into tumors can be enhanced by relief of immunosuppression. Our work also suggests a positive feedback model in which intratumoral effector activity from newly entering TCD8 is needed to maintain HRL expression and sustain entry of newly arising effectors. Work conducted by others has suggested that pro-angiogenic factors such as VEGF may limit TCD8 infiltration by multiple mechanisms, including disorganized vascular structure, interference with cytokine signaling in endothelial cells to induce HRL upregulation, and suppression of intratumoral immunity. However, how these different aspects of angiogenesis influence direct TCD8 effector entry into tumors remains to be determined. There is also still little understanding of the roles that innate immune cells and intratumoral Ag play in augmenting entry of TCD8 effectors. This multi-PI R01 application is a collaboration between two investigators with complementary expertise. The Engelhard lab has identified: subpopulations of TCD8 effectors based on homing receptor (HR) expression pattern; the patterns of HRL expression on tumor vasculature; and the HR/HRL pairs that mediate TCD8 effector migration into tumors. The Kelly lab has focused on developing tools and using engineering approaches to study the role of the endothelium in disease. Using phage display technology, they identified hornerin, a novel non-VEGF induced protein overexpressed on tumor vasculature. Hornerin knockdown leads to vessel normalization and increased perfusion without loss of blood vessels. Hornerin may be an ideal molecule to attenuate in order to promote vessel normalization without the confounding pleiotropic effects observed when inhibiting VEGF. These two investigators will use these novel tools and expertise to understand how the direct entry of TCD8 into tumors is regulated by intratumoral Ag, tumor vasculature, and tumor microenvironment. Aim 1 will determine the impact of intratumoral immunoregulatory mechanisms on HRL expression on tumor vasculature and on exogenous TCD8 effector trafficking. Aim 2 will determine the impact of pro-angiogenic factors and tumor vessel normalization on HRL expression on tumor vasculature and on exogenous TCD8 effector trafficking. Aim 3 will determine the role of innate immune cells and intratumoral Ag in promoting TCD8 effector trafficking into tumors. Overall, this work will illuminate factors that limit the migration of tumor-specific T cells into tumors after vaccination or adoptive transfer, and suggest approaches to enhance their infiltration and subsequent therapeutic efficacy. This information may be used in conjunction with active vaccination or adoptive transfer-based immunotherapies to enhance their efficacy in a broader spectrum of cancer patients.