Effector CD8 T-cell Entry into Subcutaneous and Intraperitoneal B16 Melanoma Tumors

Although CD8+ T cells are critical for controlling tumors, how they are recruited and home to primary and metastatic lesions is incompletely understood. We characterized the homing receptor (HR) ligands on tumor vasculature to determine what drives HR ligand expression and the requirements for T cell entry. The anatomic location of B16-OVA tumors affected the expression of E-selectin, Mucosal Addressin Cell Adhesion Molecule 1 (MAdCAM-1), and Vascular Cell Adhesion Molecule 1 (VCAM-1), whereas the HR ligands C-X-C motif chemokine ligand 9 (CXCL9) and Intracellular Adhesion Molecule 1 (ICAM-1) were expressed on the vasculature regardless of location. Expression of these molecules differed based on anatomic location in other implantable tumors as well, indicating that differences in expression were not tumor intrinsic. CXCL9 and VCAM-1 expression was significantly reduced in B16F1 tumors that did not express the strong antigen Ovalbumin and when B16-OVA tumors were grown in Rag1-/- mice indicating that expression was driven by adaptive immune cells. Repletion of Rag1-/- mice with CD8 T-cells from WT and IFNg-/- animals revealed that IFNg-secreting CD8 T-cells were sufficient to drive VCAM-1 and CXCL9 expression on B16-OVA tumor vasculature. VCAM-1 and CXCL9/10 enabled CD8+ T-cell effectors expressing a4b1 integrin and CXCR3 to enter both subcutaneous and peritoneal tumors, whereas E-Selectin enabled E-Selectin Ligand+ effectors to enter subcutaneous tumors. However, MAdCAM-1 did not mediate a4b7+ effector entry into peritoneal tumors. These data establish the relative importance of certain HRs expressed on activated effectors and certain HR ligands expressed on tumor vasculature in the effective immune control of tumors.
We also addressed whether the melanoma therapy, anti-CTLA-4, impacted HR ligand expression on tumor-associated vasculature and CD8 T-cell accumulation in tumors. Treating tumors with the checkpoint blockade inhibitor anti-CTLA4 led to a significant increase in both the number of CD8 T-cells in the tumor and the expression of the HR ligands VCAM-1 and ICAM-1 on the tumor vasculature. Addition of peptide vaccine in Incomplete Freund’s Adjuvant (IFA) to the checkpoint blockade treatment sequestered CD8 T-cells at the vaccination site and did not result in increased CD8 T-cell accumulation or HR ligand expression on the tumor vasculature. These data suggest that current first-line therapies have the potential to improve CD8 T-cell trafficking to tumors, but combinatorial therapies need to be evaluated and therapies may be improved by boosting ligand expression on the vasculature prior to treatment.