Abstract B102: Releasing oxygen-restricted CTL function: Insights from live intratumoral imaging

Poor tumor vascularization is an obstacle to immunotherapy by CTLs. It impairs tumor infiltration but also introduces hypoxia, known to interfere with T cell migration. It is yet unknown how suboptimal vascularization affects CTL migration and function within tumors. To study this question, we combined immunohistochemistry of human melanoma samples with two-photon imaging in live mice. Orthotopically implanted B16-OVA tumors were studied after adoptive transfer of in-vitro matured antigen-specific OT-I CTLs. In patients, CD8 T cells concentrated around peripheral vessels in the tumor and sparsely infiltrated avascular areas. This pattern was clearest in poorly-infiltrated tumors. In mice, OT-I CTLs, as well as polyclonal CTLs, entered tumors through inflamed intratumoral vessels, and crawled only in oxygenated areas within 50 µm of flowing blood vessels. Gradually, CTLs formed dense perivascular swarms, which eliminated nearby target cells leading to patchy tumor clearance around blood vessels. Occluding intratumoral blood vessels triggered immediate arrest of CTL motility, which was quickly reversed when flow was resumed. Immunohistology indicated that CTLs avoided hypoxic tumor areas. Live CTL imaging in vitro showed deceleration under hypoxic conditions and when oxidative phosphorylation was blocked. To circumvent intratumoral CTL dysfunction we attempted to increase vascular density by implanting tumors in matrices containing bFGF. bFGF-laced tumors were more easily rejected after transfer of CTLs and displayed delayed growth in untreated mice but were not affected in mice deficient in CD8 T-cells. CTLs infiltrated such tumors in normal numbers, but displayed enhanced motility in highly-vascular tumors, suggesting that enhanced rejection resulted from improved intratumoral CTL migration. Taken together, the results suggest that hypoxia limits CTL function away from blood vessels, and that alleviating it may synergize with immunotherapy. Citation Format: Tali Feferman, Yoav Manaster, Yael Gruper, Zohar Shipony, Guy Shakhar. Releasing oxygen-restricted CTL function: Insights from live intratumoral imaging. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr B102.