Introduction: Immunotherapies that harness CTLs to eradicate solid tumors, melanoma in particular, have re-emerged as potent clinical options in recent years. Improving the dispersion and cytotoxicity of intratumoral CTLs may further benefit patients. To gain insight into the regulatory processes that limit CTL activity in melanoma we investigated the influence of PD-1 signaling and Tregs on CTL behavior.
Methodology: To mimic adoptive cell therapy we transferred mice bearing B16.OVA melanoma tumors with OT-I CTLs. To study Treg-mediated regulation, we used FOXP3-GFP-LuciDTR-4 host mice, in which 95% of the cells expressing Foxp3 could be depleted using diphtheria toxin. To study PD-1 mediated regulation we injected anti PD-1 to WT host mice. Tumor rejection was monitored for 2-5 weeks.
Two-photon imaging of live mice was used to follow the behavior of CTLs and Tregs within tumors. Time-lapse confocal microscopy was used to follow the influence of PD-1 signaling on the behavior of CTLs and their interaction with B16-Ova cells.
Results: Tumor rejection was closely regulated by Tregs as tumors were densely infiltrated by these cells and their depletion dramatically accelerated tumor rejection. Interactions between CTLs and Tregs seemed indirect, as they did not form conjugates. Following their transfer, antigen-specific OT-I CTLs, as well as polyclonal CTLs, entered murine tumors through selected ICAM-decorated intratumoral vessels. As CTLs accumulated, Treg numbers in the tumors dropped and CTLs could be observed conjugating tumor cells and killing them. Treatment with anti PD-1 increased CTL velocities and accelerated killing of B16.OVA cells in vitro, and was mirrored in vivo by enhanced tumor rejection.
Whereas CTL migration and cytolysis was limited to oxygenated tumor areas within 50 µm of flowing blood vessels, Tregs exhibited higher velocities, were not affected by the proximity of blood vessels, and penetrated deeper into avascular areas.
Conclusions: Our findings suggest that regulatory mechanisms influence CTL efficiency partly by affecting their migration. CTLs, more than Tregs, depend on oxygen for intratumoral locomotion and cytolysis. This disparity allows tumor cells to escape CTL lysis in deep hypoxic tumor niches where high Treg to CTL ratios favor their survival. Thus, differential motility and dependence on oxygen among CTLs and Tregs can dictate the efficiency of CTL-based therapies of melanoma.
Citation Format: Tali Feferman, Natalia G. Zabolinsky, Yoav Manaster, Anat Hutzler, Guy Shakhar. Imaging regulatory mechanisms that limit intratumoral CTL function in a mouse melanoma model. [abstract]. In: Proceedings of the AACR Special Conference: Tumor Immunology and Immunotherapy: A New Chapter; December 1-4, 2014; Orlando, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2015;3(10 Suppl):Abstract nr B06.
- ©2015 American Association for Cancer Research.