Abstract Radiation therapy (RT) is administered to around 50% of all cancer patients making it one of the most important cancer treatments. In addition to the direct cytoreductive effect of RT there is increasing evidence that radiation-induces immunogenic tumor cell death. Despite the immunogenicity of RT-induced tumor cell death, RT delivered to tumours in the clinic rarely generates therapeutic systemic anti-cancer immune responses or ‘abscopal effects’. Here we show that tumor infiltrating CD8+ cytotoxic T lymphocytes (CTL) have increased expression of PD-1 following RT in vivo. Moreover, our data demonstrate that treatment of established syngeneic tumors with RT leads to upregulation of tumor cell expression of PD-L1 in vivo but not when cells are irradiated in vitro. Using depleting antibodies we determined that the depletion of CD8+ T cells but not CD4+ T cells or NK cells could abrogate this RT-induced increase in tumor cell expression of PD-L1 in vivo. Furthermore, silencing of IFNγR1 using ShRNA confirmed that this process was dependent on CD8+ T cell production of IFNγ suggesting an adaptive upregulation of PD-L1 following RT occurs in response to CTL activation. This novel finding suggests that the immunogenicity of RT may be limited via the PD-L1/PD-1 signalling axis and may contribute to treatment failure. We next sought to determine whether blockade of the PD-1/PD-L1 signalling axis could enhance the therapeutic response to RT. Our study demonstrates that administration of either an anti-PD-1 or anti-PD-L1 mAb in combination with RT leads to substantially improved survival when compared to either monotherapy alone with approximately 60% of treated mice undergoing a complete response. In addition, our data reveal that combination therapy generates long-term immunological memory in mice that have undergone complete response protecting against tumor rechallenge. This is the first report to our knowledge that demonstrates adaptive resistance through the upregulation of tumour cell PD-L1 expression in response to an RT-induced CTL response. This study demonstrates the potential for enhancing the efficacy of conventional RT through blockade of the PD-1/PD-L1 axis and warrants clinical evaluation. Citation Format: Simon J. Dovedi, Graznya Lipowska-Bhalla, Eleanor Cheadle, Edmund Poon, Michelle Morrow, Ross Stewart, Robert Wilkinson, Jamie Honeychurch, Timothy Illidge. The antitumor immune response generated by radiation therapy may be limited by tumor cell adaptive resistance and can be circumvented by PD-L1 blockade. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5034. doi:10.1158/1538-7445.AM2014-5034