Resistance to Radiotherapy and PD-L1 Blockade Is Mediated by TIM-3 Upregulation and Regulatory T-Cell Infiltration

Ayman Oweida, Mohammad K. Hararah, Andy Phan, David Binder, Shilpa Bhatia, Shelby Lennon, Sanjana Bukkapatnam, Benjamin Van Court, Nomin Uyanga, Laurel Darragh, Hyun Min Kim, David Raben, Aik Choon Tan, Lynn Heasley, Eric Clambey, Raphael Nemenoff, Sana D. Karam

Radiation therapy (RT) can transform the immune landscape and render poorly immunogenic tumors sensitive to PD-L1 inhibition. Here we established that the response to combined RT and PD-L1 inhibition is transient and investigated mechanisms of resistance. Mechanisms of resistance to RT and PD-L1 blockade were investigated in orthotopic murine HNSCC tumors using mass cytometry and whole genome sequencing. Mice were treated with anti-PD-L1 or anti-TIM-3 alone and in combination with and without RT. Tumor growth and survival were assessed. Flow cytometry was used to assess phenotypic and functional changes in intratumoral T cell populations. Depletion of regulatory T cells was performed using anti-CD25 antibody. We show that the immune checkpoint receptor, TIM-3, is upregulated on CD8 T cells and Tregs in tumors treated with RT and PD-L1 blockade. Treatment with anti-TIM-3 concurrently with anti-PD-L1 and RT led to significant tumor growth delay, enhanced T cell cytotoxicity, decreased Tregs and improved survival in orthotopic models of head and neck squamous cell carcinoma. Despite this treatment combination, the response was not durable and analysis of relapsed tumors revealed resurgence of Tregs. Targeted Treg depletion, however, restored anti-tumor immunity in mice treated with RT and dual immune checkpoint blockade and resulted in tumor rejection and induction of immunologic memory. These data reveal multiple layers of immunoregulation that can promote tumorigenesis, and the therapeutic potential of sequential targeting to overcome tumor resistance mechanisms. We propose that targeted Treg inhibitors may be critical for achieving durable tumor response with combined radiotherapy and immunotherapy.