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Abstract
Purpose: Radiotherapy (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.Experimental Design: Mechanisms of resistance to RT and PD-L1 blockade were investigated in orthotopic murine head and neck squamous cell carcinoma (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 (Treg) was performed using anti-CD25 antibody.Results: 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 HNSCC. Despite this treatment combination, the response was not durable, and analysis of relapsed tumors revealed resurgence of Tregs. Targeted Treg depletion, however, restored antitumor immunity in mice treated with RT and dual immune checkpoint blockade and resulted in tumor rejection and induction of immunologic memory.Conclusions: These data reveal multiple layers of immune regulation 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.

Author information

Oweida A1, Hararah MK2, Phan A1, Binder D1, Bhatia S1, Lennon S1, Bukkapatnam S1, Van Court B1, Uyanga N1, Darragh L1, Kim HM3, Raben D1, Tan AC3, Heasley L4, Clambey E5, Nemenoff R6, Karam SD7.
1
Department of Radiation Oncology, University of Colorado Denver, Aurora, Colorado.
2
Department of Otolaryngology and Head and Neck Surgery, University of Colorado Denver - Anschutz Medical Campus, Aurora, Colorado.
3
Division of Medical Oncology, University of Colorado Denver, Aurora, Colorado.
4
Department of Craniofacial Biology, University of Colorado Denver, Aurora, Colorado.
5
Department of Anesthesiology, University of Colorado Denver, Aurora, Colorado.
6
Department of Medicine, University of Colorado Denver, Aurora, Colorado.
7
Department of Radiation Oncology, University of Colorado Denver, Aurora, Colorado. sana.karam@ucdenver.edu.