Resistance to radiotherapy and PD-L1 blockade is mediated by TIM-3 upregulation in anti PD-L1 refractory head and neck cancer Ayman Oweida, Mohammed Harara, Andy Phan, David Raben, Lynn Heasley, Eric Clambey, Raphael Nemenoff, Sana D Karam University of Colorado Denver, Anschutz Medical Campus, USA Background: Clinical trials targeting the programmed-death ligand axis (PD-1/PD-L1) show that a majority of head and neck squamous cell carcinoma (HNSCC) patients are resistant to PD-1/PD-L1 inhibition. Here, we characterize the immune landscape of HNSCC tumors during radiotherapy (RT) and PD-L1 treatment and interrogate mechanisms of resistance. Methods: Murine HNSCC cells were injected into the right buccal mucosa of mice. We performed mass cytometry (CyTOF) to characterize the tumor immune microenvironment at early and late stages of response to RT and anti PD-L1. For therapeutic studies, mice were randomized to IgG, anti PD-L1, anti TIM-3, RT or combinations of RT, PD-L1 and TIM-3. Mechanistic experiments were performed on tumors harvested 72 hours after treatment. Tumors were assessed for levels of activated T-cells (CD44+IFNg+) and regulatory T-cells (FoxP3+CD4+) by flow cytometry and T-cell infiltration by immunohistochemistry.
Results: Tumors treated with RT and PD-L1 blockade significantly upregulated TIM-3 expression on CD4 and CD8 T-cells. Targeting TIM-3 concurrently with PD-L1 and RT lead to a significant tumor growth delay, enhanced T-cell cytotoxicity and improved survival. In addition, dual targeting of PD-L1 and TIM-3 in combination with RT significantly decreased the proportion of regulatory T-cells. However, the response to dual checkpoint blockade and RT was not durable and all tumors eventually relapsed. Analysis of relapsed tumors revealed decreased T-cell infiltration, re-emergence of regulatory T-cells and decreased proportion of cytotoxic and helper T-cells.
Conclusion: Our study shows upregulation of TIM-3 in response to RT and PD-L1 inhibition. However, dual targeting of TIM-3 and PD-L1 with RT did not provide a durable response in two genetically distinct HNSSCC tumor models. In light of the increased number of clinical trials employing dual immune checkpoint blockade, our findings reveal the complexity of tumor immune evasion mechanisms and underscore the need for multimodality targeting of HNSCCs.
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