Tumor-specific T cells in head and neck cancer have rescuable functionality and can be identified through single-cell co-culture

Background: Human papillomavirus (HPV)-negative head and neck squamous cell carcinoma (HNSCC) remains a treatment-resistance disease with limited response to immunotherapy. While T cells in HNSCC are known to display phenotypic dysfunction, whether they retain rescuable functional capacity and tumor-killing capability remains unclear. Methods: To investigate the functionality and tumor-specificity of tumor-infiltrating lymphocytes (TILs) across HNSCCs, malignant cell lines and TILs were derived from 31 HPV-negative HNSCCs at the time of standard surgical resection. T cell functional capacity was evaluated through ex vivo expansion, immunophenotyping, and IsoLight single-cell proteomics. Tumor-specificity was investigated through both bulk and single-cell tumor-TIL co-culture. Results: TILs could be successfully generated from 24 patients (77%), including both previously untreated and radiation recurrent HNSCCs. We demonstrate that across HNSCCs, TILs express multiple exhaustion markers but maintain a predominantly effector memory phenotype. After ex vivo expansion, TILs retain immunogenic functionality even from radiation-resistant, exhausted, and T cell-depleted disease. We further demonstrate tumor-specificity of T cells across HNSCC patients through patient-matched malignant cell-T cell co-culture. Finally, we use optofluidic technology to establish an autologous single tumor cell-single T cell co-culture platform for HNSCC. Cells derived from three HNSCC patients underwent single-cell co-culture which enabled identification and visualization of individual tumor-killing TILs in real-time in all patients. Conclusions: These studies show that cancer-specific T cells exist across HNSCC patients with rescuable immunogenicity and can be identified on a single-cell level. These data lay the foundation for development of patientspecific T cell immunotherapies in HNSCC.