Related Experiment Video
Updated: Jul 4, 2025
Analysis of Human T Cell Activity in an Allogeneic Co-Culture Setting of Pre-Treated Tumor Cells
Published on: March 7, 2025
Tumor-specific T cells in head and neck cancer have rescuable functionality and can be identified through single-cell
Joseph Zenga1, Musaddiq Awan1, Anne Frei2
1Cancer Center, Medical College of Wisconsin, Milwaukee, WI, United States; Department of Otolaryngology, Medical College of Wisconsin, Milwaukee, WI, United States; Department of Radiation Oncology, Medical College of Wisconsin, Milwaukee, WI, United States.
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 patient-specific T cell immunotherapies in HNSCC.
Insights
T cells in human papillomavirus (HPV)-negative head and neck squamous cell carcinoma (HNSCC) show dysfunction but retain tumor-killing ability. These cancer-specific T cells can be identified at the single-cell level for future immunotherapy development.
Area of Science:
- Immunology
- Oncology
- Biotechnology
Background:
- Human papillomavirus (HPV)-negative head and neck squamous cell carcinoma (HNSCC) is a treatment-resistant cancer with poor immunotherapy response.
- T cells within HNSCC tumors often exhibit phenotypic dysfunction, but their functional capacity and tumor-killing potential remain largely unknown.
Purpose of the Study:
- To investigate the functionality and tumor-specificity of tumor-infiltrating lymphocytes (TILs) in HPV-negative HNSCC.
- To determine if dysfunctional T cells in HNSCC retain rescuable anti-tumor capabilities.
Main Methods:
- Derived malignant cell lines and TILs from 31 HPV-negative HNSCC patients.
- Assessed T cell function via ex vivo expansion, immunophenotyping, and single-cell proteomics (IsoLight).
- Investigated tumor-specificity using bulk and single-cell co-culture, and developed an optofluidic single-cell co-culture platform.
Main Results:
- Successfully generated TILs from 77% of patients, including those with radiation-recurrent HNSCC.
- TILs displayed exhaustion markers but retained an effector memory phenotype and immunogenic functionality post-expansion.
- Demonstrated tumor-specificity of T cells and identified individual tumor-killing TILs in real-time using single-cell co-culture.
Conclusions:
- Cancer-specific T cells with rescuable immunogenicity exist in HNSCC patients.
- These T cells can be identified at the single-cell level, paving the way for patient-specific T cell immunotherapies.
