(A516) CD137 Activation-Induced T Cell Assay for TCR-Engineered Therapy in Head and Neck Cancer

Introduction: Head and neck squamous cell carcinoma (HNSCC) is an aggressive malignancy increasingly in need of precision therapies, with TCR-engineering approaches showing promise. Peripheral blood mononuclear cells (PBMCs) are scalable platforms to engineer TCRs capable of eliciting cytotoxic responses, while tumor-infiltrating lymphocytes (TILs) help identify TCRs naturally recognizing tumor antigens. Effective engineering requires optimizing activation and reactivation for downstream CRISPR-Cas9 engineering. We refined a CD137-based flow cytometry assay to determine peak activation timelines.

Methods: Cryopreserved TILs and PBMCs were thawed, expanded in cytokine-supplemented media, cytokine-starved for 5 days to return to baseline, and CD8+ selected using CD8 MicroBeads (Miltenyi). These were divided into activated (IL7/IL15 25 μl/million cells) and non-activated control arms. Activation was assessed by double-staining for CD137 (Miltenyi) and CD107a (Miltenyi, BD, Invitrogen) at 30 minutes, 2, 5, and 24 hours, later extended to 48, 96, and 168 hours. BD FACSAria III was used to flow sort CD137-negative populations due to baseline positivity. Analysis at all time points utilized MACSQuant 16, with additional staining for CD8, CD4, and 7-AAD.

Results: Double-staining for CD137 and CD107a revealed that the latter marker failed to show significant activation ( < 10%) even at extended timepoints, across all three antibodies we tested. CD137 was chosen as the superior marker. Peak activation for PBMCs and TILs reached ~60% at 24 hours, with no significant improvement at later timepoints. We encountered significant baseline CD137 positivity, between 4-15% initially, and therefore opted to flow sort for CD137-negative cells prior to activation. This successfully eliminated baseline positivity and greatly improved precision in assessing activation.

Conclusions: This assay determines peak activation times for PBMCs and TILs. Findings will inform future T-cell engineering protocols targeting tumor-specific antigens in HNSCC.