Evaluating Neoepitope-Specific T Cell Responses

Cryopreserved TILs and expanded B cells were thawed and rested overnight in T or B cell media, respectively, at 37°C with 5% CO₂. B cells (4 × 10⁵ cells) were suspended in 1 mL complete B cell media and incubated with individual synthesized neoepitopes or pooled viral peptides (CEF Peptide Pool/HLA Class I Control; STEMCELL Technologies) at a final concentration of 0.1 μg/mL at 37°C with 5% CO₂ for 1 hour. B cells were then irradiated (2,000 rad) and washed twice in complete B cell media. Peptide-pulsed B cells (4 × 10⁴) were cocultured with TIsL (2 × 10⁴, 2:1 effector/target [E/T] ratio) overnight at 37°C with 5% CO₂. A human IFN-γ ELISpot (R&D Systems) assay was performed according to the manufacturer’s recommendations. Spot counts for the ELISpot assays were done on an Immunospot ELISpot plate reader (Cellular Technology). The ELISpot coculture assay was performed in a separate 96-well plate for each patient. Autologous expanded B cells pulsed with HPV 16 E7_11–19 peptide and peripheral blood T cells engineered to express an HPV 16 E7-specific HLA-A*02–restricted T cell receptor (TCR) from a separate patient (47 (link)) were included as an additional positive control for each assay. Each condition was performed in technical duplicate, and mean spot counts are reported. Mean post counts of 5 or higher were considered positive.