ELISpot

ELISpot assays were performed to determine IFN-γ release by activated splenocytes using a 96-well filtration plate with an Immobilon-P membrane (MultiScreen HTS, Merck, Kenilworth, NJ, USA). Plates were activated with 35% ethanol for 5 min, washed five times with sterile water, incubated overnight with rat anti-mouse IFN-γ (BD AN-18, 4 μg/mL), and blocked with RPMI medium containing 10% fetal calf serum (FCS) and penicillin-streptomycin for at least 2 hr. A single-cell suspension of 2.5 × 10⁵ splenocytes/well was plated in 100 μL of RPMI medium including 2.5 × 10⁵ tumor cells or 20 μL of tumor lysate or 200 ng of peptide. After incubating overnight at 37°C and 5% CO2, plates were washed and stained with biotinylated anti-mouse IFN-γ (BD R4-6A2, 1 μg/mL) and incubated for 2 hr, followed by streptavidin conjugate enzyme (E236, Sigma) and appropriate washing. After drying, spots were counted using the ELISpot Reader (AID, Strasberg, Germany).

The peptides H2-Q2 (TWQLNGEEL), Rab13 (SDKKNNKCL), Ndufs1 (AAVSNMVQKI), Ppat (DPYGNRPLCM), Gsta2 (LHHFNARGRM), chd2 (APLQNSLKEL), and Arhgef10 (AWIENPEEAI) were synthesized by Biomedal (Seville, Spain), and the sequence of the peptides was as reported.11 The peptide E1B192 (VNIRNCCY)22 was synthesized by GenScript USA (Piscataway, NJ, USA). The hexon peptide pool PepTivator AdV5 Hexon was purchased from Miltenyi Biotec (Auburn, CA, USA). For all peptides, purification was >75%, and they were dissolved in DMSO. The final concentration of each peptide used for stimulation was 2 μg/mL.