40 mm filter

The mouse or hamster spleens were placed in 15 mL conical tubes containing 6 ml of R10 media (RPMI-1640 with 10% fetal bovine serum, 1% penicillin–streptomycin, and 0.1% 2-mercapthoethanol). Splenocytes were isolated using the Seward Stomacher bag and machine system. Cells were then strained through a 40 mm filter (BD Bioscience), washed, and red blood cells (RBC) were lysed in 3 ml of ACK lysing buffer (Lonza) for 2 min. After washing with 1× PBS, isolated splenocytes were counted using the Vi-Cell-Blu machine (Beckman Coulter) and resuspended at 20 × 10⁶/mL concentration ready to be used for ELISpot.

Fresh OPSCC tumors and matched adjacent normal tissue samples were placed on ice following surgical resection in Dulbecco's modified eagle medium (DMEM, USA), supplemented with 1% Penicillin/Streptomycin solution (Hyclone, USA). Subsequently, all samples were gently cut into small pieces on ice and digested using a tumor dissociation kit (Miltenyi Biotec, Germany), following the built-in program 37C_h_TDK_3 for tumor tissues and program 37C_h_TDK_1 for adjacent normal tissues. After disruption, the cells were filterd through a 40 mm filter (BD Biosciences, USA) and washed with 1x Hank's balanced salt solution (HBSS, USA). The resulting single-cell suspension was centrifuged at 500G for 5 minutes and resuspended in 4% bovine serum albumin (BSA, USA). Single-cell suspensions were then stained with CD3 MicroBeads (Miltenyi Biotec, Germany) and sorted into immune (CD3⁺) cells via magnetic separation using MS Separation columns (Miltenyi Biotec, Germany). Cell number and viability were checked after staining with 0.4% trypan blue staining (cell number > 5 × 10⁵ and viability > 80%).