Tumor bearing experiments

Female C57BL/6 mice were subcutaneously (s.c.) injected on the right back with 2 × 10⁵ B16-OVA or B16 cells. On day 3 of the tumor cell inoculation, mice were randomly allocated into groups and vaccinated with 20 μg peptides or normal saline weekly for total of three times. Tumor volume was measured every other day. After five days of last immunization, mice were sacrificed. For IL-21 blocking experiment in vivo, mice were intraperitoneally (i.p.) injected 100 μg IL-21 neutralized antibody (FFA21) every eight days for total of twice.

Tumor tissue cell suspensions were stained with anti-mouse-CD45-FITC (30-F11), anti-mouse-CD3-PerCP-eFlour710 (17A2) and anti-mouse-CD8-APC (53-6.7) or Rat IgG2a-APC kappa Isotype Control (eBR2a) for tumor infiltrating CD8⁺ T cell analysis.

For intracellular cytokine staining (ICS), single cell suspension of splenocytes and draining lymph node (dLN) was stimulated with 10 μg/mL OVA_257-264 peptide in the presence of protein transport inhibitor cocktail (eBioscience, San Diego, CA, US) for 6 h. Cells were then stained with surface markers CD3 and CD8 prior to fixation and permeabilization. Permeability cells were then stained with intracellular anti-mouse-IFN-γ-APC (XMG1.2), anti-mouse-Perforin-APC (eBioMAK-D), anti-mouse-Granzyme B-PerCP-eFlour710 (NGZB) antibody or isotype control antibody. Cells were collected and suspended in PBS containing 0.5% fetal bovine serum, incubated with the corresponding antibodies for 30 min on ice, washed once with PBS and analyzed by a FACS Calibur (Becton Dickinson, Franklin Lake, NJ, USA) flow cytometry.