Trp 2

Spleens were harvested and dissociated into single cell suspension of splenocytes by manually grinding the spleen over a 40 µm filter (Falcon) and red blood cells lysed using ACK Lysing Buffer (Thermo Fisher). Cells were washed with PBS and resuspended in RPMI 1640 with l-Glutamine (Thermo Fisher Scientific), 10% fetal bovine serum (Thermo Fisher Scientific), and 1% Antibiotic–Antimycotic (Thermo Fisher Scientific). Cytokine analysis was performed using the mouse IFN-γ ELISpotPLUS Kit (Mabtech), according Manufacturer’s protocol. Briefly, plates were blocked with serum-containing culture media and stimuli and cell suspension (400,000 per well) added. B16 antigen pool used was a mixture of GP100 (KVPRNQDWL), TRP-2 (SVYDFFVWL), and TYR (YMDGTMSQV) (AnaSpec), with each having a final assay concentration of 2.5 µg/mL. Plates were wrapped in foil and incubated for 18 h at 37 °C, 5% CO₂. Following stimulation, the cells were removed, plate washed, and 1 µg/mL detection antibody added for 2 h at room temperature. Wash was repeated and 1× Streptavidin-HRP added and incubated for 1 h at room temperature. Finally, plates were washed and TMB substrate added, incubated for 4 min in the dark for spot development, then washed out using tap water. Plates were allowed to dry and counted (ZellNet Consulting).

To prepare the target cells, 50 units/mL mouse IFNγ (Prospec Protein Specialists) was added to the B16-OVA cell culture. Forty-eight hours later, the cells were fixed with 1% paraformaldehyde and washed before being added to the coculture. For immune cell stimulation, splenocytes (5 × 10⁵ cells/well) were incubated with prefixed target cells (B16-OVA, 5 × 10⁴ cells/well) or one of the following peptides at 2 μg/mL: mgp100 (refs. 24–32 (link); AnaSpec), OVA (257–264; Invitrogen), TRP-2 (180–188; AnaSpec), or GFP (118–126; Biomatik). Twenty-four hours after incubation in a round-bottom 96-well plate, the concentration of IFNγ in the medium was assessed with a mouse IFNγ DuoSet ELISA kit (R&D Systems).

For prophylactic experiments, mice were challenged with MO4 7 days after the immunization with 30 μg of ovalbumin (OVA) plus adjuvant: 10 μg of Porins or 10 μg of CTB (Sigma-Aldrich), injected s.c. in the left flank. As controls, mice were s.c. injected with OVA, Porins, CTB or PBS. For the re-challenge experiment, the tumor-free OVA + Porins immunized mice at day 28 were re-challenged with MO4 and followed up for 100 days from the first immunization. For the melanoma-associated antigen (MAAs) experiment, mice were injected with 70 nmol of the human/mouse recombinant peptides TRP-2 (AnaSpec) and gp100 (MyBioSource), with or without Porins, 10 and 3 days before challenge with the B16-F10 melanoma cells (39 (link), 40 (link)).