Rhil 2

Mouse peripheral blood mononuclear cells from pTMK/Trp53^−/− peripheral blood were isolated by density gradient centrifugation through Ficoll-Paque solution. CD8⁺ T cells from peripheral blood mononuclear cells were purified with anti-CD8 magnetic beads (STEMCELL) and stimulated with 25 μg/mL CD3/CD28 T cell Activator (Life Technologies) and 50 U/mL rhIL-2 (STEMCELL) for 3–5 days. After 2 × 10⁵ CD8⁺ T cells were incubated with 5 × 10⁵ TANs, ALNs, or PBNs for 24 h, a mixed single-cell suspension was analyzed through flow cytometry.

Four- to five-week-old male C57BL/6 mice from the BC Cancer Research Centre’s Animal Resource Centre, maintained under pathogen-free conditions, were engrafted with 3 × 10⁵ B16F10 cells ( + /− Ova transgene) by subcutaneous injection in the scruff of the neck. Groups receiving Ova vaccination were also subcutaneously injected with 300 μL sterile-filtered PBS solution containing chicken egg ovalbumin protein (MilliporeSigma) (100 µg) and the TLR3 adjuvant, poly(I:C) (InvivoGen) (10 µg) on day 16 to day 19 post initial tumor graft at ~24 h intervals. Mice were euthanized on day 21, and the tumor masses were removed, dissociated, and subject to FACS (using single-cell level purity masking) to obtain pure populations of CD8⁺ TIL. Cells were in vitro stimulated and expanded in mixed lymphocyte reactions with irradiated (50 Gy) feeder C57BL/6 splenocytes at a 100:1 feeder: TIL ratio. Stimulations were performed in single wells of 24-well plate containing 500 µL of supplemented RPMI + 100 ng/mL each of anti-CD3/28 mAb (same clones as above) and 400 IU/mL rhIL-2 (Stemcell Technologies). Media + rhIL-2 was completely replaced on day 3, and cells were continued in culture for a further 15 days, replacing half of conditioned media with fresh media + rhIL-2 every 3 days, prior to their use in experiments.