Mil 15

Mice were injected subcutaneously with 5 × 10⁵ B16F10 cells. For adoptive T cell therapy, Pmel-1 splenocytes were cultured with mIL-7 (10 ng/ml; Peprotech) and mIL-15 (10 ng/ml; Peprotech) for 6 days in the presence of 1μM of human (h) gp100_25–33 peptide, KVPRNQDWL (GenScript). Mice were treated 12–14 days after tumor inoculation with i.v. adoptive transfer of in vitro-activated 1 × 10⁶ T cells. We injected 15,000 IU recombinant human IL-2 (rhIL-2) (Peprotech, Inc) intraperitoneally once on the day of adoptive transfer and twice a day on the two following days. In some experiments, mice received 500 cGy of sublethal irradiation prior to adoptive T cell transfer to mimic the lymphodepletion. Mice were also vaccinated with 100 μl of saline containing 100 μg of hgp100 peptide, 50 μg of agonistic anti-CD40 Ab (clone FGK4.5, BioXcell), and 50 ug of poly(I:C) (InvivoGen) at the peritumoral site or 50 mg of imiquimod cream 5% (Perrigo) applied on the vaccination sites after adoptive transfer as described before (38 (link), 39 ). Tumor volumes were calculated by determining the length of short (l) and long (L) diameters (volume = l² × L/2). Experimental end points were reached when tumors exceeded 20 mm in diameter or when mice became moribund and showed signs of lateral recumbency, cachexia, lack of response to noxious stimuli, or observable weight loss.

Semiadherent Pmel-1 iPSC-derived cells on OP9-DL1 cells were harvested and filtered through a 40μm nylon mesh. CD8 expressing cells including CD4 CD8 double positive (DP) T cells and CD8 single positive (SP) T cells in Pmel-1 iPSC-derived cells, Pmel-1 splenocytes and thymocytes were isolated using anti-CD8 beads and MACS columns to eliminate OP9-DL1 cells during T cell activation. These cells (2 × 10⁶ cells) were cultured with mIL-7 (10 ng/ml) and mIL-15 (10 ng/ml; Peprotech) for 2 days in the presence of 1μM of hgp100 peptide and mitomycin-C treated splenocytes from B6 mice (5 × 10⁵ cells). These activated cells were cultured with IL-7 and IL-15 or IL-7, IL-15 and IL-2 from day 3, and used for further experiments on day 6–8.