Kvprnqdwl

Pmel-1 CD8⁺ T cells were isolated from a single cell suspension of splenocytes using anti-CD8 beads and MACS columns (Miltenyi Biotec). The sorted 1 × 10⁵ Pmel-1 CD8⁺ T cells were pulsed with 10μM of human (h)gp100_25-33 peptide, KVPRNQDWL (GenScript) in the presence of mitomycin-C treated 5 × 10⁴ splenocytes from B6 mice in T-cell reprogramming medium (23 ). After 24 hours of culture, the solution which contained SeV vectors that individually carried each of Oct3/4, Sox2, Klf4 and c-Myc was added to wells at multiplicities of infection (MOI) 20. After 24 hours of infection, the cells were collected and transferred to a 10-cm dish that contained mitomycin C-inactivated SNL feeder cells in iPSC medium. The iPSC medium was changed every other day until the colonies were picked.

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.