Mil 7

B7 (Ba/F3 cells stably transduced with MSCV-mIL7R-IRES-GFP) cells were maintained at a density <2 million cells/mL in media with 10ng/mL mIL-7 (Peprotech), as previously described [28 (link)]. B7 cells were electroporated with 1000nM Hspa9-targeting siRNAs (Thermo Scientific: D-057872-03 [siRNA 1] and D-057872-04 [siRNA 2]) or non-targeting control siRNA (Thermo Scientific: D-001206-14) using a Nucleofector Device (Lonza) program X-001 according to manufacturer’s protocols.

Pmel-1 iPSCs and mESCs were cultured using OP9 and OP9-DL1 stromal cell co-cultured system (24 (link)). In brief, 5 × 10⁴ cells were plated on a 10-cm dish containing confluent OP9 cell monolayer in OP9 medium. On day 5 of culture, cells were trypsinized off, and 5 × 10⁵ cells were transferred to a fresh culture 10-cm dish containing confluent OP9 cell monolayer in OP9 medium with the addition of hFlt3 ligand (5 ng/ml; R&D systems). On day 8 of culture and every 4 days thereafter, semiadherent stem cell-derived hematopoietic cells were collected, filtered through a 40μm nylon mesh, and were transferred onto fresh OP9-DL1 monolayers with the addition of hFlt3 ligand and mIL-7 (1 ng/ml; Peprotech). Semiadherent cells obtained from day 16 to 28 were used for further experiments.

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.