α ifn γ xmg1.2

Naive T cells were isolated by sorting CD4⁺CD25⁻CD62L^highCD44^low cells from spleens and lymph nodes, differentiated under several Th cell conditions, and analyzed as described (Yang et al., 2008a (link)). Briefly, naïve T cells (5 × 10⁵ cells/well) were cultured at 37°C (5% CO₂) in complete medium. The cells were stimulated with the plate-bound α-CD3 (1 μg/ml; BioXCell) and the soluble α-CD28 (1 μg/ml; BioXCell). For Th0, the cells were treated with 30U/ml IL-2, 5 μg/ml α-IFN-γ (XMG1.2; BioXCell) and 5 μg/ml α-IL-4 (11B11; BioXCell). For Th1, the cells were treated with 30U/ml IL-2, 10 ng/ml IL-12 (Peprotech) and 5 μg/ml α-IL-4. For Th2, the cells were treated with 30U/ml IL-2, 10 ng/ml IL-4 (Peprotech) and 5 μg/ml α-IFN-γ. For Treg, the cells were treated with 30U/ml IL-2, 1 ng/ml TGF-β1 (Peprotech), 5 μg/ml α-IFN-γ and 5 μg/ml α-IL-4. For Th17, the cells were treated with 0.5 ng/ml TGF-β1, 10 ng/ml IL-1β (Peprotech), 10 ng/ml IL-6 (Peprotech), 10 ng/ml IL-23, 5 μg/ml α-IFN-γ and 5 μg/ml α-IL-4. For Th17(β), the cells were treated with 0.5 ng/ml TGF-β1, 10 ng/ml IL-6, 5 μg/ml α-IFN-γ and 5 μg/ml α-IL-4. When indicated, 10 μg/ml α-TGF-β (1D11; BioXCell) or ¹⁰ μM TGF-β receptor kinase inhibitor (SB431542; Calbiochem) were added. For Th17(23), the cells were treated with 10 ng/ml IL-1β, 10 ng/ml IL-6, 10 ng/ml IL-23, 5 μg/ml α-IFN-γ and 5 μg/ml α-IL-4.

Adoptive transfer experiments have been described previously (22 (link)). Briefly, recipient B6 mice were given 3 × 10⁵ B16F10 melanoma tumor cells subcutaneously (s.c.) on day 0. The mice were then irradiated with 5 or 6Gy total body irradiation, as indicated in the figure legend, 6 hours prior to CD8⁺ T cell transfer. Mice received i.v. 1 × 10^6-7 Pmel-1 CD8⁺ T cells that were in vitro-vaccinated, in conjunction with bolus rhIL-2 (was administered to mice i.p. once or twice daily at 3.6 g/dose for a total of 4-6 doses). Vaccination involved co-culturing the CD8⁺ T cells with irradiated B6 splenocytes and 1uM hgp100_25-33 peptide for 6 hours. In some experiments mice were given 0.1mg neutralizing antibody i.p. every other day for a total of 5 treatments. αIL-9 (9C1), αIL-17 (17F3) and αIFN-γ (XMG1.2) were purchased from BioXCell. Experiments were performed in a blinded, randomized fashion and tumor measurements were taken over time. Vitiligo on treated mice was scored on a scale of 0-5: 0, no vitiligo (wild-type); 1, depigmentation detected; 2, >10% vitiligo; 3, >25% vitiligo; 4, >50% vitiligo; 5, >75% vitiligo. Two different investigators who were unaware of the treatment groups 5 weeks after ACT scored mice. In one set of experiments ciprofloxacin (50 mg/kg/d for 1–2 weeks; Bayer) was added to the drinking water of two days prior to irradiation.