Adoptive transfer

gp100-specific TCR transgenic Pmel female mice (Overwijk et al, 1998, 2003) (6–12-wk-old) that had yet to begin showing signs of coat depigmentation were used as donors. CD8⁺ T cells were purified from their spleens using mouse CD8a (Ly-2) MicroBeads for positive selection (Miltenyi Biotec Inc.) and were labelled with CellTrace Violet (CTV) or CFSE (Invitrogen^TM), according to the manufacturer’s suggested protocol. For adoptive transfer, the T cells were washed twice with PBS, resuspended at 0.5–1 million cells per 200 μl, and injected via tail vein into recipient animals. Recipient mice received 600 cGy total body irradiation from a ¹³⁷Cs source several hours before adoptive transfer. The day after the adoptive transfer, mice were injected with a tumor cell vaccine of 10⁶ irradiated (3,000 rads) and treated B16 WT or Trp53^−/− cells. The cells were treated in a pulse fashion with MQ 10 μM for 4 h or iodoacetamide 20 μM for 30 min or the vehicle and cultured for 48 h in fresh media before being used for the vaccine. For B16-GMCSF–based cell vaccines, preparation was performed as per the GVAX experiment (see dedicated section above). The draining lymph nodes were harvested 5 or 6 d after the vaccination.