Adoptive Transfer of Tumor-Specific T Cells

Adoptive transfers were performed as described previously [25 (link)]. The number of transferred cells and the timing of transfer and infection are stated in each figure legend. For the experiments in Fig. 6 and Suppl. Fig. 3A, OT-Is and Pmel-Is were co-transferred into mice one day before implantation of B16F0s. Recipients were co-infected with MCMV-SL8 and MCMV-gp100 5 days after tumor implantation to expand both OT-Is and Pmel-Is. For in vitro activation, OT-Is were either taken from fresh or frozen naïve splenocytes (frozen in T cell media plus 10% DMSO) and enriched via magnetic negative CD8 selection as described above. Enriched cells were activated with anti-CD3/CD28 coated Dynabeads (ThermoFisher Scientific) following the manufacturer's protocol in T cell media containing 30 U/ml rIL-2 (Biolegend). Upon transfer, OT-Is were 90–100% alive (determined by Zombie Aqua™ Fixable Viability Kit [Biolegend]), 95–100% CD44^hi (determined by anti-CD44 [clone IM7]), and had expanded 20–30 times.