Peripheral blood samples and serum were obtained from patients with metastatic melanoma under an Institutional Review Board-approved protocol.15 (link) Samples were coded with an anonymized 5-digit number and their identity was unknown to those performing the experiments. Peripheral blood mononuclear cells (PBMCs) were isolated by density gradient with 1.077 g/mL Ficoll-Paque (GE Healthcare, Chicago, Illinois, USA). For cryopreservation, PBMCs were re-suspended in human AB serum (Omega Scientific; Tarzana, California, USA) with 10% dimethyl sulfoxide (Sigma-Aldrich, St Louis, Missouri, USA), frozen at −80°C and stored in liquid nitrogen. CD3+ and CD8+ T cells were isolated using a magnetic negative selection kit (StemCell Technologies, Vancouver, British Columbia, Canada). Cells were cultured with 10% AB human serum in beta-mercaptoethanol, non-essential amino acids, HEPES, penicillin, streptomycin and gentamicin supplemented X-VIVO media (Corning, Corning, New York, USA). Serum was obtained from whole blood patient samples, centrifuged to remove cells and frozen at −80°C until time of use.