In vitro programming of T_EM cells causes an interval of Ag-independent IFN-γ release that facilitates dynamic changes in D^b expression on tumor. (A) pmel T_EM cells were restimulated with hgp100_25-33 peptide-pulsed irradiated APCs for indicated time points, isolated using lympholyte solution, and then used in overnight coculture assays against targets, including CM (□), EL-4 pulsed with 1 μM of either βgal_96-103 (○) or hgp100_25-33 (●) peptides, and B16 melanoma (■). Data are mean ± SD. (B) Up-regulation of D^b expression on B16 melanoma in response to titrated doses of IFN-γ. B16 melanoma cells were incubated with IFN-γ at doses representing 5-fold serial dilutions between 3375 and 5.4 ng/mL. After 48 hours, cells were harvested and evaluated by FACS analysis for D^b expression. Mean fluorescence intensity (MFI) after gating on live cells versus dose of IFN-γ pretreatment is displayed. Results from 1 of 3 representative experiments are shown. (C) IFN-γ production by pmel T_EM cells causes dynamic changes in the surface expression of D^b on B16 melanoma in a contact-independent manner. B16 was plated at the bottom of a 24-well Transwell plate with CM only (…), and either pmel T_EM (■) or pmel-Ifng1^−/− T_EM (▩) cells programmed for 24 hours with hgp100_25-33 peptide before being plated in the top wells. After 24 hours, B16 from the bottom well was harvested and evaluated for D^b expression by FACS analysis after gating for live cells. Results of a representative experiment are shown.