FIG 3.

Tim-3⁺ gB-CD8⁺ T cells are multifunctional after ex vivo stimulation. TGs from latently infected mice (33 to 35 dpi) were removed, processed into single-cell suspensions, stained with anti-Tim-3 antibody, and stimulated for 6 h with gB peptide-pulsed B6WT3 cells in the presence of anti-CD107a and brefeldin A. Cells were then collected and stained for viability, CD45, CD8, IFN-γ, and TNF-α. In graphs, bars represent means ± SEM (n = 24), from two pooled independent experiments. (A) Representative dot plots of IFN-γ and TNF-α responses from an individual TG that received no peptide stimulation (3rd plot from the left) and an individual TG that received gB peptide stimulation (4th plot from the left). Shaded quadrants make up the “responding CD8⁺ T cells.” Gating for CD107a is shown at the far right, with FMO represented in black and a representative sample in gray. (B) Percentage of responding CD8⁺ T cells that are positive for Tim-3. (C) Percentage of Tim-3⁺ responding CD8⁺ T cells that are multifunctional (IFN-γ⁺ TNF-α⁺ CD107a⁺).