FIGURE 4.

Enhanced cytokine production in DGKζ-deficient CD8⁺ T cells. A, 1 × 10⁶ DGKζ-deficient (solid line/solid column) or wild type (dashed line/white column) CD8⁺ T cells were incubated with α-CD3 (500A2) in the presence of brefeldin A and BD GolgiStop^TM. Five hours later, cells were surface-stained for viability, CD8, CD4, and CD44, fixed, and then stained for intracellular IFNγ (left panel), TNFα (middle panel), and IL2 (right panel). Cells were subjected to flow cytometry, and the percent CD8⁺CD44^high cells expressing each cytokine (top panel), the percent pERK positive cells normalized to the maximum per genotype (middle panel), and the mean fluorescent intensity of cytokine at the highest concentration of α-CD3 (bottom panel) were calculated from data generated with FlowJo software. B, DGKζ-deficient OT-I (solid lines) or OT-I (dotted lines) were incubated in the presence of 1 μm OVAp or altered peptide ligand as described in A, and percentage of positive cells (top panel) and mean fluorescent intensity (bottom panels) of IFNγ (left panels), TNFα (middle panels), or IL2 (right panels) were determined by flow cytometry. The EC₅₀ (amount of peptide capable of eliciting half-maximal responses) for each peptide was also calculated after titration of responses across a range of peptide concentrations (middle panels).