Figure 3.

Analysis of antigen-specific multifunctional CD4⁺ T cells. (A) Gating strategy for multifunctional CD4⁺ memory T cells. Memory CD4+ T cells (determined by increased CD44 expression) gated to show CD3⁺CD4⁺CD44⁺ T cells. For multifunctional CD4⁺ T cells analysis, cells were identified by intracellular cytokine (IFN-γ, TNF-α, and IL-2) staining based on memory CD4⁺ T cell gating. (B) The percentage of VLP-specific total cytokine (IFN-γ, TNF-α, and/or IL-2)-producing cells among splenic CD4⁺CD44⁺ memory T cells. (C) Pie charts representing the mean frequencies of cells co-expressing IFN-γ, TNF-α, and/or IL-2. The relative amounts of single-, double- and triple-functional CD4⁺CD44⁺ memory T cells are indicated as pie arcs. Means ± SD (n = 5 mice/group) shown are representative of two independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001. n.s.: not significant.