FIGURE 7.

Heterologous vaccine immunogenicity—differentiation of IFN-γ producing CD4 T cells. Panel (A) shows the gating hierarchy to assess the differentiation of IFN-γ producing CD4 T cells. After gating on IFN-γ + CD4 T cells as described in Figure 6, their differentiation was analyzed via their CD4/CD8α expression profile to distinguish naïve (CCR7⁺CD8α^–), central memory (T_CM, CCR7⁺CD8α⁺) and effector memory (T_EM, CCR7^–CD8α⁺) CD4 T cells (top right plot). Since the vast majority of CD8α⁺ IFN-γ-producing CD4 T cells belonged to the T_CM subset (d.n.s.), both T_CM and T_EM were combined in the downstream analysis into the “memory/effector” subset. Panel (B) shows the frequency of these memory/effector within IFN-γ-producing CD4 T cells according to their challenge groups—MOCK (gray), 1-4-4 (NADC30, dark blue), NC174 (red), VR2332 (green), and 1-4-2 (NADC20, light blue). The black bars represent the median values; in addition, individual data points are shown for MOCK vaccinated animals (open diamonds) and MLV vaccinated animals (filled squares). Data were statistically analyzed using a 2-way ANOVA with time and vaccination as the two parameters and Tukey’s multiple comparison test. ^****p < 0.0001, ^***p < 0.001, ^**p < 0.01.