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. 2017 Mar 27;198(9):3494–3506. doi: 10.4049/jimmunol.1600965

FIGURE 3.

FIGURE 3.

Low-dose immunizations favor increased relative, as well as absolute, numbers of high-avidity CD4 T cells but not CD8 T cells. BALB/c mice were immunized i.p. three times at 2-wk intervals, as described, with a low (0.1 nmol), medium (1 nmol), or high (10 nmol) dose of PCLUS6.1-P18 in CAF09. One week later, functional avidity was assessed by ICS and by flow cytometry. (A) CD4 T cells of high functional avidity were defined as cells producing IFN-γ after stimulation with 5 × 10−3 μM PCLUS6.1-P18 (below the EC50 in all groups to assure cells were of high avidity), and the total response was determined as the highest response observed in all groups over all stimulation concentrations. Data points depict the ratio of high avidity/total response from individual mice in the low (0.1 nmol), medium (1 nmol), and high (10 nmol) vaccine dose groups, as indicated on the x-axis. Mean and SEM of n = 3 mice per group are shown. (B) The corresponding ratios of high-avidity/total vaccine–specific CD8 T cells from the same experiments are shown. High-avidity CD8 T cells were defined as CD8 T cells that produced IFN-γ after stimulation with 5 × 10−2 μM PCLUS6.1-P18 (corresponding to <30% of the maximum response in all groups), and total response was defined as the highest response over all stimulation concentrations. Absolute numbers of high-avidity (defined as in A and B) IFN-γ–producing CD4 (C) and CD8 (D) T cells are depicted using data pooled from five similar experiments. Bars depict mean/SEM absolute numbers of high-avidity IFN-γ–producing CD4 (C) and CD8 (D) T cells that were normalized to the response in the 1-nmol vaccine dose group (set to 100%) within each experiment. High avidity was defined as IFN-γ–producing cells responding to the stimulation concentration just below the EC50 concentration. n = 11–14 per group. Amounts of high-avidity CD4 and CD8 T cells are indicated above/within the bars relative to the 1-nmol group. Data are representative of nine experiments showing similar results. *p < 0.05, **p < 0.01, one-way ANOVA and Newman–Keul posttest for multiple comparisons. ns, not significant.