FIGURE 5.

High-avidity CD4 T cells express higher cytokine levels and greater downregulation of TCR and inhibitory receptors than do their low-avidity counterparts. Mice were immunized three times i.p. with the indicated doses of PCLUS6.1-P18 in CAF09, as described previously. One week after immunizations (4 wk for CTLA-4 and Fas analyses), splenocytes were stimulated in vitro and assessed for the surface expression of various markers, as well as intracellularly for cytokine production by flow cytometry. (A) Representative line graphs show intracellular expression of IFN-γ, TNF, and IL-2 gated on IFN-γ–producing CD4 T cells from mice immunized with 0.1 nmol (high avidity; thin black line) or 10 nmol (low avidity; filled graph) PCLUS6.1-P18 in CAF09. IFN-γ expression is shown for CD4 T cells from naive mice that did not produce IFN-γ as a staining control (thick black line). (B) From the same mice in (A), TNF and IL-2 MFI for TNF⁺ and IL-2⁺ CD4 T cells, respectively. Data are shown as described in (A). (C) Surface expression of TCR components CD3ε and TCR-β, as well as CD4 coreceptor, on IFN-γ⁺CD4⁺ T cells from mice immunized with 0.1 and 10 nmol after stimulation or on naive CD4 T cells; data are shown as described in (A). (D) Surface expression of inhibitory receptor PD-1, death receptor CD95 (Fas), and CTLA-4 on IFN-γ⁺ CD4 T cells after stimulation. Filled graph (high dose): 30 nmol PCLUS6.1-P18 (low avidity); thin black line (low dose): 0.3 nmol PCLUS6.1-P18 (high avidity), thick black line: naive unstimulated CD4 T cells (control). (E) Percentage of PD-1 expression on all gated CD4 T cells (upper panel) and IFN-γ⁺ CD4 T cells (lower panel) after stimulation. No upregulation of PD-1 was observed after in vitro stimulation. (F) Bar graphs show MFI of PD-1 on all gated CD4 T cells (upper panel), as well as on IFN-γ⁺ CD4 T cells (lower panel), from the same experiment shown in (E). Bars represent mean and SEM of n = 3 mice per group immunized as indicated on the x-axis. *p < 0.05, **p < 0.01, one-way ANOVA with Newman–Keul posttest (E and F). (G) In a separate experiment, mice were immunized i.p. with a high (30 nmol) or low (0.3 nmol) dose of PCLUS6.1-P18 in CAF09 three times, as described above. Four weeks later, splenocytes were stimulated in vitro with increasing concentrations of PCLUS6.1-P18, as indicated on the x-axis. Graphs depict surface expression (MFI) of CTLA-4 (upper left panel), CD95 (Fas; lower left panel), and T-bet (right panel) on IFN-γ⁺ CD4 T cells; data points represent mean and SEM of n = 3 mice per group. Experiments were repeated at least twice with similar results. *p < 0.05, ****p < 0.0001, two-way repeated-measures ANOVA and Bonferroni correction for multiple comparisons. Pos, positive controls (PMA-ionomycin).