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. 2024 Jun 15;32(3):200835. doi: 10.1016/j.omton.2024.200835

Figure 3.

Figure 3

CD4 helper antigens promote phenotypic changes in vaccine-induced CD8+ T cells associated with improved effector function

Mice were vaccinated intradermally three times, at 3-week intervals as indicated in Figure 2. Mice were bled at the peak of the T cell response following the third vaccination (day 52), after which expression of the indicated markers on OVA-specific CD8+ T cells was measured by flow cytometry. (A) Histograms show normalized counts of cells expressing surface markers CD25, CX3CR1, KLRG1, PD-1, and LAG3 on total CD8+ T cells of mock-vaccinated mice or OVA-specific CD8+ T cells, identified by Kb/OVA-tetramer staining, in mice vaccinated with noHELP, neoHELP, or uniHELP (Figures S2C and S2D). Marker expression on (B) OVA-specific CD8+ T cells or (C) tetramer-negative CD8+ T cells of mock-, noHELP-, neoHELP-, or uniHELP-vaccinated mice. Expression was quantified as mean fluorescence intensity (MFI) or percentage of marker-positive cells, as indicated. Dots represent individual values, bars and whiskers represent mean and standard error (SEM). Data were derived from a single experiment, representative of three experiments, with 10 mice per group. Data were analyzed by one-way ANOVA followed by Tukey’s multiple comparisons test comparing noHELP, neoHELP, and uniHELP. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001. (D) CX3CR1hi, (E) KLRG1, or (F) PD-1 expression on OVA-specific CD8+ T cells monitored in blood over time on days 13, 31, and 52 post primary vaccination. Data are derived from a single experiment, representative of three, with 10 (noHELP) or 30 (neoHELP and uniHELP) mice per group. Data from tetramer-negative (TM−) CD8+ T cells from all experimental groups were pooled and plotted as CD8 as background reference. Dots and whiskers represent means and standard errors (SEM), respectively. Data were tested in mixed-effects analysis followed by Tukey’s multiple comparisons test comparing noHELP, neoHELP, and uniHELP. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001. nd, no OVA-specific T cells detected.