FIGURE 3.

The free form of ISG15 promotes CTL priming in response to vaccination. (A) Scheme of mouse ISG15 WT protein (aa 1–161) and its C-terminal truncated version, ISG15 ΔGG (aa 1–153). The minimal sequence (LRLRGG) required for its conjugation to intracellular proteins is depicted in ISG15 WT. (B) Validation of ISG15 constructs by assessment of ISGylation of intracellular proteins. pDNA encoding ISG15 WT or ISG15 ΔGG and empty control vector (EV) were expressed in Isg15^−/− BM-derived DCs, and ISGylation was assessed by Western blotting on total cell lysates. Actin served as loading control. Results are representative of multiple independent analyses with individual samples. (C–E) Mice (n = 7 per group) were vaccinated with plasmid encoding E7SH, either combined with EV, ISG15 WT, or ISG15 ΔGG as outlined in Fig. 2. (C and D) The E7-specific CTL response was followed over time in peripheral blood by flow cytometric analysis using H-2D^b/E7_49–57 tetramers and Abs to CD8, GZB, Tbet, CD44, and CD62L. (C) Quantification of the percentage of H-2D^b/E7_49–57 tetramer⁺ cells among total CD8⁺ T cells. (D) Quantification of the percentage of GZB⁺, Tbet⁺, and CD44⁺CD62L⁻ cells within H-2D^b/E7_49–57 tetramer⁺ cells. (E) On day 16, three mice per group were sacrificed, and the T cell response was assessed in dLN and spleen. Depicted are percentage of H-2D^b/E7_49–57 tetramer⁺ cells within total CD8⁺ T cells, percentage of GZB⁺ cells within tetramer⁺ cells ex vivo, and percentage of IFN-γ⁺TNF-α⁺ cells among total CD8⁺T cells after in vitro stimulation. Results are representative of two experiments. Statistical analysis was performed using one-way ANOVA (C and D) or two-way ANOVA (E) and Tukey posttest. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.