Figure 11. Tethered CD4⁺ T cell helper epitopes optimize checkpoint blockade and CTL-mediated SCC VII tumor destruction via a CD40-dependent mechanism.

(A) C3H/HeJ mice injected with 5 × 10⁵ live SCC VII-Luc/GFP cells and given 50 μg polyI:C alone or in combination with 5 μg full-length Mut_48 peptide at day 10 after challenge (black arrow). Peptide-treated mice also received anti-PD-1 at days 10, 13, and 16 (red arrow). The immunotherapy cycle repeated at day 24 (gray box). CD4⁺ and CD8⁺ T cells were depleted 1 day before each immunotherapy cycle (green arrow), and anti-CD40 was delivered as indicated (blue arrow) with resultant tumor volume kinetics (n = 6 per group). (B) Tumor-bearing C3H/HeJ mice delivered 50 μg polyI:C alone or combined with 5 μg PADRE(X), Mut_48.10, mixed PADRE(X) and Mut_48.10, or tethered PADRE(X)-AAA-Mut_48.10 peptide at day 10 after challenge (black arrow) and anti-PD-1 (red arrow) as in A with resultant tumor volume kinetics (n = 5-6 per group). All experiments were performed 2 or more times and data indicate mean ± SEM; (A and B) **P < 0.01 and ****P < 0.0001 (2-way ANOVA and Dunnett’s posthoc test relative to polyI:C); ^†P < 0.05, ^††P < 0.01, and ^†††P < 0.001 (2-way ANOVA and Dunnett’s posthoc test relative to Mut_48 + polyI:C + anti-PD-1 + anti-CD4 or anti-CD8 [blue groups]).