Figure 5. PancVAX2 recruits neoantigen-specific Th1 CD4⁺ T cells into the tumor and reduces infiltration of Tregs into the tumor.

C57BL/6 mice were implanted with 3 × 10⁶ Panc02 cells, s.c., on day 0. Mice were vaccinated s.c. at the base of the tail on day 14 and 21 with PancVAX2 (n = 5). On day 28, tumors were harvested and pooled and dissociated into a single-cell suspension, and isolated CD4 T cells were cocultured overnight at a 1:1 ratio with T2-H2-IA^b APCs pulsed with 5 µg/mL MHCII peptides. (A) Representative gating of CD4 and CD137 expression from intratumoral CD3⁺ cells after cocultured with OVAII peptide (control) or peptide 43. (B) Quantification of CD4⁺CD137⁺ T cells following restimulation with each MHCII-specific peptide. (C–G) Activated (CD137⁺) CD4 T cells were then assessed for Th1-like cytokine expression (IFN-γ, IL-2, and TNF-α) (C), cytotoxic effector cytokine expression (GzB) (D), Th2-like cytokine expression (IL-4) (E), Th17-like cytokine expression (IL-17) (F), and Treg activity (IL-10) (G). Symbols represent technical triplicates from pooled CD4 T cells. Data are shown as mean ± SD. Significance was calculated by 2-way ANOVA followed by Sidak’s multiple-comparison test. (H) C57BL/6 mice were implanted on the right flank, s.c, with 3 × 10⁶ Panc02 cells on day 0. Mice were vaccinated s.c. at the base of the tail on day 14 and 21 with CD4 Vaccine or CD8 Vaccine (n = 4). On day 28, tumors were harvested, dissociated into a single-cell suspension, and stained for flow cytometry analysis. CD45⁺CD3⁺CD4⁺ populations were assessed for the proportion of cells expressing transcription factors T-bet (Th1, left) or GATA3 (Th2, right). (I) Proportion of FOXP3⁺CD4⁺ T cells (left) and the ratio of CD45⁺CD3⁺CD8⁺ T cells to FOXP3⁺CD4⁺ T cells (right). Symbols represent technical duplicate of individual mice. Data are shown as mean ± SD. Significance was calculated by a 2-tailed unpaired t test. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001.