Figure 2. Murine pancreatic cancer MHCII predicted neoantigens are immunogenic in non–tumor-bearing mice.

(A) Pipeline to identify predicted MHCII restricted neoantigens that stimulate a CD4⁺ T cell response in vivo. (B) Non–tumor bearing C57BL/6 mice were vaccinated with 20 mer SLPs predicted to bind to H2-IA^b on days 0 and 7 (n = 3). Splenocytes from vaccinated mice were pooled on day 14, and CD4⁺ T cells were isolated using magnetic bead negative isolation kits. Isolated CD4⁺ T cells were cocultured overnight at a 1:1 ratio with I-A^b–expressing T-2 APCs pulsed with MHCII-specific peptides in an IFN-γ capture plate and assessed for reactivity by ELISpot the following day. The 20 mer neoepitopes that produced a significant immune response in the CD4 vaccinated group over the untreated group are displayed. Symbols represent technical replicates. To confirm CD4 and CD8 T cell specificity to each peptide, non–tumor-bearing C57BL/6 mice were vaccinated with PancVAX2 on days 0 and 7 (n = 4). (C and D) Splenocytes from vaccinated mice were isolated and pooled on day 14, and CD4⁺ and CD8⁺ T cells were separately sorted using magnetic bead negative isolation kits and cocultured overnight at a 1:1 ratio with matured murine BMDCs that had been pulsed with 5 μg/mL MHCI-specific (C) or MHCII-specific (D) peptides in an IFN-γ capture plate and assessed for reactivity by ELISpot the following day. Data are shown as mean ± SD. Significance was calculated by 2-way ANOVA followed by Sidak’s multiple-comparison test. *P ≤ 0.05, **P ≤ 0.01,****P ≤ 0.0001. Significance values for CD8 T cell cultures (black asterisks) and CD4 T cell cultures (blue asterisks) are shown.