Fig. 3

Simultaneous increases in IgG signals to 15mers and splenocyte IFNγ recognition of individual 8-11mer antigens. Serum and splenocytes were harvested from naïve and 4T1 autophagosome-enriched vaccine + poly-I:C vaccinated animals. Serum was run on the 15mer arrays presented previously (Fig. 2). Splenocytes were stimulated with WT and SNV versions of top predicted MHCI binding 8-11mer mutation site peptides for 48 h, then expanded on IL2 for an additional 96 h before wells were split and restimulated with either naïve splenocytes or naïve splenocytes pulsed with a second stimulation of peptide. Graphs are of the average increase in IFNγ secretion by ELISA in wells with peptide restimulation over splenocytes alone for n = 3 experiments with vaccine groups and n = 2 experiments with naïve groups. a, b Increase in average IFNγ secretion in vaccine groups upon secondary exposure to n = 15 different WT (P = 0.002) (a) and n = 15 different SNV (P = 0.005) peptides (b) by Wilcoxon matched-pairs signed rank test. c, d Simultaneous serum IgG array recognition data for 15mer peptides centered at these same mutation sites from the same n = 3 vaccine groups and n = 2 naïve groups used in splenocyte assays. Increase in average IgG signal intensity to n = 15 different WT (P = 0.01) (c) and n = 15 different SNV (P = 0.02) (d) peptides by Wilcoxon matched-pairs signed rank test. e Combined data previously presented in (a-d) plots average differences in IgG and IFNγ recognition for each of the n = 15 WT and n = 15 SNV mutation sites along with AH1. Positive values represent increased signals in vaccine groups and negative values represent increased signals in naïve groups. Values in upper-right quadrant demonstrated simultaneous increases in IgG and splenocyte IFNγ recognition of individual 4T1 mutation-site antigens in vaccine groups. However, there was no significant overall correlation of these increases in recognition (P = 0.6) by Pearson correlation coefficient