Fig. 4

Simultaneous increases in IgG signals to 15mers and CD8+ IFNγ recognition of individual 8-11mer antigens. Serum and CD4-depleted 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). CD4-depleted 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 naïve splenocytes pulsed with a second stimulation of peptide. Graphs are of the IFNγ secretion for each individual paired experiment from n = 3 paired replicates with vaccine and naïve groups, each involving n = 15 different WT and n = 15 SNV peptide experiments. a, b Increase in IFNγ secretion in vaccine groups upon secondary exposure to n = 45 paired experiments with WT peptides (P < 0.0001) (a) and n = 45 paired experiments with 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 vaccinated animal groups and n = 3 naïve groups used in splenocyte assays. Increase in average IgG signal intensity to n = 45 paired WT peptide experiments (P < 0.0001) (c) and n = 45 SNV peptide experiments (P < 0.0001) (d) 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 = 45 WT experiments and n = 45 SNV experiments. 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. There was a significant overall correlation of these increases in IgG and CD8+ IFNγ recognition (P = 0.0039) by Pearson correlation coefficient