Fig. 5

Simultaneous increases in IgG signals to 15mers and improvements in CD8+ IFNγ recognition of tumor. Serum and CD4-depleted splenocytes were harvested from 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 as presented previously (Fig. 4), then placed in empty wells or restimulated with 4T1 tumor cells. Graphs are of the IFNγ secretion for each individual paired experiment from n = 2 paired replicates with CD8+ T cells only or CD8+ T cells plus live 4T1 cells, each pair initially stimulated with one of n = 15 WT or n = 15 SNV peptides. a, b IFNγ secretion in 4T1 tumor restimulated groups demonstrated outliers, but no overall increased 4T1 recognition after primary exposure to n = 30 paired experiments with WT peptides (P = 0.65) (a), but did show overall increased 4T1 recognition after primary exposure to n = 30 paired experiments with SNV peptides (P = 0.0002) (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 = 2 vaccinated animal groups used in splenocyte assays and n = 2 naïve group controls. Increase in average IgG signal intensity to n = 30 paired WT peptide experiments (P < 0.0022) (c) and n = 30 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 = 30 WT experiments and n = 30 SNV experiments. Positive values represent increased IgG signals versus naïve controls and increased IFNγ recognition of 4T1 tumor over T cells only. Values in upper-right quadrant demonstrated increases in serum IgG recognition of that antigen, and a simultaneous ability for that antigen to improve CD8+ T cell IFNγ recognition of live 4T1 cells. However, there was no significant direct correlation of these increases in IgG and CD8+ IFNγ recognition (P = 0.95) by Pearson correlation coefficient