Fig. 6

Simultaneous vaccine-induced IgG and CD8+ IFNγ recognition of 4T1 tumor and peptides confirmed by LC-MS/MS. LC-MS/MS was performed to determine whether proteins containing the 4T1 peptides were present in live 4T1 cells and 4T1 autophagosome-enriched vaccine. Of the 15 4T1 WT and SNV pairs additionally analyzed in T cell assays, there were n = 9 WT and SNV antigen pairs from proteins confirmed in 4T1 by mass spectrometry and n = 6 WT and SNV antigen pairs not confirmed by mass spectrometry. a-d Data shown are from experiments previously presented in Fig. 4. Vaccinated animals demonstrated increased serum IgG to the peptides analyzed in T cell assays; however, there was no improved IgG response (a) to the WT and SNV peptides confirmed by mass spectrometry over unconfirmed proteins (P = 0.1) by unpaired t-test. However, confirmed presence of the antigenic protein in the vaccine by mass spectrometry (b) resulted in improved IFNγ secretion in vaccine groups upon secondary exposure to WT and SNV 4T1 8-11mer peptides (P = 0.04) by unpaired t-test. There was no significant overall correlation of these increases in IgG and CD8+ IFNγ peptide recognition (c) for peptides unconfirmed by mass spectrometry (P = 0.75), but there was a significant correlation between improvements in IgG and CD8+ IFNγ peptide recognition (d) for mass spectrometry confirmed proteins (P = 0.01) by Pearson correlation coefficient. For 4T1 tumor recognition data previously presented in Fig. 5, WT and SNV antigens from proteins confirmed in 4T1 cells and vaccine by mass spectrometry (e) produced greater CD8+ IFNγ recognition of 4T1 cells if serum from those animals also had higher IgG recognition of those antigens