Identification of a novel murine class I epitope from EGFRvIII.

a) BALB/c, C57BL/6, C3H and SJL mice were vaccinated with Lm-EGFRvIII and 7 days later tested by ICS for EGFRvIII-specific T cells using a library of overlapping peptides from EGFRvIII20-40, or a DMSO vehicle control. Spleens from three mice per strain were used for restimulation. The percentage of IFN-γ+ events within the CD3+CD8+ T cell gate are shown for each peptide; b) Splenocytes from C3H mice primed and boosted with Lm-EGFRvIII were stimulated using a library of overlapping peptides from EGFRvIII20-40, or SIINFEKL as a negative control. A subgroup of peptides within the previously identified 15-mer were used to define the optimal 9-mer peptide by ICS. c) H2Kk binding of EGFRvIII or control peptides was assessed by stabilization of surface MHC I using flow cytometry. d) Identification of EGFRvIII-specific T cells in the spleen following prime-boost vaccination with Lm-EGFRvIII using H2Kk-tetramers folded with the defined peptide EEKKGNYV.