Ovarian and breast cytotoxic T lymphocytes can recognize peptides from the amino enhancer of split protein of the Notch complex

Abstract

In this study we investigated recognition by ovarian tumor associated lymphocyte (OVTAL), and breast tumor associated lymphocytes (BRTAL), of peptides corresponding to the sequence 125–135 of the Aminoenhancer of split (AES) protein. Three of these peptides designated as G75:AES1/2 (128–135), G60: AES1/2 (127–137) and G61: AES1/2 (125–133) correspond to the wild-type AES sequence, while the fourth G76: G PLTPLPV, AES1/2 (128–135) corresponds to a variant sequence of the peptide G75 with the N-terminal Leu substituted to glycine. These sequences were chosen for study because mass-spectrometric analysis (MS) of a CTL active HPLC peptide fraction eluted from immunoaffinity precipitated HLA-A2 molecule, revealed: (a) the presence of an ion with a mass-to-charge ratio ( m/z) of 793 which was more abundant than other ions of similar masses; (b) the tentatively reconstituted sequence of the ion 793 matched the sequence of peptide G76. We found that AES peptides G75 (128–135) and G76 (128–135) (L128G) reconstituted CTL recognition at concentrations ranging between 200–500 nM. These concentrations are lower than concentrations reported to activate effector function of CTL recognizing other epithelial tumor Ag. Furthermore, analysis with cloned CD8 + T cells indicated that G75 and G76 were not cross-reactive specificities, suggesting a key role for the N-terminal residues of the variant peptide in dictating specificities. Since the AES proteins are part of a set of transcriptional repressors encoded by the Enhancer of split [E(spl)] genes, and since these repressors are activated to suppress cell differentiation in response to Notch receptors signalling, the AES peptides may represent a novel class of self-antigens that deserve further consideration as tumor Ag in epithelial cancers.