Influence of MHC class I peptide interaction on antigen presentation in normal and malignant cells

Abstract: The presentation of MHC class I peptide complexes in normal and malignant cells was studied. An HLA A11 binding motif was defined by comparing synthetic peptide analogues of a known cytotoxic T Iymphocyte (CTL) target epitope for induction of surface A11 expression and triggering of cytotoxic activity. The motif predicts the presence of hydrophobic aminoacids in position 2 (P2), small amino acids in P3 and P6 and Lys in P9. The off-rates of P2 analogues discriminated between "apparent" and "true" binders that formed complexes with a half life longer than 72 hours. The persistence of MHC-peptide complexes the surface of antigen presenting cells was shown to correlate with the immunogenicity two A11-restricted epitopes of the Epstein-Barr virus (EBV) nuclear antigen (EBNA) 4 (designated IVT and AVF, respectively). Molecular modeling and alanine scanning mutagenesis of the IVT and AVF peptides demonstrated that solvent exposed peptide side chains affect CTL recognition as well as antibody binding suggesting that allospecific antibodies recognize the complexes in a fashion similar to T cell receptors. Class I molecules are important for target recognition by T cells as well as natural killer (NK) cells. Overexpression of the c-myc oncogene increased the sensitivity of EBV transformed Iymphoblastoid cell lines (LCLs) to NK lysis. The reactivity with allospecific antibodies was significantly reduced suggesting that a different set of antigenic peptides may occupy the class I groove, resulting in inability to deliver a negative regulatory signal to NK cells. Defective presentation of endogenously expressed EBNA4 was demonstrated in A11 positive Burkitt's Iymphoma (BL) lines. The defect was not overcome by cytosolic expression of the preformed epitope. The tumor cell expressed low levels of the transporter associated with antigen processing (TAP) and behaved poorly in a streptolysin-O mediated peptide translocation assay but presentation was not restored by upregulation TAP activity following treatment with IFN-y. Efficient maturation of class I molecules to Endo H resistant species was demonstrated in pulse-chase experiments. The results localize the defect to functions downstream of the generation of antigenic peptides but upstream of TAP-dependent peptide transport and class I assembly and maturation.