PEPTIDE PRESENTATION BY THE MHC CLASS IB MOLECULE, H2-M3

The presentation of N-formylated peptides to cytotoxic T cells is restricted to the mouse class I MHC molecule, H2-M3. Previous studies have shown that M3 is unable to present unformylated peptides. We demonstrate that the unformylated ND1 peptide can sensitize M3(wt)-transfected fibroblasts for killing by ND1-specific cytotoxic T cells, At 1 mu M, both N-formylated and unformylated ND1 peptides induced equivalent levels of killing. However, the concentrations required for half maximal killing differed by 10(4)-fold, from 10-50 pM for N-formylated ND1 to 100 nM for unformylated ND1. The peptide binding groove of M3 differs from other class I molecules at three highly conserved positions: 34 (V-->Q), 167 (W-->L) and 171 (Y-->F). Site-directed mutagenesis was used to determine the importance of these changes in the presentation of N-formylated peptides by M3. Cell lines expressing the mutations Q34V, L167W or F171Y all presented the N-formylated ND1 peptide equally well to ND1-specific T cells. The N-formylated ND1 peptide was also presented by a triple mutant, containing substitutions at all three positions, Q34, L167 and F171 are therefore not required individually, nor in combination, for the presentation of N-formylated peptides by M3. However, all three point mutations did affect killing by alloreactive, M3-specific T cells, F171Y was the least damaging mutation, affecting only one of the two T cell lines tested, By contrast, both T cell lines failed to kill Q34V and L167W targets. Q34 and L167 are thus important determinants in the M3-specific CTL response.