Peptide presentation by the MHC class Ib molecule, H2-M3

Abstract

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 M3wt-transfected fibroblasts for killing by ND1-specific cytotoxic T cells. At 1 microM, 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.

Functional Roles of TAP and Tapasin in the Assembly of M3-N-Formylated Peptide Complexes

“…LPS blasts from TAP o , tapasin o , and littermate control mice and cell lines were labeled overnight with 35 S-Translabel (ICN Pharmaceuticals, Costa Mesa, CA) in the presence or absence of 10 M LemA (fMIGWII) peptide (45). Labeled cells were lysed in buffer containing 50 mM Tris (pH 7.4), 150 mM NaCl, 0.5% Nonidet P-40, 20 mM iodoacetamide, 1 mM PMSF, and 10 g/ml aprotinin.…”

“…After starvation in 3 ml of methionine/cysteine-free medium (ICN Pharmaceuticals) for 2 h, cells were pulsed with 0.5 mCi/ml 35 S-Translabel for 20 min and then chased in complete medium for various periods of time in the presence or absence of 10 M LemA peptide. Aliquots of cells for each chase point were lysed in lysis buffer.…”

“…M3 is a murine class Ib molecule with a high affinity for Nformylated peptides (34,35). The unique binding specificity of M3 restricts its ligands to peptides derived from the amino terminus of mitochondrial and bacterial proteins.…”

Variable Immunodominance Hierarchies for H2-M3-RestrictedN-Formyl Peptides Following Bacterial Infection

“…Binding by H2-M3 is highly dependent on the presence of formyl methionine at the peptide N terminus (4,5); while M3 can present nonformylated peptides, it does so 10 2 -to 10 4 -fold less efficiently (6,7). Only 13 mitochondrial proteins can supply endogenous f-Met peptides, and most of these are bound by H2-M3 with low affinity (8).…”

Presentation of peptide antigens by mouse CD1 requires endosomal localization and protein antigen processing

“…These so-called NK T cells rapidly secrete large amounts of cytokines, and they have been reported to play important immunoregulatory roles in a variety of situations (10,11). mCD1 molecules are unique in their ability to present chemically well defined peptide and nonpeptide antigens, although the nonclassical H-2M3 molecule also may be capable of this duality of function (12). It has been shown recently, however, that the great majority of mCD1 molecules purified from mammalian cells are bound to glycophosphatidyl inositol containing compounds and that bound peptides could not be detected (13).…”