Short peptides sensitize target cells to CTL specific for the MHC class Ib molecule, H2-M3.

The MHC class Ib molecule H2-M3 presents N-formylated peptides from the N terminus of proteins encoded by the mitochondrial genome to CTL. A panel of CTL specific for a peptide derived from a mitochondrial protein, either COI or ND1, was used to determine the optimal peptide length for sensitizing antigen-deficient target cells. All long-term CTL lines and most CTL clones lysed target cells sensitized with either a COI hexamer or an ND1 heptamer. Only 3 out of 12 anti-ND1 clones preferred an octamer or nonamer peptide and no CTL required to longer peptides. The CTL preference for short peptides matches a shortened groove in M3. The CTL all lysed lymphoblasts encoding the appropriate mitochondrial antigen, suggesting that these target cells express naturally processed, endogenous, formylated peptides, ranging from six to nine amino acids in length.

The alloreactive T cell response against the class Ib molecule H2-M3 is specific for high affinity peptides.

MHC class Ib molecule H2-M3 presents N-formylated peptides to CD8+ CTLs. Endogenous formylated peptides can come from the N-terminus of each of the 13 proteins encoded by the mitochondrial genome. In peptide competition assays, two of these peptides bind with high affinity, six bind with intermediate affinity, three bind with low affinity, and two do not bind measurably. Alloreactive CTLs from M3-specific, mixed lymphocyte cultures responded strongly against the two peptides with high affinity for M3, occasionally to peptides with intermediate affinity, and not at all to the rest. Long term lines and CTL clones reacted with only the high affinity peptides, demonstrating that alloreactive CTLs depend on specific peptides and that peptide affinity for class I correlates with alloantigenicity.

H2-M3, a full-service class Ib histocompatibility antigen.

H2-M3 is an MHC class Ib molecule of the mouse with a unique preference for N-formylated peptides, which may come from the N-termini of endogenous, mitochondrial proteins or foreign, bacterial proteins. The crystal structure of M3 revealed a hydrophobic peptide-binding groove with an occluded A pocket and the peptide shifted one residue relative to class Ia structures. The formyl group is held by a novel hydrogen bonding network, involving His9 on the bottom of the groove, and the side chain of the P1 methionine is lodged in the B pocket. M3 is a full-service histocompatibility (H) antigen, i.e. self-M3 can present endogenous peptides as minor H antigens and foreign, bacterial antigens in a defensive immune response to infection; and foreign M3 complexed with endogenous self-peptides.

The COI mitochondrial gene encodes a minor histocompatibility antigen presented by H2-M3.

We found that (LP x C57BL/6)F1 mice could raise a CTL response against parental C57BL/6 cells. These CTLs recognized a maternally transmitted, H2-M3wt-restricted, minor histocompatibility Ag (MiHA) that is widely distributed among many strains of mice and encoded by the COI mitochondrial gene. The wild-type MiHA is the COI N-terminal hexapeptide. Sequencing the 5' end of the COI gene in LP and C57BL/6 mice showed that the LP allele arose by a T-->C transition in the third codon, which caused substitution of threonine for isoleucine. Molecular characterization of this MiHA and the demonstration that it is presented exclusively by H2-M3: 1) support the concept that differential expression of MiHA in MHC-identical animals is caused by polymorphism of the MiHA gene proper; 2) expand our knowledge of the repertoire of self-peptides naturally presented by H2-M3 and show that this MHC class I molecule can present short endogenous peptide ligands; and 3) suggest that mitochondrial DNA mutations that modify the repertoire of H2-M3-associated mitochondrial peptides are representative of mitochondrial DNA mutations in general.

MtDNA-encoded histocompatibility antigens.

Mitochondrially encoded H antigens are by-products of a system that has evolved in vertebrates to present peptides from intracellular pathogens on the cell surface for detection by CTLs, which can lyse the infected cell. CTL lines and clones with defined specificity against mitochondrial H antigens, which can be maintained in culture for long periods, offer a unique tool in mitochondrial genetics. Expression of polymorphic mitochondrial H antigens depends on both the presence and the activity of the corresponding mitochondrial genome, and CTLs can provide strong selection against cells displaying their cognate antigen.

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 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.