Recent biochemical characterization of the T23-encoded Qa-1 molecule revealed an additional higher molecular mass species of 50 kD coprecipitated with the 48-kD Qa-1 molecule in H-2b and H-2d mouse strains. We now demonstrate that the 50-kD protein coprecipitated with Qa-1 is the class I-a antigen Ld in all H-2Ld-positive mouse strains examined. Furthers analyses of a panel of recombinants revealed that the 50-kD protein coprecipitated with Qa-1 in H-2b haplotype mouse strains is encoded or controlled by a gene centromeric to major histocompatibility complex class II I-E beta. We have designated this gene and corresponding protein product as Qsm, Qa-1 structure modifier. Both Ld and Qsm can interact with Qa-1 to form cell surface-expressed heterodimers in vivo. These Qa-1 heterodimers are not expressed in H-2k haplotype cells. The Qa-1/Ld and Qa-1/Qsm heterodimers are associated by noncovalent interactions and occur only between fully processed proteins. In addition, we show that the Qsm-encoded protein can form heterodimers with Ld as well, and that the Ld molecules participating in these interactions with Qa-1 and Qsm may be devoid of beta 2-microglobulin and/or peptide. These data represent the first demonstration that class I molecules can be expressed as heterodimers (Qa-1/Ld) on the cell surface, and map a gene (Qsm) that may potentially encode a novel class I molecule, or another protein, that associates with both Qa-1 and Ld. These interactions may enable increased levels of Qa-1 to reach the cell surface and may subsequently influence T cell recognition of Qa-1 and/or Ld molecules.

The physiological functions of the mouse telomeric major histocompatibility complex (MHC) class I molecules, including Hmt, are unknown. Hmt presents a polymorphic, N-formylated peptide encoded by the mitochondrial gene ND1 forming the cell surface maternally transmitted antigen (Mta). Because the N-formyl moiety is required for Hmt binding, we proposed that Hmt may function generally in presentation of N-formylated antigens. This hypothesis was validated by a competitive binding assay, demonstrating that synthetic N-formyl peptides from other mitochondrial genes also bound Hmt. Bacteria similarly initiate protein synthesis with N-formylmethionine; indeed, we established that Hmt can also present prokaryotic peptides in an N-formyl-dependent manner. These results indicate biochemical specialization of this MHC-peptide interaction and suggest a unique role for Hmt in prokaryotic host defenses.

The staphylococcal enterotoxins (SEs) are homologous proteins related in their capacity for stimulating both T cells and monocytes. To assess the importance of conserved structure and sequence to functional activity, the role of the disulfide loop and adjacent sequence in these toxins was evaluated. Contrary to previous reports, we demonstrate here that the disulfide loop was required for the mitogenic activity of SEA and SEB. While T cell-stimulatory activity was compromised, reduced and alkylated SEs retained major histocompatibility complex class II-binding and monocyte-stimulatory activities, suggesting that their inability to induce T cell proliferation was due to failure to interact with T cell receptor (TCR) rather than with class II molecules. Reduction and alkylation did not affect the far-ultraviolet circular dichroic spectrum of SEA, suggesting that the loss of mitogenic activity was not associated with significant changes in secondary structure. The disulfide linkage imparts considerable stability to these toxins as peptide cleavages within the loop of SEB were not associated with detectable loss of function, although cleavage in the conserved sequence outside the loop of SEA resulted in loss of mitogenic activity. This report thus establishes a functional role for a conserved element in SEs, the disulfide loop, and further indicates that their class II- and TCR-binding activities can be dissociated.

Of all the biochemically defined mouse MHC class I molecules, the Qa-1 antigens are the only ones for which a gene has not been identified. Recent evidence has suggested that Qa-1 antigens are functional class I molecules and can function as restriction elements for gamma/delta T cells. We have examined the relationship between Qa-1 and the product of gene 37, a presumed novel class I antigen encoded within the TL region. Immunoprecipitation and polyacrylamide gel electrophoresis analysis of the molecules reactive with anti-Qa-1 and anti-37 sera show that the Qa-1 molecule of Qa-1b (Qa-1.2) mouse strains is identical to the product of gene 37 on the basis of molecular weight, pI, and strain distribution. Immunodepletion, biosynthetic labeling, and tunicamycin treatment confirm that the protein encoded by gene 37 in Qa-1b mice is Qa-1.2. In contrast, the anti-37 serum was unable to recognize the Qa-1 molecule in Qa-1a strains. Given the fact that the only allele to gene 37 thus far identified in a Qa-1a strain (A/J) has a termination codon in the alpha 3 domain, our data lead us to conclude that the Qa-1 molecule expressed in Qa-1a mice is not a true allelic product of the gene 37 encoded antigen of Qa-1b mouse strains.

Maternally transmitted factor (Mtf) is a mitochondrial gene that controls the antigenic polymorphism of the MHC class I maternally transmitted antigen (Mta). Synthetic peptides from the NH2 terminus of the mitochondrially encoded NADH dehydrogenase subunit 1 (ND1) mimic Mtf peptide activity in an allele-specific manner. We show that the minimal ND1-alpha peptide length recognized by Mtaa-specific polyclonal CTLs was between 8 and 12 amino acids, while some Mtaa-specific CTL clones recognized a six amino acid peptide. The N-formyl group at the NH2 terminus of ND1 was essential for Mta activity. Competition experiments using N-substituted ND1-alpha peptides showed that an N-formyl peptide receptor on the target cell, which differs from the chemotactic peptide receptor, was required for Mta expression. The specificity of this receptor can account for the distinct immune restriction of Mta in which Mtf peptides are uniquely restricted by Hmt. It is possible that the Hmt gene product is the N-formyl peptide receptor itself and that it represents a class I antigen presentation molecule specialized for binding, transport, and immune presentation of N-formyl-peptide antigens of mitochondrial and prokaryotic origin.

Peripheral T lymphocytes activated in vitro with concanavalin A (Con A) or alloantigens express the thymus leukemia (TL) alloantigen as assessed by staining with the monoclonal antibody TL.m3 and flow cytometric analysis. The determinants detected by TL.m3 on activated cells are encoded within the Tla region and are detected as early as 48 h after activation with Con A. Several long-term cloned cytotoxic T lymphocyte lines were also examined and each expressed TL. By two-dimensional analysis, the TL isolated from activated peripheral cells was indistinguishable from that found on thymocytes and the leukemia cell line ASL-1.

The E/C alpha- and beta-subunits of intra-I-region recombinants were analyzed for primary structural variation by comparative tryptic peptide mapping. The E/C alpha-polypeptides from B10.A, B10.A (3R) and B10.A (5R) showed complete coincident elution of peptides; the E/C beta-chains from B10.A and 3R (or 5R) were approximately 40% different. This suggests that the structural gene for the E/C beta-polypeptide is within the I-A subregion.