H2-M3wt-restricted, Listeria monocytogenes-specific CD8 T cells recognize a novel, hydrophobic, protease-resistant, periodate-sensitive antigen.

Mice infected with Listeria monocytogenes (LM) generate H2-M3wt-restricted CD8 effectors which recognize a heat-killed LM-associated antigen (HAA) presented by macrophages. To characterize HAA, we extracted a bioactive component from LM using SDS or NaOH. Extracted HAA aggregated in hydrophilic solvents but dissociated in the presence of SDS into a smaller subunit which migrated in Sephadex G-200 between chymotrypsinogen (25 kDa) and cytochrome c (12.5 kDa). HAA bioactivity and size was unaffected by proteinase K under conditions which degraded virtually all detectable protein. HAA was also unaffected by other proteases, RNase and DNase, but HAA bioactivity was destroyed by periodate, an agent that degrades carbohydrates. These studies demonstrate that H2-M3wt can present a hydrophobic, non-peptide, microbial antigen, probably glycolipid in origin, to CD8 T cells.

Peptides from the amino-terminus of mouse mitochondrially encoded NADH dehydrogenase subunit 1 are potent chemoattractants.

Binding of N-formylated chemotactic peptides to specific cell surface receptors on polymorphonuclear leukocytes initiates a wide range of biological responses including migration of inflammatory cells, superoxide release, lysosomal enzyme secretion, calcium mobilization, and cellular activation. We previously established that the mouse MHC class I-b molecule H-2M3a binds peptides from the NH2-terminus of the mitochondrially encoded NADH dehydrogenase subunit 1 (ND1). Inasmuch as the N-formyl group is essential for peptide binding both to the chemotactic peptide receptor and to H-2M3a, we sought to test whether ND1 peptides can induce chemotaxis. We now show that fND1(1-12), fND1(1-8), fND1(1-5), fND1(1-4) and fND1(1-3) trigger the chemotactic receptor. Although all tested ND1 peptide derivatives were chemotactic, we found an inverse relationship between peptide length and chemotactic potency (ED50). Our data establish that mitochondrially derived peptides are potent chemotactic ligands. The release of N-formylated peptides from disintegrating mitochondria may play an important role in the inflammatory response resulting from tissue injury. By attracting the host phagocytic cells to sites of tissue breakdown, these peptides could mediate an essential first step in tissue repair and healing.

Antigen presentation by major histocompatibility complex class I-B molecules.

Class I-b genes constitute the majority of MHC class I loci. These monomorphic or oligomorphic molecules have been described in many organisms; they are best characterized in the mouse, which contains a substantial number of potentially intact genes. Two main characteristics differentiate class I-b from class I-a molecules: limited polymorphism and lower cell surface expression. These distinguishing features suggest possible generalizations regarding the evolution and function of this class. Additionally, class I-b proteins tend to have shorter cytoplasmic domains or in some cases may be secreted or may substitute a lipid anchor for the transmembrane domain. Some are also expressed in a limited distribution of cells or tissues. At least six mouse MHC class I-b molecules have been shown to present antigens to alpha beta or gamma delta T cells. Recent advances have provided insight into the physiological function of H-2M3a and have defined the natural peptide-binding motif of Qa-2. In addition, significant progress has been made toward better understanding of other class I-b molecules, including Qa-1, TL, HLA-E, HLA-G, and the MHC-unlinked class I molecule CD1. We begin this review, however, by arguing that the dichotomous categorization of MHC genes as class I-a and I-b is conceptually misleading, despite its historical basis and practical usefulness. With these reservations in mind, we then discuss antigen presentation by MHC class I-b molecules with particular attention to their structure, polymorphism, requirements for peptide antigen binding and tissue expression.

Availability of endogenous peptides limits expression of an M3a-Ld major histocompatibility complex class I chimera.

Taking advantage of our understanding of the peptide specificity of the major histocompatibility complex class I-b molecule M3a, we sought to determine why these molecules are poorly represented on the cell surface. To this end we constructed a chimeric molecule with the alpha 1 and alpha 2 domains of M3a and alpha 3 of Ld thereby allowing use of available monoclonal antibodies to quantify surface expression. Transfected, but not control, B10.CAS2 (H-2M3b) cells were lysed readily by M3a-restricted monoclonal cytotoxic T lymphocytes. Thus, the chimera bound, trafficked, and presented endogenous mitochondrial peptides. However, despite high levels of M3a-Ld mRNA, transfectants were negative by surface staining. This finding was consistent with inefficient trafficking to the cell surface. Incubation at 26 degrees C, thought to permit trafficking of unoccupied heavy (H) chains, resulted in detectable cell surface expression of chimeric molecules. Incubation with exogenous peptide at 26 degrees C (but not at 37 degrees C) greatly enhanced expression of M3a-Ld molecules in a dose-dependent manner, suggesting stabilization of unoccupied molecules. Stable association of beta 2-microglobulin with the chimeric H chain was observed in labeled cell lysates only in the presence of exogenous specific peptide, indicating that peptide is required for the formation of a ternary complex. These results indicate that surface expression of M3a-Ld is limited largely by the steady-state availability of endogenous peptides. Since most known M3a-binding peptides are N-formylated, native M3a may normally be expressed at high levels only during infection by intracellular bacteria.

Differential amino-terminal anchors for peptide binding to H-2M3a or H-2Kb and H-2Db.

We previously established that H-2M3a, the H chain of the maternally transmitted Ag (Mta), is specialized for presentation of N-formylated peptides. We hypothesized that the N-formyl group might prevent or limit the presentation of peptide Ag by H-2K and H-2D molecules. We now show by Mta- and OVA-specific CTL assays, peptide competition, and immunofluorescence analyses that N-formyl modification of four antigenic peptides inhibited their binding by either H-2Kb (OVAMet258-264, VSVNP52-59, and SVNP324-332) or H2-Db (SVNP324-332, and IVNP366-374). In contrast, N-formyl-OVAMet258-264 did bind to H2-M3a. The data imply lack of an N-formyl-binding pocket in classical MHC class I molecules and are consistent with a specialized role for H2-M3a in presentation of N-formylated peptides such as derived from intracellular prokaryotic parasites.

Biochemical specificity of H-2M3a. Stereospecificity and space-filling requirements at position 1 maintain N-formyl peptide binding.

The maternally transmitted Ag is a cell surface product of three gene products: 1) H-2M3a (formerly Hmta), a class I MHC heavy chain; 2) beta 2-microglobulin; and 3) maternally transmitted factor (Mtf), the N-terminus of the mitochondrially encoded ND1 subunit of the reduced form of nicotinamide-adenine dinucleotide dehydrogenase. This class I molecule has been shown to be an N-formyl peptide receptor. Although the N-formyl moiety is necessary for binding to M3a, it is not sufficient. We proposed that the R group of the amino acid in position 1 plays a pivotal role in peptide binding to M3a. To test this hypothesis, analogues differing in size and stereospecificity of the R group were synthesized. Substitutions with other hydrophobic amino acids such as N-formyl phenylalanine and N-formyl valine had no significant effect on the ability of these Mtf alpha analogues to sensitize target cells (M3a, Mtf beta) to M3a, Mtf alpha-specific CTL. In contrast, the nonsubstituted, N-formylated, and N-acetylated glycyl analogues of Mtf beta bound equivalently to M3a in a peptide competition assay. Moreover, the alanine analogue bound in an N-formyl-dependent manner. To determine the limitations of the putative N-formyl pocket, peptide analogues were constructed incorporating D-isomer amino acids. When formylated D-alanine or D-methionine replaced the native methionine, these peptide derivatives did not show significant binding to M3a. Therefore, the presence of a space-filling R group (greater than hydrogen) is necessary for an antigenic peptide to bind M3a in an N-formyl-dependent manner. Additionally, the ability of M3a to discriminate between the optical forms of methionine and alanine demonstrates that this N-formyl pocket is stereospecific in its ability to bind peptide. Thus, we have defined three requirements for peptide binding to M3a: an N-formyl moiety at the amino terminus of the peptide, a space-filling R group at position 1 to maintain this N-formyl specificity, and the correct stereoisomer of the first amino acid.

Kinetics of killing by monoclonal cytotoxic T lymphocytes. I. Colorimetric detection of cryptic CTL determinants on adherent target cells and survivorship analysis.

Some targets of cell-mediated cytolysis do not efficiently release 51Cr but manifestly are killed in direct viability assays. We characterize and validate an alternative and non-radioactive (colorimetric) method for measuring killing of adherent targets by monoclonal CTL. The method obviates concerns about the effects of trypsinization, is technically simple, quantitative and in some cases more sensitive than conventional 51Cr assays. Target loss obeyed first-order kinetics with respect both to [CTL] and time. These results are consistent with an exponential (Poisson) model of killing and support the use of a single kinetic parameter to describe the lytic activity of monoclonal CTL on adherent targets. When monoclonal CTL are used at appropriate effector:target ratios (less than or equal to 1:1), the residuals obtained after least squares linear regression are homoscedastic and normally distributed, justifying the use of commonly available statistical calculators or programs for the analysis of CTL data.

Specialized role for a murine class I-b MHC molecule in prokaryotic host defenses.

Although nonclassical (class I-b) gene products represent the majority of murine major histocompatibility complex (MHC) genes, the role of these relatively nonpolymorphic molecules remains uncertain. Recently, one such protein, H-2M3 (formerly designated Hmt), was shown to bind and specifically present N-formylated peptides to cytotoxic T lymphocytes. Because N-formylation is characteristic of prokaryotic proteins, this MHC molecule may be especially adapted for a role in the mammalian defense against bacterial attack. The current studies demonstrate that an MHC molecule, indistinguishable from H-2M3, presents antigens derived from the intracellular pathogen Listeria monocytogenes to Listeria-specific CD8+ cells.

Specialized functions of major histocompatibility complex class I molecules. II. Hmt binds N-formylated peptides of mitochondrial and prokaryotic origin.

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.

Specialized functions of MHC class I molecules. I. An N-formyl peptide receptor is required for construction of the class I antigen Mta.

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.

Retrovirus-mediated insertional mutagenesis: phagemid rescue of flanking DNA by selecting plasmid ori.

A method for screening recombinant lambda libraries was devised to select phage containing genomic regions containing provirus insertions of retroviruses that carry the kanamycin and G418 resistance factor neo and the origin of replication derived from pBR322 (oripBR). Such recombinants are phagemids, able to replicate as bacteriophages or as plasmids under lambda repressor control. lambda repressor was cloned into a plasmid derived from pSC101 that is compatible with pBR322-derived phagemids. A strain carrying this plasmid may be used to select phagemids derived from a single proviral insertion with 100% efficiency from complex recombinant libraries. Homologous recombination between proviral long terminal repeats was observed at a rate of 10(-4)/plaque-forming unit in recABC+ strains. Despite this frequency, intact phagemids are easily recovered as phage after temperature shift to 42 degrees C. Since oripBR itself is a selectable marker in this system, the method could be applied to recover any sequence carrying the ori sequence from pBR322.

Schistosoma mansoni: purification and characterization of a membrane-associated leucine aminopeptidase.

Membrane-associated leucine aminopeptidase (EC 3.4.11.1, LAP) has been purified to homogeneity from Schistosoma mansoni egg homogenates by a combination of ultracentrifugation, chromatofocusing, and molecular sieve chromatography. A 260-fold increase in specific activity was observed after purification. This is a metalloenzyme, containing carbohydrate moieties. Optimal enzyme activity was found at neutral pH. Enzyme activity was measured using L-leucine-7-amino-4-trifluoromethylcoumarin (L-Leu-AFC); in addition, schistosome egg LAP hydrolyzed a variety of other aminopeptidase substrates. Hydrolysis of L-Leu-AFC was inhibited by a number of aminopeptidase inhibitors, including 1,10-phenanthroline, bestatin, and amastatin.