John M. Martinko

Primate ABO glycosyltransferases: Evidence for trans-species evolution

The human ABO blood group system is controlled by alleles at a single locus on chromosome 9. The alleles encode glycosyltransferases, which add different sugar residues to the terminal part of the oligosaccharide core, thus generating the A or B antigens; an allele encoding enzymatically inactive protein is responsible for the blood group O. The A and B antigens are present not only in humans, but also in many other primate species and it has been proposed that the AB polymorphism was established long before these species diverged. Here we provide molecular evidence for the trans-species evolution of the AB polymorphism. Polymerase-chain reaction (PCR) amplification and sequencing has revealed that the critical substitutions differentiating the A and B genes occurred before the divergence of the lineages leading to humans, chimpanzees, gorillas, and orangutans. This polymorphism is therefore at least 13 million years old and is most likely maintained by selection. Comparison of the sequences derived from different species indicates that the difference in enzymatic activities between the A and B transferases is caused by two single nucleotide substitutions responsible for Leu-Met and Gly-Ala replacement at positions 265 and 267 in the polypeptide chains, respectively.

Disulfide Bond Engineering to Trap Peptides in the MHC Class I Binding Groove

Immunodominant peptides in CD8 T cell responses to pathogens and tumors are not always tight binders to MHC class I molecules. Furthermore, antigenic peptides that bind weakly to the MHC can be problematic when designing vaccines to elicit CD8 T cells in vivo or for the production of MHC multimers for enumerating pathogen-specific T cells in vitro. Thus, to enhance peptide binding to MHC class I, we have engineered a disulfide bond to trap antigenic peptides into the binding groove of murine MHC class I molecules expressed as single-chain trimers or SCTs. These SCTs with disulfide traps, termed dtSCTs, oxidized properly in the endoplasmic reticulum, transited to the cell surface, and were recognized by T cells. Introducing a disulfide trap created remarkably tenacious MHC/peptide complexes because the peptide moiety of the dtSCT was not displaced by high-affinity competitor peptides, even when relatively weak binding peptides were incorporated into the dtSCT. This technology promises to be useful for DNA vaccination to elicit CD8 T cells, in vivo study of CD8 T cell development, and construction of multivalent MHC/peptide reagents for the enumeration and tracking of T cells—particularly when the antigenic peptide has relatively weak affinity for the MHC.

Humoral Immune Response in Mice Over-expressing or Deficient in Growth Hormone

Effects of growth hormone (GH) levels on the humoral immune response were investigated in metallothionein I (MT)-bovine (b) GH-transgenic (tg) and GH-deficient Ames dwarf (Prop1 dt–/–) mice. Four-month-old mice were given primary and secondary injections of either normal saline or tetanus toxoid (TT) to induce specific antibody (Ab) production. MT-bGH-tg mice with high peripheral levels of bGH produced less TT-specific Ab than normal nontransgenic (Ntg) littermates, df, or nondwarf (Ndf) control mice. Titers reached maximum levels at 3–4 weeks postprimary immunization (PPI) and declined gradually through 24 weeks PPI in all groups of mice. Peripheral CD4+ and CD8+ T cell populations were significantly lower in tg than in Ntg, df, or Ndf mice. No significant differences were found in B cell numbers between tg, Ntg, or df mice. T helper 2 (Th2) cell populations were significantly greater in df mice compared to Ntg control mice. No significant differences were found in CD4+:CD8+ T cell ratios, interleukin (IL)-4 concentrations or interferon (IFN)-γ levels between tg, Ntg, df, and Ndf mice. No patterns of significant sexual dimorphism were found for any of the immune parameters studied. Elevated levels of corticosterone were investigated as a possible immunosuppressant mechanism responsible for low Ab responses in the tg mice. Ab production was not enhanced by decreasing corticosterone in tg mice. Thus, high endogenous GH levels inhibit specific Ab production and peripheral T cell populations but not peripheral B cell numbers, Th2 cell populations, or IL-4 or IFN-gamma production. Elevated corticosterone levels do not appear to be responsible for suppressed humoral immune responses. Low levels of endogenous GH do not inhibit specific Ab production but may contribute to increased peripheral Th2 cell numbers.

Human Major Histocompatibility Complex (MHC) Class I Molecules with Disulfide Traps Secure Disease-related Antigenic Peptides and Exclude Competitor Peptides

The ongoing discovery of disease-associated epitopes detected by CD8 T cells greatly facilitates peptide-based vaccine approaches and the construction of multimeric soluble recombinant proteins (e.g. tetramers) for isolation and enumeration of antigen-specific CD8 T cells. Related to these outcomes of epitope discovery is the recent demonstration that MHC class I/peptide complexes can be expressed as single chain trimers (SCTs) with peptide, β2m and heavy chain connected by linkers to form a single polypeptide chain. Studies using clinically relevant mouse models of human disease have shown that SCTs expressed by DNA vaccination are potent stimulators of cytotoxic T lymphocytes. Their vaccine efficacy has been attributed to the fact that SCTs contain a preprocessed and preloaded peptide that is stably displayed on the cell surface. Although SCTs of HLA class I/peptide complexes have been previously reported, they have not been characterized for biochemical stability or susceptibility to exogenous peptide binding. Here we demonstrate that human SCTs remain almost exclusively intact when expressed in cells and can incorporate a disulfide trap that dramatically excludes the binding of exogenous peptides. The mechanistic and practical applications of these findings for vaccine development and T cell isolation/enumeration are discussed.

Effects of exam stress on mood, cortisol, and immune functioning: Influences of neuroticism and smoker-non-smoker status

Abstract In a number of studies, neuroticism, depression and stress have been reported to be positively correlated with each other, with serum cortisol concentration and with smoking. The same factors are inversely related to measures of immune system functioning. The present study assessed in smokers and non-smokers the effects of the presumed stress of final examinations on moods, cortisol and immune system functioning. Subjects were college students selected because they reported feeling reliable high degrees of stress during examinations. Immune system functioning (natural killer cell cytotoxic activity [NKCA], and ConA and PHA lymphocyte proliferation), serum cortisol concentration and mood were assessed in 19 smokers and 23 non-smokers. The findings indicate that exam stress was associated with large increases in reported tension and slightly increased symptoms of depression. Further, T lymphocyte proliferation in response to ConA, but not to PHA, was suppressed during the exam period, while changes in NKCA during exams were associated with an interaction of smoker status and neuroticism. There was also a neuroticism by stress interaction for negative mood assessed by the Profile of Mood States such that those individuals who scored high on measures of neuroticism were higher in negative affect at baseline and postexam periods, but not during the exam period. Smokers had higher serum cortisol concentrations than non-smokers across conditions and scored higher in Beck Depression Inventory-assessed symptoms of depression. Cortisol did not vary as a function of stress and was not correlated with changes in immune functioning, with depression or with negative moods. Serum cortisol and beta-endorphin concentrations were not associated with immune functioning or habitual nicotine intake (plasma nicotine and cotinine concentrations). Among smokers, exam stress did not result in elevated plasma nicotine, cotinine or caffeine concentrations.

The H-2Kkml mutation: A single nucleotide substitution is responsible for multiple functional differences in a class I MHC molecule

Nucleotide sequence analysis of mRNA from the H-2K locus of the CBA.M523 mouse, which has the class I murine MHC mutation H-2Kkml, has established the only alteration to be at the codon for amino acid position 152 as compared to the sequence of standard Kk from both the AKR and CBA inbred mouse lines. Complete sequence information for the nucleotides coding for amino acids 1-292, which includes all of the extracellular protein domains, demonstrated an A----C alteration in the codon for amino acid 152 as compared to the standard Kk sequence, changing Asp (GAT) in Kkml. The GCT codon occurring in Kkml may be the result of a gene conversion in Kkml. The GCT codon occurring in Kkml may be the result of a gene conversion event because a potential donor gene, the pH-2III pseudogene of H-2k, is transcribed in the CBA.M523 mouse and has a GCT codon at amino acid position 152. This sequence information obtained for Kkml also demonstrates that Kk gene transcripts from two genetically distinct inbred mouse lines, CBA and AKR, are completely identical. Finally, several other murine and human class I MHC variants have similar alterations at amino acid position 152 which result in altered biological functions. This information suggests that amino acid 152 is an important part of a T-cell-recognized antigenic determinant on MHC class I antigens.

The H-ZKkm1 mutation: Nucleotide sequence and comparative analysis

Abstract Nucleotide sequence analysis of mRNA from the class I murine MHC mutant H-2Kkml has established a site of mutation to be at the codon for amino acid position 152. Complete sequence information for the nucleotides coding for amino acids 136–163 demonstrates an A → C alteration at the codon for amino acid 152, changing Asp (GAT) in Kk to Ala (GCT) in Kkml . Several other murine and human class I MHC variants have similar alterations at amino acid position 152, resulting in altered biological activity. Finally, the pH-2III pseudogene of the H-2k haplotype has a GCT codon at amino acid position 152, suggesting that the GCT codon occurring in Kkml is the result of a gene conversion event.

A murine cell line defective in expression of several class I molecules

Class I major histocompatibil i ty complex (MHC) antigens are expressed as membrane-integrated proteins on the surface of most mammal ian cells. The mouse normally expresses from two to three class I antigens encoded by the classical t ransplantat ion regions K and D in each haplotype that has been investigated (Klein et al. 1983). w e describe here a cell line, ABM-3, that was isolated from Abelson virus-treated (CBA.M523 x BALB/c)F 1 cells (H-2kml/H-2d). The unique proper ty of this cell line is that it does not express H-2K kin, H-2K d, H-2D d, or H-2D k. However, the H-2L a molecule is expressed on the cell surface. CBA.M523 mice carrying the H-2 kin1 mutat ion were obtained as breeding pairs f rom Dr. James Forman, Southwestern Medical School, Dallas, Texas. Successive generations of CBA.M523 mice were maintained by brother-sister matings of offspring of these original breeding pairs. BALB/c mice were obtained from the Jackson Labora tory , Bar Harbor , Maine. The (CBA.M523 x BALB/c)F1 mice were bred and maintained in the vivarium at the Southern Illinois University at Carbondale, Illinois. An Abelson virus-producing cell line, 54-C12, was obtained from Dr. N a o m i Rosenberg, Tufts University, Boston, Massachusetts. The A N C cell line with the H2k/I-I-2 d haplotype, is an Abelson virus-transformed cell line from (CBA/N x BALB/c)F 1 bone mar row cells (Bluestone et al. 1984). Transformation of (CBA.M523 x BALB/c)F 1 spleen and bone mar row cells was performed according to the methods of Rosenberg and co-workers (1975) and Rosenberg and Baltimore (1976). Spleen and bone mar row cells were isolated from 6-week-old H-2km/H 2 a mice, infected with Abelson virus-containing culture

The deduced amino-acid sequence of opsin from rabbit rod photoreceptors

The amino acid (aa) sequence of rabbit opsin from rod photoreceptor cells was determined by direct aa sequencing and conceptual translation from the cDNA. The cDNA (1198 bp) containing the complete coding region encodes a 348-aa opsin protein. Of the 16 rod cell opsins that are known, rabbit opsin is most similar to human opsin (96.3% identity at the aa level).

The H-2K kml Mutation: A Single Nucleotide Substitution Causes Multiple Functional Differences in a Murine Class I MHC Molecule

The polymorphic MHC class I glycoproteins are implicated in a number of immunologic reactions involving T-cell recognition. Among these polymorphic molecules in the murine H-2 complex, a number of mutants have been identified. One of these mutants was mapped to the H-2K k allele in the CBA inbred mouse strain and resulted in graft rejection and graft versus host reactions. This mutant was designated H-2K kml and the mouse strain carrying it was designated CBA.M523 (1). Analysis of the structural variation in the H-2K kml molecule identified a single nucleotide substitution at codon 152, resulting in a change from Asp (GAT) in Kk to Ala (GCT) in K kml (2). This report documents the completion of the nucleotide sequence coding for the extracellular domains of the altered K kml and compares the known K k coding sequence from the AKR strain (3) to that of the CBA strain which is ancestral to the CBA.M523 mutant-bearing strain.