Mouse cytomegalovirus (MCMV), a β-herpesvirus, encodes the m145 family of glycoproteins. Several members of this family are predicted to adopt the MHC-I fold although their amino acid sequences exhibit less than 30% identity to classical MHC-I (MHC-Ia) proteins. Our aim was to determine how related the viral proteins are to MHCIa and characterize them in terms of cellular expression, structure and function. We studied the cellular localization of FLAG-tagged m17, M37, m145, m151, m152, m153 and m155 in transfected mouse fibroblasts. Flow cytometry analysis of transfected cells showed that M37, m145, m151 and m153 localize predominantly to the cell surface, whereas the majority of m17, m152 and m155 remain inside the cell. MHC-Ia proteins require assembly with β2-microglobulin (β2m) and peptide for stable cell surface expression. Transient transfection studies with β2m- or transporter associated with antigen (TAP)-deficient cell lines revealed that M37, m145, m151 and m153 could be expressed stably at the cell surface in the absence of β2m or TAP expression. To generate protein material for crystallization screening we evaluated both bacterial and insect cell expression systems. Although most m145 family members could be expressed in bacteria in insoluble inclusion bodies, none of the proteins could be accurately refolded. Since M37, m151 and m153 are cell surface molecules with the potential to bind receptors on host cells, we focused our structure determination efforts on them and evaluated their expression in Drosophila S2 insect cells. The extracellular domains of all three proteins expressed at significant levels, however, m151 tended to aggregate and precipitate over time. M37 and m153 were stable and could easily be purified to homogeneity. Size exclusion chromatography and SDS-PAGE analysis of m153 suggested that it forms a non-covalent homodimer. Analytical ultracentrifugation experiments confirmed this observation and provided an estimated molecular mass of 78.8 kDa. Enzymatic and mass spectrometry analyses showed that insect-expressed m153 is highly glycosylated. We tested a wide range of crystallization conditions for m153. It formed very fragile crystals and after optimization we obtained several that diffracted to 2.3 Å. To determine the structure of m153, we prepared a seleno-methionine derivative in insect cells, collected data on a single crystal and solved the phases by the single anomalous dispersion method. The m153 model was refined at 2.4 Å resolution to final Rcryst and Rfree of 23% and 27.9%, respectively. The m153 homodimer is formed by two MHC-I-like heavy chains, each consisting of two α-helices arranged on a platform of seven β-strands and an Ig-like α3 domain. The monomers are arranged “head-to-tail”, with the α1α2 platform domain of one chain interacting with the Ig-like α3 domain of the other. The α1 and α2 helices are closely juxtaposed and do not form a peptide binding groove. Three N-linked carbohydrate residues were visualized in the crystal structure. Major deviations from the MHC-I fold include an extended N-terminus, which originates next to the α3 domain, and an elongated α2 helix (designated H2b) that reaches down towards the α3 domain. In addition, m153 has two unique disulfide bonds, one between strands of the platform domain and another that links the extended N-terminus to the H2b helix. Both unique disulfide bonds were verified by mass spectrometry. The canonical Ig-fold disulfide bond is present in the α3 domain. Alanine mutation of four amino acids involved in interface hydrogen bonds abolished m153 dimer formation, validating the dimer interface visualized in the structure. The crystal structure of m153, together with the recently reported m157 structure, confirms the MHC-I fold for the MCMV m145 family and highlights shared structural features in the m145 family. We have demonstrated dimerization of full-length m153 in mammalian cells by bimolecular fluorescence complementation and co-immunoprecipitation studies. Further, we have shown that m153 is expressed at the surface of MCMV-infected cells at early times after infection. To initiate a search for host ligands of m153, we generated a reporter cell line by introducing an m153-human zeta chain fusion protein into 43.1 cells that contain an NFAT-driven GFP construct. While a variety of mouse cell lines were unable to stimulate the m153-reporter cells, coculture with mouse splenocytes specifically induced GFP production in m153-reporters but not in the parental or control reporter cell lines. We identified conventional CD11c+ and plasmacytoid dendritic cells (DCs) as the most potent m153-reporter cell stimulating populations in the spleen. The stimulation was shown to be m153-specific, dose- and cell contact-dependent. DCs derived from bonemarrow cultures also potently stimulated the m153-reporter cells. Macrophages and NK cells exhibited weaker stimulation of the reporter cells, indicating lower levels of ligand or that only small subsets of the cells express a ligand. DCs from several mouse strains, but not from the rat, stimulated m153-reporter cells. We evaluated DC surface phenotype and migratory capacity after coculture with m153-reporter cells or on m153-coated plates, but could not detect any changes induced specifically by the presence of m153.

Transcriptional activation of Major Histocompatability Complex (MHC) I and II molecules by the cytokine interferon gamma (IFN-g) is a key step in cell-mediated immunity against pathogens and tumors. Following IFN-g induction, JAK/STAT signaling triggers activation of MHC genes. Recent evidence suggests suppression of MHC I and II expression on multiple tumor types plays important roles in tumor immunoevasion. One such tumor is malignant melanoma, the leading cause of skin cancer related deaths. Despite awareness of MHC expression defects, the molecular mechanisms by which melanoma cells suppress MHC and escape from immune-mediated destruction remain unknown. Here we analyze dysregulation of the JAK/STAT pathway and its role in suppression of MHC II in melanoma cell lines at the Radial Growth Phase (RGP), the Vertical Growth Phase (VGP) and the Metastatic Phase (MET). RGP and VGP cells express both MHC II and the MHC master regulator, the Class II Transactivator (CIITA). MET cells lack not only MHC II and CIITA, but also both STAT 1 and the STAT 1 coactivator, the Interferon Response Factor (IRF) 1. Our studies have implicated that the suppression of MHCII on the cell surface of metastatic melanoma is due to silencing at the level of STAT1 transcription. Furthermore, we determined that silencing of STAT1 is, in part, due to hemi-methylation of the STAT1 promoter.

Kin-recognition abilities, first demonstrated 25 years ago in toad tadpoles, now appear to be widespread among amphibians. In some vertebrates kin recognition is based, at least in part, on highly polymorphic major histocompatibility complex (MHC) genes. Besides protecting animals from disease resistance, MHC genes regulate social behaviour. They allow relatives to recognise one another so that they can cooperate for mutual benefit. These two seemingly distinct functions of MHC genes may be integrally related, because animals need to outbreed to optimise the immune systems of their offspring. The ability to discriminate MHC-type is therefore likely to facilitate kin discrimination in tadpoles. I tested association preferences of African clawed-frog (Xenopus laevis) tadpoles in a laboratory choice apparatus. As in other anuran species, I found that tadpoles at earlier developmental stages preferentially associate with unfamiliar siblings over unfamiliar non-siblings but that this preference reverses during development. Tadpoles approaching metamorphosis demonstrated a reversal in their preference; they preferentially school with non-kin rather than kin. The ontogenetic switch in larval schooling preferences coincides with the onset of thyroid hormone (TH) controlled development and may be indicative of decreased fitness benefits associated with schooling with kin at later developmental stages. These may result from an increase in intraspecific competition, predation, or disease susceptibilities of prometamorphic individuals. Alternatively, the kin avoidance behaviours observed at later larval stages might reflect disassociative behaviour that facilitates inbreeding avoidance at reproductive maturity. This is the first study to find a shift from an association preference for kin to non-kin during amphibian larval development. Using allele-specific PCR techniques to MHC-type tadpoles, I tested association preferences among siblings based on shared MHC haplotypes. By using only full siblings in experimental tests, I controlled for genetic variation elsewhere in the genome that might influence schooling preferences. I found that X. laevis tadpoles discriminate among familiar full siblings based on differences at MHC genes. Subjects from four families preferentially schooled with MHC-identical siblings over those with which they shared no or one haplotype. Furthermore, the strength of tadpoles’ MHC-assortative schooling preferences significantly correlated with amino acid differences in the peptide-binding region (PBR) of both the MHC class I and II loci. Since MHC-PBR polymorphisms determine the pool of peptides that can serve as ligands for MHC molecules, these findings support the hypothesis that MHC peptide ligands mediate MHC type discrimination. As test subjects were equally familiar with all stimulus groups, tadpole discrimination appears to involve a self-referent genetic recognition mechanism whereby individuals compare their own MHC type with those of conspecifics. I also found that non-MHC-linked genetic differences contribute to tadpole association preferences in tests that contrast MHC and kinship. Tadpoles did not discriminate between MHC-similar non-siblings and MHC-dissimilar siblings and preferentially associated with MHC-dissimilar non-siblings rather than MHC-similar non-siblings. Although the MHC may be not solely responsible for the genetically determined cues that direct tadpole association preferences, it certainly is important in facilitating discrimination among conspecifics in X. laevis tadpoles. MHC-based discrimination may be retained through ontogeny and serve to maintain MHC-polymorphisms by facilitating disassortative mating.

Zugl.: Hannover, Tierärztliche Hochsch., Diss., 2005

Diss. (sammanfattning) Stockholm : Karol. inst. / Härtill 6 uppsatser.

Konstanz, Univ., Diss., 2002.

München, Techn. Univ., Diss., 2002.

Major histocompatibility complex class I (MHC-I) proteins are responsible for the presentation of intracellular protein fragments on the cellular surface and are thus the primary method for the broader immune system to recognize and respond to intracellular deformity or infection. A successful immune response against intracellular pathogens relies upon effective epitope presentation by MHC-I and recognition of this epitope by cytotoxic cluster of differentiation 8 positive (CD8+) T cells. Although MHC-I mediated immunity is a powerful mechanism which might resist infection by pathogens, many such pathogens have evolved methods of eluding or suppressing MHC-I mediated immune responses and are thereby able to persist within our cells. Close study of the fine details of the functionality of the MHC-I mediated antigen presentation system may yield important clues about how to best move forward in the quest to eliminate these problematic diseases. Although diseases such as tuberculosis, malaria, human immunodeficiency virus (HIV), and cancer may differ in many fundamental ways, it has become clear that the future of treating these elusive pathogens must involve utilization of the tools provided in the human immune system.

No description available.

A RIDGE (Region of IncreaseD Gene Expression), as defined by previous studies, is a consecutive set of active genes on a chromosome that span a region around 110 kbp long. This study investigated RIDGE formation by focusing on the well-defined, immunological important MHC locus. Macrophages were assayed for gene expression levels using the Affymetrix MG-U74Av2 chip are were either 1) uninfected, 2) primed with IFN-g, 3) viral activated with mCMV, or 4) both primed and viral activated. Gene expression data from these conditions was studied using data structures and new software developed for the visualisation and handling of structured functional genomic data. Specifically, the data was used to study RIDGE structures and investigate whether physically linked genes were also functionally related, and exhibited co-expression and potentially co-regulation. A greater number of RIDGEs with a greater number of members than expected by chance were found. Observed RIDGEs featured functional associations between RIDGE members (mainly explored via GO, UniProt, and Ingenuity), shared upstream control elements (via PROMO, TRANSFAC, and ClustalW), and similar gene expression profiles. Furthermore RIDGE formation cannot be explained by sequence duplication events alone. When the analysis was extended to the entire mouse genome, it became apparent that known genomic loci (for example the protocadherin loci) were more likely to contain more and longer RIDGEs. RIDGEs outside such loci tended towards single-gene RIDGEs unaffected by the conditions of study. New RIDGEs were also uncovered in the cascading response to IFNg priming and mCMV infection, as found by investigating an extensive time series during the first 12 hours after treatment. Existing RIDGEs were found to be elongated having more members the further the cascade progress.