Down-regulation of MHC class I by the Marek's disease virus (MDV) UL49.5 gene product mildly affects virulence in a haplotype-specific fashion

Marek's disease is a devastating neoplastic disease of chickens caused by Marek's disease virus (MDV). MDV down-regulates surface expression of MHC class I molecules, although the mechanism has remained elusive. MDV harbors a UL49.5 homolog that has been shown to down-regulate MHC class I expression in other Varicelloviruses. Using in vitro assays, we showed that MDV pUL49.5 down-regulates MHC class I directly and identified its cytoplasmic tail as essential for this function. In vivo, viruses lacking the cytoplasmic tail of pUL49.5 showed no differences in MD pathogenesis compared to revertant viruses in highly susceptible chickens of the B¹⁹B¹⁹ MHC class I haplotype, while there was a mild reduction in pathogenic potential of the deletion viruses in chickens more resistant to MD pathogenesis (MHC:B²¹B²¹). We concluded that the pathogenic effect of MHC class I down-regulation mediated by pUL49.5 is small because virus immune evasion possibly requires more than one viral protein.     

Coexpression network based on natural variation in human gene expression reveals gene interactions and functions

Genes interact in networks to orchestrate cellular processes. Analysis of these networks provides insights into gene interactions and functions. Here, we took advantage of normal variation in human gene expression to infer gene networks, which we constructed using correlations in expression levels of more than 8.5 million gene pairs in immortalized B cells from three independent samples. The resulting networks allowed us to identify biological processes and gene functions. Among the biological pathways, we found processes such as translation and glycolysis that co-occur in the same subnetworks. We predicted the functions of poorly characterized genes, including CHCHD2 and TMEM111, and provided experimental evidence that TMEM111 is part of the endoplasmic reticulum-associated secretory pathway. We also found that IFIH1, a susceptibility gene of type 1 diabetes, interacts with YES1, which plays a role in glucose transport. Furthermore, genes that predispose to the same diseases are clustered nonrandomly in the coexpression network, suggesting that networks can provide candidate genes that influence disease susceptibility. Therefore, our analysis of gene coexpression networks offers information on the role of human genes in normal and disease processes.The functions of many human genes are unknown. It is not unusual that when one searches the literature on a gene, one fails to find any papers that provide information on its biological roles. Identifying gene function is difficult, especially if no hints, such as homologies to known genes, are available to direct the search. However, since genes work by interacting with other genes, we may learn about their functions through their neighboring genes (Stuart et al. 2003; Ayroles et al. 2009). Identifying gene function is increasingly important; in the last several years, genome-wide association studies (GWAS) have identified DNA variants that are associated with common complex diseases. But for many of these studies, the functional links between the susceptibility genes and the diseases are unknown.In this study, we used correlations in expression levels of more than 8.5 million human gene pairs in immortalized B cells from three data sets to infer gene coexpression networks. The resulting gene networks were based on correlations between genes that were found reproducibly in the three data sets. This provided us with gene networks in which we had high confidence in the gene correlations. We then used the networks to identify key biological processes and interactions among those processes in our cells. Then, we identified the functions of 36 human genes with no known functions and four genes that have been implicated in GWAS as susceptibility genes for common human diseases, including IFIH1, which was recently found to be associated with type 1 diabetes.     

Tunneling nanotubes enable intercellular transfer of MHC class I molecules

Carefully orchestrated intercellular communication is an essential prerequisite for an effective immune response. In recent years tunneling nanotubes (TNT) have emerged as a novel mechanism of cell–cell communication. These long membrane protrusions can establish cytoplasmic continuity between distant cells and enable the exchange of cellular components. In the present study we addressed the question whether these structures can facilitate the intercellular transfer of MHC class I molecules. We found a transmembrane HLA-A2-EGFP but not a soluble HLA-G1s-EGFP fusion protein to be effectively transferred between HeLa cells. Inhibition of actin polymerization significantly reduced the HLA-A2 transfer rate, indicating that transfer is dependent on tunneling nanotubes, whose de novo formation requires actin polymerization. Furthermore, overexpression of the nanotube-inducing protein LST1 promoted transfer of HLA-A2. Moreover, LST1 protein expression is enhanced in antigen presenting cells. Our results indicate that tunneling nanotubes can mediate transfer of MHC class I molecules between distant cells.     

Cloning, distribution and up-regulation of the teleost fish MHC class II alpha suggests a role for granulocytes as antigen-presenting cells

The major histocompatibility complex (MHC) class II alpha chain gene of the teleost fish gilthead seabream (Sparus aurata), Spau-DAA, has been characterized. We cloned, sequenced and studied its polymorphism, before evaluating its expression in resting seabream leucocytes, tissues and tumor cells as well as in primed leucocytes. A complete allele was obtained by overlapping sequence fragments obtained by RT-PCR. The full-length Spau-DAA*101 comprises 1840 bp with a 5'-UTR region of 84 bp, an ORF of 729 bp and a 3'-UTR of 1027 bp. The putative protein of 242 residues shows homology with known MHC class II alpha genes, varying from 71 to 28% in other fish and humans, respectively. The protein sequence showed all the important features: leader peptide, alpha1, alpha2 and CP/TM/CYT regions, conserved cysteines and N-glycosylation site. The phylogenetic tree shows that it is included in the cluster containing the Percomorpha subclass and far from the human and shark genes. It is polymorphic, as seen when we sequenced the complete ORF of 11 alleles showing most of the amino acidic changes in the alpha1 domain, where the peptide-binding region (PBR) is found. Spau-DAA mRNA expression was mainly found in peritoneal exudate leucocytes, head-kidney, spleen, thymus and gill. Minor expression was detected in gut, brain, liver and PBLs. RT-PCR expression studies in isolated leucocyte subpopulations revealed, for the first time in the literature, that acidophilic granulocytes show high MHC class II gene expression. Apart from these granulocytes lymphocytes also express the Spau-DAA gene, although other cell types may also do the same. Finally, incubation of head-kidney leucocytes with yeast cells or pathogenic bacteria up-regulates Spau-DAA gene expression whilst incubation with ConA, ConA+LPS or PHA does not. The possible involvement of the seabream MHC class II alpha gene in the fish defence and antigen presentation are discussed.     

CIITA-induced occupation of MHC class II promoters is independent of the cooperative stabilization of the promoter-bound multi-protein complexes

Precise regulation of MHC class II expression plays a crucial role in the control of the immune response. The transactivator CIITA behaves as a master controller of constitutive and inducible MHC class II gene activation, but its exact mechanism of action is not known. Activation of MHC class II promoters requires binding of at least three distinct multi-protein complexes (RFX, X2BP and NF-Y). It is known that the stability of this binding results from cooperative interactions between these proteins. We show here that expression of CIITA in MHC class II- cells triggers occupation of the promoters by these complexes. This observation raised the possibility that the effect of CIITA on promoter occupation is mediated by an effect on the cooperative stabilization of the DNA-bound multi-protein complexes. We show, however, that the presence of CIITA does not affect the stability of the higher-order protein complex formed on DNA by RFX, X2BP and NF-Y. This suggests other mechanisms for CIITA-induced promoter occupancy, such as an effect on chromatin structure leading to increased accessibility of MHC class II promoters. This ability of CIITA to facilitate promoter occupation is undissociable from its transactivation potential. Finally, we conclude that this effect of CIITA is cell-type specific, since expression of CIITA is not required for normal occupation of MHC class II promoters in B lymphocytes.     

Altered MHC class I expression in dorsolateral prefrontal cortex of nonsmoker patients with schizophrenia

Schizophrenia (SZ) is a psychiatric disease with plausible neurodevelopmental etiology. Although genetic studies show significant association of immune molecules loci such as major histocompatibility complex (MHC) class I with SZ, it is not clear whether these immune molecules are involved in the pathology observed in SZ brains. MHC class I and the classical pathway components of complement system (C1q and C3) have been shown to regulate brain neuronal maturation and function. We have examined the expression of MHC class I and complement protein C3 in two frontal cortical regions of postmortem brains of SZ patients. Since cigarette smoking may modulate MHC class I protein expression and a higher rate of smoking is observed in SZ patients, we studied the expression of MHC class I and C3 in relation to the presence of smoking. We found that MHC class I protein expression is reduced in the dorsolateral prefrontal cortex (DLPFC) but not in the orbitofrontal cortex (OFC) of nonsmoker SZ patients. We did not observe SZ-associated changes in C3 mRNA expression. Our exploratory research suggests a potential involvement of MHC class I in SZ and implies that smoking might modulate its expression.     

Involvement of Thyroid Hormones in the Expression of MHC class I Antigens During Ontogeny in Xenopus

The major histocompatibility complex (MHC) is a cluster of genes encoding products central to all major functions of the vertebrate immune system. Evidence for an MHC can be found in all vertebrate groups that have been examined except the jawless fishes. Expression of MHC class I and class II antigens early in ontogeny is critically important for development of T lymphocytes capable of discriminating self from nonself. Because of this essential role in T-cell development, the ontogeny of MHC expression in the South African clawed frog, Xenopus laevis, was studied. Previous studies of MHC class I expression in Xenopus laevis suggested that class I antigens are virtually absent from tadpole tissues until climax of metamorphosis. We therefore examined the possible role of thyroid hormones (TH) in the induction of class I. By flow cytometry, a small amount of class I expression was detectable on splenocytes and erythrocytes in untreated frogs at prometamorphic stages 55-58, and the amount increased significantly at the conclusion of metamorphic climax. Thus, metamorphosis is associated with increased intensity of class I expression. Neither inhibition nor acceleration of metamorphosis altered the timing of onset of class I expression. However, inhibition of metamorphosis prevented the increase in class I expression characteristic of adult cell populations. Because expression was not accelerated in TH-treated frogs or delayed in metamorphosis-inhibited frogs, it is unlikely that TH are the direct developmental cues that induce expression, although they seem to be required for the upregulation of class I expression occurring at metamorphosis. Differences in the pattern of expression in different subpopulations of cells suggest a complex pattern of regulation of expression of class I antigens during ontogeny.     

EpsinR,a target for pyrenocine B,role in endogenous MHC‐II‐restricted antigen presentation

While the presentation mechanism of antigenic peptides derived from exogenous proteins by MHC class II molecules is well understood, relatively little is known about the presentation mechanism of endogenous MHC class II‐restricted antigens. We therefore screened a chemical library of 200 compounds derived from natural products to identify inhibitors of the presentation of endogenous MHC class II‐restricted antigens. We found that pyrenocine B, a compound derived from the fungus Pyrenochaeta terrestris, inhibits presentation of endogenous MHC class II‐restricted minor histocompatibility antigen IL‐4 inducible gene 1 (IL4I1) by primary dendritic cells (DCs). Phage display screening and surface plasmon resonance (SPR) analysis were used to investigate the mechanism of suppressive action by pyrenocine B. EpsinR, a target molecule for pyrenocine B, mediates endosomal trafficking through binding of soluble N‐ethylmaleimide‐sensitive factor attachment protein receptors (SNAREs). Lentiviral‐mediated short hairpin (sh) RNA downregulation of EpsinR expression in DCs resulted in a decrease in the responsiveness of CD4⁺ T cells. Our data thus suggest that EpsinR plays a role in antigen presentation, which provides insight into the mechanism of presentation pathway of endogenous MHC class II‐restricted antigen.     

Structural and functional characteristics of a dominant‐negative isoform of porcine MHC class II transactivator

The MHC class II transactivator, CIITA, is critical for MHC class II gene expression in all species studied to date. We isolated an interferon (IFN)‐γ‐inducible isoform of porcine CIITA (pCIITA′) encoding a protein of 566 amino acids (aa) with significant homology to human CIITA (hCIITA). Analysis indicated that pCIITA′ lacks the entire GTP‐binding domain that is important for nuclear translocation and activation of target genes by hCIITA. In pCIITA′ this region is replaced by a 14‐aa motif with homology to several signalling peptide sequences. Expression of pCIITA′ in porcine (ST‐IOWA) and human (HeLa) cell lines resulted in suppression of IFN‐γ‐stimulated MHC class II gene expression, at the protein and mRNA levels. We also identified two IFN‐γ‐inducible variants of hCIITA, hCIITAlo and hCIITA′ from Hela cells, both exhibiting dominant‐negative suppression of MHC class II gene expression. Interestingly, hCIITA′ encodes a predicted protein of 546 aa with a strikingly similar organization to pCIITA′ including the 14‐aa GTP‐binding domain‐replacement motif in which 10 out of 14 amino acids are identical to the pig sequence. Expression of hCIITA′ and hCIITAlo sequences in Hela cells suppressed IFN‐γ‐induced MHC class II gene expression. hCIITAlo, a predicted 303‐aa protein with deleted GTP‐binding and carboxy‐terminal domain, displayed a more subtle suppression of IFN‐γ‐induced MHC class II expression. These in vitro data indicate that there may be a role in vivo for isoforms of CIITA that can suppress full‐length CIITA‐mediated MHC class II gene expression. Both humans and now, potentially, pigs are candidate donors for organ and tissue allografts and xenografts, respectively. Regulation of MHC class II gene expression by manipulation of CIITA isoform expression in humans and pigs may provide a useful strategy for attenuation of T‐cell‐mediated cellular rejection.     

Cell-mediated immune functions in a patient with MHC class II deficiency

This report focuses on cell-mediated immune functions in a patient with MHC class II deficiency. The patient described presented with a case of "classical" MHC class II deficiency (T and B cells within the normal range, normal lymphocyte proliferation in response to stimulation with mitogens, gene encoding for MHC class II present, no expression of MHC class II). The absence of MHC class II expression resulted in an incapability of the patient's antigen-presenting cells to function as accessory cells in the presentation of soluble protein antigens, while accessory functions required for the induction of alloantigen-induced lymphocyte proliferation or for the generation of cytotoxic T cells in response to an allostimulus were normal. The patient's T cells responded normally to alloantigenic stimulation and also had the capacity to develop antigen-specific cytotoxic functions. However, the T cells were completely naive with respect to activation by soluble protein antigens, even after presentation by accessory cells derived from the patient's healthy histoidentical brother. In this context it was interesting to note that the patient's CD4-positive cells showed a normal pattern of expression of the 4B4 marker, a marker generally present on memory T cells. These data make it tempting to speculate that in the absence of MHC class II, other cell surface structures may at least partially take over immune functions normally under the control of the MHC class II complex.