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  1. MHC II on Transfused Murine Blood is Not Required for Alloimmunization Against MHC I

    PubMed Central

    Gilson, Christopher R.; Cadwell, Chantel M.; Smith, Nicole H.; Hendrickson, Jeanne E.; Zimring, James C.

    2010-01-01

    Background and Objectives Transfusion of allogeneic platelet products can result in antibodies against donor MHC I antigens, leading to a refractory state to subsequent platelet transfusions. However, there is disagreement in the field regarding the molecular mechanisms of humoral alloimmunization. One hypothesis states that donor MHC II is a requirement for alloimmunization. However, other studies have suggested that donor MHC I is alone sufficient and MHC II is not required. Materials and Methods We utilized a mouse model of anti-MHC I alloimmunization to transfused blood, which employed donors with a complete deletion of all MHC II genes. BALB/c (H-2d) recipients were transfused with blood from either C57BL/6 (H-2b) or MHC II null donors on a C57BL/6 background. Anti-MHC I alloimmunization was monitored by indirect immunofluorescence. Results Recipients of either wild type or MHC II null blood produced equivalent humoral responses against donor MHC I antigens. However, there was variation in the relative amounts of IgG subclasses. Conclusion These data reject the hypothesis that donor MHC II expression is required for alloimmunization to MHC I antigens. PMID:20546207

  2. Achieving stability through editing and chaperoning: regulation of MHC class II peptide binding and expression.

    PubMed

    Busch, Robert; Rinderknecht, Cornelia H; Roh, Sujin; Lee, Andrew W; Harding, James J; Burster, Timo; Hornell, Tara M C; Mellins, Elizabeth D

    2005-10-01

    In antigen-presenting cells (APCs), loading of major histocompatibility complex class II (MHC II) molecules with peptides is regulated by invariant chain (Ii), which blocks MHC II antigen-binding sites in pre-endosomal compartments. Several molecules then act upon MHC II molecules in endosomes to facilitate peptide loading: Ii-degrading proteases, the peptide exchange factor, human leukocyte antigen-DM (HLA-DM), and its modulator, HLA-DO (DO). Here, we review our findings arguing that DM stabilizes a globally altered conformation of the antigen-binding groove by binding to a lateral surface of the MHC II molecule. Our data imply changes in the interactions between specificity pockets and peptide side chains, complementing data from others that suggest DM affects hydrogen bonds. Selective weakening of peptide/MHC interactions allows DM to alter the peptide repertoire. We also review our studies in cells that highlight the ability of several factors to modulate surface expression of MHC II molecules via post-Golgi mechanisms; these factors include MHC class II-associated Ii peptides (CLIP), DM, and microbial products that modulate MHC II traffic from endosomes to the plasma membrane. In this context, we discuss possible mechanisms by which the association of some MHC II alleles with autoimmune diseases may be linked to their low CLIP affinity.

  3. MHC class II super-enhancer increases surface expression of HLA-DR and HLA-DQ and affects cytokine production in autoimmune vitiligo

    PubMed Central

    Cavalli, Giulio; Hayashi, Masahiro; Jin, Ying; Yorgov, Daniel; Santorico, Stephanie A.; Holcomb, Cherie; Rastrou, Melinda; Erlich, Henry; Tengesdal, Isak W.; Dagna, Lorenzo; Neff, C. Preston; Palmer, Brent E.; Spritz, Richard A.; Dinarello, Charles A.

    2016-01-01

    Genetic risk for autoimmunity in HLA genes is most often attributed to structural specificity resulting in presentation of self-antigens. Autoimmune vitiligo is strongly associated with the MHC class II region. Here, we fine-map vitiligo MHC class II genetic risk to three SNPs only 47 bp apart, located within a predicted super-enhancer in an intergenic region between HLA-DRB1 and HLA-DQA1, localized by a genome-wide association study of 2,853 Caucasian vitiligo patients. The super-enhancer corresponds to an expression quantitative trait locus for expression of HLA-DR and HLA-DQ RNA; we observed elevated surface expression of HLA-DR (P = 0.008) and HLA-DQ (P = 0.02) on monocytes from healthy subjects homozygous for the high-risk SNP haplotype. Unexpectedly, pathogen-stimulated peripheral blood mononuclear cells from subjects homozygous for the high-risk super-enhancer haplotype exhibited greater increase in production of IFN-γ and IL-1β than cells from subjects homozygous for the low-risk haplotype. Specifically, production of IFN-γ on stimulation of dectin-1, mannose, and Toll-like receptors with Candida albicans and Staphylococcus epidermidis was 2.5- and 2.9-fold higher in high-risk subjects than in low-risk subjects, respectively (P = 0.007 and P = 0.01). Similarly, production of IL-1β was fivefold higher in high-risk subjects than in low-risk subjects (P = 0.02). Increased production of immunostimulatory cytokines in subjects carrying the high-risk haplotype may act as an “adjuvant” during the presentation of autoantigens, tying together genetic variation in the MHC with the development of autoimmunity. This study demonstrates that for risk of autoimmune vitiligo, expression level of HLA class II molecules is as or more important than antigen specificity. PMID:26787888

  4. Ubiquitination by March-I prevents MHC class II recycling and promotes MHC class II turnover in antigen-presenting cells

    PubMed Central

    Cho, Kyung-Jin; Walseng, Even; Ishido, Satoshi; Roche, Paul A.

    2015-01-01

    MHC class II (MHC-II)-dependent antigen presentation by antigen-presenting cells (APCs) is carefully controlled to achieve specificity of immune responses; the regulated assembly and degradation of antigenic peptide–MHC-II complexes (pMHC-II) is one aspect of such control. In this study, we have examined the role of ubiquitination in regulating pMHC-II biosynthesis, endocytosis, recycling, and turnover in APCs. By using APCs obtained from MHC-II ubiquitination mutant mice, we find that whereas ubiquitination does not affect pMHC-II formation in dendritic cells (DCs), it does promote the subsequent degradation of newly synthesized pMHC-II. Acute activation of DCs or B cells terminates expression of the MHC-II E3 ubiquitin ligase March-I and prevents pMHC-II ubiquitination. Most importantly, this change results in very efficient pMHC-II recycling from the surface of DCs and B cells, thereby preventing targeting of internalized pMHC-II to lysosomes for degradation. Biochemical and functional assays confirmed that pMHC-II turnover is suppressed in MHC-II ubiquitin mutant DCs or by acute activation of wild-type DCs. These studies demonstrate that acute APC activation blocks the ubiquitin-dependent turnover of pMHC-II by promoting efficient pMHC-II recycling and preventing lysosomal targeting of internalized pMHC-II, thereby enhancing pMHC-II stability for efficient antigen presentation to CD4 T cells. PMID:26240324

  5. Ubiquitination by March-I prevents MHC class II recycling and promotes MHC class II turnover in antigen-presenting cells.

    PubMed

    Cho, Kyung-Jin; Walseng, Even; Ishido, Satoshi; Roche, Paul A

    2015-08-18

    MHC class II (MHC-II)-dependent antigen presentation by antigen-presenting cells (APCs) is carefully controlled to achieve specificity of immune responses; the regulated assembly and degradation of antigenic peptide-MHC-II complexes (pMHC-II) is one aspect of such control. In this study, we have examined the role of ubiquitination in regulating pMHC-II biosynthesis, endocytosis, recycling, and turnover in APCs. By using APCs obtained from MHC-II ubiquitination mutant mice, we find that whereas ubiquitination does not affect pMHC-II formation in dendritic cells (DCs), it does promote the subsequent degradation of newly synthesized pMHC-II. Acute activation of DCs or B cells terminates expression of the MHC-II E3 ubiquitin ligase March-I and prevents pMHC-II ubiquitination. Most importantly, this change results in very efficient pMHC-II recycling from the surface of DCs and B cells, thereby preventing targeting of internalized pMHC-II to lysosomes for degradation. Biochemical and functional assays confirmed that pMHC-II turnover is suppressed in MHC-II ubiquitin mutant DCs or by acute activation of wild-type DCs. These studies demonstrate that acute APC activation blocks the ubiquitin-dependent turnover of pMHC-II by promoting efficient pMHC-II recycling and preventing lysosomal targeting of internalized pMHC-II, thereby enhancing pMHC-II stability for efficient antigen presentation to CD4 T cells.

  6. Generation of MHC class II:peptide ligands for CD4 T cell allorecognition of MHC Class II molecules

    PubMed Central

    Leddon, Scott A.; Sant, Andrea J.

    2011-01-01

    Purpose of review The molecular and cellular mechanisms that underlie allorecognition of MHC class II molecules has been the subject much debate and experimentation in recent decades. In this review, we discuss several aspects of MHC class II structure, peptide acquisition and TcR-MHC:peptide interactions that have particular relevance to recognition of cells bearing allogeneic class II molecules. Recent findings First, MHC polymorphism is heavily biased toward those amino acids that influence stable peptide binding by MHC class II. Second, the peptide repertoire presented by class II molecules is highly diverse and can be edited substantially by the molecular catalyst HLA-DM and by tissue-specific expression of HLA-DO, stress and cytokines. Third, T cell receptor docking onto MHC peptide typically involves substantial contacts with the bound peptide in the MHC class II molecule. Finally, there is increasing evidence that T cell recognition of MHC is in part germline-encoded through T cell receptor V region contacts with MHC class II alpha helices. Summary Together, these conclusions support the view that allorecognition of MHC class II molecules is likely to parallel key aspects of conventional CD4 T cell recognition, with allele-dependent variation in peptide representation accounting in large part for the high precursor frequency of alloreactive CD4 T cells PMID:20616724

  7. Impaired synthesis of erythropoietin, glutamine synthetase and metallothionein in the skin of NOD/SCID/gamma(c)(null) and Foxn1 nu/nu mice with misbalanced production of MHC class II complex.

    PubMed

    Danielyan, L; Verleysdonk, S; Buadze, M; Gleiter, C H; Buniatian, G H

    2010-06-01

    Most skin pathologies are characterized by unbalanced synthesis of major histocompatability complex II (MHC-II) proteins. Healthy skin keratinocytes simultaneously produce large amounts of MHC-II and regeneration-supporting proteins, e.g. erythropoietin (EPO), EPO receptor (EPOR), glutamine synthetase (GS) and metallothionein (MT). To investigate the level of regeneration-supporting proteins in the skin during misbalanced production of MHC-II, skin sections from nonobese diabetic/severe combined immunodeficient (NOD/SCID)/gamma (c) (null) and or Foxn1 nu/nu mice which are a priory known to under- and over-express MHC II, respectively, were used. Double immunofluorescence analysis of NOD/SCID/gamma (c) (null) skin sections showed striking decrease in expression of MHC-II, EPO, GS and MT. In Foxn1 nu/nu mouse skin, GS was strongly expressed in epidermis and in hair follicles (HF), which lacked EPO. In nude mouse skin EPO and MHC-II were over-expressed in dermal fibroblasts and they were completely absent from cortex, channel, medulla and keratinocytes surrounding the HF, suggest a role for EPO in health and pathology of hair follicle. The level of expression of EPO and GS in both mutant mice was confirmed by results of Western blot analyses. Strong immunoresponsiveness of EPOR in the hair channels of NOD/SCID/gamma (c) (null) mouse skin suggests increased requirements of skin cells for EPO and possible benefits of exogenous EPO application during disorders of immune system accompanied by loss MHC-II in skin cells.

  8. Predicting MHC-II binding affinity using multiple instance regression

    PubMed Central

    EL-Manzalawy, Yasser; Dobbs, Drena; Honavar, Vasant

    2011-01-01

    Reliably predicting the ability of antigen peptides to bind to major histocompatibility complex class II (MHC-II) molecules is an essential step in developing new vaccines. Uncovering the amino acid sequence correlates of the binding affinity of MHC-II binding peptides is important for understanding pathogenesis and immune response. The task of predicting MHC-II binding peptides is complicated by the significant variability in their length. Most existing computational methods for predicting MHC-II binding peptides focus on identifying a nine amino acids core region in each binding peptide. We formulate the problems of qualitatively and quantitatively predicting flexible length MHC-II peptides as multiple instance learning and multiple instance regression problems, respectively. Based on this formulation, we introduce MHCMIR, a novel method for predicting MHC-II binding affinity using multiple instance regression. We present results of experiments using several benchmark datasets that show that MHCMIR is competitive with the state-of-the-art methods for predicting MHC-II binding peptides. An online web server that implements the MHCMIR method for MHC-II binding affinity prediction is freely accessible at http://ailab.cs.iastate.edu/mhcmir. PMID:20855923

  9. Major Histocompatibility Complex (MHC) Class I and MHC Class II Proteins: Conformational Plasticity in Antigen Presentation

    PubMed Central

    Wieczorek, Marek; Abualrous, Esam T.; Sticht, Jana; Álvaro-Benito, Miguel; Stolzenberg, Sebastian; Noé, Frank; Freund, Christian

    2017-01-01

    Antigen presentation by major histocompatibility complex (MHC) proteins is essential for adaptive immunity. Prior to presentation, peptides need to be generated from proteins that are either produced by the cell’s own translational machinery or that are funneled into the endo-lysosomal vesicular system. The prolonged interaction between a T cell receptor and specific pMHC complexes, after an extensive search process in secondary lymphatic organs, eventually triggers T cells to proliferate and to mount a specific cellular immune response. Once processed, the peptide repertoire presented by MHC proteins largely depends on structural features of the binding groove of each particular MHC allelic variant. Additionally, two peptide editors—tapasin for class I and HLA-DM for class II—contribute to the shaping of the presented peptidome by favoring the binding of high-affinity antigens. Although there is a vast amount of biochemical and structural information, the mechanism of the catalyzed peptide exchange for MHC class I and class II proteins still remains controversial, and it is not well understood why certain MHC allelic variants are more susceptible to peptide editing than others. Recent studies predict a high impact of protein intermediate states on MHC allele-specific peptide presentation, which implies a profound influence of MHC dynamics on the phenomenon of immunodominance and the development of autoimmune diseases. Here, we review the recent literature that describe MHC class I and II dynamics from a theoretical and experimental point of view and we highlight the similarities between MHC class I and class II dynamics despite the distinct functions they fulfill in adaptive immunity. PMID:28367149

  10. Conservation of MHC class II DOA sequences among carnivores.

    PubMed

    Soll, S J; Stewart, B S; Lehman, N

    2005-03-01

    We obtained the nucleotide sequence for most of the major histocompatibility complex (MHC) class II DOA locus for Weddell, leopard, northern elephant, and southern elephant seals and from the coyote and compared them to all known DOA data available to date. We found generally low levels of interspecific polymorphisms, providing further support for stabilizing selection acting on the DOA locus. This suggests that DO gene products play a substantial functional role in the regulation of antigen presentation. A seven-amino-acid motif of VWRLPEF was found to be conserved across all DOA sequences and may be a DO-specific recognition element.

  11. Blocking MHC class II on human endothelium mitigates acute rejection

    PubMed Central

    Abrahimi, Parwiz; Qin, Lingfeng; Chang, William G.; Bothwell, Alfred L.M.; Tellides, George; Saltzman, W. Mark; Pober, Jordan S.

    2016-01-01

    Acute allograft rejection is mediated by host CD8+ cytotoxic T lymphocytes (CTL) targeting graft class I major histocompatibility complex (MHC) molecules. In experimental rodent models, rejection requires differentiation of naive CD8+ T cells into alloreactive CTL within secondary lymphoid organs, whereas in humans, CTL may alternatively develop within the graft from circulating CD8+ effector memory T cells (TEM) that recognize class I MHC molecules on graft endothelial cells (EC). This latter pathway is poorly understood. Here, we show that host CD4+ TEM, activated by EC class II MHC molecules, provide critical help for this process. First, blocking HLA-DR on EC lining human artery grafts in immunodeficient mice reduces CD8+ CTL development within and acute rejection of the artery by adoptively transferred allogeneic human lymphocytes. Second, siRNA knockdown or CRISPR/Cas9 ablation of class II MHC molecules on EC prevents CD4+ TEM from helping CD8+ TEM to develop into CTL in vitro. Finally, implanted synthetic microvessels, formed from CRISPR/Cas9-modified EC lacking class II MHC molecules, are significantly protected from CD8+ T cell–mediated destruction in vivo. We conclude that human CD8+ TEM–mediated rejection targeting graft EC class I MHC molecules requires help from CD4+ TEM cells activated by recognition of class II MHC molecules. PMID:26900601

  12. DNA sequence of the Peromyscus leucopus MHC class II gene Aa (MhcPeleAa)

    SciTech Connect

    Crew, M.D.; Bates, L.M.

    1996-09-01

    The genus Peromyscus has been extensively studied by populations biologists and ecologists for over eighty years, with P. leucopus (the white-footed mouse) being one of the most intensively investigated species. Polymorphic major histocompatibility complex (MHC) genes have proven useful in population genetic studies and might be helpful in understanding the population dynamics of Peromyscus species which are ubiquitously distributed over North and Central America. Polymorphism of P. leucopus MHC (MhcPele) class II genes was evident by restriction fragment length polymorphism (RFLP) analyses using human and mouse probes and Pele class II loci exhibited degrees of polymorphism similar to H2 class II genes (A-like>E-like). 8 refs., 2 figs.

  13. MHC2SKpan: a novel kernel based approach for pan-specific MHC class II peptide binding prediction

    PubMed Central

    2013-01-01

    Background Computational methods for the prediction of Major Histocompatibility Complex (MHC) class II binding peptides play an important role in facilitating the understanding of immune recognition and the process of epitope discovery. To develop an effective computational method, we need to consider two important characteristics of the problem: (1) the length of binding peptides is highly flexible; and (2) MHC molecules are extremely polymorphic and for the vast majority of them there are no sufficient training data. Methods We develop a novel string kernel MHC2SK (MHC-II String Kernel) method to measure the similarities among peptides with variable lengths. By considering the distinct features of MHC-II peptide binding prediction problem, MHC2SK differs significantly from the recently developed kernel based method, GS (Generic String) kernel, in the way of computing similarities. Furthermore, we extend MHC2SK to MHC2SKpan for pan-specific MHC-II peptide binding prediction by leveraging the binding data of various MHC molecules. Results MHC2SK outperformed GS in allele specific prediction using a benchmark dataset, which demonstrates the effectiveness of MHC2SK. Furthermore, we evaluated the performance of MHC2SKpan using various benckmark data sets from several different perspectives: Leave-one-allele-out (LOO), 5-fold cross validation as well as independent data testing. MHC2SKpan has achieved comparable performance with NetMHCIIpan-2.0 and outperformed NetMHCIIpan-1.0, TEPITOPEpan and MultiRTA, being statistically significant. MHC2SKpan can be freely accessed at http://datamining-iip.fudan.edu.cn/service/MHC2SKpan/index.html. PMID:24564280

  14. Quantitative analysis of peptide-MHC class II interaction.

    PubMed

    Fleckenstein, B; Jung, G; Wiesmüller, K H

    1999-12-01

    The tremendous progress in the field of basic immunology and immunochemistry made in the last decade has significantly advanced our understanding of antigen processing and presentation by MHC class I and II proteins. In this review different techniques to study peptide interaction with MHC class II molecules are summarized and their impact on the elucidation of quantitative parameters, like affinities or kinetic data, is discussed. A recently introduced method based on synthetic combinatorial peptide libraries allows to quantify the binding contribution of each amino acid residue in a class II ligand and is presented in more detail. As this knowledge is fundamental for current investigations in modern medicine, e.g. for novel immune system based therapy concepts, further aspects like the design of new high affinity MHC class II ligands and the prediction of peptide antigens are discussed.

  15. Molecular characterization of MHC class II region in guinea fowl.

    PubMed

    Singh, S K; Mathew, J; Gupta, J; Mehra, S; Goyal, G; Sharma, D

    2010-12-01

    1. The MHC class II gene was amplified, cloned and sequenced in guinea fowl. 2. The NumeMHC II sequence of 754 nucleotides included complete exon 1 (91 nt), exon 2 (270 nt), exon 3 (282 nt) and exon 4 (110 nt). 3. The size of β(1) and β(2), domains were 89 and 93 amino acids, respectively in guinea fowl. 4. High amino acid variability (38·2%) was observed within guinea fowl in β(1) domain, while in β(2) domain, amino acid variability (6·3%) was low. 5. Among poultry species, the percent amino acid identity between guinea fowl and chicken, quail, pheasant and duck was 38·8, 42·2, 44·4 and 58·8 in β(1) domain; and 13·8, 17·0, 13·8 and 27·6 in β(2) domain, respectively. 6. Sequence alignment with mammalian and avian MHC showed that many of the conserved features of MHC class II glycoprotein was conserved in guinea fowl. 7. Within-species genetic distances (Poisson correction) based on cumulative amino acid variability in β(1) domain and β(2) domains was 0·141 in guinea fowl. 8. Guinea fowl showed low and similar genetic distances with all the poultry species (0·255-0·268) except duck (0·456). 9. Guinea fowl made separate branch within the major cluster having chicken, quail and pheasant, showing equal distance from these poultry species, whereas duck MHC II clustered separately.

  16. Defective MHC class II expression in an MHC class II deficiency patient is caused by a novel deletion of a splice donor site in the MHC class II transactivator gene.

    PubMed

    Peijnenburg, A; Van den Berg, R; Van Eggermond, M J; Sanal, O; Vossen, J M; Lennon, A M; Alcaïde-Loridan, C; Van den Elsen, P J

    2000-01-01

    MHC class II deficiency patients are mutated for transcription factors that regulate the expression of major histocompatibility complex (MHC) class II genes. Four complementation groups (A-D) are defined and the gene defective in group A has been shown to encode the MHC class II transactivator (CIITA). Here, we report the molecular characterization of a new MHC class II deficiency patient, ATU. Cell fusion experiments indicated that ATU belongs to complementation group A. Subsequent mutation analysis revealed that the CIITA mRNA lacked 84 nucleotides. This deletion was the result of the absence of a splice donor site in the CIITA gene of ATU. As a result of this novel homozygous genomic deletion, ATU CIITA failed to transactivate MHC class II genes. Furthermore, this truncated CIITA of ATU did not display a dominant negative effect on CIITA-mediated transactivation of various isotypic MHC class II promoters.

  17. New Design of MHC Class II Tetramers to Accommodate Fundamental Principles of Antigen Presentation

    PubMed Central

    Landais, Elise; Romagnoli, Pablo A.; Corper, Adam L.; Shires, John; Altman, John D.; Wilson, Ian A.; Garcia, K. Christopher; Teyton, Luc

    2009-01-01

    Direct identification and isolation of antigen-specific T cells became possible with the development of “MHC tetramers”, based on fluorescent avidins displaying biotinylated peptide-MHC (pMHC) complexes. This approach, extensively used for MHC class I–restricted T cells, has met very limited success with MHC class II tetramers (pMHCT-2) for the detection of CD4+ specific T cells. In addition, a very large number of these reagents while capable of specifically activating T cells after being coated on solid support, are still unable to stain. In order to try to understand this puzzle and design usable tetramers, we examined each parameter critical for the production of pMHCT-2 using the I-Ad-OVA system as a model. Through this process the geometry of pMHC display by avidin tetramers was examined, as well as the stability of recombinant MHC molecules. However, we discovered that the most important factor limiting the reactivity of pMHCT-2 was the display of peptides. Indeed, long peptides, as presented by MHC class II molecules, can be bound to I-A/HLA-DQ molecules in more than one register as suggested by structural studies. This mode of anchorless peptide binding allows the selection of a broader repertoire on single peptides and should favor anti-infectious immune responses. Thus, beyond the technical improvements that we propose, the redesign of pMHCT-2 will give us the tools to evaluate the real size of the CD4 repertoire and help us in the production and testing of new vaccines. PMID:19923463

  18. MHC class II DR allelic diversity in bighorn sheep

    USDA-ARS?s Scientific Manuscript database

    We hypothesized that decreased diversity and/or unique polymorphisms in MHC class II alleles of bighorn sheep (BHS, Ovis canadensis) are responsible for lower titer of antibodies against Mannheimia haemolytica leukotoxin, in comparison to domestic sheep (DS, Ovis aries). To test this hypothesis, DRA...

  19. Contrasting patterns of selection between MHC I and II across populations of Humboldt and Magellanic penguins.

    PubMed

    Sallaberry-Pincheira, Nicole; González-Acuña, Daniel; Padilla, Pamela; Dantas, Gisele P M; Luna-Jorquera, Guillermo; Frere, Esteban; Valdés-Velásquez, Armando; Vianna, Juliana A

    2016-10-01

    The evolutionary and adaptive potential of populations or species facing an emerging infectious disease depends on their genetic diversity in genes, such as the major histocompatibility complex (MHC). In birds, MHC class I deals predominantly with intracellular infections (e.g., viruses) and MHC class II with extracellular infections (e.g., bacteria). Therefore, patterns of MHC I and II diversity may differ between species and across populations of species depending on the relative effect of local and global environmental selective pressures, genetic drift, and gene flow. We hypothesize that high gene flow among populations of Humboldt and Magellanic penguins limits local adaptation in MHC I and MHC II, and signatures of selection differ between markers, locations, and species. We evaluated the MHC I and II diversity using 454 next-generation sequencing of 100 Humboldt and 75 Magellanic penguins from seven different breeding colonies. Higher genetic diversity was observed in MHC I than MHC II for both species, explained by more than one MHC I loci identified. Large population sizes, high gene flow, and/or similar selection pressures maintain diversity but limit local adaptation in MHC I. A pattern of isolation by distance was observed for MHC II for Humboldt penguin suggesting local adaptation, mainly on the northernmost studied locality. Furthermore, trans-species alleles were found due to a recent speciation for the genus or convergent evolution. High MHC I and MHC II gene diversity described is extremely advantageous for the long-term survival of the species.

  20. MHC class I and MHC class II DRB gene variability in wild and captive Bengal tigers (Panthera tigris tigris).

    PubMed

    Pokorny, Ina; Sharma, Reeta; Goyal, Surendra Prakash; Mishra, Sudanshu; Tiedemann, Ralph

    2010-10-01

    Bengal tigers are highly endangered and knowledge on adaptive genetic variation can be essential for efficient conservation and management. Here we present the first assessment of allelic variation in major histocompatibility complex (MHC) class I and MHC class II DRB genes for wild and captive tigers from India. We amplified, cloned, and sequenced alpha-1 and alpha-2 domain of MHC class I and beta-1 domain of MHC class II DRB genes in 16 tiger specimens of different geographic origin. We detected high variability in peptide-binding sites, presumably resulting from positive selection. Tigers exhibit a low number of MHC DRB alleles, similar to other endangered big cats. Our initial assessment-admittedly with limited geographic coverage and sample size-did not reveal significant differences between captive and wild tigers with regard to MHC variability. In addition, we successfully amplified MHC DRB alleles from scat samples. Our characterization of tiger MHC alleles forms a basis for further in-depth analyses of MHC variability in this illustrative threatened mammal.

  1. Why does the immune system of Atlantic cod lack MHC II?

    PubMed

    Star, Bastiaan; Jentoft, Sissel

    2012-08-01

    MHC II, a major feature of the adaptive immune system, is lacking in Atlantic cod, and there are different scenarios (metabolic cost hypothesis or functional shift hypothesis) that might explain this loss. The lack of MHC II coincides with an increased number of genes for MHC I and Toll-like receptors (TLRs).

  2. Tolerance to MHC class II disparate allografts through genetic modification of bone marrow

    PubMed Central

    Jindra, Peter T.; Tripathi, Sudipta; Tian, Chaorui; Iacomini, John; Bagley, Jessamyn

    2012-01-01

    Induction of molecular chimerism through genetic modification of bone marrow is a powerful tool for the induction of tolerance. Here we demonstrate for the first time that expression of an allogeneic MHC class II gene in autologous bone marrow cells, resulting in a state of molecular chimerism, induces tolerance to MHC class II mismatched skin grafts, a stringent test of transplant tolerance. Reconstitution of recipients with syngeneic bone marrow transduced with retrovirus encoding H-2I-Ab (I-Ab) resulted the long-term expression of the retroviral gene product on the surface of MHC class II-expressing bone marrow derived cell types. Mechanistically, tolerance was maintained by the presence of regulatory T cells, which prevented proliferation and cytokine production by alloreactive host T cells. Thus, the introduction of MHC class II genes into bone marrow derived cells through genetic engineering results in tolerance. These results have the potential to extend the clinical applicability of molecular chimerism for tolerance induction. PMID:22833118

  3. MHC Class II haplotypes of Colombian Amerindian tribes

    PubMed Central

    Yunis, Juan J.; Yunis, Edmond J.; Yunis, Emilio

    2013-01-01

    We analyzed 1041 individuals belonging to 17 Amerindian tribes of Colombia, Chimila, Bari and Tunebo (Chibcha linguistic family), Embera, Waunana (Choco linguistic family), Puinave and Nukak (Maku-Puinave linguistic families), Cubeo, Guanano, Tucano, Desano and Piratapuyo (Tukano linguistic family), Guahibo and Guayabero (Guayabero Linguistic Family), Curripaco and Piapoco (Arawak linguistic family) and Yucpa (Karib linguistic family). for MHC class II haplotypes (HLA-DRB1, DQA1, DQB1). Approximately 90% of the MHC class II haplotypes found among these tribes are haplotypes frequently encountered in other Amerindian tribes. Nonetheless, striking differences were observed among Chibcha and non-Chibcha speaking tribes. The DRB1*04:04, DRB1*04:11, DRB1*09:01 carrying haplotypes were frequently found among non-Chibcha speaking tribes, while the DRB1*04:07 haplotype showed significant frequencies among Chibcha speaking tribes, and only marginal frequencies among non-Chibcha speaking tribes. Our results suggest that the differences in MHC class II haplotype frequency found among Chibcha and non-Chibcha speaking tribes could be due to genetic differentiation in Mesoamerica of the ancestral Amerindian population into Chibcha and non-Chibcha speaking populations before they entered into South America. PMID:23885196

  4. MHC Class II haplotypes of Colombian Amerindian tribes.

    PubMed

    Yunis, Juan J; Yunis, Edmond J; Yunis, Emilio

    2013-07-01

    We analyzed 1041 individuals belonging to 17 Amerindian tribes of Colombia, Chimila, Bari and Tunebo (Chibcha linguistic family), Embera, Waunana (Choco linguistic family), Puinave and Nukak (Maku-Puinave linguistic families), Cubeo, Guanano, Tucano, Desano and Piratapuyo (Tukano linguistic family), Guahibo and Guayabero (Guayabero Linguistic Family), Curripaco and Piapoco (Arawak linguistic family) and Yucpa (Karib linguistic family). for MHC class II haplotypes (HLA-DRB1, DQA1, DQB1). Approximately 90% of the MHC class II haplotypes found among these tribes are haplotypes frequently encountered in other Amerindian tribes. Nonetheless, striking differences were observed among Chibcha and non-Chibcha speaking tribes. The DRB1*04:04, DRB1*04:11, DRB1*09:01 carrying haplotypes were frequently found among non-Chibcha speaking tribes, while the DRB1*04:07 haplotype showed significant frequencies among Chibcha speaking tribes, and only marginal frequencies among non-Chibcha speaking tribes. Our results suggest that the differences in MHC class II haplotype frequency found among Chibcha and non-Chibcha speaking tribes could be due to genetic differentiation in Mesoamerica of the ancestral Amerindian population into Chibcha and non-Chibcha speaking populations before they entered into South America.

  5. Mechanistic understanding and significance of small peptides interaction with MHC class II molecules for therapeutic applications.

    PubMed

    Afridi, Saifullah; Hoessli, Daniel C; Hameed, Muhammad Waqar

    2016-07-01

    Major histocompatibility complex (MHC) class II molecules are expressed by antigen-presenting cells and stimulate CD4(+) T cells, which initiate humoral immune responses. Over the past decade, interest has developed to therapeutically impact the peptides to be exposed to CD4(+) T cells. Structurally diverse small molecules have been discovered that act on the endogenous peptide exchanger HLA-DM by different mechanisms. Exogenously delivered peptides are highly susceptible to proteolytic cleavage in vivo; however, it is only when successfully incorporated into stable MHC II-peptide complexes that these peptides can induce an immune response. Many of the small molecules so far discovered have highlighted the molecular interactions mediating the formation of MHC II-peptide complexes. As potential drugs, these small molecules open new therapeutic approaches to modulate MHC II antigen presentation pathways and influence the quality and specificity of immune responses. This review briefly introduces how CD4(+) T cells recognize antigen when displayed by MHC class II molecules, as well as MHC class II-peptide-loading pathways, structural basis of peptide binding and stabilization of the peptide-MHC complexes. We discuss the concept of MHC-loading enhancers, how they could modulate immune responses and how these molecules have been identified. Finally, we suggest mechanisms whereby MHC-loading enhancers could act upon MHC class II molecules.

  6. Sibling rivalry: competition between MHC class II family members inhibits immunity.

    PubMed

    Denzin, Lisa K; Cresswell, Peter

    2013-01-01

    Peptide loading of major histocompatibility complex (MHC) class II molecules in the endosomes and lysosomes of antigen-presenting cells is catalyzed by human leukocyte antigen-DM (HLA-DM) and modulated by HLA-DO. In a structural study in this issue, Guce et al. show that HLA-DO is an MHC class II mimic and functions as a competitive and essentially irreversible inhibitor of HLA-DM activity, thereby inhibiting MHC class II antigen presentation.

  7. A Case of Probable MHC Class II Deficiency with Disseminated BCGitis.

    PubMed

    Alyasin, Soheyla; Abolnezhadian, Farhad; Khoshkhui, Maryam

    2015-09-01

    Major histocompatibility complex (MHC) class II deficiency is a primary immunodeficiency disease characterized by abnormality of MHC class II molecules surface expression on peripheral blood lymphocytes and monocytes. Clinical manifestations include extreme susceptibility to viral, bacterial, and fungal infections but the immunodeficiency is not as severe as SCID (severe combined immunodeficiency), as evidenced by failure to develop disseminated infection after BCG vaccination. Therefore, MHC II deficiency with BCGosis, that is disseminated BCGitis, is not reported commonly. We report an interesting case of BCGosis after vaccination that was diagnosed to have probable MHC II deficiency.

  8. MHC class II-assortative mate choice in European badgers (Meles meles).

    PubMed

    Sin, Yung Wa; Annavi, Geetha; Newman, Chris; Buesching, Christina; Burke, Terry; Macdonald, David W; Dugdale, Hannah L

    2015-06-01

    The major histocompatibility complex (MHC) plays a crucial role in the immune system, and in some species, it is a target by which individuals choose mates to optimize the fitness of their offspring, potentially mediated by olfactory cues. Under the genetic compatibility hypothesis, individuals are predicted to choose mates with compatible MHC alleles, to increase the fitness of their offspring. Studies of MHC-based mate choice in wild mammals are under-represented currently, and few investigate more than one class of MHC genes. We investigated mate choice based on the compatibility of MHC class I and II genes in a wild population of European badgers (Meles meles). We also investigated mate choice based on microsatellite-derived pairwise relatedness, to attempt to distinguish MHC-specific effects from genomewide effects. We found MHC-assortative mating, based on MHC class II, but not class I genes. Parent pairs had smaller MHC class II DRB amino acid distances and smaller functional distances than expected from random pairings. When we separated the analyses into within-group and neighbouring-group parent pairs, only neighbouring-group pairs showed MHC-assortative mating, due to similarity at MHC class II loci. Our randomizations showed no evidence of genomewide-based inbreeding, based on 35 microsatellite loci; MHC class II similarity was therefore the apparent target of mate choice. We propose that MHC-assortative mate choice may be a local adaptation to endemic pathogens, and this assortative mate choice may have contributed to the low MHC genetic diversity in this population.

  9. Neurons Preferentially Respond to Self-MHC Class I Allele Products Regardless of Peptide Presented

    PubMed Central

    Escande-Beillard, Nathalie; Washburn, Lorraine; Zekzer, Dan; Wu, Zhongqi-Phyllis; Eitan, Shoshy; Ivkovic, Sonja; Lu, Yuxin; Dang, Hoa; Middleton, Blake; Bilousova, Tina V.; Yoshimura, Yoshitaka; Evans, Christopher J.; Joyce, Sebastian; Tian, Jide; Kaufman, Daniel L.

    2010-01-01

    Studies of mice lacking MHC class I (MHC I)-associated proteins have demonstrated a role for MHC I in neurodevelopment. A central question arising from these observations is whether neuronal recognition of MHC I has specificity for the MHC I allele product and the peptide presented. Using a well-established embryonic retina explant system, we observed that picomolar levels of a recombinant self-MHC I molecule inhibited neurite outgrowth. We then assessed the neurobiological activity of a panel of recombinant soluble MHC Is, consisting of different MHC I heavy chains with a defined self- or nonself-peptide presented, on cultured embryonic retinas from mice with different MHC I haplotypes. We observed that self-MHC I allele products had greater inhibitory neuroactivity than nonself-MHC I molecules, regardless of the nature of the peptide presented, a pattern akin to MHC I recognition by some innate immune system receptors. However, self-MHC I molecules had no effect on retinas from MHC I-deficient mice. These observations suggest that neuronal recognition of MHC I may be coordinated with the inherited MHC I alleles, as occurs in the innate immune system. Consistent with this notion, we show that MHC I and MHC I receptors are coexpressed by precursor cells at the earliest stages of retina development, which could enable such coordination. PMID:20018625

  10. MHC Class II Structural Requirements for the Association with Igα/β, and Signaling of Calcium Mobilization and Cell Death

    PubMed Central

    Jin, Lei; Stolpa, John C.; Young, Ryan M.; Pugh-Bernard, Aimee E.; Refaeli, Yosef; Cambier, John C.

    2008-01-01

    Summary Emerging evidence indicates that in addition to their well-characterized role in antigen presentation, MHC II molecules transmit signals that induce death of APCs. Appropriately timed APC death is important for prevention of autoimmunity. Though the exact mechanism of MHC II-mediated cell death signaling is unknown, the response appears independent of caspase activation and does not involve Fas-FasL interaction. Here we investigated MHC II structural requirements for mediation of cell death signaling in a murine B cell lymphoma. We found that neither the transmembrane spanning regions nor the cytoplasmic tails of MHC II, which are required for MHC II-mediated cAMP production and PKC activation, are required for the death response. However, mutations in the connecting peptide region of MHC II α chain (αCP), but not the β chain (βCP), resulted in significant impairment of the death response. The αCP mutant was also unable to mediate calcium mobilization responses, and did not associate with Igα/β. Knock-down of Igβ by shRNA eliminated the MHC II mediated calcium response but not cell death. We propose that MHC II mediates cell death signaling via association with an undefined cell surface protein(s), whose interaction is partially dependent on αCP region. PMID:18194817

  11. MHC class II tetramers made from isolated recombinant α and β chains refolded with affinity-tagged peptides.

    PubMed

    Braendstrup, Peter; Justesen, Sune; Osterbye, Thomas; Nielsen, Lise Lotte Bruun; Mallone, Roberto; Vindeløv, Lars; Stryhn, Anette; Buus, Søren

    2013-01-01

    Targeting CD4+ T cells through their unique antigen-specific, MHC class II-restricted T cell receptor makes MHC class II tetramers an attractive strategy to identify, validate and manipulate these cells at the single cell level. Currently, generating class II tetramers is a specialized undertaking effectively limiting their use and emphasizing the need for improved methods of production. Using class II chains expressed individually in E. coli as versatile recombinant reagents, we have previously generated peptide-MHC class II monomers, but failed to generate functional class II tetramers. Adding a monomer purification principle based upon affinity-tagged peptides, we here provide a robust method to produce class II tetramers and demonstrate staining of antigen-specific CD4+ T cells. We also provide evidence that both MHC class II and T cell receptor molecules largely accept affinity-tagged peptides. As a general approach to class II tetramer generation, this method should support rational CD4+ T cell epitope discovery as well as enable specific monitoring and manipulation of CD4+ T cell responses.

  12. EpsinR, a target for pyrenocine B, role in endogenous MHC-II-restricted antigen presentation.

    PubMed

    Shishido, Tatsuya; Hachisuka, Masami; Ryuzaki, Kai; Miura, Yuko; Tanabe, Atsushi; Tamura, Yasuaki; Kusayanagi, Tomoe; Takeuchi, Toshifumi; Kamisuki, Shinji; Sugawara, Fumio; Sahara, Hiroeki

    2014-11-01

    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.

  13. Rheumatoid Rescue of Misfolded Cellular Proteins by MHC Class II Molecules: A New Hypothesis for Autoimmune Diseases.

    PubMed

    Arase, Hisashi

    2016-01-01

    Misfolded proteins localized in the endoplasmic reticulum are degraded promptly and thus are not transported outside cells. However, misfolded proteins in the endoplasmic reticulum are rescued from protein degradation upon association with major histocompatibility complex (MHC) class II molecules and are transported to the cell surface by MHC class II molecules without being processed to peptides. Studies on the misfolded proteins rescued by MHC class II molecules have revealed that misfolded proteins associated with MHC class II molecules are specific targets for autoantibodies produced in autoimmune diseases. Furthermore, a strong correlation has been observed between autoantibody binding to misfolded proteins associated with MHC class II molecules and the autoimmune disease susceptibility conferred by each MHC class II allele. These new insights into MHC class II molecules suggest that misfolded proteins rescued from protein degradation by MHC class II molecules are recognized as "neo-self" antigens by immune system and are involved in autoimmune diseases as autoantibody targets.

  14. Characterization and expression of MHC class II alpha and II beta genes in mangrove red snapper (Lutjanus argentimaculatus).

    PubMed

    Wang, Tianyan; Tan, Shangjin; Cai, Zhonghua

    2015-12-01

    The major histocompatibility complex (MHC) class II plays a key role in adaptive immunity by presenting foreign peptides to CD4(+) T cells and by triggering the adaptive immune response. While the structure and function of MHC class II have been well characterized in mammalian, limited research has been done on fishes. In this study, we characterized the gene structure and expression of MHC class II α (Lunar-DAA) and II β (Lunar-DAB) of mangrove red snapper (Lutjanus argentimaculatus). Both genes shared, respectively, a high similarity and typical features with other vertebrate MHC class II α and II β. The phylogenetic analysis of the deduced peptides revealed that both Lunar-DAA and Lunar-DAB were located in the teleost subclass. Western blotting analyses indicated that both MHC class II α and II β were expressed ubiquitously in immune-related cells, tissues and organs, and that MHC class II α and II β chains existed mainly as heterodimers. While it was highly expressed in gills, thymus, head kidney (HK), spleen, head kidney macrophage and spleen leucocytes, MHC class II β chain was expressed with a low abundance in skin, intestine, stomach and heart. The highest expression of MHC class II β in thymus confirmed the conclusion that thymus is one of the primary lymphoid organs in fishes. The detection of MHC class II αβ dimers in HK macrophages and spleen leucocytes indicated that HK macrophages and spleen leucocytes play a critical role in the adaptive immunity in fishes. All these results provide valuable information for understanding the structure of MHC class II α and II β and their function in immune responses.

  15. An analysis of the sensitivity and specificity of MHC-I and MHC-II immunohistochemical staining in muscle biopsies for the diagnosis of inflammatory myopathies.

    PubMed

    Rodríguez Cruz, Pedro M; Luo, Yue-Bei; Miller, James; Junckerstorff, Reimar C; Mastaglia, Frank L; Fabian, Victoria

    2014-12-01

    Although there have been several previous reports of immunohistochemical staining for MHC antigens in muscle biopsies, there appears to be a lack of consensus about its routine use in the diagnostic evaluation of biopsies from patients with suspected inflammatory myopathy. Positive MHC-I staining is nonspecific but is widely used as a marker for inflammatory myopathy, whilst the role of MHC-II staining is not clearly defined. We investigated the sensitivity and specificity of MHC-I and MHC-II immunostaining for the diagnosis of inflammatory myopathy in a large group of biopsies from a single reference laboratory. Positive staining for MHC-I was found to have a high sensitivity in biopsies from patients with inflammatory myopathy but a very low specificity, as it was also common in other non-inflammatory myopathies and neurogenic disorders. On the other hand, MHC-II positivity had a much higher specificity in all major subgroups of inflammatory myopathy, especially inclusion body myositis. The findings indicate that the combination of MHC-I and MHC-II staining results in a higher degree of specificity for the diagnosis of inflammatory myopathy and that in biopsies with inflammation, positive MHC-II staining strongly supports the diagnosis of an immune-mediated myopathy. We recommend that immunohistochemical staining for both MHC-I and MHC-II should be included routinely in the diagnostic evaluation of muscle biopsies from patients with suspected inflammatory myopathy. However, as the sensitivity and interpretation of MHC staining may depend on the technique used, further studies are needed to compare procedures in different centres and develop standardised protocols. Copyright © 2014 Elsevier B.V. All rights reserved.

  16. Astrocyte cytolysis by MHC class II-specific mouse T cell clones.

    PubMed

    Reder, A T; Lascola, C D; Flanders, S A; Maimone, D; Jensen, M A; Skias, D D; Lancki, D W

    1993-08-01

    The brain is "immunologically privileged," in part because class I and II MHC antigens are not normally present on glia or neurons. However, under certain conditions such as transplantation, glial cells express MHC proteins at levels sufficient for glia to become targets of immune responses. Cultured astrocytes expressing very low levels of MHC class I protein are killed efficiently by MHC class I antigen-specific CTL. Mouse brain allografts, however, are rejected by CD4+ T cells that are likely to be class II MHC-specific. The level of expression of MHC class II antigen needed to trigger specific killing of astrocytes by CD4+ T cells, independent of exogenous antigen, has not been studied. We examined the role of glial class II MHC in the lysis of cultured neonatal mouse astrocytes by an alloreactive murine CD4+ CTL alone. IFN-gamma induced functionally relevant increases in MHC class II antigen on target cells. Astrocytes were lysed by the CD4+ clone only when class II MHC antigens reached levels readily detectable by flow cytometry. MHC class II expression and lysis increased when astrocytes were coincubated with IFN-gamma and TNF-alpha. Conversely, lysis decreased when class II expression was downregulated by IFN-alpha/beta or dbcAMP. Cytolysis by CD4+ clones was blocked by antibodies to CD4 and LFA-1 on T cells, and to ICAM-1 and class II molecules on astrocytes. The role of LFA-1 in CD4+ cell-mediated lysis was greater than that of LFA-1/ICAM-1 in CD8+ T cell-mediated lysis. CD4+ cells may lyse activated astrocytes when the immune privilege of the brain is compromised as in transplantation, tumors, and inflammatory diseases.

  17. Impact of MHC Class II Incompatibility on Localization of Mononuclear Cell Infiltrates to the Bronchiolar Compartment of Orthotopic Lung Allografts

    PubMed Central

    Nakashima, Shinji; Soong, T. Rinda; Fox-Talbot, Karen; Qian, Zhiping; Rahimi, Salma; Wasowska, Barbara A.; Rohde, Charles A.; Chen, Sabrina; Garcia, Joe G.N.; Baldwin, William M.

    2005-01-01

    Chronic pathological changes in transplanted lungs are unique because they center on the airways. We examined the relative role of MHC class I and II antigens in causing bronchial pathology in orthotopic lung transplants to rats maintained on cyclosporin A (CsA). Transplants mismatched for MHC class II antigens had significantly more peri-bronchiolar infiltrates than MHC class I incompatible transplants. No significant increase in infiltrates was found in lung transplants incompatible for MHC class I plus II antigens compared to MHC class II antigens alone. Immunohistochemistry demonstrated that MHC class II antigen expression was confined to macrophages in MHC class I incompatible transplants, but was upregulated on bronchial epithelium in transplants with MHC class II incompatibilities. Vascular endothelium was notably devoid of MHC class II antigen expression in all transplants. However, both peri-bronchial and peri-vascular infiltrates were frequently cuffed by alveolar macrophages and type II pneumocytes that expressed MHC class II antigens. PCR analysis demonstrated that IFN-γ and regulated on activation, normal T cells expressed and secreted (RANTES) were upregulated in MHC class II incompatible transplants. Thus, MHC class II incompatible orthotopic lung transplants in rats maintained on CsA immunosuppression undergo a bronchiolcentric upregulation of alloantigens. PMID:15760392

  18. Shark Class II Invariant Chain Reveals Ancient Conserved Relationships with Cathepsins and MHC Class II

    PubMed Central

    Criscitiello, Michael F.; Ohta, Yuko; Eubanks, Jeannine O.; Chen, Patricia L.; Flajnik, Martin F.

    2011-01-01

    The invariant chain (Ii) is the critical third chain required for the MHC class II heterodimer to be properly guided through the cell, loaded with peptide, and expressed on the surface of antigen presenting cells. Here, we report the isolation of the nurse shark Ii gene, and the comparative analysis of Ii splice variants, expression, genomic organization, predicted structure, and function throughout vertebrate evolution. Alternative splicing to yield Ii with and without the putative protease-protective, thyroglobulin-like domain is as ancient as the MHC-based adaptive immune system, as our analyses in shark and lizard further show conservation of this mechanism in all vertebrate classes except bony fish. Remarkable coordinate expression of Ii and class II was found in shark tissues. Conserved Ii residues and cathepsin L orthologs suggest their long co-evolution in the antigen presentation pathway, and genomic analyses suggest 450 million years of conserved Ii exon/intron structure. Other than an extended linker preceding the thyroglobulin-like domain in cartilaginous fish, the Ii gene and protein are predicted to have largely similar physiology from shark to man. Duplicated Ii genes found only in teleosts appear to have become sub-functionalized, as one form is predicted to play the same role as that mediated by Ii mRNA alternative splicing in all other vertebrate classes. No Ii homologs or potential ancestors of any of the functional Ii domains were found in the jawless fish or lower chordates. PMID:21996610

  19. Shark class II invariant chain reveals ancient conserved relationships with cathepsins and MHC class II.

    PubMed

    Criscitiello, Michael F; Ohta, Yuko; Graham, Matthew D; Eubanks, Jeannine O; Chen, Patricia L; Flajnik, Martin F

    2012-03-01

    The invariant chain (Ii) is the critical third chain required for the MHC class II heterodimer to be properly guided through the cell, loaded with peptide, and expressed on the surface of antigen presenting cells. Here, we report the isolation of the nurse shark Ii gene, and the comparative analysis of Ii splice variants, expression, genomic organization, predicted structure, and function throughout vertebrate evolution. Alternative splicing to yield Ii with and without the putative protease-protective, thyroglobulin-like domain is as ancient as the MHC-based adaptive immune system, as our analyses in shark and lizard further show conservation of this mechanism in all vertebrate classes except bony fish. Remarkable coordinate expression of Ii and class II was found in shark tissues. Conserved Ii residues and cathepsin L orthologs suggest their long co-evolution in the antigen presentation pathway, and genomic analyses suggest 450 million years of conserved Ii exon/intron structure. Other than an extended linker preceding the thyroglobulin-like domain in cartilaginous fish, the Ii gene and protein are predicted to have largely similar physiology from shark to man. Duplicated Ii genes found only in teleosts appear to have become sub-functionalized, as one form is predicted to play the same role as that mediated by Ii mRNA alternative splicing in all other vertebrate classes. No Ii homologs or potential ancestors of any of the functional Ii domains were found in the jawless fish or lower chordates. Copyright © 2011 Elsevier Ltd. All rights reserved.

  20. Transport of misfolded endoplasmic reticulum proteins to the cell surface by MHC class II molecules

    PubMed Central

    Jiang, Yan; Arase, Noriko

    2013-01-01

    Nascent MHC class II molecules are associated with the invariant chain and are transported to the endolysosomal pathway, where MHC class II molecules acquire peptide antigens. On the other hand, misfolded endoplasmic reticulum (ER) proteins are generally degraded in the cells and are neither expressed on the cell surface nor secreted. Here, we found that MHC class II molecules associate with some misfolded ER proteins via the peptide-binding groove in competition with invariant chain. The misfolded proteins associated with MHC class II molecules are transported intact to the cell surface without processing to peptides. Furthermore, these complexes efficiently stimulate antigen-specific B cells. These findings reveal that MHC class II molecules function as a chaperone for the cell surface expression of misfolded ER proteins. In addition, we suggest that MHC class II molecules present not only peptides but also intact host-cell-derived proteins on the cell surface. These findings provide new insights into the function of MHC class II molecules. PMID:23334921

  1. Protein arginine methyltransferase 1 (PRMT1) represses MHC II transcription in macrophages by methylating CIITA

    PubMed Central

    Fan, Zhiwen; Li, Jianfei; Li, Ping; Ye, Qing; Xu, Huihui; Wu, Xiaoyan; Xu, Yong

    2017-01-01

    Efficient presentation of alien antigens triggers activation of T lymphocytes and robust host defense against invading pathogens. This pathophysiological process relies on the expression of major histocompatibility complex (MHC) molecules in antigen presenting cells such as macrophages. Aberrant MHC II transactivation plays a crucial role in the pathogenesis of atherosclerosis. Class II transactivator (CIITA) mediates MHC II induction by interferon gamma (IFN-γ). CIITA activity can be fine-tuned at the post-translational level, but the mechanisms are not fully appreciated. We investigated the role of protein arginine methyltransferase 1 (PRMT1) in this process. We report here that CIITA interacted with PRMT1. IFN-γ treatment down-regulated PRMT1 expression and attenuated PRMT1 binding on the MHC II promoter. Over-expression of PRMT1 repressed MHC II promoter activity while PRMT1 depletion enhanced MHC II transactivation. Mechanistically, PRMT1 methylated CIITA and promoted CIITA degradation. Therefore, our data reveal a previously unrecognized role for PRMT1 in suppressing CIITA-mediated MHC II transactivation. PMID:28094290

  2. Secretory granules of mast cells accumulate mature and immature MHC class II molecules.

    PubMed

    Vincent-Schneider, H; Théry, C; Mazzeo, D; Tenza, D; Raposo, G; Bonnerot, C

    2001-01-01

    Bone marrow-derived mast cells as well as dendritic cells, macrophages and B lymphocytes express major histocompatibility complex (MHC) class II molecules. In mast cells, the majority of MHC class II molecules reside in intracellular cell type-specific compartments, secretory granules. To understand the molecular basis for the localisation of MHC class II molecules in secretory granules, MHC class II molecules were expressed, together with the invariant chain, in the mast cell line, RBL-2H3. Using electron and confocal microscopy, we observed that in RBL-2H3 cells, mature and immature class II molecules accumulate in secretory granules. Two particular features of class II transport accounted for this intracellular localization: first, a large fraction of newly synthesized MHC class II molecules remained associated with invariant chain fragments. This defect, resulting in a slower rate of MHC class II maturation, was ascribed to a low cathepsin S activity. Second, although a small fraction of class II dimers matured (i.e. became free of invariant chain), allowing their association with antigenic peptides, they were retained in secretory granules. As a consequence of this intracellular localization, cell surface expression of class II molecules was strongly increased by cell activation stimuli which induced the release of the contents of secretory granules. Our results suggest that antigen presentation, and thereby antigen specific T cell stimulation, are regulated in mast cells by stimuli which induce mast cell activation.

  3. Interspecific hybridization increases MHC class II diversity in two sister species of newts.

    PubMed

    Nadachowska-Brzyska, Krystyna; Zieliński, Piotr; Radwan, Jacek; Babik, Wiesław

    2012-02-01

    Our understanding of the evolutionary mechanisms generating variation within the highly polymorphic major histocompatibility complex (MHC) genes remains incomplete. Assessing MHC variation across multiple populations, of recent and ancient divergence, may facilitate understanding of geographical and temporal aspects of variation. Here, we applied 454 sequencing to perform a large-scale, comprehensive analysis of MHC class II in the closely related, hybridizing newts, Lissotriton vulgaris (Lv) and Lissotriton montandoni (Lm). Our study revealed an extensive (299 alleles) geographically structured polymorphism. Populations at the southern margin of the Lv distribution, inhabited by old and distinct lineages (southern Lv), exhibited moderate MHC variation and strong population structure, indicating little gene flow or extensive local adaptation. Lissotriton vulgaris in central Europe and the northern Balkans (northern Lv) and almost all Lm populations had a high MHC variation. A much higher proportion of MHC alleles was shared between Lm and northern Lv than between Lm and southern Lv. Strikingly, the average pairwise F(ST) between northern Lv and Lm was significantly lower than between northern and southern Lv for MHC, but not for microsatellites. Thus, high MHC variation in Lm and northern Lv may result from gene flow between species. We hypothesize that the interspecific exchange of MHC genes may be facilitated by frequency-dependent selection. A marginally significant correlation between the MHC and microsatellite allelic richness indicates that demographic factors may have contributed to the present-day pattern of MHC variation, but unequivocal signatures of adaptive evolution in MHC class II sequences emphasize the role of selection on a longer timescale.

  4. Cross-species association of quail invariant chain with chicken and mouse MHC II molecules.

    PubMed

    Chen, Fangfang; Wu, Chao; Pan, Ling; Xu, Fazhi; Liu, Xuelan; Yu, Weiyi

    2013-05-01

    There are different degrees of similarity among vertebrate invariant chains (Ii). The aim of this study was to determine the relationship between quail and other vertebrate Ii MHC class II molecules. The two quail Ii isoforms (qIi-1, qIi-2) were cloned by RACE, and qRT-PCR analysis of different organs showed that their expression levels were positively correlated with MHC II gene (B-LB) transcription levels. Confocal microscopy indicated that quail full-length Ii co-localized with MHC II of quail, chicken or mouse in 293FT cells co-transfected with both genes. Immunoprecipitation and western blotting further indicated that these aggregates corresponded to polymers of Ii and MHC class II molecules. This cross-species molecular association of quail Ii with chicken and mouse MHC II suggests that Ii molecules have a high structural and functional similarity and may thereby be used as potential immune carriers across species. Copyright © 2013 Elsevier Ltd. All rights reserved.

  5. A central role for HSC70 in regulating antigen trafficking and MHC class II presentation.

    PubMed

    Deffit, Sarah N; Blum, Janice S

    2015-12-01

    Cells rely on multiple intracellular trafficking pathways to capture antigens for proteolysis. The resulting peptides bind to MHC class II molecules to promote CD4(+) T cell recognition. Endocytosis enhances the capture of extracellular and cell surface bound antigens for processing and presentation, while autophagy pathways shunt cytoplasmic and nuclear antigens for presentation in the context of MHC class II molecules. Understanding how physiological changes and cellular stress alter antigen trafficking and the repertoire of peptides presented by class II molecules remains challenging, yet important in devising novel approaches to boost immune responses to pathogens and tumors. An abundant, constitutively expressed cytoplasmic chaperone, HSC70 plays a central role in modulating antigen transport within cells to control MHC class II presentation during nutrient stress. HSC70 may serve as a molecular switch to modulate endocytic and autophagy pathways, impacting the source of antigens delivered for MHC class II presentation during cellular stress.

  6. Cellular misfolded proteins rescued from degradation by MHC class II molecules are possible targets for autoimmune diseases.

    PubMed

    Arase, Noriko; Arase, Hisashi

    2015-11-01

    The major function of major histocompatibility complex (MHC) class II molecules is the presentation of peptide antigens to helper T cells. However, when misfolded proteins are associated with MHC class II molecules in the endoplasmic reticulum, they are transported to the cell surface by MHC class II molecules without processing to peptides. Of note, misfolded proteins complexed with MHC class II molecules are specifically recognized by autoantibodies produced in patients with autoimmune diseases such as rheumatoid arthritis and antiphospholipid syndrome. Furthermore, autoantibody binding to misfolded proteins complexed with MHC class II molecules is associated with the susceptibility to autoimmune diseases conferred by each MHC class II allele. Therefore, misfolded proteins rescued from degradation by MHC class II molecules may be recognized as 'neo-self' antigens by the immune system and be involved in the pathogenicity of autoimmune diseases.

  7. The multigenic structure of the MHC locus contributes to positive selection efficiency: a role for MHC class II gene-specific restriction.

    PubMed

    Monteiro, Miguel Caetano; Couceiro, Sofia; Penha-Gonçalves, Carlos

    2005-12-01

    The study of T cell positive selection in the thymus has long been focused on the specificity of the MHC-TCR interactions, making use of genetically manipulated mice that display TCR specificities or selecting peptides of limited diversity. However, little is known on the role of the MHC molecules irrespective of the peptide specificity and the implications of MHC multigenic structure in thymic positive selection have not been addressed. Here, we investigated the effect of MHC class II genetic configuration on the positive selection efficiency of naturally generated pre-selection repertoires in the mouse thymus. Analysis of positively selected thymocyte populations in MHC-congenic and -transgenic mice revealed that expression of I-E molecule in the thymic cortex increases positive selection efficiency of CD4 cells by approximately 50%. We show that increments in positive selection attributable to either the I-A and I-E genes are not due to increased MHC class II expression in the thymic cortex and are not affected by the number of MHC alleles. Collectively, our findings imply that MHC class II gene-restricted TCR specificities significantly contribute to positive selection efficiency, introducing the notion that multigenic structure of the MHC locus serves to increase selection of non-overlapping TCR repertoires.

  8. MHC2MIL: a novel multiple instance learning based method for MHC-II peptide binding prediction by considering peptide flanking region and residue positions

    PubMed Central

    2014-01-01

    Background Computational prediction of major histocompatibility complex class II (MHC-II) binding peptides can assist researchers in understanding the mechanism of immune systems and developing peptide based vaccines. Although many computational methods have been proposed, the performance of these methods are far from satisfactory. The difficulty of MHC-II peptide binding prediction comes mainly from the large length variation of binding peptides. Methods We develop a novel multiple instance learning based method called MHC2MIL, in order to predict MHC-II binding peptides. We deem each peptide in MHC2MIL as a bag, and some substrings of the peptide as the instances in the bag. Unlike previous multiple instance learning based methods that consider only instances of fixed length 9 (9 amino acids), MHC2MIL is able to deal with instances of both lengths of 9 and 11 (11 amino acids), simultaneously. As such, MHC2MIL incorporates important information in the peptide flanking region. For measuring the distances between different instances, furthermore, MHC2MIL explicitly highlights the amino acids in some important positions. Results Experimental results on a benchmark dataset have shown that, the performance of MHC2MIL is significantly improved by considering the instances of both 9 and 11 amino acids, as well as by emphasizing amino acids at key positions in the instance. The results are consistent with those reported in the literature on MHC-II peptide binding. In addition to five important positions (1, 4, 6, 7 and 9) for HLA(human leukocyte antigen, the name of MHC in Humans) DR peptide binding, we also find that position 2 may play some roles in the binding process. By using 5-fold cross validation on the benchmark dataset, MHC2MIL outperforms two state-of-the-art methods of MHC2SK and NN-align with being statistically significant, on 12 HLA DP and DQ molecules. In addition, it achieves comparable performance with MHC2SK and NN-align on 14 HLA DR molecules. MHC2MIL

  9. Protein sorting within the MHC class II antigen-processing pathway.

    PubMed

    Marks, M S

    1998-01-01

    Major histocompatibility complex (MHC) class II molecules are required for the presentation of antigenic peptides that are derived predominantly from internalized proteins. The assembly of MHC class II/peptide complexes occurs within endosomal compartments of antigen-presenting cells (APCs). Therefore, for assembly to occur, MHC class II molecules, foreign proteins, and accessory molecules must be sorted to appropriate intracellular sites. My laboratory is trying to understand how proteins are sorted to various antigen-processing compartments as well as to conventional endosomal organelles. Using chimeric marker proteins and a variety of biochemical and genetic approaches, we are addressing the specificity of protein sorting and the mechanisms by which sorting signals are deciphered. By using a similar chimeric protein approach to target endogenous proteins to distinct compartments, we hope to address the role of processing events in each compartment in the generation of MHC class II ligands.

  10. HLA-DM is localized to conventional and unconventional MHC class II-containing endocytic compartments.

    PubMed

    Pierre, P; Denzin, L K; Hammond, C; Drake, J R; Amigorena, S; Cresswell, P; Mellman, I

    1996-03-01

    HLA-DM molecules remove invariant (Ii) chain peptides from newly synthesized MHC class II complexes. Their localization may thus delineate compartments, e.g., MIIC, specialized for loading peptides onto class II molecules. In murine A20 B cells, however, DM is not restricted to specialized endosomal class II-containing vesicles (CIIV). Although DM was found in CIIV, it was also found throughout the endocytic pathway, principally in lysosomes devoid of class II molecules. In human lymphoblasts, HLA-DM was found in structures indistinguishable from late endosomes or lysosomes, although in these cells the lysosomes contained MHC class II molecules. Thus, the distribution of HLA-DM does not necessarily identify specialized class II compartments. Many "MIIC" may represent conventional lysosomes that accumulate MHC class II and HLA-DM in a number of cell types.

  11. Evidence for multiple MHC class II β loci in New Zealand's critically endangered kakapo, Strigops habroptilus.

    PubMed

    Knafler, Gabrielle J; Fidler, Andrew; Jamieson, Ian G; Robertson, Bruce C

    2014-02-01

    Immunologically important genes of the major histocompatibility complex (MHC) have been characterized in a number of avian species with the general finding of considerable variation in size and structural organization among organisms. A range of nonpasserines which represent early-diverging Neoave lineages have been described as having only one MHC class II β locus potentially leading to the conclusion that this is the ancestral condition. Here, we examine the monotypic, early-diverging, critically endangered kakapo, Strigops habroptilus, for allelic variation at MHC class II β exon 2, as part of species' recovery efforts. We found two to four confirmed sequence variants per individual indicating the presence of more than one MHC class II β locus. Given the kakapo's basal evolutionary status, evidence for multiple MHC class II β loci seems to counter the proposed mono-locus history of modern birds. However, MHC gene duplication, maintenance, and loss among and within bird species may confound avian relationships making it difficult to elucidate the ancestral state. This study adds essential data for disentangling the course of MHC structural evolution in birds.

  12. Extensive characterization of the immunophenotype and pattern of cytokine production by distinct subpopulations of normal human peripheral blood MHC II+/lineage− cells

    PubMed Central

    Almeida, J; Bueno, C; Alguero, M C; Sanchez, M L; Cañizo, M C; Fernandez, M E; Vaquero, J M; Laso, F J; Escribano, L; San Miguel, J F; Orfao, A

    1999-01-01

    Dendritic cells (DC) represent the most powerful professional antigen-presenting cells (APC) in the immune system. The aim of the present study was to analyse, on a single-cell basis by multiparametric flow cytometry with simultaneous four-colour staining and a two-step acquisition procedure, the immunophenotypic profile and cytokine production of DC from 67 normal whole peripheral blood (PB) samples. Two clearly different subsets of HLA-II+/lineage− were identified on the basis of their distinct phenotypic characteristics: one DC subset was CD33strong+ and CD123dim+ (0.16 ± 0.06% of the PB nucleated cells and 55.9 ± 11.9% of all PB DC) and the other, CD33dim+ and CD123strong+ (0.12 ± 0.04% of PB nucleated cells and 44.53 ± 11.5% of all PB DC). Moreover, the former DC subpopulation clearly showed higher expression of the CD13 myeloid-associated antigen, the CD29 and CD58 adhesion molecules, the CD2, CD5 and CD86 costimulatory molecules, the CD32 IgG receptor and the CD11c complement receptor. In addition, these cells showed stronger HLA-DR and HLA-DQ expression and a higher reactivity for the IL-6 receptor α-chain (CD126) and for CD38. In contrast, the CD123strong+/CD33dim+ DC showed a stronger reactivity for the CD4 and CD45RA molecules, whereas they did not express the CD58, CD5, CD11c and CD13 antigens. Regarding cytokine production, our results show that while the CD33strong+/CD123dim+ DC are able to produce significant amounts of inflammatory cytokines, such as IL-1β (97 ± 5% of positive cells), IL-6 (96 ± 1.1% of positive cells), IL-12 (81.5 ± 15.5% of positive cells) and tumour necrosis factor-alpha (TNF-α) (84 ± 22.1% of positive cells) as well as chemokines such as IL-8 (99 ± 1% of positive cells), the functional ability of the CD123strong+/CD33dim+ DC subset to produce cytokines under the same conditions was almost null. Our results therefore clearly show the presence of two distinct subsets of DC in normal human PB, which differ not only in

  13. MHC II expression in the CNS after long-term demyelination

    SciTech Connect

    Cannella, B.; Aquino, D.A.; Raine, C.S.

    1995-07-01

    The ability of chronically demyelinated central nervous system (CNS) tissue to express major histocompatibility complex (MHC) class II molecules has been measured in mouse spinal cord cultures exposed for 1 and 3 weeks to demyelinating anti-white matter (WM) serum. From previous studies, It was known that after 3 weeks of demyelination in vitro, such cultures are incapable of remyelination. In the present report, MHC II levels were evaluated by immunocytochemistry and by Western and Northern blots. The results have shown that after both 1 and 3 weeks of exposure to myelinotoxic anti-WM serum, the cultures retained the ability to express MHC II and this could be further upregulated by incubation with interferon {gamma} (IFN{gamma}). Control groups showed increased expression of MHC II with age. By immunocytochemistry, all groups of cultures expressed high levels of MHC II and all groups showed upregulation after IFN{gamma} treatment. Anti-WM-treated cultures demonstrated slightly higher levels of MHC II than controls. Morphologically, the MHC II expression was associated with the surface of astrocytes. Semiquantitative analysis by Western blotting confirmed the increase in class II MHC expression in the long-term treated cultures after IFN{gamma} exposure, revealing no differences between anti-WM-treated and complement-treated cultures. This was also supported by Northern blotting which showed similar mRNA levels in both groups. These findings suggest that long-term demyelinated CNS tissue still possesses the ability to interact with CD4{sup +} T cells, observations of significance to the expansion of the chronic multiple sclerosis lesion. 50 refs., 6 figs., 2 tabs.

  14. Angiotensin-converting enzyme affects the presentation of MHC class II antigens.

    PubMed

    Zhao, Tuantuan; Bernstein, Kenneth E; Fang, Jianmin; Shen, Xiao Z

    2017-07-01

    Antigen processing and presentation through the MHC class II pathway is critical for activating T helper cells. Angiotensin-converting enzyme (ACE) is a carboxyl peptidase expressed by antigen-presenting cells. By analysis of ACE null (knockout), wild-type, and ACE-overexpressing (ACE10) mice and the antigen-presenting cells derived from these mice, we found that ACE has a physiological role in the processing of peptides for MHC class II presentation. The efficiency of presenting MHC class II epitopes from ovalbumin (OVA) and hen egg lysosome is markedly affected by cellular ACE levels. Mice overexpressing ACE in myeloid cells have a much more vigorous CD4(+) T-cell and antibody response when immunized with OVA. ACE is present in the endosomal pathway where MHC class II peptide processing and loading occur. The efficiency of MHC class II antigen presentation can be altered by ACE overexpression or ACE pharmacological inhibition. Thus, ACE is a dynamic participant in processing MHC class II peptides. Manipulation of ACE expression by antigen-presenting cells may prove to be a novel strategy to alter the immune response.

  15. IRF-4-mediated CIITA transcription is blocked by KSHV encoded LANA to inhibit MHC II presentation.

    PubMed

    Cai, Qiliang; Banerjee, Shuvomoy; Cervini, Amanda; Lu, Jie; Hislop, Andrew D; Dzeng, Richard; Robertson, Erle S

    2013-10-01

    Peptides presentation to T cells by MHC class II molecules is of importance in initiation of immune response to a pathogen. The level of MHC II expression directly influences T lymphocyte activation and is often targeted by various viruses. Kaposi's sarcoma-associated herpesvirus (KSHV) encoded LANA is known to evade MHC class I peptide processing, however, the effect of LANA on MHC class II remains unclear. Here, we report that LANA down-regulates MHC II expression and presentation by inhibiting the transcription of MHC II transactivator (CIITA) promoter pIII and pIV in a dose-dependent manner. Strikingly, although LANA knockdown efficiently disrupts the inhibition of CIITA transcripts from its pIII and pIV promoter region, the expression of HLA-DQβ but no other MHC II molecules was significantly restored. Moreover, we revealed that the presentation of HLA-DQβ enhanced by LANA knockdown did not help LANA-specific CD4+ T cell recognition of PEL cells, and the inhibition of CIITA by LANA is independent of IL-4 or IFN-γ signaling but dependent on the direct interaction of LANA with IRF-4 (an activator of both the pIII and pIV CIITA promoters). This interaction dramatically blocked the DNA-binding ability of IRF-4 on both pIII and pIV promoters. Thus, our data implies that LANA can evade MHC II presentation and suppress CIITA transcription to provide a unique strategy of KSHV escape from immune surveillance by cytotoxic T cells.

  16. Immunological Functions of the Membrane Proximal Region of MHC Class II Molecules

    PubMed Central

    Harton, Jonathan; Jin, Lei; Hahn, Amy; Drake, Jim

    2016-01-01

    Major histocompatibility complex (MHC) class II molecules present exogenously derived antigen peptides to CD4 T cells, driving activation of naïve T cells and supporting CD4-driven immune functions. However, MHC class II molecules are not inert protein pedestals that simply bind and present peptides. These molecules also serve as multi-functional signaling molecules delivering activation, differentiation, or death signals (or a combination of these) to B cells, macrophages, as well as MHC class II-expressing T cells and tumor cells. Although multiple proteins are known to associate with MHC class II, interaction with STING (stimulator of interferon genes) and CD79 is essential for signaling. In addition, alternative transmembrane domain pairing between class II α and β chains influences association with membrane lipid sub-domains, impacting both signaling and antigen presentation. In contrast to the membrane-distal region of the class II molecule responsible for peptide binding and T-cell receptor engagement, the membrane-proximal region (composed of the connecting peptide, transmembrane domain, and cytoplasmic tail) mediates these “non-traditional” class II functions. Here, we review the literature on the function of the membrane-proximal region of the MHC class II molecule and discuss the impact of this aspect of class II immunobiology on immune regulation and human disease. PMID:27006762

  17. MHC evolution in three salmonid species: a comparison between class II alpha and beta genes.

    PubMed

    Gómez, Daniela; Conejeros, Pablo; Marshall, Sergio H; Consuegra, Sofia

    2010-08-01

    The genes of the major histocompatibility complex (MHC) are amongst the most variable in vertebrates and represent some of the best candidates to study processes of adaptive evolution. However, despite the number of studies available, most of the information on the structure and function of these genes come from studies in mammals and birds in which the MHC class I and II genes are tightly linked and class II alpha exhibits low variability in many cases. Teleost fishes are among the most primitive vertebrates with MHC and represent good organisms for the study of MHC evolution because their class I and class II loci are not physically linked, allowing for independent evolution of both classes of genes. We have compared the diversity and molecular mechanisms of evolution of classical MH class II alpha and class II beta loci in farm populations of three salmonid species: Oncorhynchus kisutch, Oncorhynchus mykiss and Salmo salar. We found single classical class II loci and high polymorphism at both class II alpha and beta genes in the three species. Mechanisms of evolution were common for both class II genes, with recombination and point mutation involved in generating diversity and positive selection acting on the peptide-binding residues. These results suggest that the maintenance of variability at the class IIalpha gene could be a mechanism to increase diversity in the MHC class II in salmonids in order to compensate for the expression of one single classical locus and to respond to a wider array of parasites.

  18. Hyperexpression of interferon-gamma-induced MHC class II genes associated with reorganization of the cytoskeleton.

    PubMed Central

    Ulevitch, R. J.; Kline, L.; Schreiber, R. D.; Pingel, J.; Amaldi, I.; Reith, W.; Mach, B.

    1991-01-01

    Class I and class II major histocompatibility complex (MHC) gene products are key recognition units in the induction and regulation of the immune response. Expression of class I and class II may be constitutive or inducible by cytokines such as interferon-gamma (IFN-gamma). A key step in the induction of MHC genes is recognition of IFN-gamma by its membrane receptor. The work described here examines the regulation of the occupied IFN-gamma receptor by the cytoskeleton. To do this the authors have used the fungal metabolites dihydrocytochalasin B (DHCB) and cytochalasin D (CD), substances that bind to actin filaments and thereby disrupt the cytoskeleton. The authors have studied the effect of DHCB and CD on IFN-gamma-induced MHC gene expression in 143 B cells, a human osteosarcoma-derived cell line. Herein the authors demonstrate that alterations in the cytoskeleton induced by DHCB and CD can lead to increases in IFN-gamma-induced MHC gene expression. Dihydrocytochalasin B added up to 3 hours after IFN-gamma results in a threefold to sixfold increase in levels of class II mRNA while producing minimal enhancement of class I gene expression. In contrast, glyceraldehyde-3-phosphate dehydrogenase mRNA expression was unaltered by IFN-gamma or by the cytochalasins. The increased amount of class II mRNA can be accounted for by a concomitant increase in transcription rate of this gene. Studies using 125I-IFN-gamma demonstrate that the occupied IFN-gamma receptor associates with a Triton X-100 insoluble fraction of 143 B cells and that DHCB and CD markedly inhibit this association. The results described here provide evidence that is consistent with the hypothesis that the activity of the occupied IFN-gamma receptor may be modulated by interactions with the cytoskeleton of the cell. This receptor may be one of a group of plasma membrane receptors that are sensitive to the action of cytochalasins after ligand binding. Images Figure 1 Figure 2 PMID:1907805

  19. Regulation of MHC II and CD1 antigen presentation: from ubiquity to security.

    PubMed

    Gelin, Catherine; Sloma, Ivan; Charron, Dominique; Mooney, Nuala

    2009-02-01

    MHC class II and CD1-mediated antigen presentation on various APCs [B cells, monocytes, and dendritic cells (DC)] are subject to at least three distinct levels of regulation. The first one concerns the expression and structure of the antigen-presenting molecules; the second is based on the extracellular environment and signals of danger detected. However, a third level of regulation, which has been largely overlooked, is determined by lateral associations between antigen-presenting molecules and other proteins, their localization in specialized microdomains within the plasma membrane, and their trafficking pathways. This review focuses on features common to MHC II and CD1 molecules in their ability to activate specific T lymphocytes with the objective of addressing one basic question: What are the mechanisms regulating antigen presentation by MHC II and CD1 molecules within the same cell? Recent studies in immature DC, where MHC II and CD1 are coexpressed, suggest that the invariant chain (Ii) regulates antigen presentation by either protein. Ii could therefore favor MHC II or CD1 antigen presentation and thereby discriminate between antigens.

  20. The genetic architecture of the MHC class II region in British Texel sheep.

    PubMed

    Ali, Alsagher O A; Stear, Abigail; Fairlie-Clarke, Karen; Brujeni, Gholamreza Nikbakht; Isa, N Mahiza Md; Salisi, M Shahrom Bin; Donskow-Łysoniewska, Katarzyna; Groth, David; Buitkamp, Johannes; Stear, Michael J

    2017-03-01

    Understanding the structure of the major histocompatibility complex, especially the number and frequency of alleles, loci and haplotypes, is crucial for efficient investigation of the way in which the MHC influences susceptibility to disease. Nematode infection is one of the most important diseases suffered by sheep, and the class II region has been repeatedly associated with differences in susceptibility and resistance to infection. Texel sheep are widely used in many different countries and are relatively resistant to infection. This study determined the number and frequency of MHC class II genes in a small flock of Texel sheep. There were 18 alleles at DRB1, 9 alleles at DQA1, 13 alleles at DQB1, 8 alleles at DQA2 and 16 alleles at DQB2. Several haplotypes had no detectable gene products at DQA1, DQB1 or DQB2, and these were defined as null alleles. Despite the large numbers of alleles, there were only 21 distinct haplotypes in the population. The relatively small number of observed haplotypes will simplify finding disease associations because common haplotypes provide more statistical power but complicate the discrimination of causative mutations from linked marker loci.

  1. The overlooked "nonclassical" functions of major histocompatibility complex (MHC) class II antigens in immune and nonimmune cells.

    PubMed

    Altomonte, M; Pucillo, C; Maio, M

    1999-06-01

    Besides their "classical" antigenic peptide-presenting activity, major histocompatibility complex (MHC) class II antigens can activate different cellular functions in immune and nonimmune cells. However, this "nonclassical" role and its functional consequences are still substantially overlooked. In this review, we will focus on these alternative functional properties of MHC class II antigens, to reawaken attention to their present and foreseeable immunobiologic and pathogenetic implications. The main issues that will be addressed concern 1) the role of MHC class II molecules as basic components of exchangeable oligomeric protein complexes with intracellular signaling ability; 2) the nonclassical functions of MHC class II antigens in immune cells; 3) the pathogenetic role of MHC class II antigens in inflammatory/autoimmune and infectious disease; and 4) the functional role of MHC class II antigens in solid malignancies.

  2. Susceptibility of amphibians to chytridiomycosis is associated with MHC class II conformation

    PubMed Central

    Bataille, Arnaud; Cashins, Scott D.; Grogan, Laura; Skerratt, Lee F.; Hunter, David; McFadden, Michael; Scheele, Benjamin; Brannelly, Laura A.; Macris, Amy; Harlow, Peter S.; Bell, Sara; Berger, Lee; Waldman, Bruce

    2015-01-01

    The pathogenic chytrid fungus Batrachochytrium dendrobatidis (Bd) can cause precipitous population declines in its amphibian hosts. Responses of individuals to infection vary greatly with the capacity of their immune system to respond to the pathogen. We used a combination of comparative and experimental approaches to identify major histocompatibility complex class II (MHC-II) alleles encoding molecules that foster the survival of Bd-infected amphibians. We found that Bd-resistant amphibians across four continents share common amino acids in three binding pockets of the MHC-II antigen-binding groove. Moreover, strong signals of selection acting on these specific sites were evident among all species co-existing with the pathogen. In the laboratory, we experimentally inoculated Australian tree frogs with Bd to test how each binding pocket conformation influences disease resistance. Only the conformation of MHC-II pocket 9 of surviving subjects matched those of Bd-resistant species. This MHC-II conformation thus may determine amphibian resistance to Bd, although other MHC-II binding pockets also may contribute to resistance. Rescuing amphibian biodiversity will depend on our understanding of amphibian immune defence mechanisms against Bd. The identification of adaptive genetic markers for Bd resistance represents an important step forward towards that goal. PMID:25808889

  3. Susceptibility of amphibians to chytridiomycosis is associated with MHC class II conformation.

    PubMed

    Bataille, Arnaud; Cashins, Scott D; Grogan, Laura; Skerratt, Lee F; Hunter, David; McFadden, Michael; Scheele, Benjamin; Brannelly, Laura A; Macris, Amy; Harlow, Peter S; Bell, Sara; Berger, Lee; Waldman, Bruce

    2015-04-22

    The pathogenic chytrid fungus Batrachochytrium dendrobatidis (Bd) can cause precipitous population declines in its amphibian hosts. Responses of individuals to infection vary greatly with the capacity of their immune system to respond to the pathogen. We used a combination of comparative and experimental approaches to identify major histocompatibility complex class II (MHC-II) alleles encoding molecules that foster the survival of Bd-infected amphibians. We found that Bd-resistant amphibians across four continents share common amino acids in three binding pockets of the MHC-II antigen-binding groove. Moreover, strong signals of selection acting on these specific sites were evident among all species co-existing with the pathogen. In the laboratory, we experimentally inoculated Australian tree frogs with Bd to test how each binding pocket conformation influences disease resistance. Only the conformation of MHC-II pocket 9 of surviving subjects matched those of Bd-resistant species. This MHC-II conformation thus may determine amphibian resistance to Bd, although other MHC-II binding pockets also may contribute to resistance. Rescuing amphibian biodiversity will depend on our understanding of amphibian immune defence mechanisms against Bd. The identification of adaptive genetic markers for Bd resistance represents an important step forward towards that goal.

  4. High-throughput engineering and analysis of peptide binding to class II MHC

    PubMed Central

    Jiang, Wei; Boder, Eric T.

    2010-01-01

    Class II major histocompatibility complex (MHC-II) proteins govern stimulation of adaptive immunity by presenting antigenic peptides to CD4+ T lymphocytes. Many allelic variants of MHC-II exist with implications in peptide presentation and immunity; thus, high-throughput experimental tools for rapid and quantitative analysis of peptide binding to MHC-II are needed. Here, we present an expression system wherein peptide and MHC-II are codisplayed on the surface of yeast in an intracellular association-dependent manner and assayed by flow cytometry. Accordingly, the relative binding of different peptides and/or MHC-II variants can be assayed by genetically manipulating either partner, enabling the application of directed evolution approaches for high-throughput characterization or engineering. We demonstrate the application of this tool to map the side-chain preference for peptides binding to HLA-DR1 and to evolve novel HLA-DR1 mutants with altered peptide-binding specificity. PMID:20622157

  5. Studying MHC class II presentation of immobilized antigen by B lymphocytes.

    PubMed

    Yuseff, M I; Lennon-Dumenil, A M

    2013-01-01

    The ability of B lymphocytes to capture external antigens (Ag) and present them as peptide fragments, loaded on Major Histocompatibility complex (MHC) class II molecules, to CD4(+) T cells is a crucial part of the adaptive immune response. This allows T-B cooperation, a cellular communication that is required for B cells to develop into germinal centers (GC) and form mature high-affinity antibody producing cells and to further develop B cell memory. MHC class II antigen presentation by B lymphocytes is a multistep process involving (1) Recognition and capture of external Ag by B lymphocytes through their B cell receptor (BCR); (2) Ag processing, which comprises the degradation of Ag in internal compartments within the B cell and loading of the corresponding peptide fragments on MHC class II molecules and (3) Presentation of MHC II-peptide complexes to CD4(+) T cells. Here, we describe how to study MHC class II antigen presentation by B lymphocytes at these three major levels.

  6. The biogenesis of the MHC class II compartment in human I-cell disease B lymphoblasts

    PubMed Central

    1996-01-01

    The localization and intracellular transport of major histocompatibility complex (MHC) class II molecules nd lysosomal hydrolases were studied in I-Cell Disease (ICD) B lymphoblasts, which possess a mannose 6-phosphate (Man-6-P)-independent targeting pathway for lysosomal enzymes. In the trans-Golgi network (TGN), MHC class II- invariant chain complexes colocalized with the lysosomal hydrolase cathepsin D in buds and vesicles that lacked markers of clathrin-coated vesicle-mediated transport. These vesicles fused with the endocytic pathway leading to the formation of "early" MHC class II-rich compartments (MIICs). Similar structures were observed in the TGN of normal beta lymphoblasts although they were less abundant. Metabolic labeling and subcellular fractionation experiments indicated that newly synthesized cathepsin D and MHC class II-invariant chain complexes enter a non-clathrin-coated vesicular structure after their passage through the TGN and segregation from the secretory pathway. These vesicles were also devoid of the cation-dependent mannose 6-phosphate (Man-6-P) receptor, a marker of early and late endosomes. These findings suggest that in ICD B lymphoblasts the majority of MHC class II molecules are transported directly from the TGN to "early" MIICs and that acid hydrolases cam be incorporated into MIICs simultaneously by a Man-6-P-independant process. PMID:8603911

  7. Analysis of cDNA coding MHC class II beta chain of the chimpanzee (Pan troglodytes).

    PubMed

    Hatta, Yuki; Kanai, Tomoko; Matsumoto, Yoshitsugu; Kyuwa, Shigeru; Hayasaka, Ikuo; Yoshikawa, Yasuhiro

    2002-04-01

    The chimpanzee (Pan troglodytes, Patr) is the closest zoological living relative of humans and shares approximately 98.6% genetic homology to human beings. Although major histocompatibility complex (MHC) plays a critical role in T cell-mediated immune responses in vertebrates, the information on Patr MHC remains at a relatively poor level. Therefore, we attempted to isolate Patr MHC class II genes and determine their nucleotide sequences. The cDNAs encoding Patr MHC class II DP, DQ and DR beta chains were isolated from the cDNA library of a chimpanzee B lymphocyte cell line Bch261. As a result of screening, the clone 6-3-1 as a representative of Patr DP clone, clone 30-1 as a Patr DQ clone, and clones 4-7-1 and 55-1 having different sequences as Patr DR clones were detected. The clone 6-3-1 consisted of 1,062 nucleotides including an open reading frame (ORF) of 777 bp. In the same way, clone 30-1 consisted of 1,172 nucleotides including ORF of 786 bp, clones 4-7-1 and 55-1 consisted of 1,163 nucleotides including ORF of 801 bp. Except for five nucleotide changes, clones 4-7-1 and 55-1 were the same sequence. By comparison with the nucleotide sequences already reported on chimpanzee MHC class II beta 1 genes, clones 6-3-1, 30-1, 4-7-1 and 55-1 were classified as PatrDPB1*16, PatrDQB1*0302, PatrDRB1*0201 and PatrDRB1*0204, respectively. This is the first report to describe complete cDNA sequences of Patr DP and DQ molecules. The nucleotide sequence data of Patr MHC class II genes obtained in this study will be useful for the genotyping of Patr MHC class II genes in individual chimpanzees.

  8. CDw78 defines MHC class II-peptide complexes that require Ii chain-dependent lysosomal trafficking, not localization to a specific tetraspanin membrane microdomain.

    PubMed

    Poloso, Neil J; Denzin, Lisa K; Roche, Paul A

    2006-10-15

    MHC class II molecules (MHC-II) associate with detergent-resistant membrane microdomains, termed lipid rafts, which affects the function of these molecules during Ag presentation to CD4+ T cells. Recently, it has been proposed that MHC-II also associates with another type of membrane microdomain, termed tetraspan microdomains. These microdomains are defined by association of molecules to a family of proteins that contain four-transmembrane regions, called tetraspanins. It has been suggested that MHC-II associated with tetraspanins are selectively identified by a mAb to a MHC-II determinant, CDw78. In this report, we have re-examined this issue of CDw78 expression and MHC-II-association with tetraspanins in human dendritic cells, a variety of human B cell lines, and MHC-II-expressing HeLa cells. We find no correlation between the expression of CDw78 and the expression of tetraspanins CD81, CD82, CD53, CD9, and CD37. Furthermore, we find that the relative amount of tetraspanins bound to CDw78-reactive MHC-II is indistinguishable from the amount bound to peptide-loaded MHC-II. We found that expression of CDw78 required coexpression of MHC-II together with its chaperone Ii chain. In addition, analysis of a panel of MHC-II-expressing B cell lines revealed that different alleles of HLA-DR express different amounts of CDw78 reactivity. We conclude that CDw78 defines a conformation of MHC-II bound to peptides that are acquired through trafficking to lysosomal Ag-processing compartments and not MHC-II-associated with tetraspanins.

  9. Antigen-specific tumor vaccine efficacy in vivo against prostate cancer with low class I MHC requires competent class II MHC.

    PubMed

    Neeley, Yilin C; McDonagh, Kevin T; Overwijk, Willem W; Restifo, Nicholas P; Sanda, Martin G

    2002-11-01

    Cancers can escape immune recognition by means of evading class I major histocompatibility complex (MHC) -mediated recognition by cytotoxic T lymphocytes. However, immunization strategies targeting defined tumor-associated antigens have not been extensively characterized in murine prostate cancer models. Therefore, we evaluated antigen-specific, antitumor immunity after antigen-encoding vaccinia immunization against mouse prostate cancer cells expressing a model tumor-associated antigen (beta-galactosidase) and exhibiting partially deficient class I MHC. Low class I MHC expression in beta-galactosidase-expressing D7RM-1 prostate cancer cells was shown by fluorescence activated cell sorting, and deficient class I MHC-mediated antigen presentation was shown in resistance of D7RM-1 to cytolysis by beta-galactosidase-specific cytotoxic T lymphocytes (CTL). Despite partially deficient class I MHC presenting function, immunization with vaccinia encoding beta-galactosidase conferred antigen-specific protection against D7RM-1 cancer. Antigen-specific immunity was recapitulated in beta(2)m knockout mice (with deficient class I MHC and CTL function), confirming that class I MHC antigen presentation was not required for immunity against tumor partially deficient in class I MHC. Conversely, antigen-specific antitumor immunity was abrogated in A(b)beta knockout mice (with deficient class II MHC and helper T cell function), demonstrating a requirement for functional class II MHC. Resistant tumors from the otherwise effectively immunized beta(2)m knockout mice (among which tumor progression had been reduced or delayed) showed reduced target antigen expression, corroborating antigen-specificity (and showing an alternative immune escape mechanism), whereas antigen expression (like tumor growth) was unaffected among A(b)beta knockout mice. Our results demonstrate that class I MHC-restricted antigen presentation and CTL activity is neither necessary nor sufficient for antigen

  10. Differential Transmembrane Domain GXXXG Motif Pairing Impacts Major Histocompatibility Complex (MHC) Class II Structure*

    PubMed Central

    Dixon, Ann M.; Drake, Lisa; Hughes, Kelly T.; Sargent, Elizabeth; Hunt, Danielle; Harton, Jonathan A.; Drake, James R.

    2014-01-01

    Major histocompatibility complex (MHC) class II molecules exhibit conformational heterogeneity, which influences their ability to stimulate CD4 T cells and drive immune responses. Previous studies suggest a role for the transmembrane domain of the class II αβ heterodimer in determining molecular structure and function. Our previous studies identified an MHC class II conformer that is marked by the Ia.2 epitope. These Ia.2+ class II conformers are lipid raft-associated and able to drive both tyrosine kinase signaling and efficient antigen presentation to CD4 T cells. Here, we establish that the Ia.2+ I-Ak conformer is formed early in the class II biosynthetic pathway and that differential pairing of highly conserved transmembrane domain GXXXG dimerization motifs is responsible for formation of Ia.2+ versus Ia.2− I-Ak class II conformers and controlling lipid raft partitioning. These findings provide a molecular explanation for the formation of two distinct MHC class II conformers that differ in their inherent ability to signal and drive robust T cell activation, providing new insight into the role of MHC class II in regulating antigen-presenting cell-T cell interactions critical to the initiation and control of multiple aspects of the immune response. PMID:24619409

  11. Fluorogenic Probes for Monitoring Peptide Binding to Class II MHC Proteins in Living Cells

    SciTech Connect

    Venkatraman,P.; Nguyen, T.; Sainlos, M.; Bilsel, o.; Chitta, S.; Imperiali, B.; Stern, L.

    2007-01-01

    A crucial step in the immune response is the binding of antigenic peptides to major histocompatibility complex (MHC) proteins. Class II MHC proteins present their bound peptides to CD4+ T cells, thereby helping to activate both the humoral and the cellular arms of the adaptive immune response. Peptide loading onto class II MHC proteins is regulated temporally, spatially and developmentally in antigen-presenting cells1. To help visualize these processes, we have developed a series of novel fluorogenic probes that incorporate the environment-sensitive amino acid analogs 6-N,N-dimethylamino-2-3-naphthalimidoalanine and 4-N,N-dimethylaminophthalimidoalanine. Upon binding to class II MHC proteins these fluorophores show large changes in emission spectra, quantum yield and fluorescence lifetime. Peptides incorporating these fluorophores bind specifically to class II MHC proteins on antigen-presenting cells and can be used to follow peptide binding in vivo. Using these probes we have tracked a developmentally regulated cell-surface peptide-binding activity in primary human monocyte-derived dendritic cells.

  12. Expression of Functional MHC Class II Molecules By a Mouse Pro-B Cell Clone

    PubMed Central

    Fisher, Amanda G.; Meyer, Valérie; Ceredig, Rhodri

    1995-01-01

    We describe here the G12 pro-B cell clone that has been isolated from an IL-7 transgenic mouse. This clone has the phenotype B220+, BP-1+ , HSA +, CD43+ λ5+ , and CD25-, and has its Ig locus in a germline configuration. G12 cells spontaneously express cell-surface MHC class II molecules, although to a much lesser extent than the mature M12.4.1 B-cell lymphoma. G12 cells can process and present the native Hen Egg Lysozyme (HEL) to an MHC class II-restricted T-cell hybridoma. The efficiency of presentation is inferior to that obtained with M12.4.1 cells. This is the first report where a pro-B cell can serve as APC in an MHC class II-restricted presentation. PMID:9700358

  13. The nucleotide sequence of the sheep MHC class II DNA gene

    SciTech Connect

    Wright, H.; Redmond, J.; Ballingall, K.T.; Wright, F.

    1995-01-11

    The human MHC class II DNA gene was identified and sequenced by Trowsdale and Kelly. When a molecular map of the HLA-D region became available, it was shown that the HLA-DNA gene was unusual in not having a B gene partner situated within a few kilobases (kb), the nearest B gene being HLA-DPB1. The nearest unpaired B gene is HLA-DOB which is approximately 160 kb telomeric of HLA-DNA. More recently, the mouse MHC class II genes H-20A and H-20B were shown to be equivalent to the HLA-DNA and HLA-DOB genes. Moreover, the mouse genes expressed an MHC class II protein whose tissue distribution was restricted to B cells and epithelial cell of the thymic medulla. No corresponding HLA-DN protein has been reported. 21 refs., 3 figs.

  14. Human cytomegalovirus decreases constitutive transcription of MHC class II genes in mature Langerhans cells by reducing CIITA transcript levels.

    PubMed

    Lee, Andrew W; Wang, Nan; Hornell, Tara M C; Harding, James J; Deshpande, Chetan; Hertel, Laura; Lacaille, Vashti; Pashine, Achal; Macaubas, Claudia; Mocarski, Edward S; Mellins, Elizabeth D

    2011-05-01

    Human cytomegalovirus (HCMV) productively infects CD34(+) progenitor-derived, mature Langerhans-type dendritic cells (matLC) and reduces surface expression of MHC class II complexes (MHC II) by increasing intracellular retention of these molecules. To determine whether HCMV also inhibits MHC II expression by other mechanisms, we assessed mRNA levels of the class II transcriptional regulator, CIITA, and several of its target genes in infected matLC. Levels of CIITA, HLA-DRA (DRA) and DRB transcripts, and new DR protein synthesis were compared in mock-infected and HCMV-infected cells by quantitative PCR and pulse-chase immunoprecipitation analyses, respectively. CIITA mRNA levels were significantly lower in HCMV-infected matLC as compared to mock-infected cells. When assessed in the presence of Actinomycin D, the stability of CIITA transcripts was not diminished by HCMV. Analysis of promoter-specific CIITA isoforms revealed that types I, III and IV all were decreased by HCMV, a result that differs from changes after incubation of these cells with lipopolysaccharide (LPS). Exposure to UV-inactivated virus failed to reduce CIITA mRNA levels, implicating de novo viral gene expression in this effect. HCMV-infected matLC also expressed lower levels of DR transcripts and reduced DR protein synthesis rates compared to mock-infected matLC. In summary, we demonstrate that HCMV infection of a human dendritic cell subset inhibits constitutive CIITA expression, most likely at the transcriptional level, resulting in reduced MHC II biosynthesis. We suggest this represents a new mechanism of modulation of mature LC by HCMV.

  15. Human cytomegalovirus decreases constitutive transcription of MHC class II genes in mature Langerhans cells by reducing CIITA transcript levels

    PubMed Central

    Lee, Andrew W.; Wang, Nan; Hornell, Tara M.C.; Harding, James J.; Deshpande, Chetan; Hertel, Laura; Lacaille, Vashti; Pashine, Achal; Macaubas, Claudia; Mocarski, Edward S.; Mellins, Elizabeth D.

    2011-01-01

    Human cytomegalovirus (HCMV) productively infects CD34+ progenitor-derived, mature Langerhans-type dendritic cells (matLC) and reduces surface expression of MHC class II complexes (MHC II) by increasing intracellular retention of these molecules. To determine whether HCMV also inhibits MHC II expression by other mechanisms, we assessed mRNA levels of the class II transcriptional regulator, CIITA, and several of its target genes in infected matLC. Levels of CIITA, HLA-DRA (DRA) and DRB transcripts, and new DR protein synthesis were compared in mock-infected and HCMV-infected cells by quantitative PCR and pulse-chase immunoprecipitation analyses, respectively. CIITA mRNA levels were significantly lower in HCMV-infected matLC as compared to mock-infected cells. When assessed in the presence of Actinomycin D, the stability of CIITA transcripts was not diminished by HCMV. Analysis of promoter-specific CIITA isoforms revealed that types I, III and IV all were decreased by HCMV, a result that differs from changes after incubation of these cells with lipopolysaccharide (LPS). Exposure to UV-inactivated virus failed to reduce CIITA mRNA levels, implicating de novo viral gene expression in this effect. HCMV-infected matLC also expressed lower levels of DR transcripts and reduced DR protein synthesis rates compared to mock-infected matLC. In summary, we demonstrate that HCMV infection of a human dendritic cell subset inhibits constitutive CIITA expression, most likely at the transcriptional level, resulting in reduced MHC II biosynthesis. We suggest this represents a new mechanism of modulation of mature LC by HCMV. PMID:21458073

  16. MHC class II-associated proteins in B-cell exosomes and potential functional implications for exosome biogenesis.

    PubMed

    Buschow, Sonja I; van Balkom, Bas W M; Aalberts, Marian; Heck, Albert J R; Wauben, Marca; Stoorvogel, Willem

    2010-01-01

    Professional antigen-presenting cells secrete major histocompatibility complex class II (MHC II) carrying exosomes with unclear physiological function(s). Exosomes are first generated as the intraluminal vesicles (ILVs) of a specific type of multivesicular body, and are then secreted by fusion of this compartment with the plasma membrane. We have previously shown that in contrast to the sorting of MHC II at lysosomally targeted multivesicular bodies, sorting of MHC II into exosomes does not rely on MHC II ubiquitination. In search for proteins that drive the incorporation of MHC II into exosomes or functionally discriminate exosomal from plasma membrane MHC II, we first analyzed the total proteome of highly purified B cell-derived exosomes using sensitive and accurate mass spectrometry (MS), and identified 539 proteins, including known and not previously identified constituents. Using quantitative MS, we then identified a small subset of proteins that were specifically co-immunoprecipitated with MHC II from detergent-solubilized exosomes. These include HSC71, HSP90, 14-3-3ɛ, CD20 and pyruvate kinase type M2 (PKM2), and we speculate on the functionality of their interaction with exosomal MHC II.

  17. Characterization of MHC class I and II genes in a subantarctic seabird, the blue petrel, Halobaena caerulea (Procellariiformes).

    PubMed

    Strandh, Maria; Lannefors, Mimi; Bonadonna, Francesco; Westerdahl, Helena

    2011-10-01

    The great polymorphism observed in the major histocompatibility complex (MHC) genes is thought to be maintained by pathogen-mediated selection possibly combined with MHC-disassortative mating, guided by MHC-determined olfactory cues. Here, we partly characterize the MHC class I and II B of the blue petrel, Halobaena caerulea (Procellariiformes), a bird with significant olfactory abilities that lives under presumably low pathogen burdens in Subantarctica. Blue petrels are long-lived, monogamous birds which suggest the necessity of an accurate mate choice process. The species is ancestral to songbirds (Passeriformes; many MHC loci), although not to gamefowls (Galliformes; few MHC loci). Considering the phylogenetic relationships and the low subantarctic pathogen burden, we expected few rather than many MHC loci in the blue petrel. However, when we analysed partial MHC class I and class II B cDNA and gDNA sequences we found evidence for as many as at least eight MHC class I loci and at least two class II B loci. These class I and II B sequences showed classical MHC characteristics, e.g. high nucleotide diversity, especially in putative peptide-binding regions where signatures of positive selection was detected. Trans-species polymorphism was found between MHC class II B sequences of the blue petrel and those of thin-billed prion, Pachyptila belcheri, two species that diverged ∼25 MYA. The observed MHC allele richness in the blue petrel may well serve as a basis for mate choice, especially since olfactory discrimination of MHC types may be possible in this species.

  18. MHC class II β genes in the endangered Hainan Eld's deer (Cervus eldi hainanus).

    PubMed

    Liu, Hong-Yi; Xue, Fei; Wan, Qiu-Hong; Ge, Yun-Fa

    2013-01-01

    Contrary to neutral markers, the major histocompatibility complex (MHC) can reflect the fitness and adaptive potential of a given species due to its association with the immune system. For this reason, the use of MHC in endangered wildlife management has increased greatly in recent years. Here, we isolated complementary DNA (cDNA) and genomic DNA (gDNA) sequences to characterize the MHC class II β genes in Hainan Eld's deer (Cervus eldi hainanus), a highly endangered cervid, which recovered from a severe population bottleneck consisting of 26 animals. Analysis of 7 individuals revealed the presence of 3 DRB and 3 DQB putatively functional gDNA sequences. The Ceel-DRB and DQB sequences displayed high variability in exon 2, and most nonsynonymous substitutions were detected in this region. Phylogenetic analysis indicated that trans-species evolution of MHC class II β might occur in the Cervinae subfamily. Comparison of the number of sequences between gDNA and cDNA revealed that all sequences isolated from the genome were detectable in the cDNA libraries derived from different tissues (including the liver, kidney, and spleen), suggesting none of these sequences were derived from silent genes or pseudogenes. Characterization of the MHC class II β genes may lay the foundation for future studies on genetic structure, mate choice, and viability analysis in Hainan Eld's deer.

  19. Tumor-specific CD4+ T cells eradicate myeloma cells genetically deficient in MHC class II display

    PubMed Central

    Tveita, Anders; Fauskanger, Marte; Bogen, Bjarne; Haabeth, Ole Audun Werner

    2016-01-01

    CD4+ T cells have been shown to reject tumor cells with no detectable expression of major histocompatibility complex class II (MHC II). However, under certain circumstances, induction of ectopic MHC II expression on tumor cells has been reported. To confirm that CD4+ T cell-mediated anti-tumor immunity can be successful in the complete absence of antigen display on the tumor cells themselves, we eliminated MHC II on tumor cells using CRISPR/Cas9. Our results demonstrate that ablation of the relevant MHC II (I-Ed) in multiple myeloma cells (MOPC315) does not hinder rejection by tumor-specific CD4+ T cells. These findings provide conclusive evidence that CD4+ T cells specific for tumor antigens can eliminate malignant cells in the absence of endogenous MHC class II expression on the tumor cells. This occurs through antigen uptake and indirect presentation on tumor-infiltrating macrophages. PMID:27626487

  20. Isolation and characterization of three class II MHC genomic clones from the chicken.

    PubMed

    Xu, Y X; Pitcovski, J; Peterson, L; Auffray, C; Bourlet, Y; Gerndt, B M; Nordskog, A W; Lamont, S J; Warner, C M

    1989-03-15

    A genomic library was constructed from sperm DNA from an individual of the inbred chicken line G-B2, MHC haplotype B6. The library was screened with a chicken class II probe (beta 2 exon specific) and three MHC class II beta chain genomic clones were isolated. The restriction maps of the three clones showed that each of the three clones was unique. The position of the beta chain sequence was located in each of the three genomic clones by Southern blot hybridization. Subclones containing the beta chain gene were produced from each of the genomic clones and the orientation of the leader peptide, beta 1, beta 2, transmembrane, and cytoplasmic exons was determined by Southern blot hybridization and nucleotide sequencing. The complete nucleotide sequence of two of the three subclones was determined. Comparison of the nucleotide and predicted amino acid sequences of the two subclones with other class II beta chain sequences showed that the B6 chicken beta chain genes are evolutionarily related to the class II beta chain genes from chickens of other MHC haplotypes, and to class II beta chain genes from other species. Analysis of Southern blots of B6 chicken DNA, as well as the isolation of the three beta chain genes, suggests that chickens of the B6 haplotype possess at least three MHC class II beta chain genes.

  1. Characterization, polymorphism, and evolution of MHC class II B genes in birds of prey.

    PubMed

    Alcaide, Miguel; Edwards, Scott V; Negro, Juan J

    2007-11-01

    During the last decade, the major histocompatibility complex (MHC) has received much attention in the fields of evolutionary and conservation biology because of its potential implications in many biological processes. New insights into the gene structure and evolution of MHC genes can be gained through study of additional lineages of birds not yet investigated at the genomic level. In this study, we characterized MHC class II B genes in five families of birds of prey (Accipitridae, Pandionidae, Strigidae, Tytonidae, and Falconidae). Using PCR approaches, we isolated genomic MHC sequences up to 1300 bp spanning exons 1 to 3 in 26 representatives of each raptor lineage, finding no stop codons or frameshift mutations in any coding region. A survey of diversity across the entirety of exon 2 in the lesser kestrel Falco naumanni reported 26 alleles in 21 individuals. Bayesian analysis revealed 21 positively selected amino acid sites, which suggests that the MHC genes described here are functional and probably expressed. Finally, through interlocus comparisons and phylogenetic analysis, we also discuss genetic evidence for concerted and transspecies evolution in the raptor MHC.

  2. Single-Molecule Motions of MHC Class II Rely on Bound Peptides

    PubMed Central

    Kozono, Haruo; Matsushita, Yufuku; Ogawa, Naoki; Kozono, Yuko; Miyabe, Toshihiro; Sekiguchi, Hiroshi; Ichiyanagi, Kouhei; Okimoto, Noriaki; Taiji, Makoto; Kanagawa, Osami; Sasaki, Yuji C.

    2015-01-01

    The major histocompatibility complex (MHC) class II protein can bind peptides of different lengths in the region outside the peptide-binding groove. Peptide-flanking residues (PFRs) contribute to the binding affinity of the peptide for MHC and change the immunogenicity of the peptide/MHC complex with regard to T cell receptor (TCR). The mechanisms underlying these phenomena are currently unknown. The molecular flexibility of the peptide/MHC complex may be an important determinant of the structures recognized by certain T cells. We used single-molecule x-ray analysis (diffracted x-ray tracking (DXT)) and fluorescence anisotropy to investigate these mechanisms. DXT enabled us to monitor the real-time Brownian motion of the peptide/MHC complex and revealed that peptides without PFRs undergo larger rotational motions than peptides with PFRs. Fluorescence anisotropy further revealed that peptides without PFRs exhibit slightly larger motions on the nanosecond timescale. These results demonstrate that peptides without PFRs undergo dynamic motions in the groove of MHC and consequently are able to assume diverse structures that can be recognized by T cells. PMID:25606683

  3. SYK regulates macrophage MHC-II expression via activation of autophagy in response to oxidized LDL

    PubMed Central

    Choi, Soo-Ho; Gonen, Ayelet; Diehl, Cody J; Kim, Jungsu; Almazan, Felicidad; Witztum, Joseph L; Miller, Yury I

    2015-01-01

    Adaptive immunity, which plays an important role in the development of atherosclerosis, is mediated by major histocompatibility complex (MHC)-dependent antigen presentation. In atherosclerotic lesions, macrophages constitute an important class of antigen-presenting cells that activate adaptive immune responses to oxidized low-density lipoprotein (OxLDL). It has been reported that autophagy regulates adaptive immune responses by enhancing antigen presentation to MHC class II (MHC-II). In a previous study, we have demonstrated that SYK (spleen tyrosine kinase) regulates generation of reactive oxygen species (ROS) and activation of MAPK8/JNK1 in macrophages. Because ROS and MAPK8 are known to regulate autophagy, in this study we investigated the role of SYK in autophagy, MHC-II expression and adaptive immune response to OxLDL. We demonstrate that OxLDL induces autophagosome formation, MHC-II expression, and phosphorylation of SYK in macrophages. Gene knockout and pharmacological inhibitors of NOX2 and MAPK8 reduced OxLDL-induced autophagy. Using bone marrow-derived macrophages isolated from wild-type and myeloid-specific SYK knockout mice, we demonstrate that SYK regulates OxLDL-induced ROS generation, MAPK8 activation, BECN1-BCL2 dissociation, autophagosome formation and presentation of OxLDL-derived antigens to CD4+ T cells. ldlr−/− syk−/− mice fed a high-fat diet produced lower levels of IgG to malondialdehyde (MDA)-LDL, malondialdehyde-acetaldehyde (MAA)-LDL, and OxLDL compared to ldlr−/− mice. These results provide new insights into the mechanisms by which SYK regulates MHC-II expression via autophagy in macrophages and may contribute to regulation of adaptive immune responses in atherosclerosis. PMID:25946330

  4. Cutting edge: requirement of MARCH-I-mediated MHC II ubiquitination for the maintenance of conventional dendritic cells.

    PubMed

    Ohmura-Hoshino, Mari; Matsuki, Yohei; Mito-Yoshida, Mari; Goto, Eiji; Aoki-Kawasumi, Masami; Nakayama, Manabu; Ohara, Osamu; Ishido, Satoshi

    2009-12-01

    MARCH-I (membrane-associated RING-CH I) has been suggested as a physiological E3 ubiquitin ligase for both MHC class II (MHC II) and B7-2. In this study, we show that MARCH-I-mediated MHC II ubiquitination is necessary for the maintenance of conventional dendritic cell (cDC) functions in the steady state. MARCH-I-deficient cDCs accumulated MHC II and B7-2 and exhibited low Ag-presenting ability for exogenous Ags and low cytokine-producing ability upon stimulation in vivo. Importantly, MHC II, but not B7-2, was required for impaired cDC function induced by loss of MARCH-I in vivo. Moreover, MHC II knockin mice whose MHC II was not ubiquitinated showed dysfunction of cDC similar to that of MARCH-I knockout mice. These results suggest that the accumulation of MHC II resulting from loss of ubiquitination caused cDC abnormality; therefore, MARCH-I may function as a housekeeper of cDC in the steady state.

  5. Molecular characterization of MHC class II in the Australian invasive cane toad reveals multiple splice variants.

    PubMed

    Lillie, Mette; Cui, Jian; Shine, Richard; Belov, Katherine

    2016-07-01

    The cane toad has gained notoriety for its invasion across the Australian landscape, with significant impacts on the native Australian fauna. The invasion has accelerated over time, with invading cane toads adapted for highly dispersive traits. This, however, has come at the cost of the immune system, with lower investment in some immune functions. To investigate the cane toad's immunogenetics, we characterized four major histocompatibility complex (MHC) class IIA and three MHC class IIB loci. Preliminary observations suggest very low allelic diversity at all loci. We also observed various splice isoforms. One isoform seen at one class IIA and two class IIB loci was missing exon 2, which is essential to peptide binding and presentation. The other isoform, observed at a class IIA locus, is likely to be a soluble MHC product. These results may suggest a significant role of alternative splicing of MHC loci in the Australian cane toad.

  6. Spectrum of MHC Class II Variability in Darwin’s Finches and Their Close Relatives

    PubMed Central

    Sato, Akie; Tichy, Herbert; Grant, Peter R.; Grant, B. Rosemary; Sato, Tetsuji; O’hUigin, Colm

    2011-01-01

    The study describes >400 major histocompatibility complex (MHC) class II B exon 2 and 114 intron 2 sequences of 36 passerine bird species, 13 of which belong to the group of Darwin’s finches (DFs) and the remaining 23 to close or more distant relatives of DFs in Central and South America. The data set is analyzed by a combination of judiciously selected statistical methods. The analysis reveals that reliable information concerning MHC organization, including the assignment of sequences to loci, and evolution, as well as the process of species divergence, can be obtained in the absence of genomic sequence data, if the analysis is taken several steps beyond the standard phylogenetic tree construction approach. The main findings of the present study are these: The MHC class II B region of the passerine birds is as elaborate in its organization, divergence, and genetic diversity as the MHC of the eutherian mammals, specifically the primates. Hence, the reported simplicity of the fowl MHC is an oddity. With the help of appropriate markers, the divergence of the MHC genes can be traced deep in the phylogeny of the bird taxa. Transspecies polymorphism is rampant at many of the bird MHC loci. In this respect, the DFs behave as if they were a single, genetically undifferentiated population. There is thus far no indication of alleles that could be considered species, genus, or even DF group specific. The implication of these findings is that DFs are in the midst of adaptive radiations, in which morphological differentiation into species is running ahead of genetic differentiation in genetic systems such as the MHC or the mitochondrial DNA. The radiations are so young that there has not been enough time to sort out polymorphisms at most of the loci among the morphologically differentiating species. These findings parallel those on Lake Victoria haplochromine fishes. Several of the DF MHC allelic lineages can be traced back to the MHC genes of the species Tiaris obscura

  7. Extensive sharing of MHC class II alleles between rhesus and cynomolgus macaques.

    PubMed

    Doxiadis, Gaby G M; Rouweler, Annemiek J M; de Groot, Natasja G; Louwerse, Annet; Otting, Nel; Verschoor, Ernst J; Bontrop, Ronald E

    2006-05-01

    In contrast to rhesus monkeys, substantial knowledge on cynomolgus monkey major histocompatibility complex (MHC) class II haplotypes is lacking. Therefore, 17 animals, including one pedigreed family, were thoroughly characterized for polymorphic Mhc class II region genes as well as their mitochondrial DNA (mtDNA) sequences. Different cynomolgus macaque populations appear to exhibit unique mtDNA profiles reflecting their geographic origin. Within the present panel, 10 Mafa-DPB1, 14 Mafa-DQA1, 12 Mafa-DQB1, and 35 Mafa-DRB exon 2 sequences were identified. All of these alleles cluster into lineages that were previously described for rhesus macaques. Moreover, about half of the Mafa-DPB1, Mafa-DQA1, and Mafa-DQB1 alleles and one third of the Mafa-DRB exon 2 sequences are identical to rhesus macaque orthologues. Such a high level of Mhc class II allele sharing has not been reported for primate species. Pedigree analysis allowed the characterization of nine distinct Mafa class II haplotypes, and seven additional ones could be deduced. Two of these haplotypes harbor a duplication of the Mafa-DQB1 locus. Despite extensive allele sharing, rhesus and cynomolgus monkeys do not appear to possess identical Mhc class II haplotypes, thus illustrating that new haplotypes were generated after speciation by recombination-like processes.

  8. Comparison of the transcriptional regulation of classical and non-classical MHC class II genes.

    PubMed

    Hake, Sandra B; Tobin, Helen M; Steimle, Viktor; Denzin, Lisa K

    2003-09-01

    The class II transactivator (CIITA) regulates expression of the classical and non-classical MHC class II genes, HLA-DR, -DP, -DQ and -DM, but not the B cell-specific HLA-DO (DO). Here we show that only HLA-DR expression is completely dependent on CIITA, since residual expression of HLA-DM, -DP and the beta chain of DQ was observed in CIITA-deficient RJ2.2.5 cells. Although DO shows a unique expression pattern compared to other MHC class II genes, prolonged IFN-gamma treatment of HeLa cells induced DOB expression. Similar to all MHC class II promoters, the DOB promoter contains the highly conserved W, X1, and Y boxes in addition to a putative OCT box. Mutational analysis of the DOB promoter demonstrated that the X1, Y and OCT boxes are necessary for maximum promoter activity.Furthermore, our results demonstrate that CREB-1, RFXANK and Oct-2 occupy the DOB promoter in vivo, However, CIITA and Bob-1 were only minimally recruited. Finally, fusion of Bjab, a DOB-negative B cell line, with.174 B cells that lack the complete MHC class II region (including the DO genes), lead to DO expression. These data indicate that the expression of DO is regulated by an unidentified factor in B cells.

  9. Cordyceps militaris Enhances MHC-restricted Antigen Presentation via the Induced Expression of MHC Molecules and Production of Cytokines

    PubMed Central

    Shin, Seulmee; Park, Yoonhee; Kim, Seulah; Oh, Hee-Eun; Ko, Young-Wook; Han, Shinha; Lee, Seungjeong; Lee, Chong-Kil; Cho, Kyunghae

    2010-01-01

    Background Cordyceps militarys water extract (CME) has been reported to exert antitumor and immunomodulatory activities in vivo and in vitro. However, the therapeutic mechanism has not yet been elucidated. In this study, we examined the effects of CME on the antigen presenting function of antigen presenting cells (APCs). Methods Dendritic cells (DCs) were cultured in the presence of CME, and then allowed to phagocytose microspheres containing ovalbumin (OVA). After washing and fixing the efficacy of OVA, peptide presentation by DCs were evaluated using CD8 and CD4 T cells. Also, we confirmed the protein levels of proinflammatory cytokines through western blot analysis. Results CME enhanced both MHC class I and class II-restricted presentation of OVA in DCs. In addition, the expression of both MHC class I and II molecules was enhanced, but there was no changes in the phagocytic activity of exogenous OVA. Furthermore, CME induced the protein levels of iNOS, COX-2, proinflammatory cytokines, and nuclear p65 in a concentration-dependent manner, as determined by western blot. Conclusion These results provide an understanding of the mechanism of the immuno-enhancing activity of CME on the induction of MHC-restricted antigen presentation in relation to their actions on APCs. PMID:20844738

  10. Mutant MHC class II epitopes drive therapeutic immune responses to cancer

    PubMed Central

    Kreiter, Sebastian; Vormehr, Mathias; van de Roemer, Niels; Diken, Mustafa; Löwer, Martin; Diekmann, Jan; Boegel, Sebastian; Schrörs, Barbara; Vascotto, Fulvia; Castle, John C.; Tadmor, Arbel D.; Schoenberger, Stephen P.; Huber, Christoph; Türeci, Özlem; Sahin, Ugur

    2016-01-01

    Tumour-specific mutations are ideal targets for cancer immunotherapy as they lack expression in healthy tissues and can potentially be recognized as neo-antigens by the mature T-cell repertoire. Their systematic targeting by vaccine approaches, however, has been hampered by the fact that every patient’s tumour possesses a unique set of mutations (‘the mutanome’) that must first be identified. Recently, we proposed a personalized immunotherapy approach to target the full spectrum of a patient’s individual tumour-specific mutations1. Here we show in three independent murine tumour models that a considerable fraction of non-synonymous cancer mutations is immunogenic and that, unexpectedly, the majority of the immunogenic mutanome is recognized by CD4+ T cells. Vaccination with such CD4+ immunogenic mutations confers strong antitumour activity. Encouraged by these findings, we established a process by which mutations identified by exome sequencing could be selected as vaccine targets solely through bioinformatic prioritization on the basis of their expression levels and major histocompatibility complex (MHC) class II-binding capacity for rapid production as synthetic poly-neo-epitope messenger RNA vaccines. We show that vaccination with such polytope mRNA vaccines induces potent tumour control and complete rejection of established aggressively growing tumours in mice. Moreover, we demonstrate that CD4+ T cell neo-epitope vaccination reshapes the tumour microenvironment and induces cytotoxic T lymphocyte responses against an independent immunodominant antigen in mice, indicating orchestration of antigen spread. Finally, we demonstrate an abundance of mutations predicted to bind to MHC class II in human cancers as well by employing the same predictive algorithm on corresponding human cancer types. Thus, the tailored immunotherapy approach introduced here may be regarded as a universally applicable blueprint for comprehensive exploitation of the substantial neo

  11. Multiple expressed MHC class II loci in salmonids; details of one non-classical region in Atlantic salmon (Salmo salar)

    PubMed Central

    Harstad, Håvard; Lukacs, Morten F; Bakke, Hege G; Grimholt, Unni

    2008-01-01

    Background In teleosts, the Major Histocompatibility Complex (MHC) class I and class II molecules reside on different linkage groups as opposed to tetrapods and shark, where the class I and class II genes reside in one genomic region. Several teleost MHC class I regions have been sequenced and show varying number of class I genes. Salmonids have one major expressed MHC class I locus (UBA) in addition to varying numbers of non-classical genes. Two other more distant lineages are also identifyed denoted L and ZE. For class II, only one major expressed class II alpha (DAA) and beta (DAB) gene has been identified in salmonids so far. Results We sequenced a genomic region of 211 kb encompassing divergent MHC class II alpha (Sasa-DBA) and beta (Sasa-DBB) genes in addition to NRGN, TIPRL, TBCEL and TECTA. The region was not linked to the classical class II genes and had some synteny to genomic regions from other teleosts. Two additional divergent and expressed class II sequences denoted DCA and DDA were also identified in both salmon and trout. Expression patterns and lack of polymorphism make these genes non-classical class II analogues. Sasa-DBB, Sasa-DCA and Sasa-DDA had highest expression levels in liver, hindgut and spleen respectively, suggestive of distinctive functions in these tissues. Phylogenetic studies revealed more yet undescribed divergent expressed MHC class II molecules also in other teleosts. Conclusion We have characterised one genomic region containing expressed non-classical MHC class II genes in addition to four other genes not involved in immune function. Salmonids contain at least two expressed MHC class II beta genes and four expressed MHC class II alpha genes with properties suggestive of new functions for MHC class II in vertebrates. Collectively, our data suggest that the class II is worthy of more elaborate studies also in other teleost species. PMID:18439319

  12. Tolerance to solid organ transplants through transfer of MHC class II genes

    PubMed Central

    Sonntag, Kai-C.; Emery, David W.; Yasumoto, Akihiko; Haller, Gary; Germana, Sharon; Sablinski, Tomasz; Shimizu, Akira; Yamada, Kazuhiko; Shimada, Hideaki; Arn, Scott; Sachs, David H.; LeGuern, Christian

    2001-01-01

    Donor/recipient MHC class II matching permits survival of experimental allografts without permanent immunosuppression, but is not clinically applicable due to the extensive polymorphism of this locus. As an alternative, we have tested a gene therapy approach in a preclinical animal model to determine whether expression of allogeneic class II transgenes (Tg’s) in recipient bone marrow cells would allow survival of subsequent Tg-matched renal allografts. Somatic matching between donor kidney class II and the recipient Tg’s, in combination with a short treatment of cyclosporine A, prolonged graft survival with DR and promoted tolerance with DQ. Class II Tg expression in the lymphoid lineage and the graft itself were sequentially implicated in this tolerance induction. These results demonstrate the potential of MHC class II gene transfer to permit tolerance to solid organ allografts. PMID:11134181

  13. Variation in MHC class II B genes in marbled murrelets: implications for delineating conservation units

    Treesearch

    C. Vásquez-Carrillo; V. Friesen; L. Hall; M.Z. Peery

    2013-01-01

    Conserving genetic variation is critical for maintaining the evolutionary potential and viability of a species. Genetic studies seeking to delineate conservation units, however, typically focus on characterizing neutral genetic variation and may not identify populations harboring local adaptations. Here, variation at two major histocompatibility complex (MHC) class II...

  14. Coalescence of B cell receptor and invariant chain MHC II in a raft-like membrane domain.

    PubMed

    Hauser, Julian T; Lindner, Robert

    2014-11-01

    The BCR binds antigen for processing and subsequent presentation on MHC II molecules. Polyvalent antigen induces BCR clustering and targeting to endocytic processing compartments, which are also accessed by Ii-MHC II. Here, we report that clustered BCR is able to team up with Ii-MHC II already at the plasma membrane of mouse B-lymphocytes. Colocalization of BCR and Ii-MHC II on the cell surface required clustering of both types of molecules. The clustering of only one type did not trigger the recruitment of the other. Ii-bound MIF (a ligand of Ii) also colocalized with clustered BCR upon oligomerization of MIF on the surface of the B cell. Abundant surface molecules, such as B220 or TfnR, did not cocluster with the BCR. Some membrane raft-associated molecules, such as peptide-loaded MHC II, coclustered with the BCR, whereas others, such as GM1, did not. The formation of a BCR- and Ii-MHC II-containing membrane domain by antibody-mediated clustering was independent of F-actin and led to the coendocytosis of its constituents. With a rapid Brij 98 extraction method, it was possible to capture this membrane domain biochemically as a DRM. Ii and clustered BCR were present on the same DRM, as shown by immunoisolation. The coalescence of BCR and Ii-MHC II increased tyrosine phosphorylation, indicative of enhanced BCR signaling. Our work suggests a novel role for MIF and Ii-MHC II in BCR-mediated antigen processing.

  15. Characterisation of class II B MHC genes from a ratite bird, the little spotted kiwi (Apteryx owenii).

    PubMed

    Miller, Hilary C; Bowker-Wright, Gemma; Kharkrang, Marie; Ramstad, Kristina

    2011-04-01

    Major histocompatibility complex (MHC) genes are important for vertebrate immune response and typically display high levels of diversity due to balancing selection from exposure to diverse pathogens. An understanding of the structure of the MHC region and diversity among functional MHC genes is critical to understanding the evolution of the MHC and species resilience to disease exposure. In this study, we characterise the structure and diversity of class II MHC genes in little spotted kiwi Apteryx owenii, a ratite bird representing the basal avian lineage (paleognaths). Results indicate that little spotted kiwi have a more complex MHC structure than that of other non-passerine birds, with at least five class II MHC genes, three of which are expressed and likely to be functional. Levels of MHC variation among little spotted kiwi are extremely low, with 13 birds assayed having nearly identical MHC genotypes (only two genotypes containing four alleles, three of which are fixed). These results suggest that recent genetic drift due to a species-wide bottleneck of at most seven birds has overwhelmed past selection for high MHC diversity in little spotted kiwi, potentially leaving the species highly susceptible to disease.

  16. Expressed MHC class II genes in sea otters (Enhydra lutris) from geographically disparate populations.

    PubMed

    Bowen, L; Aldridge, B M; Miles, A K; Stott, J L

    2006-05-01

    The major histocompatibility complex (MHC) is central to maintaining the immunologic vigor of individuals and populations. Classical MHC class II genes were targeted for partial sequencing in sea otters (Enhydra lutris) from populations in California, Washington, and Alaska. Sequences derived from sea otter peripheral blood leukocyte mRNAs were similar to those classified as DQA, DQB, DRA, and DRB in other species. Comparisons of the derived amino acid compositions supported the classification of these as functional molecules from at least one DQA, DQB, and DRA locus and at least two DRB loci. While limited in scope, phylogenetic analysis of the DRB peptide-binding region suggested the possible existence of distinct clades demarcated by geographic region. These preliminary findings support the need for additional MHC gene sequencing and expansion to a comprehensive study targeting additional otters.

  17. Expressed MHC class II genes in sea otters (Enhydra lutris) from geographically disparate populations

    USGS Publications Warehouse

    Bowen, L.; Aldridge, B.M.; Miles, A.K.; Stott, J.L.

    2006-01-01

    The major histocompatibility complex (MHC) is central to maintaining the immunologic vigor of individuals and populations. Classical MHC class II genes were targeted for partial sequencing in sea otters (Enhydra lutris) from populations in California, Washington, and Alaska. Sequences derived from sea otter peripheral blood leukocyte mRNAs were similar to those classified as DQA, DQB, DRA, and DRB in other species. Comparisons of the derived amino acid compositions supported the classification of these as functional molecules from at least one DQA, DQB, and DRA locus and at least two DRB loci. While limited in scope, phylogenetic analysis of the DRB peptide-binding region suggested the possible existence of distinct clades demarcated by geographic region. These preliminary findings support the need for additional MHC gene sequencing and expansion to a comprehensive study targeting additional otters. ?? 2006 Blackwell Munksgaard.

  18. Influence of kinship and MHC class II genotype on visual traits in zebrafish larvae (Danio rerio).

    PubMed

    Hinz, Cornelia; Gebhardt, Katharina; Hartmann, Alexander K; Sigman, Lauren; Gerlach, Gabriele

    2012-01-01

    Kin recognition can drive kin selection and the evolution of social behaviour. In zebrafish (Danio rerio, Hamilton 1822), kin recognition is based on olfactory and visual imprinting processes. If larvae are exposed to visual and chemical cues of kin at day 5 and 6 post fertilization they will recognize kin throughout life, while exposure to non-kin fails to trigger any recognition. Chemical imprinting signals are transcribed by polymorphic genes of the major histocompatibility complex (MHC) code; however, the underlying mechanism for visual imprinting remains unclear. Here we provide evidence for the existence of family-specific differences in morphometry and pigmentation pattern of six day old zebrafish larvae. While rump, tail and body pigmentation were dependent on relatedness, iris pigmentation and morphometry were also influenced by MHC class II genotype. Our study revealed that the MHC not only influences the chemical signature of individuals, but also their visual appearance.

  19. Development of MHC class I and II B primers in common carp and its molecular characterization.

    PubMed

    Jia, Zhiying; Chi, Xifeng; Li, Chitao; Shi, Lianyu

    2010-08-01

    The major histocompatibility complex (MHC) has an important role in immune response and is known as the most polymorphic locus in vertebrates. We developed three pairs of polymerase chain reaction primers of the alpha-2 domain (exon 3) of MHC class I and the beta-2 (exon 3) and beta-3 domains (exon 4) of MHC class II B gene in the German mirror common carp (Cyprinus carpio L.). We analyzed the three loci in a population of 65 individuals that had suffered the serious disease of gill rot. Five to six variable nucleotide sites and two to six variable amino acid sites (71.43%) were detected in the exon sequence of the sampled populations, indicating that many of them corresponded to amino acids involved in antigen recognition. Deviation from Hardy-Weinberg equilibrium and linkage disequilibrium were differentially found in some loci, which will be important for further study of disease resistance/susceptibility and population evolution.

  20. Large-scale MHC class II genotyping of a wild lemur population by next generation sequencing.

    PubMed

    Huchard, Elise; Albrecht, Christina; Schliehe-Diecks, Susanne; Baniel, Alice; Roos, Christian; Kappeler, Peter M; Peter, Peter M Kappeler; Brameier, Markus

    2012-12-01

    The critical role of major histocompatibility complex (MHC) genes in disease resistance, along with their putative function in sexual selection, reproduction and chemical ecology, make them an important genetic system in evolutionary ecology. Studying selective pressures acting on MHC genes in the wild nevertheless requires population-wide genotyping, which has long been challenging because of their extensive polymorphism. Here, we report on large-scale genotyping of the MHC class II loci of the grey mouse lemur (Microcebus murinus) from a wild population in western Madagascar. The second exons from MHC-DRB and -DQB of 772 and 672 individuals were sequenced, respectively, using a 454 sequencing platform, generating more than 800,000 reads. Sequence analysis, through a stepwise variant validation procedure, allowed reliable typing of more than 600 individuals. The quality of our genotyping was evaluated through three independent methods, namely genotyping the same individuals by both cloning and 454 sequencing, running duplicates, and comparing parent-offspring dyads; each displaying very high accuracy. A total of 61 (including 20 new) and 60 (including 53 new) alleles were detected at DRB and DQB genes, respectively. Both loci were non-duplicated, in tight linkage disequilibrium and in Hardy-Weinberg equilibrium, despite the fact that sequence analysis revealed clear evidence of historical selection. Our results highlight the potential of 454 sequencing technology in attempts to investigate patterns of selection shaping MHC variation in contemporary populations. The power of this approach will nevertheless be conditional upon strict quality control of the genotyping data.

  1. Independent evolution of functional MHC class II DRB genes in New World bat species.

    PubMed

    Schad, Julia; Voigt, Christian C; Greiner, Sabine; Dechmann, Dina K N; Sommer, Simone

    2012-07-01

    Genes of the major histocompatibility complex (MHC) play a pivotal role in the vertebrate immune system and are attractive markers for functional, fitness-related, genetic variation. Although bats (Chiroptera) represent the second largest mammalian order and are prone to various emerging infectious diseases, little is known about MHC evolution in bats. In the present study, we examined expressed MHC class II DRB sequences (exons 1 to 4) of New World bat species, Saccopteryx bilineata, Carollia perspicillata, Noctilio albiventris and Noctilio leporinus (only exon 2). We found a wide range of copy number variation of DRB loci with one locus detected in the genus Noctilio and up to ten functional loci observed in S. bilineata. Sequence variation between alleles of the same taxa was high with evidence for positive selection. We found statistical support for recombination or gene conversion events among sequences within the same but not between bat species. Phylogenetic relationships among DRB alleles provided strong evidence for independent evolution of the functional MHC class II DRB genes in the three investigated species, either by recent gene duplication, or homogenization of duplicated loci by frequent gene conversion events. Phylogenetic analysis of all available chiropteran DRB exon 2 sequences confirmed their monophyletic origin within families, but revealed a possible trans-species mode of evolution pattern in congeneric bat species, e.g. within the genera Noctilio and Myotis. This is the first study investigating phylogenetic relationships of MHC genes within bats and therefore contributes to a better understanding of MHC evolution in one of the most dominant mammalian order.

  2. Major histocompatibility complex class II (MHC II) expression during the development of human fetal cerebral occipital lobe, cerebellum, and hematopoietic organs.

    PubMed

    Wierzba-Bobrowicz, T; Kosno-Kruszewska, E; Gwiazda, E; Lechowicz, W

    2000-01-01

    In adults, under physiological conditions proteins of the major histocompatibility complex, class II (MHC II) molecules are synthesized and then presented on the surface of the cells known under a common name as antigen presenting cells (APCs). Dendritic cells (DCs), microglia, macrophages, ameboid microglia and lymphocytes B are qualified as APCs. The aim of present study was to evaluate the expression of MHC II molecules in the central nervous system (CNS) and hematopoietic organs during the fetal development. Observations were made on the cerebral occipital lobe, cerebellum, thymus, spleen and liver of 30 normal human fetuses, between 11 and 22 week of gestation (GW). Histological, histochemical and immunohistochemical techniques were used to identify cells with expression of MHC II molecules. In the brain, MHC II molecules were detected on macrophages/ameboid microglia in meninges, choroid plexus and single cells of ramified microglia in deeper layers of the cortex and white matter. In the other organs besides macrophages and dendritic cells, MHC II molecules were also immunopositive in thymic epithelial cells, and in the spleen and liver also in other cells of stroma and lobule. The expression of MHC II molecules on so extensive population of cells, at an early stage of the fetal development, may evidence their significant involvement in histogenesis and morphogenesis. It seems that in adults the complex of MHC II with protein is originated from the foreign antigen. On the contrary, during normal fetal development the complex of MHC II with protein origins most probably from the fetus own structures.

  3. CD4+ T-cell activation for immunotherapy of malignancies using Ii-Key/MHC class II epitope hybrid vaccines.

    PubMed

    Xu, Minzhen; Kallinteris, Nikoletta L; von Hofe, Eric

    2012-04-16

    Active immunotherapy is becoming a reality in the treatment of malignancies. Peptide-based vaccines represent a simple, safe, and economic basis for cancer immunotherapeutics development. However, therapeutic efficacy has been disappointing. Some of the reasons for this, such as selection of patients with advanced disease and ignorance of the delayed activity of many immunotherapeutic vaccines, have hampered the entire field of cancer immunotherapy over the last decade. Another reason for this may be that most peptide regimens historically have focused on activation of CD8+ cytotoxic T lymphocytes, having little or only indirect CD4+ T helper (Th) cell activation. We review here evidence for the importance of specific CD4+ Th activation in cancer immunotherapy and the use of Ii-Key technology to accomplish this. Ii-Key (LRMK), a portion of the MHC class II-associated invariant chain (Ii protein), facilitates the direct charging of peptide epitopes onto MHC class II molecules. Directly linking Ii-Key to MHC class II peptide epitopes greatly enhances their potency in activating CD4+ T-cells. The Ii-Key hybrid AE37, generated by linking LRMK to the known HER2 MHC class II epitope HER2 (aa 776-790), has been shown to generate robust, long lasting HER2-specific immune responses both in patients with breast and prostate cancer. Interim data from a phase II study of AE37 in breast cancer patients suggest a possible improvement in clinical outcome. The Ii-Key hybrid technology is compared to other methods for enhancing the potency of peptide immunotherapy for cancer. Copyright © 2012 Elsevier Ltd. All rights reserved.

  4. Characterization of genetic polymorphism of novel MHC B-LB II alleles in Chinese indigenous chickens.

    PubMed

    Xu, Rifu; Li, Kui; Chen, Guohong; Xu, Hui; Qiang, Bayangzong; Li, Changchun; Liu, Bang

    2007-02-01

    Genetic polymorphism of the major histocompatibility complex (MHC) B-LB II gene was studied by amplification of exon 2 using PCR, followed by cloning and DNA sequencing in eight indigenous Chinese chicken populations. To reveal the genetic variation of the B-LB II gene, 37 types of patterns detected by PCR-SSCP were investigated first, which would be used to screen novel B-LB II sequences within the breeds. The types of PCR-SSCP patterns and final sequencing allowed for the identification of 31 novel MHC B-LB II alleles from 30 unrelated individuals of Chinese chickens that were sampled. These are the first designators for the alleles of chicken MHC B-LB II gene based on the rule of assignment for novel mammalian alleles. Sequence alignment of the 31 B-LB II alleles revealed a total of 68 variable sites in the fragment of exon 2, of which 51 parsimony informative and 17 singleton variable sites were observed. Among the polymorphic sites, the nucleotide substitutions in the first and second positions of the codons accounted for 36.76% and 35.29%, respectively. The sequence similarities between the alleles were estimated to be 90.6%-99.5%. The relative frequencies of synonymous and nonsynonymous nucleotide substitutions within the region were 2.92%+/-0.94% and 14.64%+/-2.67%, respectively. These results indicated that the genetic variation within exon 2 appeared to have largely arisen by gene recombination and balancing selection. Alignment of the deduced amino acid sequences of the beta1 domain coded by exon 2 revealed 6 synonymous mutations and 27 nonsynonymous substitutions at the 33 disparate sites. In particular, the nonsynonymous substitutions at the putative peptide-binding sites are considered to be associated with immunological specificity of MHC B-LB II molecule in Chinese native chickens. These results can provide a molecular biological basis for the study of disease resistance in chicken breeding.

  5. Characterization and evolution of MHC class II B genes in Ardeid birds.

    PubMed

    Li, Li; Zhou, Xiaopin; Chen, Xiaolin

    2011-06-01

    Major histocompatibility complex (MHC) is a multi-gene family that is very suitable to investigate a wide range of open questions in evolutionary ecology. In this study, we characterized two expressed MHC class II B genes (DAB1 and DAB2) in the Grey Heron (Aves: Ardea cinerea). We further developed the primer pairs to amplify and sequence two MHC class II B loci in ten ardeid birds. Phylogenetic analysis revealed that different parts of the genes showed different evolutionary patterns. The exon 2 sequences tended to cluster two gene-specific lineages. In each lineage, exon 2 sequences from several species showed closer relationships than sequences within species, and two shared identical alleles were found between species (Egretta sacra and Nycticorax nycticorax; Egretta garzetta and Bubulcus ibis), supporting the hypothesis of trans-species polymorphism. In contrast, the species-specific intron 2 plus partial exon 3 tree suggested that DAB1 and DAB2 were subject to concerted evolution. GENECONV analyses showed the gene exchange played an important role in the ardeid MHC evolution.

  6. Minimal conformational plasticity enables TCR cross-reactivity to different MHC class II heterodimers

    PubMed Central

    Holland, Christopher J.; Rizkallah, Pierre J.; Vollers, Sabrina; Calvo-Calle, J. Mauricio; Madura, Florian; Fuller, Anna; Sewell, Andrew K.; Stern, Lawrence J.; Godkin, Andrew; Cole, David K.

    2012-01-01

    Successful immunity requires that a limited pool of αβ T-cell receptors (TCRs) provide cover for a vast number of potential foreign peptide antigens presented by ‘self’ major histocompatibility complex (pMHC) molecules. Structures of unligated and ligated MHC class-I-restricted TCRs with different ligands, supplemented with biophysical analyses, have revealed a number of important mechanisms that govern TCR mediated antigen recognition. HA1.7 TCR binding to the influenza hemagglutinin antigen (HA306–318) presented by HLA-DR1 or HLA-DR4 represents an ideal system for interrogating pMHC-II antigen recognition. Accordingly, we solved the structure of the unligated HA1.7 TCR and compared it to both complex structures. Despite a relatively rigid binding mode, HA1.7 T-cells could tolerate mutations in key contact residues within the peptide epitope. Thermodynamic analysis revealed that limited plasticity and extreme favorable entropy underpinned the ability of the HA1.7 T-cell clone to cross-react with HA306–318 presented by multiple MHC-II alleles. PMID:22953050

  7. MHC class II expression and potential antigen-presenting cells in the retina during experimental autoimmune uveitis.

    PubMed

    Lipski, Deborah A; Dewispelaere, Rémi; Foucart, Vincent; Caspers, Laure E; Defrance, Matthieu; Bruyns, Catherine; Willermain, François

    2017-07-18

    Controversy exists regarding which cell types are responsible for autoantigen presentation in the retina during experimental autoimmune uveitis (EAU) development. In this study, we aimed to identify and characterize the retinal resident and infiltrating cells susceptible to express major histocompatibility complex (MHC) class II during EAU. EAU was induced in C57BL/6 mice by adoptive transfer of autoreactive lymphocytes from IRBP1-20-immunized animals. MHC class II expression was studied by immunostainings on eye cryosections. For flow cytometry (FC) analysis, retinas were dissected and enzymatically digested into single-cell suspensions. Three MHC class II(+) retinal cell populations were sorted by FC, and their RNA processed for RNA-Seq. Immunostainings demonstrate strong induction of MHC class II expression in EAU, especially in the inner retina at the level of inflamed vessels, extending to the outer retinal layers and the subretinal space in severely inflamed eyes. Most MHC class II(+) cells express the hematopoietic marker IBA1. FC quantitative analyses demonstrate that MHC class II induction significantly correlates with disease severity and is associated with upregulation of co-stimulatory molecule expression. In particular, most MHC class II(hi) cells express co-stimulatory molecules during EAU. Further phenotyping identified three MHC class II(+) retinal cell populations: CD45(-)CD11b(-) non-hematopoietic cells with low MHC class II expression and CD45(+)CD11b(+) hematopoietic cells with higher MHC class II expression, which can be further separated into Ly6C(+) and Ly6C(-) cells, possibly corresponding to infiltrating macrophages and resident microglia. Transcriptome analysis of the three sorted populations leads to a clear sample clustering with some enrichment in macrophage markers and microglial cell markers in Ly6C(+) and Ly6C(-) cells, respectively. Functional annotation analysis reveals that both hematopoietic cell populations are more competent in

  8. A Triad of Molecular Regions Contribute to the Formation of Two Distinct MHC Class II Conformers

    PubMed Central

    Drake, Lisa A.; Drake, James R.

    2016-01-01

    MHC class II molecules present antigen-derived peptides to CD4 T cells to drive the adaptive immune response. Previous work has established that class II αβ dimers can adopt two distinct conformations, driven by the differential pairing of transmembrane domain GxxxG dimerization motifs. These class II conformers differ in their ability to be loaded with antigen-derived peptide and to effectively engage CD4 T cells. Motif 1 (M1) paired I-Ak class II molecules are efficiently loaded with peptides derived from the processing of B cell receptor-bound antigen, have unique B cell signaling properties and high T cell stimulation activity. The 11-5.2 mAb selectively binds M1 paired I-Ak class II molecules. However, the molecular determinants of 11-5.2 binding are currently unclear. Here, we report the ability of a human class II transmembrane domain to drive both M1 and M2 class II conformer formation. Protease sensitivity analysis further strengthens the idea that there are conformational differences between the extracellular domains of M1 and M2 paired class II. Finally, MHC class II chain alignments and site directed mutagenesis reveals a triad of molecular regions that contributes to 11-5.2 mAb binding. In addition to transmembrane GxxxG motif domain pairing, 11-5.2 binding is influenced directly by α chain residue Glu-71 and indirectly by the region around the inter-chain salt bridge formed by α chain Arg-52 and β chain Glu-86. These findings provide insight into the complexity of 11-5.2 mAb recognition of the M1 paired I-Ak class II conformer and further highlight the molecular heterogeneity of peptide-MHC class II complexes that drive T cell antigen recognition. PMID:27148821

  9. Identification of MHC class II restricted T-cell-mediated reactivity against MHC class I binding Mycobacterium tuberculosis peptides.

    PubMed

    Wang, Mingjun; Tang, Sheila T; Stryhn, Anette; Justesen, Sune; Larsen, Mette V; Dziegiel, Morten H; Lewinsohn, David M; Buus, Søren; Lund, Ole; Claesson, Mogens H

    2011-04-01

    Major histocompatibility complex (MHC) class I restricted cytotoxic T lymphocytes (CTL) are known to play an important role in the control of Mycobacterium tuberculosis infection so identification of CTL epitopes from M. tuberculosis is of importance for the development of effective peptide-based vaccines. In the present work, bioinformatics technology was employed to predict binding motifs of 9mer peptides derived from M. tuberculosis for the 12 HLA-I supertypes. Subsequently, the predicted peptides were synthesized and assayed for binding to HLA-I molecules in a biochemically based system. The antigenicity of a total of 157 peptides with measured affinity for HLA-I molecules of K(D) ≤ 500 nM were evaluated using peripheral blood T cells from strongly purified protein derivative reactive healthy donors. Of the 157 peptides, eight peptides (5%) were found to induce T-cell responses. As judged from blocking with HLA class I and II subtype antibodies in the ELISPOT assay culture, none of the eight antigenic peptides induced HLA class I restricted CD8(+) T-cell responses. Instead all responses were blocked by pan-HLA class II and anti-HLA-DR antibodies. In addition, CD4(+) T-cell depletion before the 10 days of expansion, resulted in total loss of reactivity in the ELISPOT culture for most peptide specificities. FACS analyses with intracellular interferon-γ staining of T cells expanded in the presence of M. tuberculosis peptides confirmed that the responsive cells were indeed CD4(+). In conclusion, T-cell immunity against HLA-I binding 9mer M. tuberculosis-derived peptides might in many cases turn out to be mediated by CD4(+) T cells and restricted by HLA-II molecules. The use of 9mer peptides recognized by both CD8(+) and CD4(+) T cells might be of importance for the development of future M. tuberculosis peptide-based vaccines.

  10. Contrasting evolutionary histories of MHC class I and class II loci in grouse--effects of selection and gene conversion.

    PubMed

    Minias, P; Bateson, Z W; Whittingham, L A; Johnson, J A; Oyler-McCance, S; Dunn, P O

    2016-05-01

    Genes of the major histocompatibility complex (MHC) encode receptor molecules that are responsible for recognition of intracellular and extracellular pathogens (class I and class II genes, respectively) in vertebrates. Given the different roles of class I and II MHC genes, one might expect the strength of selection to differ between these two classes. Different selective pressures may also promote different rates of gene conversion at each class. Despite these predictions, surprisingly few studies have looked at differences between class I and II genes in terms of both selection and gene conversion. Here, we investigated the molecular evolution of MHC class I and II genes in five closely related species of prairie grouse (Centrocercus and Tympanuchus) that possess one class I and two class II loci. We found striking differences in the strength of balancing selection acting on MHC class I versus class II genes. More than half of the putative antigen-binding sites (ABS) of class II were under positive or episodic diversifying selection, compared with only 10% at class I. We also found that gene conversion had a stronger role in shaping the evolution of MHC class II than class I. Overall, the combination of strong positive (balancing) selection and frequent gene conversion has maintained higher diversity of MHC class II than class I in prairie grouse. This is one of the first studies clearly demonstrating that macroevolutionary mechanisms can act differently on genes involved in the immune response against intracellular and extracellular pathogens.

  11. Contrasting evolutionary histories of MHC class I and class II loci in grouse—effects of selection and gene conversion

    PubMed Central

    Minias, P; Bateson, Z W; Whittingham, L A; Johnson, J A; Oyler-McCance, S; Dunn, P O

    2016-01-01

    Genes of the major histocompatibility complex (MHC) encode receptor molecules that are responsible for recognition of intracellular and extracellular pathogens (class I and class II genes, respectively) in vertebrates. Given the different roles of class I and II MHC genes, one might expect the strength of selection to differ between these two classes. Different selective pressures may also promote different rates of gene conversion at each class. Despite these predictions, surprisingly few studies have looked at differences between class I and II genes in terms of both selection and gene conversion. Here, we investigated the molecular evolution of MHC class I and II genes in five closely related species of prairie grouse (Centrocercus and Tympanuchus) that possess one class I and two class II loci. We found striking differences in the strength of balancing selection acting on MHC class I versus class II genes. More than half of the putative antigen-binding sites (ABS) of class II were under positive or episodic diversifying selection, compared with only 10% at class I. We also found that gene conversion had a stronger role in shaping the evolution of MHC class II than class I. Overall, the combination of strong positive (balancing) selection and frequent gene conversion has maintained higher diversity of MHC class II than class I in prairie grouse. This is one of the first studies clearly demonstrating that macroevolutionary mechanisms can act differently on genes involved in the immune response against intracellular and extracellular pathogens. PMID:26860199

  12. Contrasting evolutionary histories of MHC class I and class II loci in grouse—Effects of selection and gene conversion

    USGS Publications Warehouse

    Minias, Piotr; Bateson, Zachary W; Whittingham, Linda A; Johnson, Jeff A.; Oyler-McCance, Sara J.; Dunn, Peter O

    2016-01-01

    Genes of the major histocompatibility complex (MHC) encode receptor molecules that are responsible for recognition of intracellular and extracellular pathogens (class I and class II genes, respectively) in vertebrates. Given the different roles of class I and II MHC genes, one might expect the strength of selection to differ between these two classes. Different selective pressures may also promote different rates of gene conversion at each class. Despite these predictions, surprisingly few studies have looked at differences between class I and II genes in terms of both selection and gene conversion. Here, we investigated the molecular evolution of MHC class I and II genes in five closely related species of prairie grouse (Centrocercus and Tympanuchus) that possess one class I and two class II loci. We found striking differences in the strength of balancing selection acting on MHC class I versus class II genes. More than half of the putative antigen-binding sites (ABS) of class II were under positive or episodic diversifying selection, compared with only 10% at class I. We also found that gene conversion had a stronger role in shaping the evolution of MHC class II than class I. Overall, the combination of strong positive (balancing) selection and frequent gene conversion has maintained higher diversity of MHC class II than class I in prairie grouse. This is one of the first studies clearly demonstrating that macroevolutionary mechanisms can act differently on genes involved in the immune response against intracellular and extracellular pathogens.

  13. Selection and function of CD4+ T lymphocytes in transgenic mice expressing mutant MHC class II molecules deficient in their interaction with CD4.

    PubMed

    Gilfillan, S; Shen, X; König, R

    1998-12-15

    Interactions of the T cell coreceptors, CD4 and CD8, with MHC molecules participate in regulating thymocyte development and T lymphocyte activation and differentiation to memory T cells. However, the exact roles of these interactions in normal T cell development and function remain unclear. CD4 interacts with class II MHC7 molecules via several noncontiguous regions in both the class II MHC alpha- and beta-chains. We have introduced a double mutation that disrupts interaction with CD4 into the I-A(beta)k gene and used this construct to generate transgenic mice expressing only mutant class II MHC. Although CD4+ thymocytes matured to the single-positive stage in these mice, their frequency was reduced by threefold compared with that of wild-type transgenics. Positive selection of CD4+ T cells in the mutant transgenic mice may have been mediated by TCRs with a higher than usual affinity for class II MHC/Ag complexes. In A(beta)k mutant transgenics, peripheral CD4+ lymphocytes promoted B cell differentiation to plasma cells. These CD4+ T cells also secreted IFN-gamma in response to various stimuli (e.g., protein Ag, bacterial superantigen, and alloantigen), but were deficient in IL-2 secretion. Interactions between CD4 and class II MHC molecules appeared to regulate lymphokine production, with a strong bias toward IFN-gamma and against IL-2 in the absence of these interactions. Our results have implications for the manipulation of T cell-dependent immune responses.

  14. Contrasting patterns of selection acting on MHC class I and class II DRB genes in the Alpine marmot (Marmota marmota).

    PubMed

    Kuduk, K; Johanet, A; Allainé, D; Cohas, A; Radwan, J

    2012-08-01

    The major histocompatibility complex (MHC) genes code for proteins that play a critical role in the immune system response. The MHC genes are among the most polymorphic genes in vertebrates, presumably due to balancing selection. The two MHC classes appear to differ in the rate of evolution, but the reasons for this variation are not well understood. Here, we investigate the level of polymorphism and the evolution of sequences that code for the peptide-binding regions of MHC class I and class II DRB genes in the Alpine marmot (Marmota marmota). We found evidence for four expressed MHC class I loci and two expressed MHC class II loci. MHC genes in marmots were characterized by low polymorphism, as one to eight alleles per putative locus were detected in 38 individuals from three French Alps populations. The generally limited degree of polymorphism, which was more pronounced in class I genes, is likely due to bottleneck the populations undergone. Additionally, gene duplication within each class might have compensated for the loss of polymorphism at particular loci. The two gene classes showed different patterns of evolution. The most polymorphic of the putative loci, Mama-DRB1, showed clear evidence of historical positive selection for amino acid replacements. However, no signal of positive selection was evident in the MHC class I genes. These contrasting patterns of sequence evolution may reflect differences in selection pressures acting on class I and class II genes.

  15. Decreased monocyte class II MHC expression following major abdominal surgery in children is related to operative stress.

    PubMed

    McHoney, M; Klein, N J; Eaton, S; Pierro, A

    2006-04-01

    Monocyte class II major histocompatibility complex (MHC) expression is necessary for antigen presentation and stimulation of T-cells. The aim of this study was to correlate monocyte class II MHC response to operative stress in children and the possible influence of cytokines in the postoperative period. We studied 21 children undergoing elective abdominal surgery. Operative stress score (OSS) was calculated. Monocyte class II MHC expression was measured preoperatively, immediately after surgery, 24 and 48 h postoperatively, using flow cytometry. Class II MHC is expressed as mean fluorescence intensity (MFI) of monocytes expressing MHC (mean +/- SD). Cytokine levels (interleukins 1ra, 6, and 10, and tumor necrosis factor-alpha) were also measured. Data between time points were compared using repeated measures ANOVA. There was an immediate postoperative decrease in class II MHC expression, with lowest levels 24 h postoperatively (preoperative 50 +/- 23.6, 24 h 18.2 +/- 9.4, P < 0.0001 vs. preoperative). At 48 h there was partial recovery in class II MHC, but levels were still significantly lower than preoperative (23.9 +/- 11.1, P < 0.001). The degree of monocyte depression was related to the magnitude of operative stress. Patients who had OSS <10 displayed some recovery in expression at 48 h 25.5 +/- 11.1), whereas in patients with OSS > or = 10 (severe surgical stress), expression further decreased at 48 h (MFI 14.0 +/- 0.1). There was an elevation of interleukin-1ra in the immediate postoperative period in both groups. There was no elevation in the other cytokines. Abdominal surgery in children decreases monocyte MHC expression. Class II MHC depression was related to magnitude of surgical trauma, implying that more severe immuneparesis follows surgery of greater magnitude. This may predispose to postoperative infection.

  16. The common marmoset: a new world primate species with limited Mhc class II variability.

    PubMed

    Antunes, S G; de Groot, N G; Brok, H; Doxiadis, G; Menezes, A A; Otting, N; Bontrop, R E

    1998-09-29

    The common marmoset (Callithrix jacchus) is a New World primate species that is highly susceptible to fatal infections caused by various strains of bacteria. We present here a first step in the molecular characterization of the common marmoset's Mhc class II genes by nucleotide sequence analysis of the polymorphic exon 2 segments. For this study, genetic material was obtained from animals bred in captivity as well as in the wild. The results demonstrate that the common marmoset has, like other primates, apparently functional Mhc-DR and -DQ regions, but the Mhc-DP region has been inactivated. At the -DR and -DQ loci, only a limited number of lineages were detected. On the basis of the number of alleles found, the -DQA and -B loci appear to be oligomorphic, whereas only a moderate degree of polymorphism was observed for two of three Mhc-DRB loci. The contact residues in the peptide-binding site of the Caja-DRB1*03 lineage members are highly conserved, whereas the -DRB*W16 lineage members show more divergence in that respect. The latter locus encodes five oligomorphic lineages whose members are not observed in any other primate species studied, suggesting rapid evolution, as illustrated by frequent exchange of polymorphic motifs. All common marmosets tested were found to share one monomorphic type of Caja-DRB*W12 allele probably encoded by a separate locus. Common marmosets apparently lack haplotype polymorphism because the number of Caja-DRB loci present per haplotype appears to be constant. Despite this, however, an unexpectedly high number of allelic combinations are observed at the haplotypic level, suggesting that Caja-DRB alleles are exchanged frequently between chromosomes by recombination, promoting an optimal distribution of limited Mhc polymorphisms among individuals of a given population. This peculiar genetic make up, in combination with the limited variability of the major histocompatability complex class II repertoire, may contribute to the common

  17. [Planar molecular arrangements aid the design of MHC class II binding peptides].

    PubMed

    Cortés, A; Coral, J; McLachlan, C; Benítez, R; Pinilla, L

    2017-01-01

    The coupling between peptides and MHC-II proteins in the human immune system is not well understood. This work presents an evidence-based hypothesis of a guiding intermolecular force present in every human MHC-II protein (HLA-II). Previously, we examined the spatial positions of the fully conserved residues in all HLA-II protein types. In each one, constant planar patterns were revealed. These molecular planes comprise of amino acid groups of the same chemical species (for example, Gly) distributed across the protein structure. Each amino acid plane has a unique direction and this directional element offers spatial selectivity. Constant within all planes, too, is the presence of an aromatic residue possessing electrons in movement, leading the authors to consider that the planes generate electromagnetic fields that could serve as an attractive force in a single direction. Selection and attraction between HLA-II molecules and antigen peptides would, therefore, be non-random, resulting in a coupling mechanism as effective and rapid as is clearly required in the immune response. On the basis of planar projections onto the HLA-II groove, modifications were made by substituting the key residues in the class II-associated invariant chain peptide-a peptide with a universal binding affinity-resulting in eight different modified peptides with affinities greater than that of the unmodified peptide. Accurate and reliable prediction of MHC class II-binding peptides may facilitate the design of universal vaccine-peptides with greatly enhanced binding affinities. The proposed mechanisms of selection, attraction and coupling between HLA-II and antigen peptides are explained further in the paper.

  18. Antigen-B Cell Receptor Complexes Associate with Intracellular major histocompatibility complex (MHC) Class II Molecules*

    PubMed Central

    Barroso, Margarida; Tucker, Heidi; Drake, Lisa; Nichol, Kathleen; Drake, James R.

    2015-01-01

    Antigen processing and MHC class II-restricted antigen presentation by antigen-presenting cells such as dendritic cells and B cells allows the activation of naïve CD4+ T cells and cognate interactions between B cells and effector CD4+ T cells, respectively. B cells are unique among class II-restricted antigen-presenting cells in that they have a clonally restricted antigen-specific receptor, the B cell receptor (BCR), which allows the cell to recognize and respond to trace amounts of foreign antigen present in a sea of self-antigens. Moreover, engagement of peptide-class II complexes formed via BCR-mediated processing of cognate antigen has been shown to result in a unique pattern of B cell activation. Using a combined biochemical and imaging/FRET approach, we establish that internalized antigen-BCR complexes associate with intracellular class II molecules. We demonstrate that the M1-paired MHC class II conformer, shown previously to be critical for CD4 T cell activation, is incorporated selectively into these complexes and loaded selectively with peptide derived from BCR-internalized cognate antigen. These results demonstrate that, in B cells, internalized antigen-BCR complexes associate with intracellular MHC class II molecules, potentially defining a site of class II peptide acquisition, and reveal a selective role for the M1-paired class II conformer in the presentation of cognate antigen. These findings provide key insights into the molecular mechanisms used by B cells to control the source of peptides charged onto class II molecules, allowing the immune system to mount an antibody response focused on BCR-reactive cognate antigen. PMID:26400081

  19. Low genetic variation in the MHC class II DRB gene and MHC-linked microsatellites in endangered island populations of the leopard cat (Prionailurus bengalensis) in Japan.

    PubMed

    Saka, Toshinori; Nishita, Yoshinori; Masuda, Ryuichi

    2017-07-09

    Isolated populations of the leopard cat (Prionailurus bengalensis) on Tsushima and Iriomote islands in Japan are classified as subspecies P. b. euptilurus and P. b. iriomotensis, respectively. Because both populations have decreased to roughly 100, an understanding of their genetic diversity is essential for conservation. We genotyped MHC class II DRB exon 2 and MHC-linked microsatellite loci to evaluate the diversity of MHC genes in the Tsushima and Iriomote cat populations. We detected ten and four DRB alleles in these populations, respectively. A phylogenetic analysis showed DRB alleles from both populations to be closely related to those in other felid DRB lineages, indicating trans-species polymorphism. The MHC-linked microsatellites were more polymorphic in the Tsushima than in the Iriomote population. The MHC diversity of both leopard cat populations is much lower than in the domestic cat populations on these islands, probably due to inbreeding associated with founder effects, geographical isolation, or genetic drift. Our results predict low resistance of the two endangered populations to new pathogens introduced to the islands.

  20. Equine bone marrow-derived mesenchymal stromal cells are heterogeneous in MHC class II expression and capable of inciting an immune response in vitro

    PubMed Central

    2014-01-01

    Introduction The horse is a valuable species to assess the effect of allogeneic mesenchymal stromal cells (MSCs) in regenerative treatments. No studies to date have examined recipient response to major histocompatibility complex (MHC)-mismatched equine MSCs. The purposes of this study were to immunophenotype MSCs from horses of known MHC haplotype and to compare the immunogenicity of MSCs with differing MHC class II expression. Methods MSCs and peripheral blood leukocytes (PBLs) were obtained from Thoroughbred horses (n = 10) of known MHC haplotype (ELA-A2, -A3, and -A9 homozygotes). MSCs were cultured through P8; cells from each passage (P2 to P8) were cryopreserved until used. Immunophenotyping of MHC class I and II, CD44, CD29, CD90, LFA-1, and CD45RB was performed by using flow cytometry. Tri-lineage differentiation assays were performed to confirm MSC multipotency. Recombinant equine IFN-γ was used to stimulate MHC class II negative MSCs in culture, after which expression of MHC class II was re-examined. To assess the ability of MHC class II negative or positive MSCs to stimulate an immune response, modified one-way mixed leukocyte reactions (MLRs) were performed by using MHC-matched and mismatched responder PBLs and stimulator PBLs or MSCs. Proliferation of gated CFSE-labeled CD3+ responder T cells was evaluated via CFSE attenuation by using flow cytometry and reported as the number of cells in the proliferating T-cell gate. Results MSCs varied widely in MHC class II expression despite being homogenous in terms of “stemness” marker expression and ability to undergo trilineage differentiation. Stimulation of MHC class II negative MSCs with IFN-γ resulted in markedly increased expression of MHC class II. MLR results revealed that MHC-mismatched MHC class II-positive MSCs caused significantly increased responder T-cell proliferation compared with MHC-mismatched MHC class II-negative and MHC-matched MSCs, and equivalent to that of the positive control of

  1. MHC class II B diversity in blue tits: a preliminary study

    PubMed Central

    Aguilar, Juan Rivero-de; Schut, Elske; Merino, Santiago; Martínez, Javier; Komdeur, Jan; Westerdahl, Helena

    2013-01-01

    In this study, we partly characterize major histocompatibility complex (MHC) class II B in the blue tit (Cyanistes caeruleus). A total of 22 individuals from three different European locations: Spain, The Netherlands, and Sweden were screened for MHC allelic diversity. The MHC genes were investigated using both PCR-based methods and unamplified genomic DNA with restriction fragment length polymorphism (RFLP) and southern blots. A total of 13 different exon 2 sequences were obtained independently from DNA and/or RNA, thus confirming gene transcription and likely functionality of the genes. Nine out of 13 alleles were found in more than one country, and two alleles appeared in all countries. Positive selection was detected in the region coding for the peptide binding region (PBR). A maximum of three alleles per individual was detected by sequencing and the RFLP pattern consisted of 4–7 fragments, indicating a minimum number of 2–4 loci per individual. A phylogenetic analysis, demonstrated that the blue tit sequences are divergent compared to sequences from other passerines resembling a different MHC lineage than those possessed by most passerines studied to date. PMID:23919136

  2. MHC class II presentation of gp100 epitopes in melanoma cells requires the function of conventional endosomes, and is influenced by melanosomes1

    PubMed Central

    Robila, Valentina; Ostankovitch, Marina; Altrich-VanLith, Michelle L.; Theos, Alexander C.; Drover, Sheila; Marks, Michael S.; Restifo, Nicholas; Engelhard, Victor H.

    2009-01-01

    Many human solid tumors express MHC II molecules, and proteins normally localized to melanosomes give rise to MHC II restricted epitopes in melanoma. However, the pathways by which this occurs have not been defined. We analyzed the processing of one such epitope, gp10044-59, derived from gp100/Pmel17. In melanomas that have down-regulated components of the melanosomal pathway, but constitutively express HLA-DR*0401, the majority of gp100 is sorted to LAMP-1hi/MHC II+ late endosomes. Using mutant gp100 molecules with altered intracellular trafficking, we demonstrate that endosomal localization is necessary for gp10044-59 presentation. By depletion of the AP2 adaptor protein using siRNA, we demonstrate that gp100 protein internalized from the plasma membrane to such endosomes is a major source for gp10044-59 epitope production. Gp100 trapped in early endosomes gives rise to epitopes that are indistinguishable from those produced in late endosomes but their production is less sensitive to inhibition of lysosomal proteases. In melanomas containing melanosomes, gp100 is underrepresented in late endosomes, and accumulates in stage II melanosomes devoid of MHC II molecules. Gp10044-59 presentation is dramatically reduced, and processing occurs entirely in early endosomes / stage I melanosomes. This suggests that melanosomes are inefficient antigen processing compartments. Thus, melanoma de-differentiation may be accompanied by increased presentation of MHC II restricted epitopes from gp100 and other melanosome-localized proteins, leading to enhanced immune recognition. PMID:19017974

  3. Isolation and characterization of a MHC class II DRB locus in the European water vole (Arvicola terrestris).

    PubMed

    Oliver, Matthew K; Piertney, Stuart B

    2006-06-01

    In so-called model species, such as human and mouse, genes of the major histocompatibility complex (MHC) are characterized by extremely high levels of polymorphism, and it is considered that such diversity is maintained by balancing selection. ;There is now a recognized need to expand studies into nonmodel species to examine whether high MHC diversity is mirrored in natural populations, and to determine the ecological, ethological, and evolutionary processes that underpin balancing selection. To address such issues, a necessary prerequisite is the ability to characterize diversity at a single, expressed, polymorphic MHC locus on which selection may be acting. Here, we provide the first description of allelic diversity at exon 2 of an MHC class II DRB locus in the European water vole (Arvicola terrestris), characterize variation across four natural populations, and test whether the patterns of variation are consistent with the effects of balancing selection. Using single-strand conformation polymorphism analysis and subsequent DNA sequencing of gel excisions, five DRB alleles were resolved, each with a unique amino acid sequence, among 100 individuals from four geographically distinct populations. Reverse transcription polymerase chain reaction confirmed that the alleles were products from an expressed locus. Intra-allelic amino acid differences were high (10.5-33.3%), and the nonsynonymous substitution rate exceeded the synonymous substitution rate for the functional peptide-binding region (d (N):d (S)=3.91 and P<0.005). Phylogenetic comparison of resolved alleles with closely related homologues indicated that each allele represented a unique lineage preserved across speciation events. These results indicate that balancing selection has maintained diversity of DRB allelic lineages and amino acid function over evolutionary time scales, but may be less effective at preserving alleles in contemporary populations where stochastic microevolutionary processes may

  4. DPA1*02012: A DPA1*0201-related Mhc class II allele in West Africa

    SciTech Connect

    Meyer, C.G.; May, J.; Spauke, D.; Schnittger, L.

    1994-12-31

    DNA techniques such as sequence-specific oligonucleotide probe (SSOP) hybridizations, restriction-fragment length polymorphism (RFLP) analyses, and DNA sequencing have greatly supported the characterization of Mhc class II allelic polymorphism. Here the authors describe a DPA 1 allele which has been identified in two male individuals from Liberia and Benin, West Africa, during a survey study on Mhc class II associations with the different manifestations after infection with Onchocerca volvulus. 4 refs., 1 fig.

  5. Codominance of TLR2-dependent and TLR2-independent modulation of MHC class II in Mycobacterium tuberculosis infection in vivo.

    PubMed

    Kincaid, Eleanor Z; Wolf, Andrea J; Desvignes, Ludovic; Mahapatra, Sebabrata; Crick, Dean C; Brennan, Patrick J; Pavelka, Martin S; Ernst, Joel D

    2007-09-01

    Mycobacterium tuberculosis is an exceptionally successful human pathogen. A major component of this success is the ability of the bacteria to infect immunocompetent individuals and to evade eradication by an adaptive immune response that includes production of the macrophage-activating cytokine, IFN-gamma. Although IFN-gamma is essential for arrest of progressive tuberculosis, it is insufficient for efficacious macrophage killing of the bacteria, which may be due to the ability of M. tuberculosis to inhibit selected macrophage responses to IFN-gamma. In vitro studies have determined that mycobacterial lipoproteins and other components of the M. tuberculosis cell envelope, acting as agonists for TLR2, inhibit IFN-gamma induction of MHC class II. In addition, M. tuberculosis peptidoglycan and IL-6 secreted by infected macrophages inhibit IFN-gamma induction of MHC class II in a TLR2-independent manner. To determine whether TLR2-dependent inhibition of macrophage responses to IFN-gamma is quantitatively dominant over the TLR2-independent mechanisms in vivo, we prepared mixed bone marrow chimeric mice in which the hemopoietic compartment was reconstituted with a mixture of TLR(+/+) and TLR2(-/-) cells. When the chimeric mice were infected with M. tuberculosis, the expression of MHC class II on TLR2(+/+) and TLR2(-/-) macrophages from the lungs of individual infected chimeric mice was indistinguishable. These results indicate that TLR2-dependent and -independent mechanisms of inhibition of responses to IFN-gamma are equivalent in vivo, and that M. tuberculosis uses multiple pathways to abrogate the action of an important effector of adaptive immunity. This work was supported by National Institutes of Health Grants AI 065357-AI 020010.

  6. Type I interferon drives a distinctive dendritic cell maturation phenotype that allows continued class II MHC synthesis and antigen processing

    PubMed Central

    Simmons, Daimon P.; Wearsch, Pamela A.; Canaday, David H.; Meyerson, Howard J.; Liu, Yi C.; Wang, Ying; Boom, W. Henry; Harding, Clifford V.

    2012-01-01

    Microbial molecules or cytokines can stimulate dendritic cell (DC) maturation, which involves DC migration to lymph nodes and enhanced presentation of Ag to launch T cell responses. Microbial Toll-like receptor (TLR) agonists are the most studied inducers of DC maturation, but type I interferon (IFN-I) also promotes DC maturation. In response to TLR stimulation, DC maturation involves a burst of Ag processing with enhanced expression of peptide-MHC-II complexes and co-stimulator molecules. Subsequently, MHC-II synthesis and expression in intracellular vacuolar compartments is inhibited, decreasing Ag processing function. This limits presentation to a cohort of Ags kinetically associated with the maturation stimulus and excludes presentation of Ags subsequently experienced by the DC. In contrast, our studies show that IFN-I enhances DC expression of MHC-II and co-stimulatory molecules without a concomitant inhibition of subsequent MHC-II synthesis and Ag processing. Expression of mRNA for MHC-II and the transcription factor CIITA is inhibited in DCs treated with TLR agonists but maintained in cells treated with IFN-I. Following stimulation with IFN-I, MHC-II expression is increased on the plasma membrane but is also maintained in intracellular vacuolar compartments, consistent with sustained Ag processing function. These findings suggest that IFN-I drives a distinctive DC maturation program that enhances Ag presentation to T cells without a shutdown of Ag processing, allowing continued sampling of Ags for presentation. PMID:22371391

  7. Negative regulation by HLA-DO of MHC class II-restricted antigen processing.

    PubMed

    Denzin, L K; Sant'Angelo, D B; Hammond, C; Surman, M J; Cresswell, P

    1997-10-03

    HLA-DM is a major histocompatibility complex (MHC) class II-like molecule that facilitates antigen processing by catalyzing the exchange of invariant chain-derived peptides (CLIP) from class II molecules for antigenic peptides. HLA-DO is a second class II-like molecule that physically associates with HLA-DM in B cells. HLA-DO was shown to block HLA-DM function. Purified HLA-DM-DO complexes could not promote peptide exchange in vitro. Expression of HLA-DO in a class II+ and DM+, DO- human T cell line caused the accumulation of class II-CLIP complexes, indicating that HLA-DO blocked DM function in vivo and suggesting that HLA-DO is an important modulator of class II-restricted antigen processing.

  8. Selective development of CD4+ T cells in transgenic mice expressing a class II MHC-restricted antigen receptor.

    PubMed

    Kaye, J; Hsu, M L; Sauron, M E; Jameson, S C; Gascoigne, N R; Hedrick, S M

    1989-10-26

    T lymphocytes are predisposed to recognition of foreign protein fragments bound to cell-surface molecules encoded by the major histocompatibility complex (MHC). There is now compelling evidence that this specificity is a consequence of a selection process operating on developing T lymphocytes in the thymus. As a result of this positive selection, thymocytes that express antigen receptors with a threshold affinity for self MHC-encoded glycoproteins preferentially emigrate from the thymus and seed peripheral lymphoid organs. The specificity for both foreign antigen and MHC molecules is imparted by the alpha and beta chains of the T-cell antigen receptor (TCR). Two other T-cell surface proteins, CD4 and CD8, which bind non-polymorphic regions of class II and class I MHC molecules respectively, are also involved in these recognition events and play an integral role in thymic selection. In order to elucidate the developmental pathways of class II MHC-restricted T cells in relation to these essential accessory molecules, we have produced TCR-transgenic mice expressing a receptor specific for a fragment of pigeon cytochrome c and the Ek (class II MHC) molecule. The transgenic TCR is expressed on virtually all T cells in mice expressing Ek. The thymuses of these mice contain an abnormally high percentage of mature CD4+CD8- cells. In addition, the peripheral T-cell population is almost exclusively CD4+, demonstrating that the MHC specificity of the TCR determines the phenotype of T cells during selection in the thymus.

  9. IL-33 promotes MHC class II expression in murine mast cells.

    PubMed

    Ito, Tomonobu; Egusa, Chizu; Maeda, Tatsuo; Numata, Takafumi; Nakano, Nobuhiro; Nishiyama, Chiharu; Tsuboi, Ryoji

    2015-09-01

    Mast cells (MCs), recognized as tissue-resident cells of hematopoietic origin, are involved in cellular and pathological manifestations of allergic disorders including atopic dermatitis. IL-33, a member of the IL-1 cytokine family, activates Th2-type immune responses, and promotes the degranulation and maturation of MCs. However, it is uncertain whether IL-33 treatment induces mature mast cells to acquire the characteristics of the monocyte-dendritic cell lineage.We investigated the effect of IL-33 on the MHC class II expression and function of murine mast cells. IL-33-treated mature murine bone marrow-derived mast cells (BMMCs) were analyzed by FACS, real-time PCR, chromatin immunoprecipitation (ChIP) assay, and Western blotting. The morphology and degranulation activity of BMMCs and T-cell activation by BMMCs were also examined. BMMCs treated with IL-33 for 10 days induced cell surface expression of the MHC class II protein, whereas the expression of FcεRI and c-kit was not affected by IL-33. The expression of CIITA, driven from pIII and pIV, was up-regulated in IL-33-treated BMMCs. The amount of PU.1 mRNA and protein significantly increased in IL-33-treated BMMCs. The ChIP assay showed PU.1 binding to CIITA pIII, and enhanced histone acetylation due to IL-33 treatment. Syngeneic T cells were activated by co-culture with IL-33-treated BMMCs, although the expression of the co-stimulatory molecules, CD40, CD80, CD86, and PDL-1, was not detected. Mast cells express MHC class II after prolonged exposure to IL-33, probably due to enhanced recruitment of PU.1 to CIITA pIII, resulting in transactivation of CIITA and MHC class II. IL-33 is an important cytokine in allergic disorders. Mast cells have the ability to express MHC class II after prolonged exposure to IL-33 in a murine model. IL-33 holds a key to understanding the etiology of atopic dermatitis.

  10. pRB is required for interferon-gamma-induction of the MHC class II abeta gene.

    PubMed

    Zhu, X; Pattenden, S; Bremner, R

    1999-09-02

    pRB is required for IFN-gamma-induction of MHC class II in human tumor cell lines, providing a potential link between tumor suppressors and the immune system. However, other genes, such as cyclin D1, show pRB-dependency only in tumor cells, so by analogy, pRB may not be necessary for cII-regulation in normal cells. Here, we demonstrate that induction of the mouse MHC class II I-A heterodimer is normal in RB+/+ mouse embryonic fibroblasts (MEFs), but deficient in RB-/- MEFs. Inducibility is restored in RB-/- MEFs stably transfected with wild type RB cDNA or infected with an adenovirus expressing pRB. Thus, involvement of pRB in MHC class II expression is conserved in the mouse and is not an aberrant feature of tumorigenic, aneuploid, human tumor cells. Although cII genes are generally induced in a coordinate fashion, suggesting a common mechanism, we found that pRB was specifically required for induction of the Abeta, but not Aalpha or other MHC cII genes including Ebeta, Ii and H2-Malpha. Finally, IFN-gamma-induction of class II transactivator (CIITA), was pRB-independent, suggesting that pRB works downstream of this master-regulator of MHC class II expression.

  11. Dendritic cell preactivation impairs MHC class II presentation of vaccines and endogenous viral antigens

    PubMed Central

    Young, Louise J.; Wilson, Nicholas S.; Schnorrer, Petra; Mount, Adele; Lundie, Rachel J.; La Gruta, Nicole L.; Crabb, Brendan S.; Belz, Gabrielle T.; Heath, William R.; Villadangos, Jose A.

    2007-01-01

    When dendritic cells (DCs) encounter signals associated with infection or inflammation, they become activated and undergo maturation. Mature DCs are very efficient at presenting antigens captured in association with their activating signal but fail to present subsequently encountered antigens, at least in vitro. Such impairment of MHC class II (MHC II) antigen presentation has generally been thought to be a consequence of down-regulation of endocytosis, so it might be expected that antigens synthesized by the DCs themselves (for instance, viral antigens) would still be presented by mature DCs. Here, we show that DCs matured in vivo could still capture and process soluble antigens, but were unable to present peptides derived from these antigens. Furthermore, presentation of viral antigens synthesized by the DCs themselves was also severely impaired. Indeed, i.v. injection of pathogen mimics, which caused systemic DC activation in vivo, impaired the induction of CD4 T cell responses against subsequently encountered protein antigens. This immunosuppressed state could be reversed by adoptive transfer of DCs loaded exogenously with antigens, demonstrating that impairment of CD4 T cell responses was due to lack of antigen presentation rather than to overt suppression of T cell activation. The biochemical mechanism underlying this phenomenon was the down-regulation of MHC II–peptide complex formation that accompanied DC maturation. These observations have important implications for the design of prophylactic and therapeutic DC vaccines and contribute to the understanding of the mechanisms causing immunosuppression during systemic blood infections. PMID:17978177

  12. MHC class II genes in European wolves: a comparison with dogs.

    PubMed

    Seddon, Jennifer M; Ellegren, Hans

    2002-10-01

    The genome of the grey wolf, one of the most widely distributed land mammal species, has been subjected to both stochastic factors, including biogeographical subdivision and population fragmentation, and strong selection during the domestication of the dog. To explore the effects of drift and selection on the partitioning of MHC variation in the diversification of species, we present nine DQA, 10 DQB, and 17 DRB1 sequences of the second exon for European wolves and compare them with sequences of North American wolves and dogs. The relatively large number of class II alleles present in both European and North American wolves attests to their large historical population sizes, yet there are few alleles shared between these regions at DQB and DRB1. Similarly, the dog has an extensive array of class II MHC alleles, a consequence of a genetically diverse origin, but allelic overlap with wolves only at DQA. Although we might expect a progression from shared alleles to shared allelic lineages during differentiation, the partitioning of diversity between wolves and dogs at DQB and DRB1 differs from that at DQA. Furthermore, an extensive region of nucleotide sequence shared between DRB1 and DQB alleles and a shared motif suggests intergenic recombination may have contributed to MHC diversity in the Canidae.

  13. MHC Class II tetramers and the pursuit of antigen-specific T cells: define, deviate, delete.

    PubMed

    Mallone, Roberto; Nepom, Gerald T

    2004-03-01

    Selective expansion and activation of a very small number of antigen-specific CD4(+) T cells is a remarkable and essential property of the adaptive immune response. Antigen-specific T cells were until recently identified only indirectly by functional assays, such as antigen-induced cytokine secretion and proliferation. The advent of MHC Class II tetramers has added a pivotal tool to our research armamentarium, allowing the definition of allo- and autoimmune responses in deeper detail. Rare antigen-specific CD4(+) cells can now be selectively identified, isolated and characterized. The same tetramer reagents also provide a new mean of stimulating T cells, more closely reproducing the MHC-peptide/TCR interaction. This property allows the use of tetramers to direct T cells toward the more desirable outcome, that is, activation (in malignancies and infectious diseases) or Th2/T regulatory cell deviation, anergy and deletion (in autoimmune diseases). These experimental approaches hold promise for diagnostic, prognostic and therapeutic applications.

  14. Conditional MHC class I ligands and peptide exchange technology for the human MHC gene products HLA-A1, -A3, -A11, and -B7.

    PubMed

    Bakker, Arnold H; Hoppes, Rieuwert; Linnemann, Carsten; Toebes, Mireille; Rodenko, Boris; Berkers, Celia R; Hadrup, Sine Reker; van Esch, Wim J E; Heemskerk, Mirjam H M; Ovaa, Huib; Schumacher, Ton N M

    2008-03-11

    Major histocompatibility complex (MHC) class I multimer technology has become an indispensable immunological assay system to dissect antigen-specific cytotoxic CD8(+) T cell responses by flow cytometry. However, the development of high-throughput assay systems, in which T cell responses against a multitude of epitopes are analyzed, has been precluded by the fact that for each T cell epitope, a separate in vitro MHC refolding reaction is required. We have recently demonstrated that conditional ligands that disintegrate upon exposure to long-wavelength UV light can be designed for the human MHC molecule HLA-A2. To determine whether this peptide-exchange technology can be developed into a generally applicable approach for high throughput MHC based applications we set out to design conditional ligands for the human MHC gene products HLA-A1, -A3, -A11, and -B7. Here, we describe the development and characterization of conditional ligands for this set of human MHC molecules and apply the peptide-exchange technology to identify melanoma-associated peptides that bind to HLA-A3 with high affinity. The conditional ligand technology developed here will allow high-throughput MHC-based analysis of cytotoxic T cell immunity in the vast majority of Western European individuals.

  15. HLA-DM induces CLIP dissociation from MHC class II alpha beta dimers and facilitates peptide loading.

    PubMed

    Denzin, L K; Cresswell, P

    1995-07-14

    Human leukocyte antigen DM (HLA-DM) molecules are structurally related to classical MHC class II molecules and reside in the lysosome-like compartment where class II-restricted antigen processing is thought to occur. Mutant cell lines lacking HLA-DM are defective in antigen processing and accumulate class II molecules associated with a nested set of invariant chain-derived peptides (class II-associated invariant chain peptides, CLIP). Here we show that HLA-DM catalyzes the dissociation of CLIP from MHC class II-CLIP complexes in vitro and facilitates the binding of antigenic peptides. The reaction has an acidic pH optimum, consistent with its occurrence in a lysosome-like compartment in vivo. Antibody blocking experiments suggest that a transient interaction between HLA-DM and the MHC class II-CLIP complex is required.

  16. Genetic Contribution of MHC Class II Genes in Susceptibility to West Nile Virus Infection.

    PubMed

    Sarri, Constantina A; Markantoni, Maria; Stamatis, Costas; Papa, Anna; Tsakris, Athanasios; Pervanidou, Danai; Baka, Agoritsa; Politis, Constantina; Billinis, Charalambos; Hadjichristodoulou, Christos; Mamuris, Zissis

    2016-01-01

    WNV is a zoonotic neurotropic flavivirus that has recently emerged globally as a significant cause of viral encephalitis. The last five years, 624 incidents of WNV infection have been reported in Greece. The risk for severe WNV disease increases among immunosuppressed individuals implying thus the contribution of the MHC locus to the control of WNV infection. In order to investigate a possible association of MHC class II genes, especially HLA-DPA1, HLA-DQA1, HLA-DRB1, we examined 105 WNV patients, including 68 cases with neuroinvasive disease and 37 cases with mild clinical phenotype, collected during the period from 2010 to2013, and 100 control individuals selected form the Greek population. Typing was performed for exon 2 for all three genes. DQA1*01:01 was considered to be "protective" against WNV infection (25.4% vs 40.1%, P = 0.004) while DQA1*01:02 was associated with increased susceptibility (48.0% vs 32.1%, P = 0.003). Protection against neuroinvasion was associated with the presence of DRB1*11:02 (4.99% vs 0.0%, P = 0.018). DRB1*16:02 was also absent from the control cohort (P = 0.016). Three additional population control groups were used in order to validate our results. No statistically significant association with the disease was found for HLA-DPA alleles. The results of the present study provide some evidence that MHC class II is involved in the response to WNV infection, outlining infection "susceptibility" and "CNS-high-risk" candidates. Furthermore, three new alleles were identified while the frequency of all alleles in the study was compared with worldwide data. The characterization of the MHC locus could help to estimate the risk for severe WNV cases in a country.

  17. Genetic Contribution of MHC Class II Genes in Susceptibility to West Nile Virus Infection

    PubMed Central

    Sarri, Constantina A.; Markantoni, Maria; Stamatis, Costas; Papa, Anna; Tsakris, Athanasios; Pervanidou, Danai; Baka, Agoritsa; Politis, Constantina; Billinis, Charalambos; Hadjichristodoulou, Christos; Mamuris, Zissis

    2016-01-01

    WNV is a zoonotic neurotropic flavivirus that has recently emerged globally as a significant cause of viral encephalitis. The last five years, 624 incidents of WNV infection have been reported in Greece. The risk for severe WNV disease increases among immunosuppressed individuals implying thus the contribution of the MHC locus to the control of WNV infection. In order to investigate a possible association of MHC class II genes, especially HLA-DPA1, HLA-DQA1, HLA-DRB1, we examined 105 WNV patients, including 68 cases with neuroinvasive disease and 37 cases with mild clinical phenotype, collected during the period from 2010 to2013, and 100 control individuals selected form the Greek population. Typing was performed for exon 2 for all three genes. DQA1*01:01 was considered to be "protective" against WNV infection (25.4% vs 40.1%, P = 0.004) while DQA1*01:02 was associated with increased susceptibility (48.0% vs 32.1%, P = 0.003). Protection against neuroinvasion was associated with the presence of DRB1*11:02 (4.99% vs 0.0%, P = 0.018). DRB1*16:02 was also absent from the control cohort (P = 0.016). Three additional population control groups were used in order to validate our results. No statistically significant association with the disease was found for HLA-DPA alleles. The results of the present study provide some evidence that MHC class II is involved in the response to WNV infection, outlining infection "susceptibility" and "CNS-high-risk" candidates. Furthermore, three new alleles were identified while the frequency of all alleles in the study was compared with worldwide data. The characterization of the MHC locus could help to estimate the risk for severe WNV cases in a country. PMID:27812212

  18. Direct binding of a myasthenia gravis related epitope to MHC class II molecules on living murine antigen-presenting cells.

    PubMed Central

    Mozes, E; Dayan, M; Zisman, E; Brocke, S; Licht, A; Pecht, I

    1989-01-01

    MHC gene products present antigenic epitopes to the antigen receptor on T cells. Nevertheless, direct binding of such epitopes to MHC class II proteins on normal living antigen-presenting cells (APCs) has not yet been demonstrated. We have previously shown a significant difference in the ability of T cells of myasthenia gravis (MG) patients to proliferate in response to the synthetic peptide p195-212 of the human acetylcholine receptor (AChR) alpha-subunit in comparison to healthy controls. The observed proliferative responses correlated significantly with HLA-DR5. Moreover, lymph node cells of various mouse strains that were primed with the T cell epitope, p195-212, were found to proliferate to different extents. To investigate these observations further, we designed an assay for direct binding of p195-212 to MHC class II proteins on the surface of freshly prepared splenic adherent cells. Binding of a biotinylated p195-212 was monitored using phycoerythrin-avidin by flow cytometry. Fifteen to sixty per cent of the cells were labeled following incubation with the biotinylated peptide. Binding was observed only to splenic adherent cells derived from mouse strains of which T cells were capable of proliferating in response to p195-212. The binding specificity, in terms of epitope structure and its site of interaction on the cells, was shown by its inhibition with an excess of the unlabeled peptide or with the relevant monoclonal anti-I-A antibodies. These results constitute the first direct evidence for the specific binding of a T cell epitope to live APC. PMID:2480232

  19. MHC class II up-regulation and co-localization with Fas in experimental models of immune-mediated bone marrow failure

    PubMed Central

    Erie, Andrew J.; Samsel, Leigh; Takaku, Tomoiku; Desierto, Marie J.; Keyvanfar, Keyvan; McCoy, J. Philip; Young, Neal S.; Chen, Jichun

    2011-01-01

    Objective To test the hypothesis that gamma interferon (IFN-γ) promotes MHC class II expression on bone marrow (BM) cell targets that facilitates T cell-mediated BM destruction in immune-mediated BM failure. Materials and Methods Allogeneic lymph node (LN) cells were infused into MHC or minor histocompatibility antigen (minor-H) mismatched hosts to induce BM failure. MHC class II and Fas expression and cell apoptosis were analyzed by flow cytometry. MHC class II-Fas co-localization was detected by ImageStream Imaging Flow Cytometry and other cell-cell associations were visualized by confocal microscopy. T cell-mediated BM cell apoptosis and effects of IFN-γ on MHC class II-Fas co-localization on normal BM cells were studied using cell culture in vitro followed by conventional and imaging flow cytometry. Results BM failure animals had significantly up-regulated MHC class II expression on CD4−CD8−CD11b−CD45R− residual BM cells and significantly increased MHC class II-Fas co-localization on BM CD150+ and CD34+ hematopoietic cells. MHC class II+Fas+ BM cells were closely associated with CD4+ T cells in the BM of affected animals, and they were significantly more responsive to T-cell mediated cell apoptosis relative to MHC class II−Fas− BM cells. Infusion of IFN-γ-deficient LN cells into minor-H mismatched recipients resulted in no MHC class II-Fas up-regulation and no clinically overt BM failure. Treatment with recombinant IFN-γ significantly increased both MHC class II-Fas co-expression and co-localization on normal BM cells. Conclusion Elevation of the inflammatory cytokine IFN-γ stimulated MHC class II expression and MHC class II-Fas co-localization, which may facilitate T-cell mediated cell destruction. PMID:21635935

  20. Recognition of core and flanking amino acids of MHC class II-bound peptides by the T cell receptor.

    PubMed

    Sant'Angelo, Derek B; Robinson, Eve; Janeway, Charles A; Denzin, Lisa K

    2002-09-01

    CD4 T cells recognize peptides bound to major histocompatibility complex (MHC) class II molecules. Most MHC class II molecules have four binding pockets occupied by amino acids 1, 4, 6, and 9 of the minimal peptide epitope, while the residues at positions 2, 3, 5, 7, and 8 are available to interact with the T cell receptor (TCR). In addition MHC class II bound peptides have flanking residues situated outside of this peptide core. Here we demonstrate that the flanking residues of the conalbumin peptide bound to I-A(k) have no effect on recognition by the D10 TCR. To study the role of peptide flanks for recognition by a second TCR, we determined the MHC and TCR contacting amino acids of the I-A(b) bound Ealpha peptide. The Ealpha peptide is shown to bind I-A(b) using four alanines as anchor residues. TCR recognition of Ealpha peptides with altered flanking residues again suggested that, in general, no specific interactions occurred with the peptide flanks. However, using an HLA-DM-mediated technique to measure peptide binding to MHC class II molecules, we found that the peptide flanking residues contribute substantially to MHC binding.

  1. Human cytomegalovirus alters localization of MHC class II and dendrite morphology in mature Langerhans cells.

    PubMed

    Lee, Andrew W; Hertel, Laura; Louie, Ryan K; Burster, Timo; Lacaille, Vashti; Pashine, Achal; Abate, Davide A; Mocarski, Edward S; Mellins, Elizabeth D

    2006-09-15

    Hemopoietic stem cell-derived mature Langerhans-type dendritic cells (LC) are susceptible to productive infection by human CMV (HCMV). To investigate the impact of infection on this cell type, we examined HLA-DR biosynthesis and trafficking in mature LC cultures exposed to HCMV. We found decreased surface HLA-DR levels in viral Ag-positive as well as in Ag-negative mature LC. Inhibition of HLA-DR was independent of expression of unique short US2-US11 region gene products by HCMV. Indeed, exposure to UV-inactivated virus, but not to conditioned medium from infected cells, was sufficient to reduce HLA-DR on mature LC, implicating particle binding/penetration in this effect. Reduced surface levels reflected an altered distribution of HLA-DR because total cellular HLA-DR was not diminished. Accumulation of HLA-DR was not explained by altered cathepsin S activity. Mature, peptide-loaded HLA-DR molecules were retained within cells, as assessed by the proportion of SDS-stable HLA-DR dimers. A block in egress was implicated, as endocytosis of surface HLA-DR was not increased. Immunofluorescence microscopy corroborated the intracellular retention of HLA-DR and revealed markedly fewer HLA-DR-positive dendritic projections in infected mature LC. Unexpectedly, light microscopic analyses showed a dramatic loss of the dendrites themselves and immunofluorescence revealed that cytoskeletal elements crucial for the formation and maintenance of dendrites are disrupted in viral Ag-positive cells. Consistent with these dendrite effects, HCMV-infected mature LC exhibit markedly reduced chemotaxis in response to lymphoid chemokines. Thus, HCMV impedes MHC class II molecule trafficking, dendritic projections, and migration of mature LC. These changes likely contribute to the reduced activation of CD4+ T cells by HCMV-infected mature LC.

  2. Characterization of bovine MHC class II DRB3 diversity in South American Holstein cattle populations.

    PubMed

    Takeshima, S-N; Giovambattista, G; Okimoto, N; Matsumoto, Y; Rogberg-Muñoz, A; Acosta, T J; Onuma, M; Aida, Y

    2015-12-01

    Holstein cattle dominate the global milk production industry because of their outstanding milk production, however, this breed is susceptible to tropical endemic pathogens and suffers from heat stress and thus fewer Holstein populations are raised in tropical areas. The bovine major histocompatibility complex (BoLA)-DRB3 class II gene is used as a marker for disease and immunological traits, and its polymorphism has been studied extensively in Holstein cattle from temperate and cold regions. We studied the genetic diversity of the BoLA-DRB3 gene in South American Holstein populations to determine whether tropical populations have diverged from those bred in temperate and cold regions by selection and/or crossbreeding with local native breeds. We specifically studied Exon 2 of this gene from 855 South American Holstein individuals by a polymerase chain reaction (PCR) sequence-based typing method. We found a high degree of gene diversity at the allelic (Na > 20 and He > 0.87) and molecular (π > 0.080) levels, but a low degree of population structure (FST = 0.009215). A principal components analysis and tree showed that the Bolivian subtropical population had the largest genetic divergence compared with Holsteins bred in temperate or cold regions, and that this population was closely related to Bolivian Creole cattle. Our results suggest that Holstein genetic divergence can be explained by selection and/or gene introgression from local germplasms. This is the first examination of BoLA-DRB3 in Holsteins adapted to tropical environments, and contributes to an ongoing effort to catalog bovine MHC allele frequencies by breed and location.

  3. Mass spectrometry analysis and quantitation of peptides presented on the MHC II molecules of mouse spleen dendritic cells

    PubMed Central

    Bozzacco, Leonia; Yu, Haiqiang; Zebroski, Henry A.; Dengjel, Jörn; Deng, Haiteng; Mojsov, Svetlana; Steinman, Ralph M.

    2011-01-01

    Major histocompatibility complex class II (MHC II) molecules are expressed on the surface of antigen presenting cells and display short bound peptide fragments derived from self and nonself antigens. These peptide-MHC complexes function to maintain immunological tolerance in the case of self antigens and initiate the CD4+ T cell response in the case of foreign proteins. Here we report the application of LC-MS/MS analysis to identify MHC II peptides derived from endogenous proteins expressed in freshly isolated murine splenic DCs. The cell number was enriched in vivo upon treatment with Flt3L-B16 melanoma cells. In a typical experiment, starting with about 5× 108 splenic DCs, we were able to reliably identify a repertoire of over 100 MHC II peptides originating from about 55 proteins localized in membrane (23%), intracellular (26%), endo-lysosomal (12%), nuclear (14%) and extracellular (25%) compartments. Using synthetic isotopically labeled peptides corresponding to the sequences of representative bound MHC II peptides, we quantified by LC-MS relative peptide abundance. In a single experiment, peptides were detected in a wide concentration range spanning from 2.5 fmol/μL to 12 pmol/μL or from approximately 13 copies to 2×105 copies per DC. These peptides were found in similar amounts on B cells where we detected about 80 peptides originating from 55 proteins distributed homogenously within the same cellular compartments as in DCs. About 90 different binding motifs predicted by the epitope prediction algorithm were found within the sequences of the identified MHC II peptides. These results set a foundation for future studies to quantitatively investigate the MHC II repertoire on DCs generated under different immunization conditions. PMID:21913724

  4. Selection, diversity and evolutionary patterns of the MHC class II DAB in free-ranging Neotropical marsupials

    PubMed Central

    Meyer-Lucht, Yvonne; Otten, Celine; Püttker, Thomas; Sommer, Simone

    2008-01-01

    Background Research on the genetic architecture and diversity of the MHC has focused mainly on eutherian mammals, birds and fish. So far, studies on model marsupials used in laboratory investigations indicated very little or even no variation in MHC class II genes. However, natural levels of diversity and selection are unknown in marsupials as studies on wild populations are virtually absent. We used two endemic South American mouse opossums, Gracilinanus microtarsus and Marmosops incanus, to investigate characteristic features of MHC selection. This study is the first investigation of MHC selection in free-ranging Neotropical marsupials. In addition, the evolutionary history of MHC lineages within the group of marsupials was examined. Results G. microtarsus showed extensive levels of MHC diversity within and among individuals as 47 MHC-DAB alleles and high levels of sequence divergence were detected at a minimum of four loci. Positively selected codon sites were identified, of which most were congruent with human antigen binding sites. The diversity in M. incanus was rather low with only eight observed alleles at presumably two loci. However, these alleles also revealed high sequence divergence. Again, positive selection was identified on specific codon sites, all congruent with human ABS and with positively selected sites observed in G. microtarsus. In a phylogenetic comparison alleles of M. incanus interspersed widely within alleles of G. microtarsus with four alleles being present in both species. Conclusion Our investigations revealed extensive MHC class II polymorphism in a natural marsupial population, contrary to previous assumptions. Furthermore, our study confirms for the first time in marsupials the presence of three characteristic features common at MHC loci of eutherian mammals, birds and fish: large allelic sequence divergence, positive selection on specific sites and trans-specific polymorphism. PMID:18534008

  5. Modulation of MHC class II transport and lysosome distribution by macrophage-colony stimulating factor in human dendritic cells derived from monocytes.

    PubMed

    Baron, C; Raposo, G; Scholl, S M; Bausinger, H; Tenza, D; Bohbot, A; Pouillart, P; Goud, B; Hanau, D; Salamero, J

    2001-03-01

    The macrophage-colony stimulating factor (M-CSF) has been already shown to affect the function of dendritic cells (DC). Therefore, the differentiation of dendritic cells into macrophages (M(PHI)) might represent a pathway which could inhibit the immune response initiated by DC. Because Major Histocompatibility Complex class II molecules (MHC-II) are crucial for DC function, we asked whether M-CSF may influence the intracellular transport of MHC-II in monocyte derived DC. We found that, at early stages, M-CSF induced first a rapid redistribution of MHC-II from the MHC-II containing compartments (MIIC) to the plasma membrane and second an increase in MHC-II synthesis as observed with LPS or TNF-(alpha). These processes were associated with the sorting of MHC-II from lysosomal membranes which underwent a drastic structural reorganization. However, in contrast to tumor necrosis factor (TNF)-(alpha) or lipopolysaccharide (LPS), M-CSF neither potentiated the allostimulatory function of DC nor allowed the stabilization of MHC-II at the cell surface, but rather increased MHC-II turnover. We conclude that the rapid modulation of MHC-II transport and distribution may participate in the inhibitory effect of M-CSF on DC function and differentiation.

  6. A comparison of the antigen-presenting capabilities of class II MHC-expressing human lung epithelial and endothelial cells.

    PubMed Central

    Cunningham, A C; Zhang, J G; Moy, J V; Ali, S; Kirby, J A

    1997-01-01

    Human lung alveolar epithelial cells constitutively express class II major histocompatibility complex (MHC). Human lung microvascular endothelial and small airway epithelial cells can be induced to express class II MHC by stimulation with the pro-inflammatory cytokine interferon-gamma. The levels of class II MHC on lung epithelial and endothelial cells were comparable to those seen on an Epstein-Barr virus (EBV)-transformed B-cell line. However, the costimulatory molecules B7-1 and B7-2 were not expressed. The ability of the class II MHC expressing human lung parenchymal cells to present alloantigen to CD4+ T lymphocytes was investigated. Freshly isolated human alveolar epithelial cells (type II pneumocytes) and monolayers of interferon-gamma-stimulated small airway epithelial and lung microvascular endothelial cells were co-cultured with allogeneic CD4+ T lymphocytes and proliferation determined by [3H]thymidine incorporation. A clear difference was observed between effects of the epithelial and endothelial cells on CD4+ T-lymphocyte activation. Alveolar and small airway epithelial cells failed to stimulate the proliferation of allogeneic CD4+ T lymphocytes whereas lung microvascular endothelial cells did stimulate proliferation. This difference could not be explained by the levels of class II MHC or the lack of B7-1 and B7-2 solely. Microvascular endothelial cells, and not alveolar or small airway epithelial cells, possess B7-independent costimulatory pathways. PMID:9301537

  7. The common marmoset: A new world primate species with limited Mhc class II variability

    PubMed Central

    Antunes, Susana G.; de Groot, Natasja G.; Brok, Herbert; Doxiadis, Gaby; Menezes, Alexandre A. L.; Otting, Nel; Bontrop, Ronald E.

    1998-01-01

    The common marmoset (Callithrix jacchus) is a New World primate species that is highly susceptible to fatal infections caused by various strains of bacteria. We present here a first step in the molecular characterization of the common marmoset’s Mhc class II genes by nucleotide sequence analysis of the polymorphic exon 2 segments. For this study, genetic material was obtained from animals bred in captivity as well as in the wild. The results demonstrate that the common marmoset has, like other primates, apparently functional Mhc-DR and -DQ regions, but the Mhc-DP region has been inactivated. At the -DR and -DQ loci, only a limited number of lineages were detected. On the basis of the number of alleles found, the -DQA and -B loci appear to be oligomorphic, whereas only a moderate degree of polymorphism was observed for two of three Mhc-DRB loci. The contact residues in the peptide-binding site of the Caja-DRB1*03 lineage members are highly conserved, whereas the -DRB*W16 lineage members show more divergence in that respect. The latter locus encodes five oligomorphic lineages whose members are not observed in any other primate species studied, suggesting rapid evolution, as illustrated by frequent exchange of polymorphic motifs. All common marmosets tested were found to share one monomorphic type of Caja-DRB*W12 allele probably encoded by a separate locus. Common marmosets apparently lack haplotype polymorphism because the number of Caja-DRB loci present per haplotype appears to be constant. Despite this, however, an unexpectedly high number of allelic combinations are observed at the haplotypic level, suggesting that Caja-DRB alleles are exchanged frequently between chromosomes by recombination, promoting an optimal distribution of limited Mhc polymorphisms among individuals of a given population. This peculiar genetic make up, in combination with the limited variability of the major histocompatability complex class II repertoire, may contribute to the common

  8. Prediction of MHC class II binding affinity using SMM-align, a novel stabilization matrix alignment method

    PubMed Central

    Nielsen, Morten; Lundegaard, Claus; Lund, Ole

    2007-01-01

    Background Antigen presenting cells (APCs) sample the extra cellular space and present peptides from here to T helper cells, which can be activated if the peptides are of foreign origin. The peptides are presented on the surface of the cells in complex with major histocompatibility class II (MHC II) molecules. Identification of peptides that bind MHC II molecules is thus a key step in rational vaccine design and developing methods for accurate prediction of the peptide:MHC interactions play a central role in epitope discovery. The MHC class II binding groove is open at both ends making the correct alignment of a peptide in the binding groove a crucial part of identifying the core of an MHC class II binding motif. Here, we present a novel stabilization matrix alignment method, SMM-align, that allows for direct prediction of peptide:MHC binding affinities. The predictive performance of the method is validated on a large MHC class II benchmark data set covering 14 HLA-DR (human MHC) and three mouse H2-IA alleles. Results The predictive performance of the SMM-align method was demonstrated to be superior to that of the Gibbs sampler, TEPITOPE, SVRMHC, and MHCpred methods. Cross validation between peptide data set obtained from different sources demonstrated that direct incorporation of peptide length potentially results in over-fitting of the binding prediction method. Focusing on amino terminal peptide flanking residues (PFR), we demonstrate a consistent gain in predictive performance by favoring binding registers with a minimum PFR length of two amino acids. Visualizing the binding motif as obtained by the SMM-align and TEPITOPE methods highlights a series of fundamental discrepancies between the two predicted motifs. For the DRB1*1302 allele for instance, the TEPITOPE method favors basic amino acids at most anchor positions, whereas the SMM-align method identifies a preference for hydrophobic or neutral amino acids at the anchors. Conclusion The SMM-align method was

  9. Genomics and polymorphism of Agph-DAB1, an Mhc class II B gene in red-winged blackbirds (Agelaius phoeniceus).

    PubMed

    Edwards, S V; Gasper, J; March, M

    1998-03-01

    To further our understanding of the evolution of avian Mhc genes at the genomic level, we screened a cosmid library made from a red-winged blackbird (Agelaius phoeniceus) with a blackbird cDNA probe and subcloned from one of the Mhc-containing cosmids a gene which we designate Agph-DAB1. The structure of the gene is similar to that found for chicken class II B genes, except that the introns are surprisingly large, ranging from 98 to over 600 bp, making this the longest avian class II B gene to date. Using primers targeted toward the introns flanking the peptide-binding region (PBR), we amplified the entirety of the second exon and determined nucleotide sequences of 41 PCR products from eight individual blackbirds. The 10 sequence types found, among which were two probable pseudogene sequences, exhibit the classic hallmarks for evolution of PBRs, namely, an excess of nonsynonymous over synonymous substitutions and evidence of gene conversion events in polymorphic subdomains. Despite these patterns and our use of intron primers, the distribution of sequences among individuals suggests that more than one locus was amplified in most individuals, and the bushlike tree of sequences provides little information as to locus-specific clusters. These results imply a complex history of gene conversion, recent duplication, or possibly, concerted evolution among multiple loci, although Agph-DAB1, the first genomic Mhc sequence from a bird other than chicken, provides important clues in the quest for locus-specific Mhc primers in birds.

  10. Low MHC class II diversity in the Tasmanian devil (Sarcophilus harrisii).

    PubMed

    Cheng, Yuanyuan; Sanderson, Claire; Jones, Menna; Belov, Katherine

    2012-07-01

    The largest remaining carnivorous marsupial, the Tasmanian devil (Sarcophilus harrisii), is currently under threat of extinction due to a fatal contagious cancer-devil facial tumour disease. Low major histocompatibility complex (MHC) class I diversity is believed to have contributed to the transmission of the tumour allograft through devil populations. Here, we report low MHC class II variability in this species, with DA β chain genes (Saha-DAB1, 2 and 3) exhibiting very limited diversity and the sole α chain gene (Saha-DAA) monomorphic. Three, six and three alleles were found at Saha-DAB1, 2 and 3, respectively, with a predominant allele found at each locus. Heterozygosity at these three loci is low in the eastern population and modestly higher in northwestern individuals. The results are indicative of a selective sweep likely due to an infectious disease resulting in the fixation of selectively favoured alleles and depletion of genetic diversity at devil class II loci. Several attempts were made to isolate the other marsupial classical class II gene family, namely, DB, resulting in only one DBB pseudogene being found. These findings further support the view that this species has a compromised capacity to respond to pathogen evolution, emerging infectious diseases and environmental changes.

  11. Diverse repertoire of the MHC class II-peptide complexes is required for presentation of viral superantigens.

    PubMed

    Golovkina, T; Agafonova, Y; Kazansky, D; Chervonsky, A

    2001-02-15

    Among other features, peptides affect MHC class II molecules, causing changes in the binding of bacterial superantigens (b-Sag). Whether peptides can alter binding of viral superantigens (v-Sag) to MHC class II was not known. Here we addressed the question of whether mutations limiting the diversity of peptides bound by the MHC class II molecules influenced the presentation of v-Sag and, subsequently, the life cycle of the mouse mammary tumor virus (MMTV). T cells reactive to v-Sag were found in mice lacking DM molecules as well as in A(b)Ep-transgenic mice in which MHC class II binding grooves were predominantly occupied by an invariant chain fragment or Ealpha(52-68) peptide, respectively. APCs from the mutant mice failed to present v-Sag, as determined by the lack of Sag-specific T cell activation, Sag-induced T cell deletion, and by the aborted MMTV infection. In contrast, mice that express I-A(b) with a variety of bound peptides presented v-Sag and were susceptible to MMTV infection. Comparison of v-Sag and b-Sag presentation by the same mutant cells suggested that presentation of v-Sag had requirements similar to that for presentation of toxic shock syndrome toxin-1. Thus, MHC class II peptide repertoire is critical for recognition of v-Sag by the T cells and affects the outcome of infection with a retrovirus.

  12. Distribution of CD163-positive cell and MHC class II-positive cell in the normal equine uveal tract.

    PubMed

    Sano, Yuto; Matsuda, Kazuya; Okamoto, Minoru; Takehana, Kazushige; Hirayama, Kazuko; Taniyama, Hiroyuki

    2016-02-01

    Antigen-presenting cells (APCs) in the uveal tract participate in ocular immunity including immune homeostasis and the pathogenesis of uveitis. In horses, although uveitis is the most common ocular disorder, little is known about ocular immunity, such as the distribution of APCs. In this study, we investigated the distribution of CD163-positive and MHC II-positive cells in the normal equine uveal tract using an immunofluorescence technique. Eleven eyes from 10 Thoroughbred horses aged 1 to 24 years old were used. Indirect immunofluorescence was performed using the primary antibodies CD163, MHC class II (MHC II) and CD20. To demonstrate the site of their greatest distribution, positive cells were manually counted in 3 different parts of the uveal tract (ciliary body, iris and choroid), and their average number was assessed by statistical analysis. The distribution of pleomorphic CD163- and MHC II-expressed cells was detected throughout the equine uveal tract, but no CD20-expressed cells were detected. The statistical analysis demonstrated the distribution of CD163- and MHC II-positive cells focusing on the ciliary body. These results demonstrated that the ciliary body is the largest site of their distribution in the normal equine uveal tract, and the ciliary body is considered to play important roles in uveal and/or ocular immune homeostasis. The data provided in this study will help further understanding of equine ocular immunity in the normal state and might be beneficial for understanding of mechanisms of ocular disorders, such as equine uveitis.

  13. Association of CD99 short and long forms with MHC class I, MHC class II and tetraspanin CD81 and recruitment into immunological synapses

    PubMed Central

    2011-01-01

    Background CD99, a leukocyte surface glycoprotein, is broadly expressed in many cell types. On the cell surface, CD99 is expressed as two distinct isoforms, a long form and a short form. CD99 has been demonstrated to play a key role in several biological processes, including the regulation of T cell activation. However, the molecular mechanisms by which CD99 participates in such processes are unclear. As CD99 contains a short cytoplasmic tail, it is unlikely that CD99 itself takes part in its multi-functions. Association of CD99 with other membrane proteins has been suggested to be necessary for exerting its functions. Results In this study, we analyzed the association of CD99 with other cell surface molecules involved in T cell activation. We demonstrate the association of MHC class I, MHC class II and tetraspanin CD81 with CD99 molecules on the cell surface. Association of CD99 with its partners was observed for both isoforms. In addition, we determined that CD99 is a lipid raft-associated membrane protein and is recruited into the immunologic synapse during T cell activation. The implication of CD99 on T cell activation was investigated. Inhibition of anti-CD3 induced T cell proliferation by an anti-CD99 monoclonal antibody was observed. Conclusions We provide evidence that CD99 directly interact and form the complex with the MHC class I and II, and tetraspanin CD81, and is functionally linked to the formation of the immunologic synapse. Upon T cell activation, CD99 engagement can inhibit T cell proliferation. We speculate that the CD99-MHC-CD81 complex is a tetraspanin web that plays an important role in T cell activation. PMID:21838920

  14. High levels of MHC class II allelic diversity in lake trout from Lake Superior

    USGS Publications Warehouse

    Dorschner, M.O.; Duris, T.; Bronte, C.R.; Burnham-Curtis, M. K.; Phillips, R.B.

    2000-01-01

    Sequence variation in a 216 bp portion of the major histocompatibility complex (MHC) II B1 domain was examined in 74 individual lake trout (Salvelinus namaycush) from different locations in Lake Superior. Forty-three alleles were obtained which encoded 71-72 amino acids of the mature protein. These sequences were compared with previous data obtained from five Pacific salmon species and Atlantic salmon using the same primers. Although all of the lake trout alleles clustered together in the neighbor-joining analysis of amino acid sequences, one amino acid allelic lineage was shared with Atlantic salmon (Salmo salar), a species in another genus which probably diverged from Salvelinus more than 10-20 million years ago. As shown previously in other salmonids, the level of nonsynonymous nucleotide substitution (d(N)) exceeded the level of synonymous substitution (d(S)). The level of nucleotide diversity at the MHC class II B1 locus was considerably higher in lake trout than in the Pacific salmon (genus Oncorhynchus). These results are consistent with the hypothesis that lake trout colonized Lake Superior from more than one refuge following the Wisconsin glaciation. Recent population bottlenecks may have reduced nucleotide diversity in Pacific salmon populations.

  15. Selection of unrelated bone marrow donors: does the current procedure warrant complete MHC class II identity?

    PubMed

    Eiermann, T H; Ballas, M; Fakler, J; Müller, C; Wölpl, A; Goldmann, S F

    1992-01-01

    Bone marrow transplantation from unrelated donors is being used increasingly for the treatment of patients with leukemia and several other hematologic disorders. Selection of unrelated bone marrow donors currently relies on serological HLA identity and negative mixed lymphocyte reactions between donor/recipient pairs. As serological HLA-DP typing is not feasible, we used the HLA-DPB1 oligonucleotide typing method to investigate whether the current selection procedure can guarantee complete MHC class II identity. In 40 consecutive patients, one third (62/193) serologically HLA-A, -B, -C, -DR and -DQ identical donors were found to be MLC-negative with a relative response below 5%. HLA-DPB1 oligonucleotide typing of these MLC-negative donors revealed that again only one third (20/62) was also identical for DP with their presumptive recipients. In the majority of pairs a disparity in graft-versus-host direction or in host-versus-graft direction of at least one allele was seen. These data indicate that, in spite of the strict MLC criteria used, the current procedure did not warrant complete MHC class II identity. This implies that oligotyping for DPB1 can improve matching and should be introduced for typing of volunteers.

  16. Exogenous antigens bind MHC class II first, and are processed by cathepsins later.

    PubMed

    Sadegh-Nasseri, Scheherazade; Kim, AeRyon

    2015-12-01

    The field of antigen processing and presentation is likely one of the most well defined areas in immunology based on decades of intense molecular and structural studies. Many molecules contributing to antigen processing and presentation have been discovered and their mechanisms of action been largely defined, yet a major question, which lies at the very core of the field has remained hard to pin down. The question is what determines immunodominance? Immunodominance is defined as a few specific epitopes being selected to represent an antigen to the immune system and provide targets for T cells. Many studies have aimed at understanding how epitopes are selected. A range of hypotheses related to the structural features of antigens, sensitivity to proteases, epitope affinity for MHC II, T cell precursor frequency, and T cell receptor affinity for peptide/MHC II have been considered. However, because of the variety of proteins and factors involved in antigen processing and enormous complexity, finding an answer has been challenging. Here we make an effort to tease out the sequence of events in antigen processing that promote selection of immunodominant epitopes for exogenous antigens. Copyright © 2015 Elsevier Ltd. All rights reserved.

  17. Trans-species polymorphism and selection in the MHC class II DRA genes of domestic sheep.

    PubMed

    Ballingall, Keith T; Rocchi, Mara S; McKeever, Declan J; Wright, Frank

    2010-06-30

    Highly polymorphic genes with central roles in lymphocyte mediated immune surveillance are grouped together in the major histocompatibility complex (MHC) in higher vertebrates. Generally, across vertebrate species the class II MHC DRA gene is highly conserved with only limited allelic variation. Here however, we provide evidence of trans-species polymorphism at the DRA locus in domestic sheep (Ovis aries). We describe variation at the Ovar-DRA locus that is far in excess of anything described in other vertebrate species. The divergent DRA allele (Ovar-DRA*0201) differs from the sheep reference sequences by 20 nucleotides, 12 of which appear non-synonymous. Furthermore, DRA*0201 is paired with an equally divergent DRB1 allele (Ovar-DRB1*0901), which is consistent with an independent evolutionary history for the DR sub-region within this MHC haplotype. No recombination was observed between the divergent DRA and B genes in a range of breeds and typical levels of MHC class II DR protein expression were detected at the surface of leukocyte populations obtained from animals homozygous for the DRA*0201, DRB1*0901 haplotype. Bayesian phylogenetic analysis groups Ovar-DRA*0201 with DRA sequences derived from species within the Oryx and Alcelaphus genera rather than clustering with other ovine and caprine DRA alleles. Tests for Darwinian selection identified 10 positively selected sites on the branch leading to Ovar-DRA*0201, three of which are predicted to be associated with the binding of peptide antigen. As the Ovis, Oryx and Alcelaphus genera have not shared a common ancestor for over 30 million years, the DRA*0201 and DRB1*0901 allelic pair is likely to be of ancient origin and present in the founding population from which all contemporary domestic sheep breeds are derived. The conservation of the integrity of this unusual DR allelic pair suggests some selective advantage which is likely to be associated with the presentation of pathogen antigen to T-cells and the

  18. Trans-Species Polymorphism and Selection in the MHC Class II DRA Genes of Domestic Sheep

    PubMed Central

    Ballingall, Keith T.; Rocchi, Mara S.; McKeever, Declan J.; Wright, Frank

    2010-01-01

    Highly polymorphic genes with central roles in lymphocyte mediated immune surveillance are grouped together in the major histocompatibility complex (MHC) in higher vertebrates. Generally, across vertebrate species the class II MHC DRA gene is highly conserved with only limited allelic variation. Here however, we provide evidence of trans-species polymorphism at the DRA locus in domestic sheep (Ovis aries). We describe variation at the Ovar-DRA locus that is far in excess of anything described in other vertebrate species. The divergent DRA allele (Ovar-DRA*0201) differs from the sheep reference sequences by 20 nucleotides, 12 of which appear non-synonymous. Furthermore, DRA*0201 is paired with an equally divergent DRB1 allele (Ovar-DRB1*0901), which is consistent with an independent evolutionary history for the DR sub-region within this MHC haplotype. No recombination was observed between the divergent DRA and B genes in a range of breeds and typical levels of MHC class II DR protein expression were detected at the surface of leukocyte populations obtained from animals homozygous for the DRA*0201, DRB1*0901 haplotype. Bayesian phylogenetic analysis groups Ovar-DRA*0201 with DRA sequences derived from species within the Oryx and Alcelaphus genera rather than clustering with other ovine and caprine DRA alleles. Tests for Darwinian selection identified 10 positively selected sites on the branch leading to Ovar-DRA*0201, three of which are predicted to be associated with the binding of peptide antigen. As the Ovis, Oryx and Alcelaphus genera have not shared a common ancestor for over 30 million years, the DRA*0201 and DRB1*0901 allelic pair is likely to be of ancient origin and present in the founding population from which all contemporary domestic sheep breeds are derived. The conservation of the integrity of this unusual DR allelic pair suggests some selective advantage which is likely to be associated with the presentation of pathogen antigen to T-cells and the

  19. Extraordinary MHC class II B diversity in a non-passerine, wild bird: the Eurasian Coot Fulica atra (Aves: Rallidae).

    PubMed

    Alcaide, Miguel; Muñoz, Joaquin; Martínez-de la Puente, Josué; Soriguer, Ramón; Figuerola, Jordi

    2014-03-01

    The major histocompatibility complex (MHC) hosts the most polymorphic genes ever described in vertebrates. The MHC triggers the adaptive branch of the immune response, and its extraordinary variability is considered an evolutionary consequence of pathogen pressure. The last few years have witnessed the characterization of the MHC multigene family in a large diversity of bird species, unraveling important differences in its polymorphism, complexity, and evolution. Here, we characterize the first MHC class II B sequences isolated from a Rallidae species, the Eurasian Coot Fulica atra. A next-generation sequencing approach revealed up to 265 alleles that translated into 251 different amino acid sequences (β chain, exon 2) in 902 individuals. Bayesian inference identified up to 19 codons within the presumptive peptide-binding region showing pervasive evidence of positive, diversifying selection. Our analyses also detected a significant excess of high-frequency segregating sites (average Tajima's D = 2.36, P < 0.05), indicative of balancing selection. We found one to six different alleles per individual, consistent with the occurrence of at least three MHC class II B gene duplicates. However, the genotypes comprised of three alleles were by far the most abundant in the population investigated (49.4%), followed by those with two (29.6%) and four (17.5%) alleles. We suggest that these proportions are in agreement with the segregation of MHC haplotypes differing in gene copy number. The most widespread segregating haplotypes, according to our findings, would contain one single gene or two genes. The MHC class II of the Eurasian Coot is a valuable system to investigate the evolutionary implications of gene copy variation and extensive variability, the greatest ever found, to the best of our knowledge, in a wild population of a non-passerine bird.

  20. Extraordinary MHC class II B diversity in a non-passerine, wild bird: the Eurasian Coot Fulica atra (Aves: Rallidae)

    PubMed Central

    Alcaide, Miguel; Muñoz, Joaquin; Martínez-de la Puente, Josué; Soriguer, Ramón; Figuerola, Jordi

    2014-01-01

    The major histocompatibility complex (MHC) hosts the most polymorphic genes ever described in vertebrates. The MHC triggers the adaptive branch of the immune response, and its extraordinary variability is considered an evolutionary consequence of pathogen pressure. The last few years have witnessed the characterization of the MHC multigene family in a large diversity of bird species, unraveling important differences in its polymorphism, complexity, and evolution. Here, we characterize the first MHC class II B sequences isolated from a Rallidae species, the Eurasian Coot Fulica atra. A next-generation sequencing approach revealed up to 265 alleles that translated into 251 different amino acid sequences (β chain, exon 2) in 902 individuals. Bayesian inference identified up to 19 codons within the presumptive peptide-binding region showing pervasive evidence of positive, diversifying selection. Our analyses also detected a significant excess of high-frequency segregating sites (average Tajima's D = 2.36, P < 0.05), indicative of balancing selection. We found one to six different alleles per individual, consistent with the occurrence of at least three MHC class II B gene duplicates. However, the genotypes comprised of three alleles were by far the most abundant in the population investigated (49.4%), followed by those with two (29.6%) and four (17.5%) alleles. We suggest that these proportions are in agreement with the segregation of MHC haplotypes differing in gene copy number. The most widespread segregating haplotypes, according to our findings, would contain one single gene or two genes. The MHC class II of the Eurasian Coot is a valuable system to investigate the evolutionary implications of gene copy variation and extensive variability, the greatest ever found, to the best of our knowledge, in a wild population of a non-passerine bird. PMID:24683452

  1. Enhanced Detection of Antigen-Specific CD4+ T Cells Using Altered Peptide Flanking Residue Peptide–MHC Class II Multimers

    PubMed Central

    Holland, Christopher J.; Dolton, Garry; Scurr, Martin; Ladell, Kristin; Schauenburg, Andrea J.; Miners, Kelly; Madura, Florian; Sewell, Andrew K.; Price, David A.

    2015-01-01

    Fluorochrome-conjugated peptide–MHC (pMHC) class I multimers are staple components of the immunologist’s toolbox, enabling reliable quantification and analysis of Ag-specific CD8+ T cells irrespective of functional outputs. In contrast, widespread use of the equivalent pMHC class II (pMHC-II) reagents has been hindered by intrinsically weaker TCR affinities for pMHC-II, a lack of cooperative binding between the TCR and CD4 coreceptor, and a low frequency of Ag-specific CD4+ T cell populations in the peripheral blood. In this study, we show that peptide flanking regions, extending beyond the central nonamer core of MHC-II–bound peptides, can enhance TCR–pMHC-II binding and T cell activation without loss of specificity. Consistent with these findings, pMHC-II multimers incorporating peptide flanking residue modifications proved superior for the ex vivo detection, characterization, and manipulation of Ag-specific CD4+ T cells, highlighting an unappreciated feature of TCR–pMHC-II interactions. PMID:26553072

  2. Lineage targeted MHC-II transgenic mice demonstrate the role of dendritic cells in bacterial driven colitis

    PubMed Central

    Maggio-Price, Lillian; Seamons, Audrey; Bielefeldt-Ohmann, Helle; Zeng, Weiping; Brabb, Thea; Ware, Carol; Lei, Mingzu; Hershberg, Robert M.

    2012-01-01

    Background Inflammatory bowel disease (IBD) pathogenesis involves an inadequately controlled immune reaction to intestinal microbiota and CD4+ T cells, dependent upon MHC class II (MHC-II) processing and presentation by antigen presenting cells (APC), play important roles The role of professional APC (macrophages and dendritic cells (DC)) and nonprofessional APC (intestinal epithelial cells (IEC)) in microbial driven intestinal inflammation remains controversial. Methods We generated transgenic animals on a MHC-II−/− genetic background in which MHC-II is expressed on a) DC via the CD11c promoter (CD11cTg) or b) IEC via the fatty acid binding protein (liver) promoter (EpithTg). These mice were crossed with Rag2−/− mice to eliminate T and B cells (CD11cTg/Rag2−/− and EpithTg/Rag2−/−). Helicobacter bilis (Hb) infection and adoptive transfer (AT) of naïve CD4+ T cells were used to trigger IBD. Results CD11cTg/Rag2−/− mice infected with Hb+AT developed severe colitis within three weeks post AT, similar to disease in positive control Rag2−/− mice infected with Hb+AT. CD11cTg/Rag2−/− mice given AT alone or Hb alone had significantly less severe colitis. In contrast, EpithTg/Rag2−/− mice infected with Hb+AT developed mild colitis by three weeks and even after 16 weeks post adoptive transfer, had only mild lesions. Conclusions MHC-II expression restricted to DCs is sufficient to induce severe colitis in the presence of T cells and a microorganism such as Hb within three weeks of adoptive transfer. Expression of MHC-II solely on IEC in the presence of a microbial trigger and T cells was insufficient for triggering severe colitis. PMID:22619032

  3. Re-Directing CD4(+) T Cell Responses with the Flanking Residues of MHC Class II-Bound Peptides: The Core is Not Enough.

    PubMed

    Holland, Christopher J; Cole, David K; Godkin, Andrew

    2013-01-01

    Recombinant αβ T cell receptors, expressed on T cell membranes, recognize short peptides presented at the cell surface in complex with MHC molecules. There are two main subsets of αβ T cells: CD8(+) T cells that recognize mainly cytosol-derived peptides in the context of MHC class I (pMHC-I), and CD4(+) T cells that recognize peptides usually derived from exogenous proteins presented by MHC class II (pMHC-II). Unlike the more uniform peptide lengths (usually 8-13mers) bound in the MHC-I closed groove, MHC-II presented peptides are of a highly variable length. The bound peptides consist of a core bound 9mer (reflecting the binding motif for the particular MHC-II type) but with variable peptide flanking residues (PFRs) that can extend from both the N- and C-terminus of the MHC-II binding groove. Although pMHC-I and pMHC-II play a virtually identical role during T cell responses (T cell antigen presentation) and are very similar in overall conformation, there exist a number of subtle but important differences that may govern the functional dichotomy observed between CD8(+) and CD4(+) T cells. Here, we provide an overview of the impact of structural differences between pMHC-I and pMHC-II and the molecular interactions with the T cell receptor including the functional importance of MHC-II PFRs. We consider how factors such as anatomical location, inflammatory milieu, and particular types of antigen presenting cell might, in theory, contribute to the quantitative (i.e., pMHC ligand frequency) as well as qualitative (i.e., variable PFR) nature of peptide epitopes, and hence offer a means of control and influence of a CD4(+) T cell response. Lastly, we review our recent findings showing how modifications to MHC-II PFRs can modify CD4(+) T cell antigen recognition. These findings may have novel applications for the development of CD4(+) T cell peptide vaccines and diagnostics.

  4. Sequence analysis of the MHC class II DPB1 gene in chimpanzees (Pan troglodytes).

    PubMed

    Bak, E-J; Ishii, Y; Omatsu, T; Kyuwa, S; Hayasaka, I; Yoshikawa, Y

    2005-06-01

    The diversity of the MHC class II region in non-human primates is a focus of biomedical research because this region plays a crucial role in the recognition of antigens in the immune system. In particular, the chimpanzee [Pan troglodytes (Patr)], which belongs to the superfamily Hominoidea, has been used as a human model for the study of diseases such as human hepatitis C virus (HCV), human hepatitis B virus (HBV) and human immunodeficiency virus (HIV) infections, to which only humans and chimpanzees are susceptible. In the present study, polymorphisms of the MHC-DPB1 gene (Patr-DPB1) in a chimpanzee colony in Japan were examined using a stepwise polymerase chain reaction (PCR) technique. In order to design a suitable primer pair which would amplify exon 2 of the Patr-DPB1 gene, a fragment of approximately 8 kb from exon 1 to exon 3 was amplified from chimpanzee genomic DNA. After designing a 500-bp primer pair at the 3' region of intron 1 and the 5' region of intron 2, analysis of DPB1 exon 2 alleles of each chimpanzee was carried out. Twenty-two chimpanzees were used in our study, and we identified seven alleles by sequence analysis on the Patr-DPB1 gene, including one new allele. The obtained nucleotide sequence patterns suggest that Patr-DPB1 alleles emerge by genetic variations such as the exchange of sequence motifs and the accumulation of point mutations.

  5. Recombination hotspots rather than population history dominate linkage disequilibrium in the MHC class II region.

    PubMed

    Kauppi, Liisa; Sajantila, Antti; Jeffreys, Alec J

    2003-01-01

    Recombination, demographic history, drift and selection influence the extent of linkage disequilibrium (LD) in the human genome, but their relative contributions remain unclear. To investigate the effect of meiotic recombination versus population history on LD, three populations with different demographic histories (UK north Europeans, Saami and Zimbabweans) were genotyped for high-frequency single-nucleotide polymorphisms (SNPs) across a 75 kb DNA segment of the MHC class II region. This region spans three well-characterized recombination hotspots and a 60 kb long LD block. Despite a high level of underlying haplotype diversity and considerable divergence in haplotype composition between populations, all three populations showed very similar patterns of LD. Surprisingly, the entire 60 kb LD block was present even in Africans, although it was relatively difficult to detect owing to a systematic deficiency of high frequency SNPs. In contrast, DNA within recombination hotspots did not show this low nucleotide diversity in Africans. Thus, while population history has some influence on LD, our findings suggest that recombination hotspots play a major global role in shaping LD patterns as well as helping to maintain localized SNP diversity in this region of the MHC.

  6. Association of an MHC Class II Haplotype with Increased Risk of Polymyositis in Hungarian Vizsla Dogs

    PubMed Central

    Massey, Jonathan; Rothwell, Simon; Rusbridge, Clare; Tauro, Anna; Addicott, Diane; Chinoy, Hector; Cooper, Robert G.; Ollier, William E. R.; Kennedy, Lorna J.

    2013-01-01

    A breed-specific polymyositis is frequently observed in the Hungarian Vizsla. Beneficial clinical response to immunosuppressive therapies has been demonstrated which points to an immune-mediated aetiology. Canine inflammatory myopathies share clinical and histological similarities with the human immune-mediated myopathies. As MHC class II associations have been reported in the human conditions we investigated whether an MHC class II association was present in the canine myopathy seen in this breed. 212 Hungarian Vizsla pedigree dogs were stratified both on disease status and degree of relatedness to an affected dog. This generated a group of 29 cases and 183 “graded” controls: 93 unaffected dogs with a first degree affected relative, 44 unaffected dogs with a second degree affected relative, and 46 unaffected dogs with no known affected relatives. Eleven DLA class II haplotypes were identified, of which, DLA-DRB1*02001/DQA1*00401/DQB1*01303, was at significantly raised frequency in cases compared to controls (OR = 1.92, p = 0.032). When only control dogs with no family history of the disease were compared to cases, the association was further strengthened (OR = 4.08, p = 0.00011). Additionally, a single copy of the risk haplotype was sufficient to increase disease risk, with the risk substantially increasing for homozygotes. There was a trend of increasing frequency of this haplotype with degree of relatedness, indicating low disease penetrance. These findings support the hypothesis of an immune-mediated aetiology for this canine myopathy and give credibility to potentially using the Hungarian Vizsla as a genetic model for comparative studies with human myositis. PMID:23457575

  7. Brucella abortus down-regulates MHC class II by the IL-6-dependent inhibition of CIITA through the downmodulation of IFN regulatory factor-1 (IRF-1).

    PubMed

    Velásquez, Lis N; Milillo, M Ayelén; Delpino, M Victoria; Trotta, Aldana; Fernández, Pablo; Pozner, Roberto G; Lang, Roland; Balboa, Luciana; Giambartolomei, Guillermo H; Barrionuevo, Paula

    2017-03-01

    Brucella abortus is an intracellular pathogen capable of surviving inside of macrophages. The success of B. abortus as a chronic pathogen relies on its ability to orchestrate different strategies to evade the adaptive CD4(+) T cell responses that it elicits. Previously, we demonstrated that B. abortus inhibits the IFN-γ-induced surface expression of MHC class II (MHC-II) molecules on human monocytes, and this phenomenon correlated with a reduction in antigen presentation. However, the molecular mechanisms, whereby B. abortus is able to down-regulate the expression of MHC-II, remained to be elucidated. In this study, we demonstrated that B. abortus infection inhibits the IFN-γ-induced transcription of MHC-II, transactivator (CIITA) and MHC-II genes. Accordingly, we observed that the synthesis of MHC-II proteins was also diminished. B. abortus was not only able to reduce the expression of mature MHC-II, but it also inhibited the expression of invariant chain (Ii)-associated immature MHC-II molecules. Outer membrane protein 19 (Omp19), a prototypical B. abortus lipoprotein, diminished the expression of MHC-II and CIITA transcripts to the same extent as B. abortus infection. IL-6 contributes to these down-regulatory phenomena. In addition, B. abortus and its lipoproteins, through IL-6 secretion, induced the transcription of the negative regulators of IFN-γ signaling, suppressor of cytokine signaling (SOCS)-1 and -3, without interfering with STAT1 activation. Yet, B. abortus lipoproteins via IL-6 inhibit the expression of IFN regulatory factor 1 (IRF-1), a critical regulatory transcription factor for CIITA induction. Overall, these results indicate that B. abortus inhibits the expression of MHC-II molecules at very early points in their synthesis and in this way, may prevent recognition by T cells establishing a chronic infection.

  8. Characterization of a non-classical MHC class II gene in the vulnerable Chinese egret (Egretta eulophotes).

    PubMed

    Lei, Wei; Fang, Wenzhen; Lin, Qingxian; Zhou, Xiaoping; Chen, Xiaolin

    2015-08-01

    Genes of the major histocompatibility complex (MHC) are valuable makers of adaptive genetic variation in evolutionary ecology research, yet the non-classical MHC genes remain largely unstudied in wild vertebrates. In this study, we have characterized the non-classical MHC class II gene, Egeu-DAB4, in the vulnerable Chinese egret (Ciconiiformes, Ardeidae, Egretta eulophotes). Gene expression analyses showed that Egeu-DAB4 gene had a restricted tissue expression pattern, being expressed in seven examined tissues including the liver, heart, kidney, esophagus, stomach, gallbladder, and intestine, but not in muscle. With respect to polymorphism, only one allele of exon 2 was obtained from Egeu-DAB4 using asymmetric PCR, indicating that Egeu-DAB4 is genetically monomorphic in exon 2. Comparative analyses showed that Egeu-DAB4 had an unusual sequence, with amino acid differences suggesting that its function may differ from those of classical MHC genes. Egeu-DAB4 gene was only found in 30.56-36.56 % of examined Chinese egret individuals. Phylogenetic analysis showed a closer relationship between Egeu-DAB4 and the DAB2 genes in nine other ardeid species. These new findings provide a foundation for further studies to clarify the immunogenetics of non-classical MHC class II gene in the vulnerable Chinese egret and other ciconiiform birds.

  9. Evolution of Mhc class II B genes in Darwin's finches and their closest relatives: birth of a new gene.

    PubMed

    Sato, A; Mayer, W E; Tichy, H; Grant, P R; Grant, B R; Klein, J

    2001-12-01

    The 15 extant species of Darwin's finches on the Galápagos and Cocos Islands are the products of an unfinished adaptive radiation from a founder flock of birds related to the South American species Tiaris obscura. Molecular characterization of their major histocompatibility complex ( Mhc) class II B genes has revealed the existence of several related groups of sequences (presumably encoded in distinct loci) from which one (group 5) stands out because of its low divergence over extended time periods. Analysis of group 5 exon 2 and intron 2 sequences has revealed that the encoding locus apparently arose 2-3 million years ago in the Tiaris group of South and Central American Thraupini. The locus shows no evidence of inactivation, but displays a very low degree of polymorphism, both in terms of number of alleles and genetic distances between alleles. Some of the polymorphism, however, appears to be trans-specific. All the observed intergenic differences can be explained by point mutations and most of the exon 2 changes represent non-synonymous substitutions, although the rate of non-synonymous and synonymous substitutions appears to be the same. The origin of the new locus is explained by the birth-and-death model of Mhc evolution with two important extensions. First, the ancestor of the group 5 genes may have arisen without new gene duplication and second, the birth of the new group may have been brought about by a switch from balancing to directional selection. The ancestor of the group 5 genes may have been a classical class II B allele (one of many) which directional selection fixed in the ancestral population and drove into the category of nonclassical genes.

  10. Bacterial Superantigens Promote Acute Nasopharyngeal Infection by Streptococcus pyogenes in a Human MHC Class II-Dependent Manner

    PubMed Central

    Kasper, Katherine J.; Zeppa, Joseph J.; Wakabayashi, Adrienne T.; Xu, Stacey X.; Mazzuca, Delfina M.; Welch, Ian; Baroja, Miren L.; Kotb, Malak; Cairns, Ewa; Cleary, P. Patrick; Haeryfar, S. M. Mansour; McCormick, John K.

    2014-01-01

    Establishing the genetic determinants of niche adaptation by microbial pathogens to specific hosts is important for the management and control of infectious disease. Streptococcus pyogenes is a globally prominent human-specific bacterial pathogen that secretes superantigens (SAgs) as ‘trademark’ virulence factors. SAgs function to force the activation of T lymphocytes through direct binding to lateral surfaces of T cell receptors and class II major histocompatibility complex (MHC-II) molecules. S. pyogenes invariably encodes multiple SAgs, often within putative mobile genetic elements, and although SAgs are documented virulence factors for diseases such as scarlet fever and the streptococcal toxic shock syndrome (STSS), how these exotoxins contribute to the fitness and evolution of S. pyogenes is unknown. Here we show that acute infection in the nasopharynx is dependent upon both bacterial SAgs and host MHC-II molecules. S. pyogenes was rapidly cleared from the nasal cavity of wild-type C57BL/6 (B6) mice, whereas infection was enhanced up to ∼10,000-fold in B6 mice that express human MHC-II. This phenotype required the SpeA superantigen, and vaccination with an MHCII binding mutant toxoid of SpeA dramatically inhibited infection. Our findings indicate that streptococcal SAgs are critical for the establishment of nasopharyngeal infection, thus providing an explanation as to why S. pyogenes produces these potent toxins. This work also highlights that SAg redundancy exists to avoid host anti-SAg humoral immune responses and to potentially overcome host MHC-II polymorphisms. PMID:24875883

  11. Presence of specific MHC Class II expressed alleles associates with clinical disease in ovine progressive pneumonia virus (OPPV) infected sheep

    USDA-ARS?s Scientific Manuscript database

    A genetic tool hypothesized to predict which OPPV infected sheep will progress to debilitating clinical disease is MHC Class II Ovis aries (Ovar)-DRB1. Previously, fifteen Ovar-DRB1 beta 1 expressed alleles were identified in a ewe-lamb flock of 32 originating from an Idaho flock using RT-PCR, clon...

  12. Supernatants of human leukocytes contain mediator, different from interferon gamma, which induces expression of MHC class II antigens

    PubMed Central

    1986-01-01

    In this report, data are presented on the regulation of MHC class II antigen expression by a mediator present in supernatants of human mixed leukocyte cultures (MLC-SN), and which is different from IFN-gamma. The capacity of supernatants to induce antigen expression did not correspond to titers of IFN-gamma. Removal of IFN-gamma using either dialysis against pH 2 or neutralizing mAb against human IFN-gamma did not abrogate the MHC class II antigen expression-inducing capacity of MLC-SN when tested on adenocarcinoma cell lines, kidney epithelial cells, and fibroblasts in vitro in an indirect immunofluorescence assay. Therefore, supernatants of human leukocytes contain a mediator, different from IFN-gamma, which induces expression of MHC class II antigens. Dose-response studies revealed that the mediator is produced after allogeneic and lectin stimulation of human leukocytes, and by unstimulated leukocytes. Activation of leukocytes resulted in increased titers of the mediator. The mediator markedly enhances expression of both HLA-DR and HLA-DQ antigens, whereas IFN-gamma had a similar effect on HLA-DR antigens, and only a minor effect on HLA-DQ antigens. Interaction of the mediator and IFN-gamma resulted in a potentiating effect of these two factors on MHC class II antigen expression. Biochemical analysis revealed a mediator, distinguishable by FPLC from IL-1, IL-2, and human IFN-gamma, and which has a molecular mass of 32 kD. PMID:2941512

  13. Genetic characterization of MHC class II DQB exon 2 variants in gayal (Bos frontalis)

    PubMed Central

    Sun, Yongke; Xi, Dongmei; Li, Guozhi; Hao, Tiantian; Chen, Yuhan; Yang, Yuai

    2014-01-01

    In the present study, exon 2 of major histocompatibility complex (MHC) class II DQB gene from 39 gayals (Bos frontalis) was isolated, characterized and compared with previously reported patterns for other bovidae. It was revealed by sequence analyses that there are 36 DQB exon 2 variants among 39 gayals. These variants exhibited a high degree of nucleotide and amino acid substitutions with most amino acid variations occurring at positions forming the peptide-binding sites (PBS). The DQB loci were analysed for patterns of synonymous (d S) and non-synonymous (d N) substitution. The gayals were observed to be under strong balancing selection in the DQB exon 2 PBS (d N = 0.094, P = 0.001). It appears that this variability among gayals could confer the ability to mount immune responses to a wide variety of peptides or pathogens. PMID:26019566

  14. Genetic characterization of MHC class II DQB exon 2 variants in gayal (Bos frontalis).

    PubMed

    Sun, Yongke; Xi, Dongmei; Li, Guozhi; Hao, Tiantian; Chen, Yuhan; Yang, Yuai

    2014-09-03

    In the present study, exon 2 of major histocompatibility complex (MHC) class II DQB gene from 39 gayals (Bos frontalis) was isolated, characterized and compared with previously reported patterns for other bovidae. It was revealed by sequence analyses that there are 36 DQB exon 2 variants among 39 gayals. These variants exhibited a high degree of nucleotide and amino acid substitutions with most amino acid variations occurring at positions forming the peptide-binding sites (PBS). The DQB loci were analysed for patterns of synonymous (dS) and non-synonymous (dN) substitution. The gayals were observed to be under strong balancing selection in the DQB exon 2 PBS (dN = 0.094, P = 0.001). It appears that this variability among gayals could confer the ability to mount immune responses to a wide variety of peptides or pathogens.

  15. Efficient vaccine against pandemic influenza: combining DNA vaccination and targeted delivery to MHC class II molecules.

    PubMed

    Grødeland, Gunnveig; Bogen, Bjarne

    2015-06-01

    There are two major limitations to vaccine preparedness in the event of devastating influenza pandemics: the time needed to generate a vaccine and rapid generation of sufficient amounts. DNA vaccination could represent a solution to these problems, but efficacy needs to be enhanced. In a separate line of research, it has been established that targeting of vaccine molecules to antigen-presenting cells enhances immune responses. We have combined the two principles by constructing DNA vaccines that encode bivalent fusion proteins; these target hemagglutinin to MHC class II molecules on antigen-presenting cells. Such DNA vaccines rapidly induce hemagglutinin-specific antibodies and T cell responses in immunized mice. Responses are long-lasting and protect mice against challenge with influenza virus. In a pandemic situation, targeted DNA vaccines could be produced and tested within a month. The novel DNA vaccines could represent a solution to pandemic preparedness in the advent of novel influenza pandemics.

  16. MHC class II genes in the European badger (Meles meles): characterization, patterns of variation, and transcription analysis.

    PubMed

    Sin, Yung Wa; Dugdale, Hannah L; Newman, Chris; Macdonald, David W; Burke, Terry

    2012-04-01

    The major histocompatibility complex (MHC) comprises many genes, some of which are polymorphic with numerous alleles. Sequence variation among alleles is most pronounced in exon 2 of the class II genes, which encodes the α1 and β1 domains that form the antigen-binding site (ABS) for the presentation of peptides. The MHC thus plays an important role in pathogen defense. European badgers (Meles meles) are a good species in which to study the MHC, as they harbor a variety of pathogens. We present the first characterization of MHC class II genes, isolated from genomic DNA (gDNA) and complementary DNA (cDNA), in the European badger. Examination of seven individuals revealed four DRB, two DQB, two DQA, and two DRA putatively functional gDNA sequences. All of these sequences, except DRA, exhibited high variability in exon 2; DRB had the highest variability. The ABS codons demonstrated high variability, due potentially to balancing selection, while non-ABS codons had lower variability. Positively selected sites were detected in DRB and DQA. Phylogenetic analysis demonstrated trans-species polymorphism of class II genes. Comparison with cDNA from whole blood revealed that only DRB had a transcription pattern reflecting the alleles that were present in the gDNA, while the other three genes had disparities between gDNA and cDNA. Only one sequence was transcribed, even though two gDNA sequences were present, from each of both DQB and DRA. Our characterization of badger MHC sequences forms a basis for further studies of MHC variability, mate choice, and pathogen resistance in this, and other, species.

  17. Ectopic expression of HLA-DO in mouse dendritic cells diminishes MHC class II antigen presentation.

    PubMed

    Fallas, Jennifer L; Tobin, Helen M; Lou, Olivia; Guo, Donglin; Sant'Angelo, Derek B; Denzin, Lisa K

    2004-08-01

    The MHC class II-like molecule HLA-DM (DM) (H-2M in mice) catalyzes the exchange of CLIP for antigenic peptides in the endosomes of APCs. HLA-DO (DO) (H-2O in mice) is another class II-like molecule that is expressed in B cells, but not in other APCs. Studies have shown that DO impairs or modifies the peptide exchange activity of DM. To further evaluate the role of DO in Ag processing and presentation, we generated transgenic mice that expressed the human HLA-DOA and HLA-DOB genes under the control of a dendritic cell (DC)-specific promoter. Our analyses of DCs from these mice showed that as DO levels increased, cell surface levels of A(b)-CLIP also increased while class II-peptide levels decreased. The presentation of some, but not all, exogenous Ags to T cells or T hybridomas was significantly inhibited by DO. Surprisingly, H-2M accumulated in DO-expressing DCs and B cells, suggesting that H-2O/DO prolongs the half-life of H-2M. Overall, our studies showed that DO expression impaired H-2M function, resulting in Ag-specific down-modulation of class II Ag processing and presentation.

  18. Linkage relationships in the bovine MHC region. High recombination frequency between class II subregions.

    PubMed

    Andersson, L; Lundén, A; Sigurdardottir, S; Davies, C J; Rask, L

    1988-01-01

    Class II genes of the bovine major histocompatibility complex (MHC) have been investigated by Southern blot analysis using human DNA probes. Previous studies revealed the presence of bovine DO beta, DQ alpha, DQ beta, DR alpha, and DR beta genes, and restriction fragment length polymorphisms for each of these genes were documented. In the present study, the presence of three additional class II genes, designated DZ alpha, DY alpha, and DY beta, are reported. DZ alpha was assumed to correspond to the human DZ alpha gene while the other two were designated DY because their relationship to human class II genes could not be firmly established. The linkage relationships among bovine class II genes and two additional loci, TCP1B and C4, were investigated by family segregation analysis and analysis of linkage disequilibrium. The results clearly indicated that all these loci belong to the same linkage group. This linkage group is divided into two subregions separated by a fairly high recombination frequency. One region includes the C4, DQ alpha, DQ beta, DR alpha, and DR beta loci and the other one is composed of the DO beta, DY alpha, DY beta, and TCP1B loci. No recombinant was observed within any of these subregions and there was a strong or fairly strong linkage disequilibrium between loci within groups. In contrast, as many as five recombinants among three different families were detected in the interval between these subregions giving a recombination frequency estimate of 0.17 +/- 0.07. The fairly high recombination frequency observed between class II genes in cattle is strikingly different from the corresponding recombination estimates in man and mouse. The finding implies either a much larger molecular distance between some of the bovine class II genes or alternatively the presence of a recombinational "hot spot" in the bovine class II region.

  19. Class II transactivator-induced MHC class II expression in pancreatic cancer cells leads to tumor rejection and a specific antitumor memory response.

    PubMed

    Ekkirala, Chaitanya Ramesh; Cappello, Paola; Accolla, Roberto S; Giovarelli, Mirella; Romero, Irene; Garrido, Cristina; Garcia-Lora, Angel Miguel; Novelli, Francesco

    2014-10-01

    The loss of major histocompatibility complex (MHC) classes I and II is a well-known mechanism by which cancer cells are able to escape from immune recognition. In this study, we analyzed the expression of antigen processing and presenting molecules in 2 cell lines derived from mouse models of pancreatic ductal adenocarcinoma (PDA) and the effects of the re-expression of MHC class II on PDA rejection. The PDA cell lines were analyzed for the expression of MHC class I, II, and antigen-processing molecules by flow cytometry or polymerase chain reaction. We generated stable PDA-MHC class II transactivator (CIITA) cells and injected them into syngeneic mice. The CD4 and CD8 T-cell role was analyzed in vitro and in vivo. Murine PDA cell lines were negative for MHC and antigen-processing molecules, but their expression was restored by exogenous interferon-γ. CIITA-tumor cells were rejected in 80% to 100% of injected mice, which also developed long-lasting immune memory. In vitro assays and immunohistochemical analyses revealed the recruitment of T effector cells and CD8 T cells into the tumor area. Overall, these data confirm that immunotherapy is a feasible therapeutic approach to recognize and target an aggressive cancer such as PDA.

  20. Distribution of CD163-positive cell and MHC class II-positive cell in the normal equine uveal tract

    PubMed Central

    SANO, Yuto; MATSUDA, Kazuya; OKAMOTO, Minoru; TAKEHANA, Kazushige; HIRAYAMA, Kazuko; TANIYAMA, Hiroyuki

    2015-01-01

    Antigen-presenting cells (APCs) in the uveal tract participate in ocular immunity including immune homeostasis and the pathogenesis of uveitis. In horses, although uveitis is the most common ocular disorder, little is known about ocular immunity, such as the distribution of APCs. In this study, we investigated the distribution of CD163-positive and MHC II-positive cells in the normal equine uveal tract using an immunofluorescence technique. Eleven eyes from 10 Thoroughbred horses aged 1 to 24 years old were used. Indirect immunofluorescence was performed using the primary antibodies CD163, MHC class II (MHC II) and CD20. To demonstrate the site of their greatest distribution, positive cells were manually counted in 3 different parts of the uveal tract (ciliary body, iris and choroid), and their average number was assessed by statistical analysis. The distribution of pleomorphic CD163- and MHC II-expressed cells was detected throughout the equine uveal tract, but no CD20-expressed cells were detected. The statistical analysis demonstrated the distribution of CD163- and MHC II-positive cells focusing on the ciliary body. These results demonstrated that the ciliary body is the largest site of their distribution in the normal equine uveal tract, and the ciliary body is considered to play important roles in uveal and/or ocular immune homeostasis. The data provided in this study will help further understanding of equine ocular immunity in the normal state and might be beneficial for understanding of mechanisms of ocular disorders, such as equine uveitis. PMID:26537548

  1. Cholesterol Corrects Altered Conformation of MHC-II Protein in Leishmania donovani Infected Macrophages: Implication in Therapy

    PubMed Central

    Chakrabarti, Saikat; Roy, Syamal

    2016-01-01

    Background Previously we reported that Kala-azar patients show progressive decrease in serum cholesterol as a function of splenic parasite burden. Splenic macrophages (MΦ) of Leishmania donovani (LD) infected mice show decrease in membrane cholesterol, while LD infected macrophages (I-MΦ) show defective T cell stimulating ability that could be corrected by liposomal delivery of cholesterol. T helper cells recognize peptide antigen in the context of class II MHC molecule. It is known that the conformation of a large number of membrane proteins is dependent on membrane cholesterol. In this investigation we tried to understand the influence of decreased membrane cholesterol in I-MΦ on the conformation of MHC-II protein and peptide-MHC-II stability, and its bearing on the antigen specific T-cell activation. Methodology/Principal Findings MΦ of CBA/j mice were infected with Leishmania donovani (I-MΦ). Two different anti-Aκ mAbs were used to monitor the status of MHC-II protein under parasitized condition. One of them (11.5–2) was conformation specific, whereas the other one (10.2.16) was not. Under parasitized condition, the binding of 11.5–2 decreased significantly with respect to the normal counterpart, whereas that of 10.2.16 remained unaltered. The binding of 11.5–2 was restored to normal upon liposomal delivery of cholesterol in I-MΦ. By molecular dynamics (MD) simulation studies we found that there was considerable conformational fluctuation in the transmembrane domain of the MHC-II protein in the presence of membrane cholesterol than in its absence, which possibly influenced the distal peptide binding groove. This was evident from the faster dissociation of the cognate peptide from peptide-MHC complex under parasitized condition, which could be corrected by liposomal delivery of cholesterol in I-MΦ. Conclusion The decrease in membrane cholesterol in I-MΦ may lead to altered conformation of MHC II, and this may contribute to a faster dissociation of

  2. pH dependence and exchange of high and low responder peptides binding to a class II MHC molecule.

    PubMed Central

    Reay, P A; Wettstein, D A; Davis, M M

    1992-01-01

    We have compared the binding kinetics of two antigenic peptides to a soluble class II MHC molecule. One of the peptides provokes a strong T cell response and the other a much weaker one. Both show greatly increased (approximately 40-fold) association rates at pH 5 in comparison to neutral pH, consistent with the low pH environment of late endosomes being most conducive to class II MHC--peptide binding. Interestingly, the weak peptide has a much faster off-rate that is significantly increased at pH 5 and it can be entirely replaced in an exchange reaction by the stronger one. This suggests that one characteristic of immunodominant peptides is that of nearly irreversible binding, such that they will be strongly selected for in the course of class II MHC transit and recycling through endosomal compartments. Modelling the parameters of this peptide exchange also suggests that a large fraction of the GPI-chimeric MHC molecules used in this study are 'empty' with respect to endogenous peptides, or else occupied with extremely weak ones, consistent with their inability to load processed peptides intracellularly. PMID:1379172

  3. In silico designing breast cancer peptide vaccine for binding to MHC class I and II: A molecular docking study.

    PubMed

    Mahdavi, Manijeh; Moreau, Violaine

    2016-12-01

    Antigenic peptides or cancer peptide vaccines can be directly delivered to cancer patients to produce immunologic responses against cancer cells. Specifically, designed peptides can associate with Major Histocompatibility Complex (MHC) class I or II molecules on the cell surface of antigen presenting cells activating anti-tumor effector mechanisms by triggering helper T cell (Th) or cytotoxic T cells (CTL). In general, high binding to MHCs approximately correlates with in vivo immunogenicity. Consequently, a molecular docking technique was run on a library of novel discontinuous peptides predicted by PEPOP from Human epidermal growth factor receptor 2 (HER2 ECD) subdomain III. This technique is expected to improve the prediction accuracy in order to identify the best MHC class I and II binder peptides. Molecular docking analysis through GOLD identified the peptide 1412 as the best MHC binder peptide to both MHC class I and II molecules used in the study. The GOLD results predicted HLA-DR4, HLA-DP2 and TCR as the most often targeted receptors by the peptide 1412. These findings, based on bioinformatics analyses, can be exploited in further experimental analyses in vaccine design and cancer therapy to find possible proper approaches providing beneficial effects.

  4. A single nomenclature and associated database for alleles at the MHC class II DRB1 locus of sheep: IPD-MHC-OLA

    USDA-ARS?s Scientific Manuscript database

    The development of standardised nomenclatures with associated databases containing reference sequences for alleles at polymorphic loci within the Major Histocompatibility Complex (MHC) has been facilitated by the development of the Immuno Polymorphism Database (IPD-MHC). Recently, included within I...

  5. Point mutations in or near the antigen-binding groove of HLA-DR3 implicate class II-associated invariant chain peptide affinity as a constraint on MHC class II polymorphism.

    PubMed

    Doebele, Robert C; Pashine, Achal; Liu, Wendy; Zaller, Dennis M; Belmares, Michael; Busch, Robert; Mellins, Elizabeth D

    2003-05-01

    During maturation of MHC II molecules, newly synthesized and assembled complexes of MHC II alphabeta dimers with invariant chain (Ii) are targeted to endosomes, where Ii is proteolyzed, leaving remnant class II-associated Ii peptides (CLIP) in the MHC II peptide binding groove. CLIP must be released, usually with assistance from the endosomal MHC II peptide exchange factor, HLA-DM, before MHC II molecules can bind endosomal peptides. Structural factors that control rates of CLIP release remain poorly understood, although peptide side chain-MHC II specificity pocket interactions and MHC II polymorphism are important. Here we report that mutations betaS11F, betaS13Y, betaQ70R, betaK71E, betaK71N, and betaR74Q, which map to the P4 and P6 pockets of the groove of HLA-DR3 molecules, as well as alphaG20E adjacent to the groove, are associated with elevated CLIP in cells. Most of these mutations increase the resistance of CLIP-DR3 complexes to dissociation by SDS. In vitro, the groove mutations increase the stability of CLIP-DR3 complexes to dissociation. Dissociation rates in the presence of DM, as well as coimmunoprecipitation of some mutant DR3 molecules with DM, are also diminished. The profound phenotypes associated with some of these point mutations suggest that the need to maintain efficient CLIP release represents a constraint on naturally occurring MHC II polymorphism.

  6. Aberrant MHC class II expression in mouse joints leads to arthritis with extraarticular manifestations similar to rheumatoid arthritis

    PubMed Central

    Kanazawa, Satoshi; Ota, Shusuke; Sekine, Chiyoko; Tada, Toyohiro; Otsuka, Takanobu; Okamoto, Takashi; Sønderstrup, Grete; Peterlin, B. Matija

    2006-01-01

    Genetic susceptibility to rheumatoid arthritis (RA) is associated with certain MHC class II molecules. To clarify the role of these determinants in RA, we generated the D1CC transgenic mouse that expressed genes involved in antigen processing and presentation by the MHC class II pathway in joints. The class II transactivator, which was transcribed from the rat collagen type II promoter and enhancer, directed the expression of these genes. In D1CC mice congenic for the H-2q (DBA/1) background, small amounts of bovine collagen type II in adjuvant induced reproducibly an inflammatory arthritis resembling RA. Importantly, these stimuli had no effect in DBA/1 mice. Eighty-nine percent of D1CC mice developed chronic disease with joint swelling, redness, and heat in association with synovial proliferation as well as pannus formation and mononuclear infiltration of synovial membranes. Granulomatous lesions resembling rheumatoid nodules and interstitial pneumonitis also were observed. As in patients with RA, anticyclic citrullinated peptide antibodies were detected during the inflammatory stage. Finally, joints in D1CC mice displayed juxtaarticular demineralization, severe joint space narrowing, and erosions, which led to ankylosis, but without the appearance of osteophytes. Thus, aberrant expression of MHC class II in joints facilitates the development of severe erosive inflammatory polyarthritis, which is very similar to RA. PMID:16980409

  7. Regulation of the class II MHC pathway in primary human monocytes by granulocyte-macrophage colony-stimulating factor.

    PubMed

    Hornell, Tara M C; Beresford, Guy W; Bushey, Alyssa; Boss, Jeremy M; Mellins, Elizabeth D

    2003-09-01

    GM-CSF stimulates the growth and differentiation of hematopoietic progenitors and also affects mature cell function. These effects have led to the use of GM-CSF as a vaccine adjuvant with promising results; however, the mechanisms underlying GM-CSF-mediated immune potentiation are incompletely understood. In this study, we investigated the hypothesis that the immune stimulatory role of GM-CSF is in part due to effects on class II MHC Ag presentation. We find that, in primary human monocytes treated for 24-48 h, GM-CSF increases surface class II MHC expression and decreases the relative level of the invariant chain-derived peptide, CLIP, bound to surface class II molecules. GM-CSF also increases expression of the costimulatory molecules CD86 and CD40, but not the differentiation marker CD1a or CD16. Furthermore, GM-CSF-treated monocytes are better stimulators in a mixed leukocyte reaction. Additional analyses of the class II pathway revealed that GM-CSF increases total protein and RNA levels of HLA-DR, DM, and DOalpha. Expression of class II transactivator (CIITA) types I and III, but not IV, transcripts increases in response to GM-CSF. Furthermore, GM-CSF increases the amount of CIITA associated with the DR promoter. Thus, our data argue that the proinflammatory role of GM-CSF is mediated in part through increased expression of key molecules involved in the class II MHC pathway via induction of CIITA.

  8. GPS-MBA: Computational Analysis of MHC Class II Epitopes in Type 1 Diabetes

    PubMed Central

    Ren, Jian; Ma, Chuang; Gao, Tianshun; Zhou, Yanhong; Yang, Qing; Xue, Yu

    2012-01-01

    As a severe chronic metabolic disease and autoimmune disorder, type 1 diabetes (T1D) affects millions of people world-wide. Recent advances in antigen-based immunotherapy have provided a great opportunity for further treating T1D with a high degree of selectivity. It is reported that MHC class II I-Ag7 in the non-obese diabetic (NOD) mouse and human HLA-DQ8 are strongly linked to susceptibility to T1D. Thus, the identification of new I-Ag7 and HLA-DQ8 epitopes would be of great help to further experimental and biomedical manipulation efforts. In this study, a novel GPS-MBA (MHC Binding Analyzer) software package was developed for the prediction of I-Ag7 and HLA-DQ8 epitopes. Using experimentally identified epitopes as the training data sets, a previously developed GPS (Group-based Prediction System) algorithm was adopted and improved. By extensive evaluation and comparison, the GPS-MBA performance was found to be much better than other tools of this type. With this powerful tool, we predicted a number of potentially new I-Ag7 and HLA-DQ8 epitopes. Furthermore, we designed a T1D epitope database (TEDB) for all of the experimentally identified and predicted T1D-associated epitopes. Taken together, this computational prediction result and analysis provides a starting point for further experimental considerations, and GPS-MBA is demonstrated to be a useful tool for generating starting information for experimentalists. The GPS-MBA is freely accessible for academic researchers at: http://mba.biocuckoo.org. PMID:22479466

  9. GPS-MBA: computational analysis of MHC class II epitopes in type 1 diabetes.

    PubMed

    Cai, Ruikun; Liu, Zexian; Ren, Jian; Ma, Chuang; Gao, Tianshun; Zhou, Yanhong; Yang, Qing; Xue, Yu

    2012-01-01

    As a severe chronic metabolic disease and autoimmune disorder, type 1 diabetes (T1D) affects millions of people world-wide. Recent advances in antigen-based immunotherapy have provided a great opportunity for further treating T1D with a high degree of selectivity. It is reported that MHC class II I-A(g7) in the non-obese diabetic (NOD) mouse and human HLA-DQ8 are strongly linked to susceptibility to T1D. Thus, the identification of new I-A(g7) and HLA-DQ8 epitopes would be of great help to further experimental and biomedical manipulation efforts. In this study, a novel GPS-MBA (MHC Binding Analyzer) software package was developed for the prediction of I-A(g7) and HLA-DQ8 epitopes. Using experimentally identified epitopes as the training data sets, a previously developed GPS (Group-based Prediction System) algorithm was adopted and improved. By extensive evaluation and comparison, the GPS-MBA performance was found to be much better than other tools of this type. With this powerful tool, we predicted a number of potentially new I-A(g7) and HLA-DQ8 epitopes. Furthermore, we designed a T1D epitope database (TEDB) for all of the experimentally identified and predicted T1D-associated epitopes. Taken together, this computational prediction result and analysis provides a starting point for further experimental considerations, and GPS-MBA is demonstrated to be a useful tool for generating starting information for experimentalists. The GPS-MBA is freely accessible for academic researchers at: http://mba.biocuckoo.org.

  10. Salmonella Typhimurium induces SPI-1 and SPI-2 regulated and strain dependent downregulation of MHC II expression on porcine alveolar macrophages

    PubMed Central

    2012-01-01

    Foodborne salmonellosis is one of the most important bacterial zoonotic diseases worldwide. Salmonella Typhimurium is the serovar most frequently isolated from persistently infected slaughter pigs in Europe. Circumvention of the host’s immune system by Salmonella might contribute to persistent infection of pigs. In the present study, we found that Salmonella Typhimurium strain 112910a specifically downregulated MHC II, but not MHC I, expression on porcine alveolar macrophages in a Salmonella pathogenicity island (SPI)-1 and SPI-2 dependent way. Salmonella induced downregulation of MHC II expression and intracellular proliferation of Salmonella in macrophages were significantly impaired after opsonization with Salmonella specific antibodies prior to inoculation. Furthermore, the capacity to downregulate MHC II expression on macrophages differed significantly among Salmonella strains, independently of strain specific differences in invasion capacity, Salmonella induced cytotoxicity and altered macrophage activation status. The fact that strain specific differences in MHC II downregulation did not correlate with the extent of in vitro SPI-1 or SPI-2 gene expression indicates that other factors are involved in MHC II downregulation as well. Since Salmonella strain dependent interference with the pig’s immune response through downregulation of MHC II expression might indicate that certain Salmonella strains are more likely to escape serological detection, our findings are of major interest for Salmonella monitoring programs primarily based on serology. PMID:22694285

  11. Salmonella Typhimurium induces SPI-1 and SPI-2 regulated and strain dependent downregulation of MHC II expression on porcine alveolar macrophages.

    PubMed

    Van Parys, Alexander; Boyen, Filip; Verbrugghe, Elin; Leyman, Bregje; Bram, Flahou; Haesebrouck, Freddy; Pasmans, Frank

    2012-06-13

    Foodborne salmonellosis is one of the most important bacterial zoonotic diseases worldwide. Salmonella Typhimurium is the serovar most frequently isolated from persistently infected slaughter pigs in Europe. Circumvention of the host's immune system by Salmonella might contribute to persistent infection of pigs. In the present study, we found that Salmonella Typhimurium strain 112910a specifically downregulated MHC II, but not MHC I, expression on porcine alveolar macrophages in a Salmonella pathogenicity island (SPI)-1 and SPI-2 dependent way. Salmonella induced downregulation of MHC II expression and intracellular proliferation of Salmonella in macrophages were significantly impaired after opsonization with Salmonella specific antibodies prior to inoculation. Furthermore, the capacity to downregulate MHC II expression on macrophages differed significantly among Salmonella strains, independently of strain specific differences in invasion capacity, Salmonella induced cytotoxicity and altered macrophage activation status. The fact that strain specific differences in MHC II downregulation did not correlate with the extent of in vitro SPI-1 or SPI-2 gene expression indicates that other factors are involved in MHC II downregulation as well. Since Salmonella strain dependent interference with the pig's immune response through downregulation of MHC II expression might indicate that certain Salmonella strains are more likely to escape serological detection, our findings are of major interest for Salmonella monitoring programs primarily based on serology.

  12. Melanoma-specific MHC-II expression represents a tumour-autonomous phenotype and predicts response to anti-PD-1/PD-L1 therapy

    PubMed Central

    Johnson, Douglas B.; Estrada, Monica V.; Salgado, Roberto; Sanchez, Violeta; Doxie, Deon B.; Opalenik, Susan R.; Vilgelm, Anna E.; Feld, Emily; Johnson, Adam S.; Greenplate, Allison R.; Sanders, Melinda E.; Lovly, Christine M.; Frederick, Dennie T.; Kelley, Mark C.; Richmond, Ann; Irish, Jonathan M.; Shyr, Yu; Sullivan, Ryan J.; Puzanov, Igor; Sosman, Jeffrey A.; Balko, Justin M.

    2016-01-01

    Anti-PD-1 therapy yields objective clinical responses in 30–40% of advanced melanoma patients. Since most patients do not respond, predictive biomarkers to guide treatment selection are needed. We hypothesize that MHC-I/II expression is required for tumour antigen presentation and may predict anti-PD-1 therapy response. In this study, across 60 melanoma cell lines, we find bimodal expression patterns of MHC-II, while MHC-I expression was ubiquitous. A unique subset of melanomas are capable of expressing MHC-II under basal or IFNγ-stimulated conditions. Using pathway analysis, we show that MHC-II(+) cell lines demonstrate signatures of ‘PD-1 signalling', ‘allograft rejection' and ‘T-cell receptor signalling', among others. In two independent cohorts of anti-PD-1-treated melanoma patients, MHC-II positivity on tumour cells is associated with therapeutic response, progression-free and overall survival, as well as CD4+ and CD8+ tumour infiltrate. MHC-II+ tumours can be identified by melanoma-specific immunohistochemistry using commercially available antibodies for HLA-DR to improve anti-PD-1 patient selection. PMID:26822383

  13. MHC class II polymorphism is associated with a canine SLE-related disease complex.

    PubMed

    Wilbe, Maria; Jokinen, Päivi; Hermanrud, Christina; Kennedy, Lorna J; Strandberg, Erling; Hansson-Hamlin, Helene; Lohi, Hannes; Andersson, Göran

    2009-08-01

    Nova Scotia duck tolling retrievers are predisposed to a SLE-related disease complex including immune-mediated rheumatic disease (IMRD) and steroid-responsive meningitis-arteritis (SRMA). IMRD involves symptoms that resemble those seen in systemic autoimmune rheumatic diseases, such as systemic lupus erythematosus, SLE, or SLE-related diseases, in humans. This disease complex involves persistent lameness, stiffness, mainly after resting, and palpable pain from several joints of extremities. The majority of affected dogs display antinuclear autoantibody (ANA)-reactivity. SRMA is manifested in young dogs with high fever and neck stiffness and can be treated with corticosteroids. We have investigated the possible role of MHC class II as a genetic risk factor in IMRD and SRMA etiology. We performed sequence-based typing of the DLA-DRB1, -DQA1, and -DQB1 class II loci in a total of 176 dogs including 51 IMRD (33 ANA-positive), 49 SRMA cases, and 78 healthy controls (two dogs were both IMRD- and SRMA-affected). Homozygosity for the risk haplotype DRB1*00601/DQA1*005011/DQB1*02001 increased the risk for IMRD (OR = 4.9; ANA-positive IMRD: OR = 7.2) compared with all other genotypes. There was a general heterozygote advantage, homozygotes had OR = 4.4 (ANA-positive IMRD: OR = 8.9) compared with all heterozygotes. The risk haplotype contains the five amino acid epitope RARAA, known as the shared epitope for rheumatoid arthritis. No association was observed for SRMA. We conclude that DLA class II is a highly significant genetic risk factor for ANA-positive IMRD. The results indicate narrow diversity of DLA II haplotypes and identify an IMRD-related risk haplotype, which becomes highly significant in homozygous dogs.

  14. A novel HURRAH protocol reveals high numbers of monomorphic MHC class II loci and two asymmetric multi-locus haplotypes in the Père David's deer.

    PubMed

    Wan, Qiu-Hong; Zhang, Pei; Ni, Xiao-Wei; Wu, Hai-Long; Chen, Yi-Yan; Kuang, Ye-Ye; Ge, Yun-Fa; Fang, Sheng-Guo

    2011-01-18

    The Père David's deer is a highly inbred, but recovered, species, making it interesting to consider their adaptive molecular evolution from an immunological perspective. Prior to this study, genomic sequencing was the only method for isolating all functional MHC genes within a certain species. Here, we report a novel protocol for isolating MHC class II loci from a species, and its use to investigate the adaptive evolution of this endangered deer at the level of multi-locus haplotypes. This protocol was designated "HURRAH" based on its various steps and used to estimate the total number of MHC class II loci. We confirmed the validity of this novel protocol in the giant panda and then used it to examine the Père David's deer. Our results revealed that the Père David's deer possesses nine MHC class II loci and therefore has more functional MHC class II loci than the eight genome-sequenced mammals for which full MHC data are currently available. This could potentially account at least in part for the strong survival ability of this species in the face of severe bottlenecking. The results from the HURRAH protocol also revealed that: (1) All of the identified MHC class II loci were monomorphic at their antigen-binding regions, although DRA was dimorphic at its cytoplasmic tail; and (2) these genes constituted two asymmetric functional MHC class II multi-locus haplotypes: DRA1*01 ∼ DRB1 ∼ DRB3 ∼ DQA1 ∼ DQB2 (H1) and DRA1*02 ∼ DRB2 ∼ DRB4 ∼ DQA2 ∼ DQB1 (H2). The latter finding indicates that the current members of the deer species have lost the powerful ancestral MHC class II haplotypes of nine or more loci, and have instead fixed two relatively weak haplotypes containing five genes. As a result, the Père David's deer are currently at risk for increased susceptibility to infectious pathogens.

  15. Genetic diversity of the MHC class-II DQA gene in brown bears (Ursus arctos) on Hokkaido, Northern Japan.

    PubMed

    Goda, Naoki; Mano, Tsutomu; Masuda, Ryuichi

    2009-08-01

    To investigate genetic diversity of a major histocompatibility complex (MHC) gene in the brown bear (Ursus arctos) population on Hokkaido Island, northern Japan, we cloned and sequenced parts of exon 2 and intron 2 of the MHC class-II DQA gene from 32 brown bears. According to strict criteria for allele identification established by mammalian MHC nomenclature committees, four DQA types (Urar-DQA*01 to Urar-DQA*04) were identified. Of the four, however, Urar-DQA*04 had a 12-bp deletion not detected in a cDNA analysis, indicating that this is a pseudogene at a distinct locus generated by gene duplication. The nucleotide sequences of the other three DQA alleles, which were expressed (because detected from cDNA), were very similar, indicating lower DQA variation In the Hokkaido brown bear population than in other mammals. We attribute this low genetic diversity to (1) some limited effect of possible balancing selection; (2) bottlenecks and inbreeding after migration and isolation of the Hokkaido brown bear population from the Eurasian Continent; (3) a much slower evolutionary rate in DQA than in other MHC genes in the Hokkaido brown bear population.

  16. Characterization of MHC class II B polymorphism in bottlenecked New Zealand saddlebacks reveals low levels of genetic diversity.

    PubMed

    Sutton, Jolene T; Robertson, Bruce C; Grueber, Catherine E; Stanton, Jo-Ann L; Jamieson, Ian G

    2013-08-01

    The major histocompatibility complex (MHC) is integral to the vertebrate adaptive immune system. Characterizing diversity at functional MHC genes is invaluable for elucidating patterns of adaptive variation in wild populations, and is particularly interesting in species of conservation concern, which may suffer from reduced genetic diversity and compromised disease resilience. Here, we use next generation sequencing to investigate MHC class II B (MHCIIB) diversity in two sister taxa of New Zealand birds: South Island saddleback (SIS), Philesturnus carunculatus, and North Island saddleback (NIS), Philesturnus rufusater. These two species represent a passerine family outside the more extensively studied Passerida infraorder, and both have experienced historic bottlenecks. We examined exon 2 sequence data from populations that represent the majority of genetic diversity remaining in each species. A high level of locus co-amplification was detected, with from 1 to 4 and 3 to 12 putative alleles per individual for South and North Island birds, respectively. We found strong evidence for historic balancing selection in peptide-binding regions of putative alleles, and we identified a cluster combining non-classical loci and pseudogene sequences from both species, although no sequences were shared between the species. Fewer total alleles and fewer alleles per bird in SIS may be a consequence of their more severe bottleneck history; however, overall nucleotide diversity was similar between the species. Our characterization of MHCIIB diversity in two closely related species of New Zealand saddlebacks provides an important step in understanding the mechanisms shaping MHC diversity in wild, bottlenecked populations.

  17. MHC II-β chain gene expression studies define the regional organization of the thymus in the developing bony fish Dicentrarchus labrax (L.).

    PubMed

    Picchietti, S; Abelli, L; Guerra, L; Randelli, E; Proietti Serafini, F; Belardinelli, M C; Buonocore, F; Bernini, C; Fausto, A M; Scapigliati, G

    2015-02-01

    MHC II-β chain gene transcripts were quantified by real-time PCR and localised by in situ hybridization in the developing thymus of the teleost Dicentrarchus labrax, regarding the specialization of the thymic compartments. MHC II-β expression significantly rose when the first lymphoid colonization of the thymus occurred, thereafter increased further when the organ progressively developed cortex and medulla regions. The evolving patterns of MHC II-β expression provided anatomical insights into some mechanisms of thymocyte selection. Among the stromal cells transcribing MHC II-β, scattered cortical epithelial cells appeared likely involved in the positive selection, while those abundant in the cortico-medullary border and medulla in the negative selection. These latter most represent dendritic cells, based on typical localization and phenotype. These findings provide further proofs that efficient mechanisms leading to maturation of naïve T cells are operative in teleosts, strongly reminiscent of the models conserved in more evolved gnathostomes.

  18. Ubiquitin-mediated fluctuations in MHC class II facilitate efficient germinal center B cell responses

    PubMed Central

    McGowan, Simon J.; Ersching, Jonatan; Ishido, Satoshi; Victora, Gabriel D.; Shin, Jeoung-Sook

    2016-01-01

    Antibody affinity maturation occurs in germinal centers (GCs) through iterative rounds of somatic hypermutation and selection. Selection involves B cells competing for T cell help based on the amount of antigen they capture and present on their MHC class II (MHCII) proteins. How GC B cells are able to rapidly and repeatedly transition between mutating their B cell receptor genes and then being selected shortly after is not known. We report that MHCII surface levels and degradation are dynamically regulated in GC B cells. Through ectopic expression of a photoconvertible MHCII-mKikGR chimeric gene, we found that individual GC B cells differed in the rates of MHCII protein turnover. Fluctuations in surface MHCII levels were dependent on ubiquitination and the E3 ligase March1. Increases in March1 expression in centroblasts correlated with decreases in surface MHCII levels, whereas CD83 expression in centrocytes helped to stabilize MHCII at that stage. Defects in MHCII ubiquitination caused GC B cells to accumulate greater amounts of a specific peptide–MHCII (pMHCII), suggesting that MHCII turnover facilitates the replacement of old complexes. We propose that pMHCII complexes are periodically targeted for degradation in centroblasts to favor the presentation of recently acquired antigens, thereby promoting the fidelity and efficiency of selection. PMID:27162138

  19. Nucleosome eviction from MHC class II promoters controls positioning of the transcription start site

    PubMed Central

    Leimgruber, Elisa; Seguín-Estévez, Queralt; Dunand-Sauthier, Isabelle; Rybtsova, Natalia; Schmid, Christoph D.; Ambrosini, Giovanna; Bucher, Philipp; Reith, Walter

    2009-01-01

    Nucleosome depletion at transcription start sites (TSS) has been documented genome-wide in multiple eukaryotic organisms. However, the mechanisms that mediate this nucleosome depletion and its functional impact on transcription remain largely unknown. We have studied these issues at human MHC class II (MHCII) genes. Activation-induced nucleosome free regions (NFR) encompassing the TSS were observed at all MHCII genes. Nucleosome depletion was exceptionally strong, attaining over 250-fold, at the promoter of the prototypical HLA-DRA gene. The NFR was induced primarily by the transcription factor complex that assembles on the conserved promoter-proximal enhancer situated upstream of the TSS. Functional analyses performed in the context of native chromatin demonstrated that displacing the NFR without altering the sequence of the core promoter induced a shift in the position of the TSS. The NFR thus appears to play a critical role in transcription initiation because it directs correct TSS positioning in vivo. Our results provide support for a novel mechanism in transcription initiation whereby the position of the TSS is controlled by nucleosome eviction rather than by promoter sequence. PMID:19264803

  20. Translational diffusion of individual class II MHC membrane proteins in cells.

    PubMed Central

    Vrljic, Marija; Nishimura, Stefanie Y; Brasselet, Sophie; Moerner, W E; McConnell, Harden M

    2002-01-01

    Single-molecule epifluorescence microscopy was used to observe the translational motion of GPI-linked and native I-E(k) class II MHC membrane proteins in the plasma membrane of CHO cells. The purpose of the study was to look for deviations from Brownian diffusion that might arise from barriers to this motion. Detergent extraction had suggested that these proteins may be confined to lipid microdomains in the plasma membrane. The individual I-E(k) proteins were visualized with a Cy5-labeled peptide that binds to a specific extracytoplasmic site common to both proteins. Single-molecule trajectories were used to compute a radial distribution of displacements, yielding average diffusion coefficients equal to 0.22 (GPI-linked I-E(k)) and 0.18 microm(2)/s (native I-E(k)). The relative diffusion of pairs of proteins was also studied for intermolecular separations in the range 0.3-1.0 microm, to distinguish between free diffusion of a protein molecule and diffusion of proteins restricted to a rapidly diffusing small domain. Both analyses show that motion is predominantly Brownian. This study finds no strong evidence for significant confinement of either GPI-linked or native I-E(k) in the plasma membrane of CHO cells. PMID:12414700

  1. Cell surface display of functional human MHC class II proteins: yeast display versus insect cell display

    PubMed Central

    Wen, Fei; Sethi, Dhruv K.; Wucherpfennig, Kai W.; Zhao, Huimin

    2011-01-01

    Reliable and robust systems for engineering functional major histocompatibility complex class II (MHCII) proteins have proved elusive. Availability of such systems would enable the engineering of peptide-MHCII (pMHCII) complexes for therapeutic and diagnostic applications. In this paper, we have developed a system based on insect cell surface display that allows functional expression of heterodimeric DR2 molecules with or without a covalently bound human myelin basic protein (MBP) peptide, which is amenable to directed evolution of DR2–MBP variants with improved T cell receptor (TCR)-binding affinity. This study represents the first example of functional display of human pMHCII complexes on insect cell surface. In the process of developing this pMHCII engineering system, we have also explored the potential of using yeast surface display for the same application. Our data suggest that yeast display is a useful system for analysis and engineering of peptide binding of MHCII proteins, but not suitable for directed evolution of pMHC complexes that bind with low affinity to self-reactive TCRs. PMID:21752831

  2. Allelic diversity at MHC class II DQ loci in buffalo (Bubalus bubalis): evidence for duplication.

    PubMed

    Niranjan, Saket K; Deb, Sitangsu M; Kumar, Subodh; Mitra, Abhijit; Sharma, Arjava; Sakaram, Durgam; Naskar, Soumen; Sharma, Deepak; Sharma, Sita R

    2010-12-01

    The genetic diversity of MHC class II DQ genes was investigated in riverine buffalo (Bubalus bubalis) by PCR-RFLP and sequencing. Highly variable regions (exons 2-3) of DQ genes were amplified from 152 buffaloes and genotyped by PCR-RFLP. Alleles identified by differential restriction patterns were sequenced for the characterization. PCR-RFLP was a rapid method to discriminate between DQA1 and duplicated DQA2 genes in buffalo, however, the method appeared to be inadequate for determining the more complicated DQB genotypes. A total of 7 and 10 alleles were identified for DQA and DQB loci, respectively. Nucleotide as well as amino acid variations among DQ alleles particularly at peptide binding regions were high. Such variations were as expected higher in DQB than DQA alleles. The phylogenetic analysis for both genes revealed the grouping of alleles into two major sub-groups with higher genetic divergence. High divergence among DQ allelic families and the isolation of two diverse DQA and DQB sequences from individual samples indicated duplication of DQ loci was similar in buffalo to other ruminants.

  3. HLA-DO as the Optimizer of Epitope Selection for MHC Class II Antigen Presentation

    PubMed Central

    Poluektov, Yuri O.; Kim, AeRyon; Hartman, Isamu Z.; Sadegh-Nasseri, Scheherazade

    2013-01-01

    Processing of antigens for presentation to helper T cells by MHC class II involves HLA-DM (DM) and HLA-DO (DO) accessory molecules. A mechanistic understanding of DO in this process has been missing. The leading model on its function proposes that DO inhibits the effects of DM. To directly study DO functions, we designed a recombinant soluble DO and expressed it in insect cells. The kinetics of binding and dissociation of several peptides to HLA-DR1 (DR1) molecules in the presence of DM and DO were measured. We found that DO reduced binding of DR1 to some peptides, and enhanced the binding of some other peptides to DR1. Interestingly, these enhancing and reducing effects were observed in the presence, or absence, of DM. We found that peptides that were negatively affected by DO were DM-sensitive, whereas peptides that were enhanced by DO were DM-resistant. The positive and negative effects of DO could only be measured on binding kinetics as peptide dissociation kinetics were not affected by DO. Using Surface Plasmon Resonance, we demonstrate direct binding of DO to a peptide-receptive, but not a closed conformation of DR1. We propose that DO imposes another layer of control on epitope selection during antigen processing. PMID:23951115

  4. Ubiquitin ligase MARCH 8 cooperates with CD83 to control surface MHC II expression in thymic epithelium and CD4 T cell selection

    PubMed Central

    Guan, Jing; La Gruta, Nicole L.; Gray, Daniel H.

    2016-01-01

    Major histocompatibility complex class II (MHC II) expression is tightly regulated, being subjected to cell type–specific mechanisms that closely control its levels at the cell surface. Ubiquitination by the E3 ubiquitin ligase MARCH 1 regulates MHC II expression in dendritic cells and B cells. In this study, we demonstrate that the related ligase MARCH 8 is responsible for regulating surface MHC II in thymic epithelial cells (TECs). March8−/− mice have elevated MHC II at the surface of cortical TECs and autoimmune regulator (AIRE)− medullary TECs (mTECs), but not AIRE+ mTECs. Despite this, thymic and splenic CD4+ T cell numbers and repertoires remained unaltered in March8−/− mice. Notably, the ubiquitination of MHC II by MARCH 8 is controlled by CD83. Mice expressing a mutated form of CD83 (Cd83anu/anu mice) have impaired CD4+ T cell selection, but deleting March8 in Cd83anu/anu mice restored CD4+ T cell selection to normal levels. Therefore, orchestrated regulation of MHC II surface expression in TECs by MARCH 8 and CD83 plays a major role in CD4+ T cell selection. Our results also highlight the specialized use of ubiquitinating machinery in distinct antigen-presenting cell types, with important functional consequences and implications for therapeutic manipulation. PMID:27503069

  5. Ubiquitin ligase MARCH 8 cooperates with CD83 to control surface MHC II expression in thymic epithelium and CD4 T cell selection.

    PubMed

    Liu, Haiyin; Jain, Reema; Guan, Jing; Vuong, Vivian; Ishido, Satoshi; La Gruta, Nicole L; Gray, Daniel H; Villadangos, Jose A; Mintern, Justine D

    2016-08-22

    Major histocompatibility complex class II (MHC II) expression is tightly regulated, being subjected to cell type-specific mechanisms that closely control its levels at the cell surface. Ubiquitination by the E3 ubiquitin ligase MARCH 1 regulates MHC II expression in dendritic cells and B cells. In this study, we demonstrate that the related ligase MARCH 8 is responsible for regulating surface MHC II in thymic epithelial cells (TECs). March8(-/-) mice have elevated MHC II at the surface of cortical TECs and autoimmune regulator (AIRE)(-) medullary TECs (mTECs), but not AIRE(+) mTECs. Despite this, thymic and splenic CD4(+) T cell numbers and repertoires remained unaltered in March8(-/-) mice. Notably, the ubiquitination of MHC II by MARCH 8 is controlled by CD83. Mice expressing a mutated form of CD83 (Cd83(anu/anu) mice) have impaired CD4(+) T cell selection, but deleting March8 in Cd83(anu/anu) mice restored CD4(+) T cell selection to normal levels. Therefore, orchestrated regulation of MHC II surface expression in TECs by MARCH 8 and CD83 plays a major role in CD4(+) T cell selection. Our results also highlight the specialized use of ubiquitinating machinery in distinct antigen-presenting cell types, with important functional consequences and implications for therapeutic manipulation. © 2016 Liu et al.

  6. Tubulation of Class II MHC Compartments Is Microtubule Dependent and Involves Multiple Endolysosomal Membrane Proteins in Primary Dendritic Cells1

    PubMed Central

    Vyas, Jatin M.; Kim, You-Me; Artavanis-Tsakonas, Katerina; Love, J. Christopher; Van der Veen, Annemarthe G.; Ploegh, Hidde L.

    2009-01-01

    Immature dendritic cells (DCs) capture exogenous Ags in the periphery for eventual processing in endolysosomes. Upon maturation by TLR agonists, DCs deliver peptide-loaded class II MHC molecules from these compartments to the cell surface via long tubular structures (endolysosomal tubules). The nature and rules that govern the movement of these DC compartments are unknown. In this study, we demonstrate that the tubules contain multiple proteins including the class II MHC molecules and LAMP1, a lysosomal resident protein, as well as CD63 and CD82, members of the tetraspanin family. Endolysosomal tubules can be stained with acidotropic dyes, indicating that they are extensions of lysosomes. However, the proper trafficking of class II MHC molecules themselves is not necessary for endolysosomal tubule formation. DCs lacking MyD88 can also form endolysosomal tubules, demonstrating that MyD88-dependent TLR activation is not necessary for the formation of this compartment. Endolysosomal tubules in DCs exhibit dynamic and saltatory movement, including bidirectional travel. Measured velocities are consistent with motor-based movement along microtubules. Indeed, nocodazole causes the collapse of endolysosomal tubules. In addition to its association with microtubules, endolysosomal tubules follow the plus ends of microtubules as visualized in primary DCs expressing end binding protein 1 (EB1)-enhanced GFP. PMID:17513769

  7. Tubulation of class II MHC compartments is microtubule dependent and involves multiple endolysosomal membrane proteins in primary dendritic cells.

    PubMed

    Vyas, Jatin M; Kim, You-Me; Artavanis-Tsakonas, Katerina; Love, J Christopher; Van der Veen, Annemarthe G; Ploegh, Hidde L

    2007-06-01

    Immature dendritic cells (DCs) capture exogenous Ags in the periphery for eventual processing in endolysosomes. Upon maturation by TLR agonists, DCs deliver peptide-loaded class II MHC molecules from these compartments to the cell surface via long tubular structures (endolysosomal tubules). The nature and rules that govern the movement of these DC compartments are unknown. In this study, we demonstrate that the tubules contain multiple proteins including the class II MHC molecules and LAMP1, a lysosomal resident protein, as well as CD63 and CD82, members of the tetraspanin family. Endolysosomal tubules can be stained with acidotropic dyes, indicating that they are extensions of lysosomes. However, the proper trafficking of class II MHC molecules themselves is not necessary for endolysosomal tubule formation. DCs lacking MyD88 can also form endolysosomal tubules, demonstrating that MyD88-dependent TLR activation is not necessary for the formation of this compartment. Endolysosomal tubules in DCs exhibit dynamic and saltatory movement, including bidirectional travel. Measured velocities are consistent with motor-based movement along microtubules. Indeed, nocodazole causes the collapse of endolysosomal tubules. In addition to its association with microtubules, endolysosomal tubules follow the plus ends of microtubules as visualized in primary DCs expressing end binding protein 1 (EB1)-enhanced GFP.

  8. Participation of the interstitium in acute immune-complex nephritis: interstitial antigen accumulation, cellular infiltrate, and MHC class II expression

    PubMed Central

    PARRA, G; HERNÁNDEZ, S; MORENO, P; RODRÍGUEZ-ITURBE, B

    2003-01-01

    Bovine serum albumin (BSA) injected into the rabbits induces acute immune complex glomerulonephritis. Since albumin is filtered and reabsorbed in the tubules, we investigated whether tubulointerstitial cells participate in the pathogenesis of this experimental condition. For this purpose, we induced immune-complex nephritis in 45 rabbits with the injection of 125I-BSA and urinary BSA excretion, glomerular and tubulointerstitial BSA accumulation, lymphocyte infiltration, proliferative activity and MHC class II antigens were examined 2, 4–5 and 6–8 days after immunization. Proteinuria developed day 6–8. BSA was found in urine from day 2 (mean ± SE; 1089 ± 339 µg/24 h) and peaked on day 4 after immunization (2249 ± 1106). BSA content (cpm/g tissue) in tubulointerstitium (TI) and glomeruli were similar at day 2 (457 ± 45 and 407 ± 75, respectively), but afterward increased significantly in TI, reaching a peak level on day 5 (1026 ± 406) while remained unchanged in glomeruli (388 ± 95). At the same time, preceding the onset of proteinuria, maximal intensity of the lymphocyte infiltration, proliferative activity and MHC class II antigen expression in tubular cells, monocytes/macrophages and interstitial cells were observed. Our study shows that antigen is excreted in the urine and concentrated in TI in association with overexpression of MHC class II molecules and lymphocyte infiltration. These findings occur prior to the development of proteinuria and suggest that the tubulointerstitial cells play a critical role in the pathogenesis of this model. PMID:12823277

  9. DMA and DMB are the only genes in the class II region of the human MHC needed for class II-associated antigen processing

    SciTech Connect

    Ceman, S.; Rudersdorf, R.A.; Petersen, J.M.

    1995-03-15

    Previous studies have shown that homozygous mutations between the LMP2 and DNA loci in the human MHC cause class II molecules to be abnormally conformed and unstable in the presence of SDS at low temperature, and impede class II-associated Ag processing and presentation. These abnormalities result from impaired ability to form intracellular class II/peptide complexes that predominate in normal cells. We show in this work that this defect results from deficient expression of either the DMA or the DMB gene. Human B-LCL.174 (DR3) cells, which have a deletion of all known expressible genes in the class II region, express transgene-encoded HLA-DR3, but have the abnormalities. Transfer of cosmid HA14, which contains the DMA and DMB genes, into .174 (DR3) cells restored normal DR3 conformation, stability in 0.4% SDS at 0{degrees}, and ability to process and present tetanus toxoid, but only when both DMA and DMB mRNAs were present. The requirement for both genetic expressions in engendering normal phenotypes was confirmed by transferring the cloned genes into .174 (DR3) cells separately or together. Because normal phenotypes were fully restored in transferent cells expressing DMA plus DMB, other genes in the {approximately} 1-mb homozygous class II region deletion in .174 (DR3) cells either do not participate in or are dispensable for apparently normal production of intracellular class II/peptide complexes. The properties of DM-deficient EBV-transformed B lymphoblastoid cell lines (LCLs) suggest ways of identifying humans in whom DM deficiency contributes to congenital immunodeficiency and malignancy. 67 refs., 5 figs., 1 tab.

  10. Semi-empirical quantum evaluation of peptide - MHC class II binding

    NASA Astrophysics Data System (ADS)

    González, Ronald; Suárez, Carlos F.; Bohórquez, Hugo J.; Patarroyo, Manuel A.; Patarroyo, Manuel E.

    2017-01-01

    Peptide presentation by the major histocompatibility complex (MHC) is a key process for triggering a specific immune response. Studying peptide-MHC (pMHC) binding from a structural-based approach has potential for reducing the costs of investigation into vaccine development. This study involved using two semi-empirical quantum chemistry methods (PM7 and FMO-DFTB) for computing the binding energies of peptides bonded to HLA-DR1 and HLA-DR2. We found that key stabilising water molecules involved in the peptide binding mechanism were required for finding high correlation with IC50 experimental values. Our proposal is computationally non-intensive, and is a reliable alternative for studying pMHC binding interactions.

  11. Mouse γδ T cells are capable of expressing MHC class II molecules, and of functioning as antigen-presenting cells⋆

    PubMed Central

    Cheng, Lan; Cui, Yan; Shao, Hui; Han, Gencheng; Zhu, Ling; Huang, Yafei; O'Brien, Rebecca L.; Born, Willi K.; Kaplan, Henry J.; Sun, Deming

    2008-01-01

    Although human and bovine γδ T cells were shown to express MHC class II antigen and function as APCs, attempts to determine if mouse γδ T cells have similar functions remained unsuccessful. We now show that γδ T cells derived from immunized mice also can be induced to express MHC class II and co-stimulatory molecules after activation in vitro, and are capable of antigen presentation. Using highly purified γδ T cells, we found that, unlike human γδ T cells, the expression of MHC class II molecules by mouse γδ T cells is limited to newly activated cells. Highest levels of MHC class II expression were seen on activated γδ T cells that had lost most surface-expressed γδ TCR while exhibiting increased levels of intracellular γδ TCR. In the absence of further stimulation, MHC class II expression gradually declined with the γδ T cells regaining their surface TCR. We also show that cytokine-activated γδ T cells can also express MHC class II antigen and exercise antigen-presenting activity. PMID:18774183

  12. MHC class II-alpha chain knockout mice support increased viral replication that is independent of their lack of MHC class II cell surface expression and associated immune function deficiencies.

    PubMed

    Alsharifi, Mohammed; Koskinen, Aulikki; Wijesundara, Danushka K; Bettadapura, Jayaram; Müllbacher, Arno

    2013-01-01

    MHCII molecules are heterodimeric cell surface proteins composed of an α and β chain. These molecules are almost exclusively expressed on thymic epithelium and antigen presenting cells (APCs) and play a central role in the development and function of CD4 T cells. Various MHC-II knockout mice have been generated including MHC-IIAα(-/-) (I-Aα(-/-)), MHC-IIAβ(-/-) (I-β(-/-)) and the double knockout (I-Aαxβ(-/-)). Here we report a very striking observation, namely that alphaviruses including the avirulent strain of Semliki Forest virus (aSFV), which causes asymptomatic infection in wild-type C57BL6/J (B6) mice, causes a very acute and lethal infection in I-Aα(-/-), but not in I-β(-/-) or I-Aαxβ(-/-), mice. This susceptibility to aSFV is associated with high virus titres in muscle, spleen, liver, and brain compared to B6 mice. In addition, I-Aα(-/-) mice show intact IFN-I responses in terms of IFN-I serum levels and IFN-I receptor expression and function. Radiation bone marrow chimeras of B6 mice reconstituted with I-Aα(-/-) bone marrow expressed B6 phenotype, whereas radiation chimeras of I-Aα(-/-) mice reconstituted with B6 bone marrow expressed the phenotype of high viral susceptibility. Virus replication experiments both in vivo and in vitro showed enhanced virus growth in tissues and cell cultures derived form I-Aα(-/-) compared to B6 mice. This enhanced virus replication is evident for other alpha-, flavi- and poxviruses and may be of great benefit to producers of viral vaccines. In conclusion, I-Aα(-/-) mice exhibit a striking susceptibility to virus infections independent of their defective MHC-II expression. Detailed genetic analysis will be carried out to characterise the underlining genetic defects responsible for the observed phenomenon.

  13. Conformational lability in the class II MHC 310 helix and adjacent extended strand dictate HLA-DM susceptibility and peptide exchange

    PubMed Central

    Painter, Corrie A.; Negroni, Maria P.; Kellersberger, Katherine A.; Zavala-Ruiz, Zarixia; Evans, James E.; Stern, Lawrence J.

    2011-01-01

    HLA-DM is required for efficient peptide exchange on class II MHC molecules, but its mechanism of action is controversial. We trapped an intermediate state of class II MHC HLA-DR1 by substitution of αF54, resulting in a protein with increased HLA-DM binding affinity, weakened MHC-peptide hydrogen bonding as measured by hydrogen-deuterium exchange mass spectrometry, and increased susceptibility to DM-mediated peptide exchange. Structural analysis revealed a set of concerted conformational alterations at the N-terminal end of the peptide-binding site. These results suggest that interaction with HLA-DM is driven by a conformational change of the MHC II protein in the region of the α-subunit 310 helix and adjacent extended strand region, and provide a model for the mechanism of DM-mediated peptide exchange. PMID:22084083

  14. Conformational lability in the class II MHC 310 helix and adjacent extended strand dictate HLA-DM susceptibility and peptide exchange.

    PubMed

    Painter, Corrie A; Negroni, Maria P; Kellersberger, Katherine A; Zavala-Ruiz, Zarixia; Evans, James E; Stern, Lawrence J

    2011-11-29

    HLA-DM is required for efficient peptide exchange on class II MHC molecules, but its mechanism of action is controversial. We trapped an intermediate state of class II MHC HLA-DR1 by substitution of αF54, resulting in a protein with increased HLA-DM binding affinity, weakened MHC-peptide hydrogen bonding as measured by hydrogen-deuterium exchange mass spectrometry, and increased susceptibility to DM-mediated peptide exchange. Structural analysis revealed a set of concerted conformational alterations at the N-terminal end of the peptide-binding site. These results suggest that interaction with HLA-DM is driven by a conformational change of the MHC II protein in the region of the α-subunit 3(10) helix and adjacent extended strand region, and provide a model for the mechanism of DM-mediated peptide exchange.

  15. In Situ Peptide-MHC-II Tetramer Staining of Antigen-Specific CD4+ T Cells in Tissues

    PubMed Central

    Cleary, P. Patrick; Skinner, Pamela J.

    2015-01-01

    The invention of peptide-MHC-tetramer technology to label antigen-specific T cells has led to an enhanced understanding of T lymphocyte biology. Here we describe the development of an in situ pMHC-II tetramer staining method to visualize antigen-specific CD4+ T cells in tissues. This method complements other methods developed that similarly use MHC class II reagents to stain antigen-specific CD4+ T cells in situ. In this study, we used group A streptococcus (GAS) expressing a surrogate peptide (2W) to inoculate C57BL/6 mice, and used fresh nasal-associated lymphoid tissues (NALT) in optimizing the in situ staining of 2W:I-Ab specific CD4+ T cells. The results showed 2W:I-Ab tetramer-binding CD4+ T cells in GAS-2W but not GAS infected mice. This method holds promise to be broadly applicable to study the localization, abundance, and phenotype of antigen-specific CD4+ T cells in undisrupted tissues. PMID:26067103

  16. MHC class II variation in the endangered European mink Mustela lutreola (L. 1761)--consequences for species conservation.

    PubMed

    Becker, L; Nieberg, C; Jahreis, K; Peters, E

    2009-04-01

    The polymorphic major histocompatibility complex (MHC) has gained a specific relevance in pathogen resistance and mate choice. Particularly the antigen-binding site (ABS), encoded by exon 2 of the DRB class II gene, exhibits numerous alleles and extensive sequence variations between alleles. A lack of MHC variability has attributed to instances such as bottleneck effects or relaxed selection pressure and has a certain impact on the long-term viability of the species concerned. As a result of seriously decreased population density during the last century, the current population of the endangered European mink (Mustela lutreola, L. 1761) has suffered from geographic isolation. In this study, we amplified a partial sequence of the MHC class II DRB exon 2 (229 bp), assessed the degree of genetic variation and compared the variability with those of other Mustelidae. As a result, nine alleles were detected in 20 investigated individuals, which differ from each other by four to 25 nucleotide substitutions (two to 11 amino acid substitutions). Whilst an equal ratio for synonymous and non-synonymous substitutions was found inside the ABS, synonymous substitutions were significantly higher than non-synonymous substitutions in the non-ABS region. Results might indicate that no positive selection exists within the ex situ population of M. lutreola, at least in the analysed fragment. In addition, phylogenetic analyses support the trans-species model of evolution.

  17. Molecular characterization of MHC class II in a nonmodel anuran species, the fire-bellied toad Bombina bombina.

    PubMed

    Hauswaldt, J Susanne; Stuckas, H; Pfautsch, S; Tiedemann, R

    2007-06-01

    While the anuran Xenopus comprises one of the best characterized nonmammalian taxa regarding the major histocompatibility complex (MHC), the organization of this gene complex has never been studied in other anurans, and information on amphibian MHC (other than Xenopus) is generally very scarce. Here, we describe the characterization of the first MHC class II B cDNA sequences from a nonmodel anuran species, the European fire-bellied toad (Bombina bombina). We isolated two transcript sequences differing substantially in amino acid composition and length within the beta2 domain. To investigate the variability of the peptide binding region in this species, we sequenced a 158-bp large fragment from wild B. bombina (n = 20) and identified eight distinct alleles. All substitutions but one were nonsynonymous, and many of the highly polymorphic sites corresponded with amino acid positions known to be involved in antigen binding. The level of variation we found in B. bombina was similar compared to that previously found in a comparable sample of a wild urodelan species, Ambystoma tigrinum, and to that found in Xenopus laevis. Based on the cDNA data and the individual's allelic diversity, we conclude that Bombina possesses at least two class II B loci. With our new beta1 primers, we were able to generate sequences in other species of anurans. We provide here a first phylogenetic analysis of this gene in amphibians.

  18. Patterns of evolution of MHC class II genes of crows (Corvus) suggest trans-species polymorphism.

    PubMed

    Eimes, John A; Townsend, Andrea K; Sepil, Irem; Nishiumi, Isao; Satta, Yoko

    2015-01-01

    A distinguishing characteristic of genes that code for the major histocompatibility complex (MHC) is that alleles often share more similarity between, rather than within species. There are two likely mechanisms that can explain this pattern: convergent evolution and trans-species polymorphism (TSP), in which ancient allelic lineages are maintained by balancing selection and retained by descendant species. Distinguishing between these two mechanisms has major implications in how we view adaptation of immune genes. In this study we analyzed exon 2 of the MHC class IIB in three passerine bird species in the genus Corvus: jungle crows (Corvus macrorhynchos japonensis) American crows (C. brachyrhynchos) and carrion crows (C. corone orientalis). Carrion crows and American crows are recently diverged, but allopatric, sister species, whereas carrion crows and jungle crows are more distantly related but sympatric species, and possibly share pathogens linked to MHC IIB polymorphisms. These patterns of evolutionary divergence and current geographic ranges enabled us to test for trans-species polymorphism and convergent evolution of the MHC IIB in crows. Phylogenetic reconstructions of MHC IIB sequences revealed several well supported interspecific clusters containing all three species, and there was no biased clustering of variants among the sympatric carrion crows and jungle crows. The topologies of phylogenetic trees constructed from putatively selected sites were remarkably different than those constructed from putatively neutral sites. In addition, trees constructed using non-synonymous substitutions from a continuous fragment of exon 2 had more, and generally more inclusive, supported interspecific MHC IIB variant clusters than those constructed from the same fragment using synonymous substitutions. These phylogenetic patterns suggest that recombination, especially gene conversion, has partially erased the signal of allelic ancestry in these species. While clustering of

  19. Patterns of evolution of MHC class II genes of crows (Corvus) suggest trans-species polymorphism

    PubMed Central

    Townsend, Andrea K.; Sepil, Irem; Nishiumi, Isao; Satta, Yoko

    2015-01-01

    A distinguishing characteristic of genes that code for the major histocompatibility complex (MHC) is that alleles often share more similarity between, rather than within species. There are two likely mechanisms that can explain this pattern: convergent evolution and trans-species polymorphism (TSP), in which ancient allelic lineages are maintained by balancing selection and retained by descendant species. Distinguishing between these two mechanisms has major implications in how we view adaptation of immune genes. In this study we analyzed exon 2 of the MHC class IIB in three passerine bird species in the genus Corvus: jungle crows (Corvus macrorhynchos japonensis) American crows (C. brachyrhynchos) and carrion crows (C. corone orientalis). Carrion crows and American crows are recently diverged, but allopatric, sister species, whereas carrion crows and jungle crows are more distantly related but sympatric species, and possibly share pathogens linked to MHC IIB polymorphisms. These patterns of evolutionary divergence and current geographic ranges enabled us to test for trans-species polymorphism and convergent evolution of the MHC IIB in crows. Phylogenetic reconstructions of MHC IIB sequences revealed several well supported interspecific clusters containing all three species, and there was no biased clustering of variants among the sympatric carrion crows and jungle crows. The topologies of phylogenetic trees constructed from putatively selected sites were remarkably different than those constructed from putatively neutral sites. In addition, trees constructed using non-synonymous substitutions from a continuous fragment of exon 2 had more, and generally more inclusive, supported interspecific MHC IIB variant clusters than those constructed from the same fragment using synonymous substitutions. These phylogenetic patterns suggest that recombination, especially gene conversion, has partially erased the signal of allelic ancestry in these species. While clustering of

  20. Evolutionary origins of retroposon lineages of Mhc class II Ab alleles.

    PubMed

    Lu, C C; Ye, Y; She, J X; Bonhomme, F; Wakeland, E K

    1996-01-01

    Major histocompatibility complex (Mhc) class II Ab genes have evolved into three distinct lineages. While lineage 2 alleles differ from lineage 1 alleles by the insertion of a retroposon in intron 2, the basis for the extremely large intron 2 in lineage 3 alleles has heretofore been undetermined. In this report, we demonstrate by nucleotide sequencing that the genomic sequences of prototypic alleles from all three lineages diverge significantly and that lineage 3 is derived from lineage 2 by two insertional events in intron 2. One insert, composed of a member of B1 short interspersed repetitive elements (SINEs), occurs 508 base pairs (bp) 3' of exon 2, and the other, 1141 bp 3' of exon 2 within the retroposon that distinguishes lineage 2 from lineage 1. To assess the evolutionary stability of these lineages and the extent of ancestral polymorphisms of Ab within Mus species, we extended our restriction site polymorphism analysis to include 86 alleles from 120 independently derived H2 haplotypes from 12 separate species and subspecies of Mus. A phylogenetic tree revealing the relationships of these Ab alleles with respect to restriction site polymorphisms, but excluding the retroposon insertions, demonstrated that these lineages have distinctive genomic structures beyond the retroposon polymorphisms. In summary, these mouse Ab genes were produced from successive retroposon insertion events. Lineage 1 and 2 were detected in a variety of Mus species, including Mus caroli, indicating that these lineages diverged more than 2 million years ago. Lineage 3 alleles were found only in the Mus musculus subspecies, suggesting that it diverged from lineage 2 more recently. These results indicate that all three lineages of Ab have persisted through several speciation events in the genus Mus.

  1. T lymphocytes from irradiation chimeras repopulated with 13-day fetal liver cells recognize antigens only in association with self-MHC products

    SciTech Connect

    Nisbet-Brown, E.; Diener, E. )

    1986-01-01

    The restriction specificities of maturing thymocytes are determined by the Class II MHC antigens expressed by non-lymphoid thymic tissues. The proliferative response of mature T lymphocytes to antigen-presenting cells (APC) and antigen requires that the APC express the same MHC antigens as the thymus in which the T cells differentiated. Thus, in the two-way bone marrow chimera (A + B----(A x B)F1), T lymphocyte populations of A and B haplotypes have each acquired the potential to recognize antigens associated with either parental haplotype. In spite of the large body of work on MHC restriction, we still do not have a clear understanding of the mechanisms which impose self restriction. The chimeric model systems used previously to study MHC restriction have used adult bone marrow cells as the source of lymphoid precursors. During normal ontogeny, T cells are derived from precursors in the fetal liver and we felt that a direct comparison of T cells from fetal liver and bone marrow-repopulated animals would shed light on the development of MHC restriction specificities during T cell ontogeny in the thymus or prethymically. We found that parental T lymphocyte populations isolated from two-way fetal liver chimeras cooperated only with syngeneic APC, while those from bone marrow chimeras cooperated with APC of either parental haplotype. This suggests that fetal liver and bone marrow may not be equivalent sources of stem cells. Our results may be due to fundamental differences between thymocyte precursors in fetal liver and bone marrow, including the time course of their expression of T cell receptor gene products.

  2. Enhanced presentation of MHC class Ia, Ib and class II-restricted peptides encapsulated in biodegradable nanoparticles: a promising strategy for tumor immunotherapy.

    PubMed

    Ma, Wenxue; Smith, Trevor; Bogin, Vladimir; Zhang, Yu; Ozkan, Cengiz; Ozkan, Mihri; Hayden, Melanie; Schroter, Stephanie; Carrier, Ewa; Messmer, Davorka; Kumar, Vipin; Minev, Boris

    2011-03-31

    Many peptide-based cancer vaccines have been tested in clinical trials with a limited success, mostly due to difficulties associated with peptide stability and delivery, resulting in inefficient antigen presentation. Therefore, the development of suitable and efficient vaccine carrier systems remains a major challenge. To address this issue, we have engineered polylactic-co-glycolic acid (PLGA) nanoparticles incorporating: (i) two MHC class I-restricted clinically-relevant peptides, (ii) a MHC class II-binding peptide, and (iii) a non-classical MHC class I-binding peptide. We formulated the nanoparticles utilizing a double emulsion-solvent evaporation technique and characterized their surface morphology, size, zeta potential and peptide content. We also loaded human and murine dendritic cells (DC) with the peptide-containing nanoparticles and determined their ability to present the encapsulated peptide antigens and to induce tumor-specific cytotoxic T lymphocytes (CTL) in vitro. We confirmed that the nanoparticles are not toxic to either mouse or human dendritic cells, and do not have any effect on the DC maturation. We also demonstrated a significantly enhanced presentation of the encapsulated peptides upon internalization of the nanoparticles by DC, and confirmed that the improved peptide presentation is actually associated with more efficient generation of peptide-specific CTL and T helper cell responses. Encapsulating antigens in PLGA nanoparticles offers unique advantages such as higher efficiency of antigen loading, prolonged presentation of the antigens, prevention of peptide degradation, specific targeting of antigens to antigen presenting cells, improved shelf life of the antigens, and easy scale up for pharmaceutical production. Therefore, these findings are highly significant to the development of synthetic vaccines, and the induction of CTL for adoptive immunotherapy.

  3. MHC class II restricted innate-like double negative T cells contribute to optimal primary and secondary immunity to Leishmania major.

    PubMed

    Mou, Zhirong; Liu, Dong; Okwor, Ifeoma; Jia, Ping; Orihara, Kanami; Uzonna, Jude Ezeh

    2014-09-01

    Although it is generally believed that CD4(+) T cells play important roles in anti-Leishmania immunity, some studies suggest that they may be dispensable, and that MHC II-restricted CD3(+)CD4(-)CD8(-) (double negative, DN) T cells may be more important in regulating primary anti-Leishmania immunity. In addition, while there are reports of increased numbers of DN T cells in Leishmania-infected patients, dogs and mice, concrete evidence implicating these cells in secondary anti-Leishmania immunity has not yet been documented. Here, we report that DN T cells extensively proliferate and produce effector cytokines (IFN-γ, TNF and IL-17) and granzyme B (GrzB) in the draining lymph nodes and spleens of mice following primary and secondary L. major infections. DN T cells from healed mice display functional characteristics of protective anti-Leishmania memory-like cells: rapid and extensive proliferation and effector cytokines production following L. major challenge in vitro and in vivo. DN T cells express predominantly (> 95%) alpha-beta T cell receptor (αβ TCR), are Leishmania-specific, restricted mostly by MHC class II molecules and display transcriptional profile of innate-like genes. Using in vivo depletion and adoptive transfer studies, we show that DN T cells contribute to optimal primary and secondary anti-Leishmania immunity in mice. These results directly identify DN T cells as important players in effective and protective primary and secondary anti-L. major immunity in experimental cutaneous leishmaniasis.

  4. Quantum chemical analysis explains hemagglutinin peptide-MHC Class II molecule HLA-DRbeta1*0101 interactions.

    PubMed

    Cárdenas, Constanza; Villaveces, José Luis; Bohórquez, Hugo; Llanos, Eugenio; Suárez, Carlos; Obregón, Mateo; Patarroyo, Manuel Elkin

    2004-10-29

    We present a new method to explore interactions between peptides and major histocompatibility complex (MHC) molecules using the resultant vector of the three principal multipole terms of the electrostatic field expansion. Being that molecular interactions are driven by electrostatic interactions, we applied quantum chemistry methods to better understand variations in the electrostatic field of the MHC Class II HLA-DRbeta1*0101-HA complex. Multipole terms were studied, finding strong alterations of the field in Pocket 1 of this MHC molecule, and weak variations in other pockets, with Pocket 1>Pocket 4>Pocket 9 approximately Pocket 7>Pocket 6. Variations produced by "ideal" amino acids and by other occupying amino acids were compared. Two types of interactions were found in all pockets: a strong unspecific one (global interaction) and a weak specific interaction (differential interaction). Interactions in Pocket 1, the dominant pocket for this allele, are driven mainly by the quadrupole term, confirming the idea that aromatic rings are important in these interactions. Multipolar analysis is in agreement with experimental results, suggesting quantum chemistry methods as an adequate methodology to understand these interactions.

  5. Anti-coreceptor antibodies profoundly affect staining with peptide-MHC class I and class II tetramers.

    PubMed

    Wooldridge, Linda; Scriba, Thomas J; Milicic, Anita; Laugel, Bruno; Gostick, Emma; Price, David A; Phillips, Rodney E; Sewell, Andrew K

    2006-07-01

    The T cell coreceptors CD8 and CD4 bind to invariable regions of peptide-MHC class I (pMHCI) and class II (pMHCII) molecules, respectively, and facilitate antigen recognition by a number of mechanisms. It is established that some antibodies (Ab) specific for the CD8 molecule, which stabilizes TCR/pMHCI interactions, can alter the binding of pMHCI tetramers to cell surface TCR. In contrast, the extremely weak pMHCII/CD4 interaction does not stabilize TCR/pMHCII interactions or contribute to cognate tetramer binding; consequently, it is assumed that anti-CD4 Ab do not affect pMHCII binding. Here, we used a panel of point-mutated HLA A2 molecules with a range of affinities for CD8 spanning over three orders of magnitude to demonstrate that anti-CD8 Ab-mediated inhibition of pMHCI tetramer binding and cognate T cell activation correlates directly with the strength of the pMHCI/CD8 interaction. Further, some anti-CD4 Ab were found to block pMHCII tetramer binding; these effects were also paralleled in T cell activation assays. In sum, these data challenge the assertion that anti-coreceptor Ab exert their effects on T cell activation and pMHC binding solely by blocking pMHC/coreceptor interactions.

  6. Genetic variation of the major histocompatibility complex (MHC class II B gene) in the threatened Hume's pheasant, Syrmaticus humiae.

    PubMed

    Chen, Weicai; Bei, Yongjian; Li, Hanhua

    2015-01-01

    Major histocompatibility complex (MHC) genes are the most polymorphic genes in vertebrates and encode molecules that play a crucial role in pathogen resistance. As a result of their diversity, they have received much attention in the fields of evolutionary and conservation biology. Here, we described the genetic variation of MHC class II B (MHCIIB) exon 2 in a wild population of Hume's pheasant (Syrmaticus humiae), which has suffered a dramatic decline in population over the last three decades across its ranges in the face of heavy exploitation and habitat loss. Twenty-four distinct alleles were found in 73 S. humiae specimens. We found seven shared alleles among four geographical groups as well as six rare MHCIIB alleles. Most individuals displayed between one to five alleles, suggesting that there are at least three MHCIIB loci of the Hume's pheasant. The dN ⁄ dS ratio at putative antigen-binding sites (ABS) was significantly greater than one, indicating balancing selection is acting on MHCIIB exon 2. Additionally, recombination and gene conversion contributed to generating MHCIIB diversity in the Hume's pheasant. One to three recombination events and seventy-five significant gene conversion events were observed within the Hume's pheasant MHCIIB loci. The phylogenetic tree and network analysis revealed that the Hume's pheasant alleles do not cluster together, but are scattered through the tree or network indicating a trans-species evolutionary mode. These findings revealed the evolution of the Hume's pheasant MHC after suffering extreme habitat fragmentation.

  7. Structural Basis for the Presentation of Tumor-associated MHC Class II-restricted Phosphopeptides to CD4+ T Cells

    PubMed Central

    Li, Yili; Depontieu, Florence R.; Sidney, John; Salay, Theresa M.; Engelhard, Victor H.; Hunt, Donald F.; Sette, Alessandro; Topalian, Suzanne L.; Mariuzza, Roy A.

    2010-01-01

    Dysregulated protein phosphorylation is a hallmark of malignant transformation. Transformation can generate major histocompatibility complex (MHC)-bound phosphopeptides that are differentially displayed on tumor cells for specific recognition by T cells. To understand how phosphorylation alters the antigenic identity of self peptides and how MHC class II molecules present phosphopeptides for CD4+ T cell recognition, we determined the crystal structure of a phosphopeptide derived from melanoma antigen recognized by T cells-1 (pMART-1), selectively expressed by human melanomas, in complex with HLA-DR1. The structure revealed that the phosphate moiety attached to the serine residue at position P5 of pMART-1 is available for direct interactions with T cell receptor (TCR), and that the peptide N-terminus adopts an unusual conformation orienting it toward TCR. This structure, combined with measurements of peptide affinity for HLA-DR1 and of peptide–MHC recognition by pMART-1-specific T cells, suggests that TCR recognition is focused on the N-terminal portion of pMART-1. This recognition mode appears to be distinct from that of foreign antigen complexes but is remarkably reminiscent of the way autoreactive TCRs engage self or altered self peptides, consistent with the tolerogenic nature of tumor–host immune interactions. PMID:20417641

  8. Structural Basis for the Presentation of Tumor-Associated MHC Class II-Restricted Phosphopeptides to CD4+ T Cells

    SciTech Connect

    Li, Y.; Depontieu, F; Sidney, J; Salay, T; Engelhard, V; Hunt, D; Sette, A; Topalian, S; Mariuzza, R

    2010-01-01

    Dysregulated protein phosphorylation is a hallmark of malignant transformation. Transformation can generate major histocompatibility complex (MHC)-bound phosphopeptides that are differentially displayed on tumor cells for specific recognition by T cells. To understand how phosphorylation alters the antigenic identity of self-peptides and how MHC class II molecules present phosphopeptides for CD4{sup +} T-cell recognition, we determined the crystal structure of a phosphopeptide derived from melanoma antigen recognized by T cells-1 (pMART-1), selectively expressed by human melanomas, in complex with HLA-DR1. The structure revealed that the phosphate moiety attached to the serine residue at position P5 of pMART-1 is available for direct interactions with T-cell receptor (TCR) and that the peptide N-terminus adopts an unusual conformation orienting it toward TCR. This structure, combined with measurements of peptide affinity for HLA-DR1 and of peptide-MHC recognition by pMART-1-specific T cells, suggests that TCR recognition is focused on the N-terminal portion of pMART-1. This recognition mode appears to be distinct from that of foreign antigen complexes but is remarkably reminiscent of the way autoreactive TCRs engage self- or altered self-peptides, consistent with the tolerogenic nature of tumor-host immune interactions.

  9. Belle II production system

    NASA Astrophysics Data System (ADS)

    Miyake, Hideki; Grzymkowski, Rafal; Ludacka, Radek; Schram, Malachi

    2015-12-01

    The Belle II experiment will record a similar quantity of data to LHC experiments and will acquire it at similar rates. This requires considerable computing, storage and network resources to handle not only data created by the experiment but also considerable amounts of simulated data. Consequently Belle II employs a distributed computing system to provide the resources coordinated by the the DIRAC interware. DIRAC is a general software framework that provides a unified interface among heterogeneous computing resources. In addition to the well proven DIRAC software stack, Belle II is developing its own extension called BelleDIRAC. BelleDIRAC provides a transparent user experience for the Belle II analysis framework (basf2) on various environments and gives access to file information managed by LFC and AMGA metadata catalog. By unifying DIRAC and BelleDIRAC functionalities, Belle II plans to operate an automated mass data processing framework named a “production system”. The Belle II production system enables large-scale raw data transfer from experimental site to raw data centers, followed by massive data processing, and smart data delivery to each remote site. The production system is also utilized for simulated data production and data analysis. Although development of the production system is still on-going, recently Belle II has prepared prototype version and evaluated it with a large scale simulated data production. In this presentation we will report the evaluation of the prototype system and future development plans.

  10. Non-neutral evolution and reciprocal monophyly of two expressed Mhc class II B genes in Leach's storm-petrel.

    PubMed

    Dearborn, Donald C; Gager, Andrea B; Gilmour, Morgan E; McArthur, Andrew G; Hinerfeld, Douglas A; Mauck, Robert A

    2015-02-01

    The major histocompatibility complex (Mhc) is subject to pathogen-mediated balancing selection and can link natural selection with mate choice. We characterized two Mhc class II B loci in Leach's storm-petrel, Oceanodroma leucorhoa, focusing on exon 2 which encodes the portion of the protein that binds pathogen peptides. We amplified and sequenced exon 2 with locus-specific nested PCR and Illumina MiSeq using individually barcoded primers. Repeat genotyping of 78 single-locus genotypes produced identical results in 77 cases (98.7%). Sequencing of messenger RNA (mRNA) from three birds confirmed expression of both loci, consistent with the observed absence of stop codons or frameshifts in all alleles. In 48 birds, we found 9 and 12 alleles at the two loci, respectively, and all 21 alleles translated to unique amino acid sequences. Unlike many studies of duplicated Mhc genes, alleles of the two loci clustered into monophyletic groups. Consistent with this phylogenetic result, interlocus gene conversion appears to have affected only two short fragments of the exon. As predicted under a paradigm of pathogen-mediated selection, comparison of synonymous and non-synonymous substitution rates found evidence of a history of positive selection at putative peptide binding sites. Overall, the results suggest that the gene duplication event leading to these two loci is not recent and that point mutations and positive selection on the peptide binding sites may be the predominant forces acting on these genes. Characterization of these loci sets the stage for population-level work on the evolutionary ecology of Mhc in this species.

  11. MHC class II DQB diversity in the Japanese black bear, Ursus thibetanus japonicus.

    PubMed

    Yasukochi, Yoshiki; Kurosaki, Toshifumi; Yoneda, Masaaki; Koike, Hiroko; Satta, Yoko

    2012-11-29

    The major histocompatibility complex (MHC) genes are one of the most important genetic systems in the vertebrate immune response. The diversity of MHC genes may directly influence the survival of individuals against infectious disease. However, there has been no investigation of MHC diversity in the Asiatic black bear (Ursus thibetanus). Here, we analyzed 270-bp nucleotide sequences of the entire exon 2 region of the MHC DQB gene by using 188 samples from the Japanese black bear (Ursus thibetanus japonicus) from 12 local populations. Among 185 of 188 samples, we identified 44 MHC variants that encoded 31 different amino acid sequences (allotypes) and one putative pseudogene. The phylogenetic analysis suggests that MHC variants detected from the Japanese black bear are derived from the DQB locus. One of the 31 DQB allotypes, Urth-DQB*01, was found to be common to all local populations. Moreover, this allotype was shared between the black bear on the Asian continent and the Japanese black bear, suggesting that Urth-DQB*01 might have been maintained in the ancestral black bear population for at least 300,000 years. Our findings, from calculating the ratio of non-synonymous to synonymous substitutions, indicate that balancing selection has maintained genetic variation of peptide-binding residues at the DQB locus of the Japanese black bear. From examination of genotype frequencies among local populations, we observed a considerably lower level of observed heterozygosity than expected. The low level of observed heterozygosity suggests that genetic drift reduced DQB diversity in the Japanese black bear due to a bottleneck event at the population or species level. The decline of DQB diversity might have been accelerated by the loss of rare variants that have been maintained by negative frequency-dependent selection. Nevertheless, DQB diversity of the black bear appears to be relatively high compared with some other endangered mammalian species. This result suggests that

  12. MHC class II DQB diversity in the Japanese black bear, Ursus thibetanus japonicus

    PubMed Central

    2012-01-01

    Background The major histocompatibility complex (MHC) genes are one of the most important genetic systems in the vertebrate immune response. The diversity of MHC genes may directly influence the survival of individuals against infectious disease. However, there has been no investigation of MHC diversity in the Asiatic black bear (Ursus thibetanus). Here, we analyzed 270-bp nucleotide sequences of the entire exon 2 region of the MHC DQB gene by using 188 samples from the Japanese black bear (Ursus thibetanus japonicus) from 12 local populations. Results Among 185 of 188 samples, we identified 44 MHC variants that encoded 31 different amino acid sequences (allotypes) and one putative pseudogene. The phylogenetic analysis suggests that MHC variants detected from the Japanese black bear are derived from the DQB locus. One of the 31 DQB allotypes, Urth-DQB*01, was found to be common to all local populations. Moreover, this allotype was shared between the black bear on the Asian continent and the Japanese black bear, suggesting that Urth-DQB*01 might have been maintained in the ancestral black bear population for at least 300,000 years. Our findings, from calculating the ratio of non-synonymous to synonymous substitutions, indicate that balancing selection has maintained genetic variation of peptide-binding residues at the DQB locus of the Japanese black bear. From examination of genotype frequencies among local populations, we observed a considerably lower level of observed heterozygosity than expected. Conclusions The low level of observed heterozygosity suggests that genetic drift reduced DQB diversity in the Japanese black bear due to a bottleneck event at the population or species level. The decline of DQB diversity might have been accelerated by the loss of rare variants that have been maintained by negative frequency-dependent selection. Nevertheless, DQB diversity of the black bear appears to be relatively high compared with some other endangered mammalian

  13. Characterization of the MHC class II region in cattle. The number of DQ genes varies between haplotypes.

    PubMed

    Andersson, L; Rask, L

    1988-01-01

    The organization of the major histocompatibility complex (MHC) class II region in cattle was investigated by Southern blot analysis using human probes corresponding to DO, DP, DQ, and DR genes. Exon-specific probes were also employed to facilitate the assessment of the number of different bovine class II genes. The results indicated the presence of single DO beta and DR alpha genes, at least three DR beta genes, while the number of DQ genes was found to vary between MHC haplotypes. Four DQ haplotypes, DQ alpha 1 beta 1 to DQ alpha 2 beta 4, possessed a single DQ alpha and a single DQ beta gene whereas both these genes were duplicated in eight other haplotypes, DQ alpha 3 beta 5 to DQ alpha 9 beta 12. No firm evidence for the presence of bovine DP genes was obtained. The same human probes were also used to investigate the genetic polymorphism of bovine class II genes. DQ alpha, DQ beta, DR alpha, DR beta, and DO beta restriction fragment length polymorphisms (RFLPs) were resolved and in particular the DQ restriction fragment patterns were highly polymorphic. Comparison of the present result with the current knowledge of the class II region in other mammalian species suggested that the DO, DP, DQ, DR, and DZ subdivision of the class II region was established already in the ancestor of mammals. The DP genes appear to be the least conserved class II genes among mammalian species and may have been lost in cattle. The degree of polymorphism of different class II genes, as revealed by RFLP analyses, shows striking similarities between species.

  14. The class I myosin Myo1e regulates TLR4-triggered macrophage spreading, chemokine release and antigen presentation via MHC class II

    PubMed Central

    Wenzel, Jens; Ouderkirk, Jessica L.; Krendel, Mira; Lang, Roland

    2014-01-01

    TLR-mediated recognition of microbial danger induces substantial changes in macrophage migration, adherence and phagocytosis. Recently, we described the LPS-regulated phosphorylation of many cytoskeleton-associated proteins by phosphoproteomics. The functional role of these cytoskeletal and motor proteins in innate immune cell responses is largely unexplored. Here, we first identified both long-tailed class I myosins Myo1e and Myo1f as important contributors to LPS-triggered macrophage spreading. Mouse bone marrow-derived macrophages and dendritic cells (DCs) deficient in Myo1e selectively secreted increased amounts of the chemokine CCL2. In addition, the cell surface expression of MHC class II (MHC-II) on both cell types was reduced in the absence of Myo1e. However, transcriptional changes in CCL2 and MHC-II were not observed in the absence of Myo1e, indicating that Myo1e regulates specific intracellular transport processes. The capacity of macrophages and DCs lacking Myo1e to stimulate antigen-specific CD4+ T-cell proliferation was impaired, consistent with the reduced MHC-II surface protein levels. Surprisingly, in Myo1e-deficient DCs, the proteolytic cleavage of endocytosed antigen was also increased. Together, our results provide evidence for a non-redundant function of the motor protein Myo1e in the regulation of TLR4-controlled, cytoskeleton-associated functional properties of macrophages and DCs, and in induction of a full MHC-II-restricted adaptive immune response. PMID:25263281

  15. MHC-class-II are expressed in a subpopulation of human neural stem cells in vitro in an IFNγ–independent fashion and during development

    PubMed Central

    Vagaska, B.; New, S. E. P.; Alvarez-Gonzalez, C.; D’Acquisto, F.; Gomez, S. G.; Bulstrode, N. W.; Madrigal, A.; Ferretti, P.

    2016-01-01

    Expression of major histocompatibility antigens class-2 (MHC-II) under non-inflammatory conditions is not usually associated with the nervous system. Comparative analysis of immunogenicity of human embryonic/fetal brain-derived neural stem cells (hNSCs) and human mesenchymal stem cells with neurogenic potential from umbilical cord (UC-MSCs) and paediatric adipose tissue (ADSCs), while highlighting differences in their immunogenicity, led us to discover subsets of neural cells co-expressing the neural marker SOX2 and MHC-II antigen in vivo during human CNS development. MHC-II proteins in hNSCs are functional, and differently regulated upon differentiation along different lineages. Mimicking an inflammatory response using the inflammatory cytokine IFNγ induced MHC-II up-regulation in both astrocytes and hNSCs, but not in UC-MSCs and ADSCs, either undifferentiated or differentiated, though IFNγ receptor expression was comparable. Together, hypoimmunogenicity of both UC-MSCs and ADSCs supports their suitability for allogeneic therapy, while significant immunogenicity of hNSCs and their progeny may at least in part underlie negative effects reported in some patients following embryonic neural cell grafts. Crucially, we show for the first time that MHC-II expression in developing human brains is not restricted to microglia as previously suggested, but is present in discrete subsets of neural progenitors and appears to be regulated independently of inflammatory stimuli. PMID:27080443

  16. Invariant chain+ N2a neuroblastoma cells stably expressing the class II MHC transactivator CIITA fail to stimulate anti-tumor immunity.

    PubMed

    Rickard, Steve; Ono, Santa Jeremy

    2008-12-01

    A promising cancer treatment strategy involves stimulation of anti-tumor immune responses. CD4(+) T cell responses are particularly desirable, as they enhance CD8(+) T cell activity and provide immune memory. The major histocompatibility complex (MHC) class II transactivator CIITA can be used to stimulate expression of MHC II on tumor cells, thereby promoting CD4(+) T cell activation. In this study, N2a neuroblastoma cells were stably transfected with CIITA. N2aCIITA cells displayed increased expression of MHC I, MHC II and invariant chain; CD80 and CD86 were expressed by neither the parental N2a cells nor by the N2aCIITA cells. All mice injected with N2aCIITA cells developed tumors. Furthermore, no increase in the numbers of T cells, natural killer cells, macrophages, or eosinophils was observed in the spleens or tumors of mice injected with N2aCIITA cells, compared to tissues from mice injected with the parental N2a cells. This absence of an anti-tumor immune response despite MHC II expression is likely due to the presence of invariant chain, in support of the MHCII(+)/Ii(-) paradigm.

  17. Stress-induced differences in primary and secondary resistance against bacterial sepsis corresponds with diverse corticotropin releasing hormone receptor expression by pulmonary CD11c+ MHC II+ and CD11c− MHC II+ APCs

    PubMed Central

    Gonzales, Xavier F.; Desmutkh, Aniket; Pulse, Mark; Johnson, Khaisha; Jones, Harlan P.

    2009-01-01

    Stress responses have been associated with altered immunity and depending upon the type of stressor, can have diverse effects on disease outcomes. As the first line of defense against potential pathogens, alterations in cellular immune responses along the respiratory tract can have a significant impact on the manifestation of local and systemic disease. Utilizing a murine model of respiratory pneumonia, the current study investigated the effects of restraint stress on the induction of primary and secondary immunity along the respiratory tract, influencing host susceptibility. Female CD-1 mice were subjected to three hours of restraint stress over a period of four days followed by primary and secondary Streptococcus pneumoniae infection via intranasal route. Stress exposure led to increased retention of bacterial carriage in the lungs, enhanced polymorphonuclear cells and a preferential decrease in pulmonary CD11c+ MHC II+ cells resulting in delayed lethality during primary infection but significant impairment of acquired immune protection after secondary infection. We also provide evidence to support a role for lung-associated corticotrophin releasing hormone regulation through peripheral CRH and diverse CRH receptor expression by MHC II+ antigen presenting cells (APCs). We conclude that repeated restraint stress has distinct influences on immune cell populations that appear to be important in the generation of innate and adaptive immune responses along the respiratory tract with the potential to influence local and systemic protection against disease pathogenesis. PMID:18166336

  18. Characterization and evolution of MHC class II B genes in Galápagos marine iguanas (Amblyrhynchus cristatus).

    PubMed

    Glaberman, Scott; Moreno, Maria A; Caccone, Adalgisa

    2009-08-01

    Major histocompatibility complex (MHC) class II molecules play a key role in the adaptive immune system of vertebrates. Class II B genes appear to evolve in a very different manner in mammals and birds. Orthology is commonly observed among mammal loci, while genes tend to cluster phylogenetically within bird species. Here we present class II B data from a representative of another major group of amniotes, the squamates (i.e. lizards, snakes, amphisbaenians), with the ultimate goal of placing mammalian and avian MHC evolution into a broader context. In this study, eight class II B cDNA sequences were obtained from the Galápagos marine iguana (Amblyrhynchus cristatus) which were divided into five locus groups, Amcr-DAB1 through -DAB5, based on similarities along most of the coding and noncoding portions of the transcribed gene. All marine iguana sequences were monophyletic with respect to class II genes from other vertebrates indicating that they originated from a common ancestral locus after squamates split from other reptiles. The beta-1 domain, which is involved in antigen binding, exhibited signatures of positive selection as well as interlocus gene conversion in both long and short tracts-a pattern also observed in birds and fish, but not in mammals. On the other hand, the beta-2 domain was divergent between gene groups, which is characteristic of mammals. Based on these results, we preliminarily show that squamate class II B genes have been shaped by a unique blend of evolutionary forces that have been observed in differing degrees in other vertebrates.

  19. Structural Basis for the Recognition of Mutant Self by a Tumor-Specific, MHC Class II-Restricted T Cell Receptor

    SciTech Connect

    Deng,L.; Langley, R.; Brown, P.; Xu, G.; Teng, L.; Wang, Q.; Gonzales, M.; Callender, G.; Nishimura, M.; et al.

    2007-01-01

    Structural studies of complexes of T cell receptor (TCR) and peptide-major histocompatibility complex (MHC) have focused on TCRs specific for foreign antigens or native self. An unexplored category of TCRs includes those specific for self determinants bearing alterations resulting from disease, notably cancer. We determined here the structure of a human melanoma-specific TCR (E8) bound to the MHC molecule HLA-DR1 and an epitope from mutant triosephosphate isomerase. The structure had features intermediate between 'anti-foreign' and autoimmune TCR-peptide-MHC class II complexes that may reflect the hybrid nature of altered self. E8 manifested very low affinity for mutant triosephosphate isomerase-HLA-DR1 despite the highly tumor-reactive properties of E8 cells. A second TCR (G4) had even lower affinity but underwent peptide-specific formation of dimers, suggesting this as a mechanism for enhancing low-affinity TCR-peptide-MHC interactions for T cell activation.

  20. The Missing Link in Epstein-Barr Virus Immune Evasion: the BDLF3 Gene Induces Ubiquitination and Downregulation of Major Histocompatibility Complex Class I (MHC-I) and MHC-II

    PubMed Central

    Quinn, Laura L.; Williams, Luke R.; White, Claire; Forrest, Calum; Rowe, Martin

    2015-01-01

    ABSTRACT The ability of Epstein-Barr virus (EBV) to spread and persist in human populations relies on a balance between host immune responses and EBV immune evasion. CD8+ cells specific for EBV late lytic cycle antigens show poor recognition of target cells compared to immediate early and early antigen-specific CD8+ cells. This phenomenon is due in part to the early EBV protein BILF1, whose immunosuppressive activity increases with lytic cycle progression. However, published data suggest the existence of a hitherto unidentified immune evasion protein further enhancing protection against late EBV antigen-specific CD8+ cells. We have now identified the late lytic BDLF3 gene as the missing link accounting for efficient evasion during the late lytic cycle. Interestingly, BDLF3 also contributes to evasion of CD4+ cell responses to EBV. We report that BDLF3 downregulates expression of surface major histocompatibility complex (MHC) class I and class II molecules in the absence of any effect upon other surface molecules screened, including CD54 (ICAM-1) and CD71 (transferrin receptor). BDLF3 both enhanced internalization of surface MHC molecules and reduced the rate of their appearance at the cell surface. The reduced expression of surface MHC molecules correlated with functional protection against CD8+ and CD4+ T cell recognition. The molecular mechanism was identified as BDLF3-induced ubiquitination of MHC molecules and their subsequent downregulation in a proteasome-dependent manner. IMPORTANCE Immune evasion is a necessary feature of viruses that establish lifelong persistent infections in the face of strong immune responses. EBV is an important human pathogen whose immune evasion mechanisms are only partly understood. Of the EBV immune evasion mechanisms identified to date, none could explain why CD8+ T cell responses to late lytic cycle genes are so infrequent and, when present, recognize lytically infected target cells so poorly relative to CD8+ T cells specific for

  1. Synaptic Clusters of MHC Class II Molecules Induced on DCs by Adhesion Molecule–mediated Initial T-Cell ScanningV⃞

    PubMed Central

    de la Fuente, Hortensia; Mittelbrunn, María; Sánchez-Martín, Lorena; Vicente-Manzanares, Miguel; Lamana, Amalia; Pardi, Ruggero; Cabañas, Carlos; Sánchez-Madrid, Francisco

    2005-01-01

    Initial adhesive contacts between T lymphocytes and dendritic cells (DCs) facilitate recognition of peptide-MHC complexes by the TCR. In this report, we studied the dynamic behavior of adhesion and Ag receptors on DCs during initial contacts with T-cells. Adhesion molecules LFA-1- and ICAM-1,3-GFP as well as MHC class II-GFP molecules were very rapidly concentrated at the DC contact area. Binding of ICAM-3, and ICAM-1 to a lesser extent, to LFA-1 expressed by mature but not immature DC, induced MHC-II clustering into the immune synapse. Also, ICAM-3 binding to DC induced the activation of the Vav1-Rac1 axis, a regulatory pathway involved in actin cytoskeleton reorganization, which was essential for MHC-II clustering on DCs. Our results support a model in which ICAM-mediated MHC-II clustering on DC constitutes a priming mechanism to enhance antigen presentation to T-cells. PMID:15872088

  2. Novel mutations within the RFX-B gene and partial rescue of MHC and related genes through exogenous class II transactivator in RFX-B-deficient cells.

    PubMed

    Nagarajan, U M; Peijnenburg, A; Gobin, S J; Boss, J M; van den elsen, P J

    2000-04-01

    MHC class II deficiency or bare lymphocyte syndrome is a severe combined immunodeficiency caused by defects in MHC-specific regulatory factors. Fibroblasts derived from two recently identified bare lymphocyte syndrome patients, EBA and FZA, were found to contain novel mutations in the RFX-B gene. RFX-B encodes a component of the RFX transcription factor that functions in the assembly of multiple transcription factors on MHC class II promoters. Unlike RFX5- and RFXAP-deficient cells, transfection of exogenous class II transactivator (CIITA) into these RFX-B-deficient fibroblasts resulted in the induction of HLA-DR and HLA-DP and, to a lesser extent, HLA-DQ. Similarly, CIITA-mediated induction of MHC class I, beta2-microglobulin, and invariant chain genes was also found in these RFX-B-deficient fibroblasts. Expression of wild-type RFX-B completely reverted the noted deficiencies in these cells. Transfection of CIITA into Ramia cells, a B cell line that does not produce a stable RFX-B mRNA, resulted in induction of an MHC class II reporter, suggesting that CIITA overexpression may partially override the RFX-B defect.

  3. MHC class II DRB diversity in raccoons (Procyon lotor) reveals associations with raccoon rabies virus (Lyssavirus).

    PubMed

    Srithayakumar, Vythegi; Castillo, Sarrah; Rosatte, Rick C; Kyle, Christopher J

    2011-02-01

    In North America, the raccoon rabies virus (RRV) is an endemic wildlife disease which causes acute encephalopathies and is a strong selective force on raccoons (Procyon lotor), with estimates of ∼85% of the population succumbing to the disease when epizootic. RRV is regarded as a lethal disease if untreated; therefore, no evolutionary response would be expected of raccoon populations. However, variable immune responses to RRV have been observed in raccoons indicating a potential for evolutionary adaptation. Studies of variation within the immunologically important major histocompatibility complex (MHC) have revealed relationships between MHC alleles and diseases in humans and other wildlife species. This enhances our understanding of how hosts and pathogens adapt and co-evolve. In this study, we used RRV as a model system to study host-pathogen interaction in raccoons from a challenge study and from four wild populations that differ in exposure times and viral lineages. We investigated the potential role of Prlo-DRB polymorphism in relation to susceptibility/resistance to RRV in 113 RRV positive and 143 RRV negative raccoons. Six alleles were found to be associated with RRV negative status and five alleles with RRV positive animals. We found variable patterns of MHC associations given the relative number of selective RRV sweeps in the studied regions and correlations between MHC diversity and RRV lineages. The allelic associations established provide insight into how the genetic variation of raccoons may affect the disease outcome and this can be used to examine similar associations between other rabies variants and their hosts.

  4. 5HT4 agonists inhibit interferon-gamma-induced MHC class II and B7 costimulatory molecules expression on cultured astrocytes.

    PubMed

    Zeinstra, Esther M; Wilczak, Nadine; Wilschut, Jan C; Glazenburg, Lisa; Chesik, Daniel; Kroese, Frans G M; De Keyser, Jacques

    2006-10-01

    A failure of tight control of MHC class II expression on astrocytes may play a role in the development of autoimmune responses in multiple sclerosis. The 5-HT(4) serotonin receptor agonists cisapride and prucalopride, at concentrations between 10(-10) M and 10(-8) M, reduced interferon-gamma-induced MHC class II immunostaining in cultured astrocytes derived from newborn Wistar rats by approximately 50-60%. The magnitude of MHC class II inhibition by 5-HT(4) agonists was comparable to that of interferon-beta. The alpha(1)-adrenergic receptor agonist phenylephrine was without effect. Cisapride (10(-9) M) also prevented interferon-gamma-induced B7-1 and B7-2 immunostaining. Our results suggest that 5-HT(4) agonists may have therapeutic potential in multiple sclerosis by inhibiting the up-regulation of immune responsiveness of astrocytes in the central nervous system.

  5. Conserved mycobacterial lipoglycoproteins activate TLR2 but also require glycosylation for MHC class II-restricted T cell activation.

    PubMed

    Sieling, Peter A; Hill, Preston J; Dobos, Karen M; Brookman, Kerry; Kuhlman, Andrew M; Fabri, Mario; Krutzik, Stephan R; Rea, Thomas H; Heaslip, Darragh G; Belisle, John T; Modlin, Robert L

    2008-05-01

    CD4(+) T cell clones derived from a leprosy lesion and patient blood were used to monitor the isolation and identification of an Ag associated with the self-limited form of the disease. Biochemical purification and genetic analysis identified the T cell Ag as a conserved mycobacterial lipoglycoprotein LprG. LprG-mediated activation of CD4(+) T cells required specific MHC class II restriction molecules and intracellular processing. Although LprG activated TLR2, this alone was not sufficient to stimulate or inhibit T cell activation. A striking finding was that the carbohydrate moieties of LprG were required for optimal T cell activation, because recombinant LprG produced in Escherichia coli, or recombinant LprG produced in Mycobacterium smegmatis and digested by alpha-mannosidase, did not activate T cells. This study demonstrates that the universe of bacterial T cell Ags includes lipoglycoproteins, which act as TLR2 ligands but also require glycosylation for MHC class II-restricted T cell activation in vivo.

  6. The Thermodynamic Mechanism of Peptide–MHC Class II Complex Formation Is a Determinant of Susceptibility to HLA-DM

    PubMed Central

    Templeton, Megan; Hoffman, Megan; Castellini, Margaret J.

    2015-01-01

    Peptides bind MHC class II molecules through a thermodynamically nonadditive process consequent to the flexibility of the reactants. Currently, how the specific outcome of this binding process affects the ensuing epitope selection needs resolution. Calorimetric assessment of binding thermodynamics for hemagglutinin 306–319 peptide variants to the human MHC class II HLA-DR1 (DR1) and a mutant DR1 reveals that peptide/DR1 complexes can be formed with different enthalpic and entropic contributions. Complexes formed with a smaller entropic penalty feature circular dichroism spectra consistent with a non–compact form, and molecular dynamics simulation shows a more flexible structure. The opposite binding mode, compact and less flexible, is associated with greater entropic penalty. These structural variations are associated with rearrangements of residues known to be involved in HLA-DR (DM) binding, affinity of DM for the complex, and complex susceptibility to DM-mediated peptide exchange. Thus, the thermodynamic mechanism of peptide binding to DR1 correlates with the structural rigidity of the complex, and DM mediates peptide exchange by “sensing” flexible complexes in which the aforementioned residues are rearranged at a higher frequency than in more rigid ones. PMID:26116504

  7. The MHC-II transactivator CIITA, a restriction factor against oncogenic HTLV-1 and HTLV-2 retroviruses: similarities and differences in the inhibition of Tax-1 and Tax-2 viral transactivators

    PubMed Central

    Forlani, Greta; Abdallah, Rawan; Accolla, Roberto S.; Tosi, Giovanna

    2013-01-01

    The activation of CD4+ T helper cells is strictly dependent on the presentation of antigenic peptides by MHC class II (MHC-II) molecules. MHC-II expression is primarily regulated at the transcriptional level by the AIR-1 gene product CIITA (class II transactivator). Thus, CIITA plays a pivotal role in the triggering of the adaptive immune response against pathogens. Besides this well known function, we recently found that CIITA acts as an endogenous restriction factor against HTLV-1 (human T cell lymphotropic virus type 1) and HTLV-2 oncogenic retroviruses by targeting their viral transactivators Tax-1 and Tax-2, respectively. Here we review our findings on CIITA-mediated inhibition of viral replication and discuss similarities and differences in the molecular mechanisms by which CIITA specifically counteracts the function of Tax-1 and Tax-2 molecules. The dual function of CIITA as a key regulator of adaptive and intrinsic immunity represents a rather unique example of adaptation of host-derived factors against pathogen infections during evolution. PMID:23986750

  8. Immune complexes (IC) down-regulate the basal and interferon-γ-induced expression of MHC Class II on human monocytes

    PubMed Central

    Barrionuevo, P; Beigier-Bompadre, M; De La Barrera, S; Alves-Rosa, M F; Fernandez, G; Palermo, M S; Isturiz, M A

    2001-01-01

    The interaction of Fc receptors for IgG (FcγRs) on monocytes/macrophages with immune complexes (IC) triggers regulatory and effector functions. Previous studies have shown that FcγR–IC interactions inhibit the IFN-γ-induced expression of MHC class II in murine macrophages. However, the mechanism(s) responsible for these effects have not been elucidated. In addition, whether this IC-dependent effect also occurs in human cells is not known. Taking into account the fact that IC and IFN-γ are frequently found in infections and autoimmune disorders, together with the crucial role MHC class II molecules play in the regulation of immune response, we explored the effect and mechanism of IC-induced MHC class II down-regulation in human peripheral blood mononuclear cells (PBMC). This effect was studied either in the presence or absence of IFN-γ. We demonstrate that IC exert a drastic inhibition of basal and IFN-γ-induced expression of MHC class II on human monocytes. This effect was mediated through the interaction of IC with both FcγRI and FcγRII. Moreover, similar results were obtained using supernatants from IC-treated PBMC. The IC-induced down-regulation of MHC class II is abrogated by pepstatin and phosphoramidon, supporting the role of aspartic protease(s) and metalloprotease(s) in this process. In parallel with MHC class II expression, antigen presentation was markedly inhibited in the presence of IC. PMID:11529917

  9. Antigen processing of glycoconjugate vaccines; the polysaccharide portion of the pneumococcal CRM(197) conjugate vaccine co-localizes with MHC II on the antigen processing cell surface.

    PubMed

    Lai, Zengzu; Schreiber, John R

    2009-05-21

    Pneumococcal (Pn) polysaccharides (PS) are T-independent (TI) antigens and do not induce immunological memory or antibodies in infants. Conjugation of PnPS to the carrier protein CRM(197) induces PS-specific antibody in infants, and memory similar to T-dependent (Td) antigens. Conjugates have improved immunogenicity via antigen processing and presentation of carrier protein with MHC II and recruitment of T cell help, but the fate of the PS attached to the carrier is unknown. To determine the location of the PS component of PnPS-CRM(197) in the APC, we separately labeled PS and protein and tracked their location. The PS of types 14-CRM(197) and 19F-CRM(197) was specifically labeled by Alexa Fluor 594 hydrazide (red). The CRM(197) was separately labeled red in a reaction that did not label PS. Labeled antigens were incubated with APC which were fixed, permeabilized and incubated with anti-MHC II antibody labeled green by Alexa Fluor 488, followed by confocal microscopy. Labeled CRM(197) was presented on APC surface and co-localized with MHC II (yellow). Labeled unconjugated 14 or 19F PS did not go to the APC surface, but PS labeled 14-CRM(197) and 19F-CRM(197) was internalized and co-localized with MHC II. Monoclonal antibody to type 14 PS bound to intracellular type 14 PS and PS-CRM(197). Brefeldin A and chloroquine blocked both CRM(197) and PS labeled 14-CRM(197) and 19F-CRM(197) from co-localizing with MHC II. These data suggest that the PS component of the CRM(197) glycoconjugate enters the endosome, travels with CRM(197) peptides to the APC surface and co-localizes with MHC II.

  10. Characterization of polymorphism within the H2-M MHC class II loci.

    PubMed

    Hermel, E; Yuan, J; Monaco, J J

    1995-01-01

    The products of the class II-like H2-M genes of the major histocompatibility complex are required for class II antigen processing. We sequenced H2-Ma and Mb from several mouse strains to determine whether these genes are polymorphic like the classical H2-A and E genes, or are oligomorphic, like H2-O. Both Mb loci appear to be transcribed and are distinct from each other. Mb1 and Mb2 differ by about 11% at the nucleotide level and are most dissimilar in their second exons (corresponding to the beta 1 domain). Relative to the published Mb1d haplotype sequence, the products of the b, g7, f, and k2 alleles of Mb1 from Mus musculus domesticus and the separate mouse species Mus spretus differ by only one to four amino acids. The majority of the changes occurred in the second exon of Mb1, in contrast to HLA-DMB, the human orthologue. Little polymorphism was seen for Mb2, and Ma was invariant in all strains tested. The similarity of the g7 allele to those from other haplotypes makes it unlikely that the M class II genes play a role in the autoimmune diabetes of NOD strain mice. The M genes are regulated in a manner similar to classical class II genes, in that they are upregulated by IFN-gamma in macrophages, and to a lesser extent by IL4 in B cells. When modeled on the crystal structure of the HLA-DR1 class II molecule, nearly all of the differences between M beta 1 and M beta 2 affect residues facing away from the putative peptide binding groove.

  11. Effects of triamcinolone acetonide on microglial morphology and quantitative expression of MHC-II in exudative age-related macular degeneration.

    PubMed

    Penfold, P L; Wong, J G; Gyory, J; Billson, F A

    2001-06-01

    Animal models, in vitro assays and pilot clinical studies suggest that intravitreal triamcinolone acetonide may be useful in the treatment of age-related macular degeneration. The present case study reports the effect of intravitreal triamcinolone acetonide injection on a subretinal neovascular lesion, microglial morphology and quantitative expression of MHC-II antigens. Triamcinolone acetonide significantly decreased MHC-II expression consistent with immunocytochemical observations which revealed condensed microglial morphology. The modulation of subretinal oedema and microglial morphology correlates with in vitro observations suggesting that downregulation of inflammatory markers and endothelial cell permeability are significant features of the mode of action of triamcinolone acetonide.

  12. Definition of an extended MHC class II-peptide binding motif for the autoimmune disease-associated Lewis rat RT1.BL molecule.

    PubMed

    Wauben, M H; van der Kraan, M; Grosfeld-Stulemeyer, M C; Joosten, I

    1997-02-01

    The Lewis rat, an inbred rat strain susceptible to several well-characterized experimental autoimmune diseases, provides a good model to study peptide-mediated immunotherapy. Peptide immunotherapy focussing on the modulation of T cell responses by interfering with TCR-peptide-MHC complex formation requires the elucidation of the molecular basis of TCR-peptide-MHC interactions for an efficient design of modulatory peptides. In the Lewis rat most autoimmune-associated CD4+ T cell responses are MHC class II RT1.BL restricted. In this study, the characteristics of RT1.BL-peptide interactions were explored. A series of substitution analogs of two Lewis rat T cell epitopes was examined in a direct peptide-MHC binding assay on isolated RT1.BL molecules. Furthermore, other autoimmune-related as well as non-disease-related T cell epitopes were tested in the binding assay. This has led to the definition of an extended RT1.BL-peptide binding motif. The RT1.BL-peptide binding motif established in this study is the first described rat MHC-peptide binding motif based on direct MHC-peptide binding experiments. To predict good or intermediate RT1.BL binding peptides, T cell epitope search profiles were deduced from this motif. The motif and search profiles will greatly facilitate the prediction of modulatory peptides based on autoimmune-associated T cell epitopes and the identification of target structures in experimental autoimmune diseases in Lewis rats.

  13. Non-classical antigen processing pathways are required for MHC class II-restricted direct tumor recognition by NY-ESO-1-specific CD4+ T cells

    PubMed Central

    Matsuzaki, Junko; Tsuji, Takemasa; Luescher, Immanuel; Old, Lloyd J.; Shrikant, Protul; Gnjatic, Sacha; Odunsi, Kunle

    2014-01-01

    Tumor antigen-specific CD4+ T cells that directly recognize cancer cells are important for orchestrating antitumor immune responses at the local tumor sites. However, the mechanisms of direct MHC class II (MHC-II) presentation of intracellular tumor antigen by cancer cells are poorly understood. We found that two functionally distinct subsets of CD4+ T cells were expanded after HLA-DPB1*04 (DP04)-binding NY-ESO-1157–170 peptide vaccination in ovarian cancer patients. While both subsets similarly recognized exogenous NY-ESO-1 protein pulsed on DP04+ target cells, only one type recognized target cells with intracellular expression of NY-ESO-1. The tumor-recognizing CD4+ T cells more efficiently recognized the short 8–9-mer peptides than the non-tumor-recognizing CD4+ T cells. In addition to endosomal/lysosomal proteases that are typically involved in MHC-II antigen presentation, several pathways in the MHC class I presentation pathways such as the proteasomal degradation and transporter-associated with antigen-processing (TAP)-mediated peptide transport were also involved in the presentation of intracellular NY-ESO-1 on MHC-II. The presentation was inhibited significantly by primaquine, a small molecule that inhibits endosomal recycling, consistent with findings that pharmacological inhibition of new protein synthesis enhances antigen presentation. Together, our data demonstrated that cancer cells selectively present peptides from intracellular tumor antigens on MHC-II by multiple non-classical antigen-processing pathways. Harnessing direct tumor-recognizing ability of CD4+ T cells could be a promising strategy to enhance antitumor immune responses in the immunosuppressive tumor microenvironment. PMID:24764581

  14. Nonclassical antigen-processing pathways are required for MHC class II-restricted direct tumor recognition by NY-ESO-1-specific CD4(+) T cells.

    PubMed

    Matsuzaki, Junko; Tsuji, Takemasa; Luescher, Immanuel; Old, Lloyd J; Shrikant, Protul; Gnjatic, Sacha; Odunsi, Kunle

    2014-04-01

    Tumor antigen-specific CD4(+) T cells that directly recognize cancer cells are important for orchestrating antitumor immune responses at the local tumor sites. However, the mechanisms of direct MHC class II (MHC-II) presentation of intracellular tumor antigen by cancer cells are poorly understood. We found that two functionally distinct subsets of CD4(+) T cells were expanded after HLA-DPB1*04 (DP04)-binding NY-ESO-1157-170 peptide vaccination in patients with ovarian cancer. Although both subsets recognized exogenous NY-ESO-1 protein pulsed on DP04(+) target cells, only one type recognized target cells with intracellular expression of NY-ESO-1. The tumor-recognizing CD4(+) T cells more efficiently recognized the short 8-9-mer peptides than the non-tumor-recognizing CD4(+) T cells. In addition to endosomal/lysosomal proteases that are typically involved in MHC-II antigen presentation, several pathways in the MHC class I presentation pathways, such as the proteasomal degradation and transporter-associated with antigen-processing-mediated peptide transport, were also involved in the presentation of intracellular NY-ESO-1 on MHC-II. The presentation was inhibited significantly by primaquine, a small molecule that inhibits endosomal recycling, consistent with findings that pharmacologic inhibition of new protein synthesis enhances antigen presentation. Together, our data demonstrate that cancer cells selectively present peptides from intracellular tumor antigens on MHC-II by multiple nonclassical antigen-processing pathways. Harnessing the direct tumor-recognizing ability of CD4(+) T cells could be a promising strategy to enhance antitumor immune responses in the immunosuppressive tumor microenvironment.

  15. Structural insights into the editing of germ-line-encoded interactions between T-cell receptor and MHC class II by Vα CDR3.

    PubMed

    Deng, Lu; Langley, Ries J; Wang, Qian; Topalian, Suzanne L; Mariuzza, Roy A

    2012-09-11

    The conserved diagonal docking mode observed in structures of T-cell receptors (TCRs) bound to peptide-MHC ligands is believed to reflect coevolution of TCR and MHC genes. This coevolution is supported by the conservation of certain interactions between the germ-line-encoded complementarity-determining region (CDR)1 and CDR2 loops of TCR and MHC. However, the rules governing these interactions are not straightforward, even when the same variable (V) region recognizes the same MHC molecule. Here, we demonstrate that the somatically generated CDR3 loops can markedly alter evolutionarily selected contacts between TCR and MHC ("CDR3 editing"). To understand CDR3 editing at the atomic level, we determined the structure of a human melanoma-specific TCR (G4) bound to the MHC class II molecule HLA-DR1 and an epitope from mutant triose phosphate isomerase (mutTPI). A comparison of the G4-mutTPI-DR1 complex with a complex involving a TCR (E8) that uses the same Vα region to recognize the same mutTPI-DR1 ligand as G4 revealed that CDR1α adopts markedly different conformations in the two TCRs, resulting in an almost entirely different set of contacts with MHC. Based on the structures of unbound G4 and E8, the distinct conformations of CDR1α in these TCRs are not induced by binding to mutTPI-DR1 but result from differences in the length and sequence of CDR3α that are transmitted to CDR1α. The editing of germ-line-encoded TCR-MHC interactions by CDR3 demonstrates that these interactions possess sufficient intrinsic flexibility to accommodate large structural variations in CDR3 and, consequently, in the TCR-binding site.

  16. Structure of the Epstein-Barr virus gp42 protein bound to the MHC class II recepter HLA-DR1

    SciTech Connect

    Mullen, M.; Haan, K.M.; Longnecker, R.; Jardetzky, T.

    2010-03-08

    Epstein-Barr virus (EBV) causes infectious mononucleosis, establishes long-term latent infections, and is associated with a variety of human tumors. The EBV gp42 glycoprotein binds MHC class II molecules, playing a critical role in infection of B lymphocytes. EBV gp42 belongs to the C-type lectin superfamily, with homology to NK receptors of the immune system. We report the crystal structure of gp42 bound to the human MHC class II molecule HLA-DR1. The gp42 binds HLA-DR1 using a surface site that is distinct from the canonical lectin and NK receptor ligand binding sites. At the canonical ligand binding site, gp42 forms a large hydrophobic groove, which could interact with other ligands necessary for EBV entry, providing a mechanism for coupling MHC recognition and membrane fusion.

  17. IOP induces upregulation of GFAP and MHC-II and microglia reactivity in mice retina contralateral to experimental glaucoma

    PubMed Central

    2012-01-01

    Background Ocular hypertension is a major risk factor for glaucoma, a neurodegenerative disease characterized by an irreversible decrease in ganglion cells and their axons. Macroglial and microglial cells appear to play an important role in the pathogenic mechanisms of the disease. Here, we study the effects of laser-induced ocular hypertension (OHT) in the macroglia, microglia and retinal ganglion cells (RGCs) of eyes with OHT (OHT-eyes) and contralateral eyes two weeks after lasering. Methods Two groups of adult Swiss mice were used: age-matched control (naïve, n = 9); and lasered (n = 9). In the lasered animals, both OHT-eyes and contralateral eyes were analyzed. Retinal whole-mounts were immunostained with antibodies against glial fibrillary acid protein (GFAP), neurofilament of 200kD (NF-200), ionized calcium binding adaptor molecule (Iba-1) and major histocompatibility complex class II molecule (MHC-II). The GFAP-labeled retinal area (GFAP-RA), the intensity of GFAP immunoreaction (GFAP-IR), and the number of astrocytes and NF-200 + RGCs were quantified. Results In comparison with naïve: i) astrocytes were more robust in contralateral eyes. In OHT-eyes, the astrocyte population was not homogeneous, given that astrocytes displaying only primary processes coexisted with astrocytes in which primary and secondary processes could be recognized, the former having less intense GFAP-IR (P < 0.001); ii) GFAP-RA was increased in contralateral (P <0.05) and decreased in OHT-eyes (P <0.001); iii) the mean intensity of GFAP-IR was higher in OHT-eyes (P < 0.01), and the percentage of the retinal area occupied by GFAP+ cells with higher intensity levels was increased in contralateral (P = 0.05) and in OHT-eyes (P < 0.01); iv) both in contralateral and in OHT-eyes, GFAP was upregulated in Müller cells and microglia was activated; v) MHC-II was upregulated on macroglia and microglia. In microglia, it was similarly expressed in contralateral

  18. Modulation of CD8+ T cell responses to AAV vectors with IgG-derived MHC class II epitopes.

    PubMed

    Hui, Daniel J; Basner-Tschakarjan, Etiena; Chen, Yifeng; Davidson, Robert J; Buchlis, George; Yazicioglu, Mustafa; Pien, Gary C; Finn, Jonathan D; Haurigot, Virginia; Tai, Alex; Scott, David W; Cousens, Leslie P; Zhou, Shangzhen; De Groot, Anne S; Mingozzi, Federico

    2013-09-01

    Immune responses directed against viral capsid proteins constitute a main safety concern in the use of adeno-associated virus (AAV) as gene transfer vectors in humans. Pharmacological immunosuppression has been proposed as a solution to the problem; however, the approach suffers from several potential limitations. Using MHC class II epitopes initially identified within human IgG, named Tregitopes, we showed that it is possible to modulate CD8+ T cell responses to several viral antigens in vitro. We showed that incubation of peripheral blood mononuclear cells with these epitopes triggers proliferation of CD4+CD25+FoxP3+ T cells that suppress killing of target cells loaded with MHC class I antigens in an antigen-specific fashion, through a mechanism that seems to require cell-to-cell contact. Expression of a construct encoding for the AAV capsid structural protein fused to Tregitopes resulted in reduction of CD8+ T cell reactivity against the AAV capsid following immunization with an adenoviral vector expressing capsid. This was accompanied by an increase in frequency of CD4+CD25+FoxP3+ T cells in spleens and lower levels of inflammatory infiltrates in injected tissues. This proof-of-concept study demonstrates modulation of CD8+ T cell reactivity to an antigen using regulatory T cell epitopes is possible.

  19. PPAR-γ signaling and IL-5 inhibition together prevent chronic rejection of MHC Class II-mismatched cardiac grafts.

    PubMed

    Chen, Yan; Li, Daxu; Tsang, Julia Yuen Shan; Niu, Na; Peng, Jiao; Zhu, Jiang; Hui, Kenneth; Xu, Aimin; Lui, Vincent Chi Hang; Lamb, Jonathan Robert; Tam, Paul Kwong Hang

    2011-06-01

    Chronic rejection can prevent long-term survival of organ transplants. Although the beneficial effects of peroxisome proliferator-activated receptor-gamma (PPAR-γ) in reducing graft rejection have been reported, the details of the underlying mechanisms remain unclear, especially in the context of modulating cellular infiltration and preventing vasculopathy and interstitial fibrosis. The therapeutic effects of the PPAR-γ agonist, rosiglitazone, combined with anti-interleukin-5 are explored in a mouse model of MHC Class II-histoincompatible cardiac transplantation. Rosiglitazone treatment alone marginally increased long-term survival and reduced CD8 T-cell infiltration and vasculopathy in the grafts. However, there was no reduction in collagen deposition and interleukin (IL)-4, IL-5 and eosinophil infiltration were increased. Anti-IL-5 antibody treatment alone reduced eosinophil infiltration and collagen deposition, but had no effect on CD8 T-cell infiltration or vasculopathy. Combined treatment with anti-IL-5 antibody and rosiglitazone prevented graft rejection. Furthermore, rosiglitazone treatment increased adiponectin receptor II expression in grafts and on dendritic cells and T cells in vitro. Graft survival correlated with increased expression in grafts of the inhibitory molecule PD-L1. The findings obtained increase the knowledge on the mode of action of rosiglitazone in promoting the survival of MHC Class II-mismatched cardiac transplants in which the CD8 T cells and eosinophils play key roles. PPAR-γ signaling combined with IL-5 blockade prevents graft rejection. Copyright © 2011 International Society for Heart and Lung Transplantation. Published by Elsevier Inc. All rights reserved.

  20. Characterization of expressed class II MHC sequences in the banner-tailed kangaroo rat (Dipodomys spectabilis) reveals multiple DRB loci.

    PubMed

    Busch, Joseph D; Waser, Peter M; DeWoody, J Andrew

    2008-11-01

    Genes of the major histocompatibility complex (MHC) are exceptionally polymorphic due to the combined effects of natural and sexual selection. Most research in wild populations has focused on the second exon of a single class II locus (DRB), but complete gene sequences can provide an illuminating backdrop for studies of intragenic selection, recombination, and organization. To this end, we characterized class II loci in the banner-tailed kangaroo rat (Dipodomys spectabilis). Seven DRB-like sequences (provisionally named MhcDisp-DRB*01 through *07) were isolated from spleen cDNA and most likely comprise > or =5 loci; this multiformity is quite unlike the situation in muroid rodents such as Mus, Rattus, and Peromyscus. In silico translation revealed the presence of important structural residues for glycosylation sites, salt bonds, and CD4+ T-cell recognition. Amino-acid distances varied widely among the seven sequences (2-34%). Nuclear DNA sequences from the Disp-DRB*07 locus (approximately 10 kb) revealed a conventional exon/intron structure as well as a number of microsatellites and short interspersed nuclear elements (B4, Alu, and IDL-Geo subfamilies). Rates of nucleotide substitution at Disp-DRB*07 are similar in both exons and introns (pi = 0.015 and 0.012, respectively), which suggests relaxed selection and may indicate that this locus is an expressed pseudogene. Finally, we performed BLASTn searches against Dipodomys ordii genomic sequences (unassembled reads) and find 90-97% nucleotide similarity between the two kangaroo rat species. Collectively, these data suggest that class II diversity in heteromyid rodents is based on polylocism and departs from the muroid architecture.

  1. Songbird genomics: analysis of 45 kb upstream of a polymorphic Mhc class II gene in red-winged blackbirds (Agelaius phoeniceus).

    PubMed

    Gasper, J S; Shiina, T; Inoko, H; Edwards, S V

    2001-07-01

    Here we present the sequence of a 45 kb cosmid containing a previously characterized poly-morphic Mhc class II B gene (Agph-DAB1) from the red-winged blackbird (Agelaius phoeniceus). We compared it with a previously sequenced cosmid from this species, revealing two regions of 7.5 kb and 13.0 kb that averaged greater than 97% similarity to each another, indicating a very recent shared duplication. We found 12 retroelements, including two chicken repeat 1 (CR1) elements, constituting 6.4% of the sequence and indicating a lower frequency of retroelements than that found in mammalian genomic DNA. Agph-DAB3, a new class II B gene discovered in the cosmid, showed a low rate of polymorphism and may be functional. In addition, we found a Mhc class II B gene fragment and three genes likely to be functional (encoding activin receptor type II, a zinc finger, and a putative gamma-filamin). Phylogenetic analysis of exon 2 alleles of all three known blackbird Mhc genes indicated strong clustering of alleles by locus, implying that large amounts of interlocus gene conversion have not occurred since these genes have been diverging. Despite this, interspecific comparisons indicate that all three blackbird Mhc genes diverged from one another less than 35 million years ago and are subject to concerted evolution in the long term. Comparison of blackbird and chicken Mhc promoter regions revealed songbird promoter elements for the first time. The high gene density of this cosmid confirms similar findings for the chicken Mhc, but the segment duplications and diversity of retroelements resembles mammalian sequences.

  2. MHC Class II Restricted Innate-Like Double Negative T Cells Contribute to Optimal Primary and Secondary Immunity to Leishmania major

    PubMed Central

    Mou, Zhirong; Liu, Dong; Okwor, Ifeoma; Jia, Ping; Orihara, Kanami; Uzonna, Jude Ezeh

    2014-01-01

    Although it is generally believed that CD4+ T cells play important roles in anti-Leishmania immunity, some studies suggest that they may be dispensable, and that MHC II-restricted CD3+CD4−CD8− (double negative, DN) T cells may be more important in regulating primary anti-Leishmania immunity. In addition, while there are reports of increased numbers of DN T cells in Leishmania-infected patients, dogs and mice, concrete evidence implicating these cells in secondary anti-Leishmania immunity has not yet been documented. Here, we report that DN T cells extensively proliferate and produce effector cytokines (IFN-γ, TNF and IL-17) and granzyme B (GrzB) in the draining lymph nodes and spleens of mice following primary and secondary L. major infections. DN T cells from healed mice display functional characteristics of protective anti-Leishmania memory-like cells: rapid and extensive proliferation and effector cytokines production following L. major challenge in vitro and in vivo. DN T cells express predominantly (> 95%) alpha-beta T cell receptor (αβ TCR), are Leishmania-specific, restricted mostly by MHC class II molecules and display transcriptional profile of innate-like genes. Using in vivo depletion and adoptive transfer studies, we show that DN T cells contribute to optimal primary and secondary anti-Leishmania immunity in mice. These results directly identify DN T cells as important players in effective and protective primary and secondary anti-L. major immunity in experimental cutaneous leishmaniasis. PMID:25233487

  3. The tetraspan protein CD82 is a resident of MHC class II compartments where it associates with HLA-DR, -DM, and -DO molecules.

    PubMed

    Hammond, C; Denzin, L K; Pan, M; Griffith, J M; Geuze, H J; Cresswell, P

    1998-10-01

    In specialized APCs, MHC class II molecules are synthesized in the endoplasmic reticulum and transported through the Golgi apparatus to organelles of the endocytic pathway collectively called MHC class II compartments (MIICs). There, the class II-associated invariant chain is degraded, and peptides derived from internalized Ag bind to empty class II in a reaction that is facilitated by the class II-like molecule HLA-DM. An mAb raised to highly purified, immunoisolated MIICs from human B lymphoblastoid cells recognized CD82, a member of the tetraspan family of integral membrane proteins. Subcellular fractionation, immunofluorescence microscopy, and immunoelectron microscopy showed that CD82 is highly enriched in MIICs, particularly in their internal membranes. Coprecipitation analysis showed that CD82 associates in MIICs with class II, DM, and HLA-DO (an inhibitor of peptide loading that binds DM). Similar experiments showed CD63, another tetraspan protein found in MIICs, also associates with these molecules in the compartment and that CD82 and CD63 associate with each other. Preclearing experiments demonstrated that both CD82 and CD63 form complexes with DM-associated class II and DM-associated DO. The ability of CD82 and CD63 to form complexes with class II, DM, and DO in MIICs suggests that the tetraspan proteins may play an important role in the late stages of MHC class II maturation.

  4. Experimental Validation of Multi-Epitope Peptides Including Promising MHC Class I- and II-Restricted Epitopes of Four Known Leishmania infantum Proteins.

    PubMed

    Agallou, Maria; Athanasiou, Evita; Koutsoni, Olga; Dotsika, Eleni; Karagouni, Evdokia

    2014-01-01

    Leishmaniasis is a significant worldwide health problem for which no vaccine exists. Activation of CD4(+) and CD8(+) T cells is crucial for the generation of protective immunity against parasite. Recent trend in vaccine design has been shifted to epitope-based vaccines that are more specific, safe, and easy to produce. In the present study, four known antigenic Leishmania infantum proteins, cysteine peptidase A (CPA), histone H1, KMP-11, and Leishmania eukaryotic initiation factor (LeIF) were analyzed for the prediction of binding epitopes to H2(d) MHC class I and II molecules, using online available algorithms. Based on in silico analysis, eight peptides including highly scored MHC class I- and II-restricted epitopes were synthesized. Peptide immunogenicity was validated in MHC compatible BALB/c mice immunized with each synthetic peptide emulsified in complete Freund's adjuvant/incomplete Freund's adjuvant. CPA_p2, CPA_p3, H1_p1, and LeIF_p6 induced strong spleen cell proliferation upon in vitro peptide re-stimulation. In addition, the majority of the peptides, except of LeIF_p1 and KMP-11_p1, induced IFN-γ secretion, while KMP-11_p1 indicated a suppressive effect on IL-10 production. CPA_p2, CPA_p3, LeIF_p3, and LeIF_p6 induced IFN-γ-producing CD4(+) T cells indicating a TH1-type response. In addition, CPA_p2, CPA_p3, and H1_p1 induced also the induction of CD8(+) T cells. The induction of peptide-specific IgG in immunized mice designated also the existence of B cell epitopes in peptide sequences. Combining immunoinformatic tools and experimental validation, we demonstrated that CPA_p2, CPA_p3, H1_p1, H1_p3, CPA_p2, LeIF_p3, and LeIF_p6 are likely to include potential epitopes for the induction of protective cytotoxic and/or TH1-type immune responses supporting the feasibility of peptide-based vaccine development for leishmaniasis.

  5. The Ia.2 Epitope Defines a Subset of Lipid Raft Resident MHC Class II Molecules Crucial to Effective Antigen Presentation1

    PubMed Central

    Busman-Sahay, Kathleen; Sargent, Elizabeth; Harton, Jonathan A.; Drake, James R.

    2016-01-01

    Previous work has established that binding of the 11-5.2 anti-I-Ak mAb, which recognizes the Ia.2 epitope on I-Ak class II molecules, elicits MHC class II signaling, whereas binding of two other anti-I-Ak mAb that recognize the Ia.17 epitope fail to elicit signaling. Using a biochemical approach, we establish that the Ia.2 epitope recognized by the widely used 11-5.2 mAb defines a subset of cell surface I-Ak molecules predominantly found within membrane lipid rafts. Functional studies demonstrate that the Ia.2 bearing subset of I-Ak class II molecules is critically necessary for effective B cell–T cell interactions especially at low antigen doses, a finding consistent with published studies on the role of raft-resident class II molecules in CD4 T cell activation. Interestingly, B cells expressing recombinant I-Ak class II molecules possessing a β chain-tethered HEL peptide lack the Ia.2 epitope and fail to partition into lipid rafts. Moreover, cells expressing Ia.2 negative tethered peptide-class II molecules are severely impaired in their ability to present both tethered peptide or peptide derived from exogenous antigen to CD4 T cells. These results establish the Ia.2 epitope as defining a lipid raft-resident MHC class II confomer vital to the initiation of MHC class II restricted B cell–T cell interactions. PMID:21543648

  6. MHC Class II–Alpha Chain Knockout Mice Support Increased Viral Replication That Is Independent of Their Lack of MHC Class II Cell Surface Expression and Associated Immune Function Deficiencies

    PubMed Central

    Alsharifi, Mohammed; Koskinen, Aulikki; Wijesundara, Danushka K.; Bettadapura, Jayaram; Müllbacher, Arno

    2013-01-01

    MHCII molecules are heterodimeric cell surface proteins composed of an α and β chain. These molecules are almost exclusively expressed on thymic epithelium and antigen presenting cells (APCs) and play a central role in the development and function of CD4 T cells. Various MHC-II knockout mice have been generated including MHC-IIAα-/- (I-Aα-/-), MHC-IIAβ-/- (I-β-/-) and the double knockout (I-Aαxβ-/-). Here we report a very striking observation, namely that alphaviruses including the avirulent strain of Semliki Forest virus (aSFV), which causes asymptomatic infection in wild-type C57BL6/J (B6) mice, causes a very acute and lethal infection in I-Aα-/-, but not in I-β-/- or I-Aαxβ-/-, mice. This susceptibility to aSFV is associated with high virus titres in muscle, spleen, liver, and brain compared to B6 mice. In addition, I-Aα-/- mice show intact IFN-I responses in terms of IFN-I serum levels and IFN-I receptor expression and function. Radiation bone marrow chimeras of B6 mice reconstituted with I-Aα-/- bone marrow expressed B6 phenotype, whereas radiation chimeras of I-Aα-/- mice reconstituted with B6 bone marrow expressed the phenotype of high viral susceptibility. Virus replication experiments both in vivo and in vitro showed enhanced virus growth in tissues and cell cultures derived form I-Aα-/- compared to B6 mice. This enhanced virus replication is evident for other alpha-, flavi- and poxviruses and may be of great benefit to producers of viral vaccines. In conclusion, I-Aα-/- mice exhibit a striking susceptibility to virus infections independent of their defective MHC-II expression. Detailed genetic analysis will be carried out to characterise the underlining genetic defects responsible for the observed phenomenon. PMID:23840854

  7. Trans-species polymorphism of the Mhc class II DRB-like gene in banded penguins (genus Spheniscus).

    PubMed

    Kikkawa, Eri F; Tsuda, Tomi T; Sumiyama, Daisuke; Naruse, Taeko K; Fukuda, Michio; Kurita, Masanori; Wilson, Rory P; LeMaho, Yvon; Miller, Gary D; Tsuda, Michio; Murata, Koichi; Kulski, Jerzy K; Inoko, Hidetoshi

    2009-05-01

    The Major Histocompatibility Complex (Mhc) class II DRB locus of vertebrates is highly polymorphic and some alleles may be shared between closely related species as a result of balancing selection in association with resistance to parasites. In this study, we developed a new set of PCR primers to amplify, clone, and sequence overlapping portions of the Mhc class II DRB-like gene from the 5'UTR end to intron 3, including exons 1, 2, and 3 and introns 1 and 2 in four species (20 Humboldt, six African, five Magellanic, and three Galapagos penguins) of penguin from the genus Spheniscus (Sphe). Analysis of gene sequence variation by the neighbor-joining method of 21 Sphe sequences and 20 previously published sequences from four other penguin species revealed overlapping clades within the Sphe species, but species-specific clades for the other penguin species. The overlap of the DRB-like gene sequence variants between the four Sphe species suggests that, despite their allopatric distribution, the Sphe species are closely related and that some shared DRB1 alleles may have undergone a trans-species inheritance because of balancing selection and/or recent rapid speciation. The new primers and PCR assays that we have developed for the identification of the DRB1 DNA and protein sequence variations appear to be useful for the characterization of the molecular evolution of the gene in closely related Penguin species and might be helpful for the assessment of the genetic health and the management of the conservation and captivity of these endangered species.

  8. Direct Activation of Human Dendritic Cells by Particle-Bound but Not Soluble MHC Class II Ligand

    PubMed Central

    Baleeiro, Renato B.; Wiesmüller, Karl-Heinz; Dähne, Lars; Lademann, Jürgen; Barbuto, José A.; Walden, Peter

    2013-01-01

    Dendritic cells (DCs) are key activators of cellular immune responses through their capacity to induce naïve T cells and sustained effector T cell responses. This capacity is a function of their superior efficiency of antigen presentation via MHC class I and class II molecules, and the expression of co-stimulatory cell surface molecules and cytokines. Maturation of DCs is induced by microbial factors via pattern recognition receptors such as Toll-like receptors, pro-inflammatory cytokines or cognate interaction with CD4+ T cells. Here we show that, unexpectedly, the PanDR helper T cell epitope PADRE, a generic T helper cell antigen presented by a large fraction of HLA-DR alleles, when delivered in particle-bound form induced maturation of human DCs. The DCs that received the particle-bound PADRE displayed all features of fully mature DCs, such as high expression of the co-stimulatory molecules CD80, CD86, CD83, the MHC-II molecule HLA-DR, secretion of high levels of the biologically active IL-12 (IL-12p70) and induction of vigorous proliferation of naïve CD4+ T cells. Furthermore, the maturation of DCs induced by particle-bound PADRE was shown to involve sphingosine kinase, calcium signaling from internal sources and downstream signaling through the MAP kinase and the p72syk pathways, and finally activation of the transcription factor NF-κB. Based on our findings, we propose that particle-bound PADRE may be used as a DC activator in DC-based vaccines. PMID:23658796

  9. Salmonella inhibits monocyte differentiation into CD11c hi MHC-II hi cells in a MyD88-dependent fashion.

    PubMed

    Rydström, Anna; Wick, Mary Jo

    2010-05-01

    Monocytes and DCs originate from a shared precursor in the bone marrow, and steady-state DCs in lymphoid organs develop directly from the precursor rather than via a monocyte intermediate. However, monocytes can differentiate into DCs in tissues such as the lung and gut mucosa and into macrophages in most tissues. As Ly6C hi monocytes accumulate in lymphoid organs during oral Salmonella infection, we investigated their ability to develop into potential DCs, identified as CD11c hi MHC-II hi cells, in infected hosts. Ly6C hi monocytes, isolated from the blood of Salmonella-infected mice, developed into CD11c hi MHC-II hi cells after culture with GM-CSF or Flt3L. In contrast, the same monocytes cultured in the presence of GM-CSF and heat-killed Salmonella did not differentiate into CD11c hi MHC-II hi cells. The bacteria-induced differentiation block was dependent on TLRs, as monocytes from MyD88-/- mice converted into CD11c hi MHC-II hi cells even in the presence of bacteria. We hypothesized that Salmonella-activated wild-type monocytes secreted mediators that inhibited differentiation of MyD88-/--derived monocytes. However, IL-6, IL-10, TNF-alpha, or IL-12p70 did not account for the inhibition. Finally, monocyte-derived CD11c hi MHC-II hi cells pulsed with OVA peptide or protein did not induce proliferation of antigen-specific CD4+ T cells but rather, suppressed the ability of DCs to activate CD4+ T cells. Overall, the data show that Ly6C hi monocytes from Salmonella-infected mice develop into CD11c hi MHC-II hi cells with poor antigen-presentation capacity when cultured ex vivo, and that monocyte exposure to Salmonella inhibits their differentiation into CD11c hi MHC-II hi cells in a MyD88-dependent fashion.

  10. MHC class II complexes sample intermediate states along the peptide exchange pathway

    PubMed Central

    Wieczorek, Marek; Sticht, Jana; Stolzenberg, Sebastian; Günther, Sebastian; Wehmeyer, Christoph; El Habre, Zeina; Álvaro-Benito, Miguel; Noé, Frank; Freund, Christian

    2016-01-01

    The presentation of peptide-MHCII complexes (pMHCIIs) for surveillance by T cells is a well-known immunological concept in vertebrates, yet the conformational dynamics of antigen exchange remain elusive. By combining NMR-detected H/D exchange with Markov modelling analysis of an aggregate of 275 microseconds molecular dynamics simulations, we reveal that a stable pMHCII spontaneously samples intermediate conformations relevant for peptide exchange. More specifically, we observe two major peptide exchange pathways: the kinetic stability of a pMHCII's ground state defines its propensity for intrinsic peptide exchange, while the population of a rare, intermediate conformation correlates with the propensity of the HLA-DM-catalysed pathway. Helix-destabilizing mutants designed based on our model shift the exchange behaviour towards the HLA-DM-catalysed pathway and further allow us to conceptualize how allelic variation can shape an individual's MHC restricted immune response. PMID:27827392

  11. Identification and characterization of the major histocompatibility complex class II DQB (MhcMath-DQB1) alleles in Tibetan macaques (Macaca thibetana).

    PubMed

    Yao, Y-F; Zhao, J-J; Dai, Q-X; Li, J-Y; Zhou, L; Wang, Y-T; Ni, Q-Y; Zhang, M-w; Xu, H-L

    2013-08-01

    Tibetan macaque (Macaca thibetana), an endangered primate species endemic to China, have been used as experimental animal model for various human diseases. Major histocompatibility complex (MHC) genes play a crucial role in the susceptibility and/or resistance to many human diseases, but little is known about Tibetan macaques. To gain an insight into the MHC background and to facilitate the experimental use of Tibetan macaques, the second exon of Mhc-DQB1 gene was sequenced in a cohort of wild Tibetan macaques living in the Sichuan province of China. A total of 23 MhcMath-DQB1 alleles were identified for the first time, illustrating a marked allelic polymorphism at the DQB1 locus for these macaques. Most of the sequences (74%) observed in this study belong to DQB1*06 (9 alleles) and DQB1*18 (8 alleles) lineages, and the rest (26%) belong to DQB1*15 (3 alleles) and DQB1*17 (3 alleles) lineages. The most frequent alleles detected among these macaques were MhcMath-DQB1*15:02:02 (17.9%), followed by Math-DQB1*06:06, 17:03 and 18:01, which were detected in 9 (16.1%) of the monkeys, respectively. Non-synonymous substitutions occurred at a significantly higher frequency than synonymous substitutions in the peptide-binding region, suggesting balancing selection for maintaining polymorphisms at the MHC class II DQB1 locus. Phylogenetic analyses confirms the trans-species model of evolution of the Mhc-DQB1 genes in non-human primates, and in particular, the extensive allele sharing is observed between Tibetan and other macaque species.

  12. Selective pressures on MHC class II genes in the guppy (Poecilia reticulata) as inferred by hierarchical analysis of population structure.

    PubMed

    Herdegen, M; Babik, W; Radwan, J

    2014-11-01

    Genes of the major histocompatibility complex, which are the most polymorphic of all vertebrate genes, are a pre-eminent system for the study of selective pressures that arise from host-pathogen interactions. Balancing selection capable of maintaining high polymorphism should lead to the homogenization of MHC allele frequencies among populations, but there is some evidence to suggest that diversifying selection also operates on the MHC. However, the pattern of population structure observed at MHC loci is likely to depend on the spatial and/or temporal scale examined. Here, we investigated selection acting on MHC genes at different geographic scales using Venezuelan guppy populations inhabiting four regions. We found a significant correlation between MHC and microsatellite allelic richness across populations, which suggests the role of genetic drift in shaping MHC diversity. However, compared to microsatellites, more MHC variation was explained by differences between populations within larger geographic regions and less by the differences between the regions. Furthermore, among proximate populations, variation in MHC allele frequencies was significantly higher compared to microsatellites, indicating that selection acting on MHC may increase population structure at small spatial scales. However, in populations that have significantly diverged at neutral markers, the population-genetic signature of diversifying selection may be eradicated in the long term by that of balancing selection, which acts to preserve rare alleles and thus maintain a common pool of MHC alleles.

  13. MHC and Evolution in Teleosts

    PubMed Central

    Grimholt, Unni

    2016-01-01

    Major histocompatibility complex (MHC) molecules are key players in initiating immune responses towards invading pathogens. Both MHC class I and class II genes are present in teleosts, and, using phylogenetic clustering, sequences from both classes have been classified into various lineages. The polymorphic and classical MHC class I and class II gene sequences belong to the U and A lineages, respectively. The remaining class I and class II lineages contain nonclassical gene sequences that, despite their non-orthologous nature, may still hold functions similar to their mammalian nonclassical counterparts. However, the fact that several of these nonclassical lineages are only present in some teleost species is puzzling and questions their functional importance. The number of genes within each lineage greatly varies between teleost species. At least some gene expansions seem reasonable, such as the huge MHC class I expansion in Atlantic cod that most likely compensates for the lack of MHC class II and CD4. The evolutionary trigger for similar MHC class I expansions in tilapia, for example, which has a functional MHC class II, is not so apparent. Future studies will provide us with a more detailed understanding in particular of nonclassical MHC gene functions. PMID:26797646

  14. Role of MHC class II expressing CD4+ T cells in proteolipid protein(91-110)-induced EAE in HLA-DR3 transgenic mice.

    PubMed

    Mangalam, Ashutosh; Rodriguez, Moses; David, Chella

    2006-12-01

    MHC class II molecules play a central role in the control of adaptive immune responses through selection of the CD4(+) T cell repertoire in the thymus and antigen presentation in the periphery. Inherited susceptibility to autoimmune disorders such as multiple sclerosis, rheumatoid arthritis and IDDM are associated with particular MHC class II alleles. Advent of HLA transgenic mice has helped us in deciphering the role of particular HLA DR and DQ class II molecules in human autoimmune diseases. In mice, the expression of class II is restricted to professional antigen-presenting cells (APC). However, in humans, class II is also expressed on T cells, unlike murine T cells. We have developed new humanized HLA class II transgenic mice expressing class II molecules not only on APC but also on a subset of CD4(+) T cells. The expression of class II on CD4(+) T cells is inducible, and class II(+) CD4(+) T cells can present antigen in the absence of APC. Further, using EAE, a well-established animal model of MS, we tested the functional significance of these class II(+) CD4(+) T cells. DR3.AEo transgenic mice were susceptible to proteolipid protein(91-110)-induced EAE and showed CNS pathology accompanied by widespread inflammation and demyelination seen in human MS patients, suggesting a role for class II(+) CD4(+) T cells in the pathogenesis.

  15. NLRC5: a newly discovered MHC class I transactivator (CITA).

    PubMed

    Meissner, Torsten B; Li, Amy; Kobayashi, Koichi S

    2012-06-01

    Major histocompatibility complex (MHC) class I and class II are crucial for the function of the human adaptive immune system. An NLR protein, CIITA (MHC class II transactivator), is a master regulator of MHC class II gene expression as well as of some of the genes involved in MHC class II antigen presentation. It has recently been discovered that another member of the NLR protein family, NLRC5, transcriptionally activates MHC class I genes, and thus acts as "CITA" (MHC class I transactivator), a counterpart to CIITA. In addition to MHC class I genes, NLRC5 can induce the expression of β2M, TAP1 and LMP2, essential components of MHC class I antigen presentation. These findings indicate that NLRC5 and CIITA are transcriptional regulators that orchestrate the concerted expression of critical components in the MHC class I and MHC class II pathways, respectively.

  16. Atheroprotective vaccination with MHC-II-restricted ApoB peptides induces peritoneal IL-10-producing CD4 T cells.

    PubMed

    Kimura, Takayuki; Tse, Kevin; McArdle, Sara; Gerhardt, Teresa; Miller, Jacqueline; Mikulski, Zbigniew; Sidney, John; Sette, Alessandro; Wolf, Dennis; Ley, Klaus

    2017-04-01

    Although immunization with major histocompatibility complex (MHC) class II-restricted apolipoprotein B (ApoB) peptides has been shown to be atheroprotective, the mechanism is unclear. Here, we investigated CD4(+) T cell populations in immunized atherosclerotic mice. Peptides (16-mers) from mouse ApoB, the core protein of low-density lipoprotein (LDL), were screened for binding to I-A(b) by computer prediction and confirmed by radiolabeled peptide competition. Three new peptides, P101 (FGKQGFFPDSVNKALY, 5.5 nM IC50), P102 (TLYALSHAVNSYFDVD, 6.8 nM), and P103 (LYYKEDKTSLSASAAS, 95 nM), were tested in an atherosclerosis model (Apoe(-/-) mice on Western diet). Immunization with each of the three peptides (1 time in complete Freund's adjuvant subcuntaneously and 4 time in incomplete Freund's adjuvant intraperitoneally) but not with adjuvant alone showed significantly reduced atherosclerotic plaques in the aortic root by serial sections and in the whole aorta by en face staining. There were no differences in body weight, LDL cholesterol, or triglycerides. Peritoneal leukocytes from ApoB peptide-immunized mice, but not control mice, secreted significant amounts of IL-10 (150 pg/ml). Flow cytometry showed that peptide immunization induced IL-10 in 10% of peritoneal CD4(+) T cells, some of which also expressed chemokine (C-C motif) receptor 5 (CCR5). Vaccination with ApoB peptides expanded peritoneal FoxP3(+) regulatory CD4(+) T cells and more than tripled the number of CCR5(+)FoxP3(+) cells. Similar trends were also seen in the draining mediastinal lymph nodes but not in the nondraining inguinal lymph nodes. We conclude that vaccination with MHC class II-restricted autologous ApoB peptides induces regulatory T cells (Tregs) and IL-10, suggesting a plausible mechanism for atheroprotection.NEW & NOTEWORTHY Vaccination against apolipoprotein B (ApoB), the protein of LDL, attracts attention as a novel approach to prevent atherosclerosis. We discovered major histocompatibility

  17. An MHC class II restriction bias to I-A in CD4 T cell responses is altered to I-E in DM-deficient mice

    PubMed Central

    Menges, Paula R.; Jenks, Scott A.; Bikoff, Elizabeth K.; Friedmann, David R.; Knowlden, Zackery A. G.; Sant, Andrea J.

    2010-01-01

    The MHC-encoded cofactor DM, catalyzes endosomal loading of peptides onto MHC class II molecules. Despite evidence from in vitro experiments that DM acts to selectively edit the repertoire of class II:peptide complexes, the consequence of DM expression in vivo, or a predictive pattern of DM activity in the specificity of CD4 T cell responses has remained unresolved. Therefore, to characterize DM function in vivo we utilized wild type (WT) or DM-deficient (DM−/−) mice of the H-2d MHC haplotype and tested the hypothesis that DM promotes narrowing of the repertoire of class II:peptide complexes displayed by APC, leading to a correspondingly selective CD4 T cell response. Surprisingly, our results indicated that DM−/− mice do not exhibit a broadened CD4 T cell response relative to WT mice, but rather shift their immunodominance pattern to new peptides, a pattern associated with a change in class II isotype-restriction. Specifically, we found that CD4 T cell responses in WT mice were primarily restricted to the I-A class II molecule, while DM−/− mice recognize peptides in the context of I-E. The observed shift in isotype-restriction appeared to be due in part to a modification in the peripheral CD4 T cell repertoire available for peptide recognition. PMID:18209058

  18. The diabetogenic mouse MHC class II molecule I-A[superscript g7] is endowed with a switch that modulates TCR affinity

    SciTech Connect

    Yoshida, Kenji; Corper, Adam L.; Herro, Rana; Jabri, Bana; Wilson, Ian A.; Teyton, Luc

    2010-07-22

    Genetic susceptibility to autoimmunity is frequently associated with specific MHC alleles. Diabetogenic MHC class II molecules, such as human HLA-DQ8 and mouse I-A{sup g7}, typically have a small, uncharged amino acid residue at position 57 of their {beta} chain ({beta}57); this results in the absence of a salt bridge between {beta}57 and Arg{alpha}76, which is adjacent to the P9 pocket of the peptide-binding groove. However, the influence of Arg{alpha}76 on the selection of the TCR repertoire remains unknown, particularly when the MHC molecule binds a peptide with a neutral amino acid residue at position P9. Here, we have shown that diabetogenic MHC class II molecules bound to a peptide with a neutral P9 residue primarily selected and expanded cells expressing TCRs bearing a negatively charged residue in the first segment of their complementarity determining region 3{beta}. The crystal structure of one such TCR in complex with I-Ag7 bound to a peptide containing a neutral P9 residue revealed that a network of favorable long-range (greater than 4 {angstrom}) electrostatic interactions existed among Arg{alpha}76, the neutral P9 residue, and TCR, which supported the substantially increased TCR/peptide-MHC affinity. This network could be modulated or switched to a lower affinity interaction by the introduction of a negative charge at position P9 of the peptide. Our results support the existence of a switch at residue {beta}57 of the I-A{sup g7} and HLA-DQ8 class II molecules and potentially link normal thymic TCR selection with abnormal peripheral behavior.

  19. The diabetogenic mouse MHC class II molecule I-A[subscript g7] is endowed with a switch that modulates TCR affinity

    SciTech Connect

    Yoshida, Kenji; Corper, Adam L.; Herro, Rana; Jabri, Bana; Wilson, Ian A.; Teyton, Luc

    2011-11-16

    Genetic susceptibility to autoimmunity is frequently associated with specific MHC alleles. Diabetogenic MHC class II molecules, such as human HLA-DQ8 and mouse I-A{sub g7}, typically have a small, uncharged amino acid residue at position 57 of their {beta} chain ({beta}57); this results in the absence of a salt bridge between {beta}57 and Arg{alpha}76, which is adjacent to the P9 pocket of the peptide-binding groove. However, the influence of Arg{alpha}76 on the selection of the TCR repertoire remains unknown, particularly when the MHC molecule binds a peptide with a neutral amino acid residue at position P9. Here, we have shown that diabetogenic MHC class II molecules bound to a peptide with a neutral P9 residue primarily selected and expanded cells expressing TCRs bearing a negatively charged residue in the first segment of their complementarity determining region 3{beta}. The crystal structure of one such TCR in complex with I-A{sub g7} bound to a peptide containing a neutral P9 residue revealed that a network of favorable long-range (greater than 4 {angstrom}) electrostatic interactions existed among Arg{alpha}76, the neutral P9 residue, and TCR, which supported the substantially increased TCR/peptide-MHC affinity. This network could be modulated or switched to a lower affinity interaction by the introduction of a negative charge at position P9 of the peptide. Our results support the existence of a switch at residue {beta}57 of the I-Ag7 and HLA-DQ8 class II molecules and potentially link normal thymic TCR selection with abnormal peripheral behavior.

  20. Recombinant Lipoprotein Rv1016c Derived from Mycobacterium tuberculosis Is a TLR-2 Ligand that Induces Macrophages Apoptosis and Inhibits MHC II Antigen Processing.

    PubMed

    Su, Haibo; Zhu, Shenglin; Zhu, Lin; Huang, Wei; Wang, Honghai; Zhang, Zhi; Xu, Ying

    2016-01-01

    TLR2-dependent cellular signaling in Mycobacterium tuberculosis-infected macrophages causes apoptosis and inhibits class II major histocompatibility complex (MHC-II) molecules antigen processing, leading to evasion of surveillance. Mycobacterium tuberculosis (MTB) lipoproteins are an important class of Toll-like receptor (TLR) ligand, and identified as specific components that mediate these effects. In this study, we identified and characterized MTB lipoprotein Rv1016c (lpqT) as a cell wall associated-protein that was exposed on the cell surface and enhanced the survival of recombinants M. smegmatis_Rv1016c under stress conditions. We found that Rv1016c lipoprotein was a novel TLR2 ligand and able to induce macrophage apoptosis in a both dose- and time-dependent manner. Additionally, apoptosis induced by Rv1016c was reserved in THP-1 cells blocked with anti-TLR-2 Abs or in TLR2(-/-) mouse macrophages, indicating that Rv1016c-induced apoptosis is dependent on TLR2. Moreover, we demonstrated that Rv1016c lipoprotein inhibited IFN-γ-induced MHC-II expression and processing of soluble antigens in a TLR2 dependent manner. Class II transactivator (CIITA) regulates MHC II expression. In this context, Rv1016c lipoprotein diminished IFN-γ-induced expression of CIITA IV through TLR2 and MAPK Signaling. TLR2-dependent apoptosis and inhibition of MHC-II Ag processing induced by Rv1016c during mycobacteria infection may promote the release of residual bacilli from apoptotic cells and decrease recognition by CD4(+) T cells. These mechanisms may allow intracellular MTB to evade immune surveillance and maintain chronic infection.

  1. HLA-DO increases bacterial superantigen binding to human MHC molecules by inhibiting dissociation of class II-associated invariant chain peptides.

    PubMed

    Pezeshki, Abdul Mohammad; Azar, Georges A; Mourad, Walid; Routy, Jean-Pierre; Boulassel, Mohamed-Rachid; Denzin, Lisa K; Thibodeau, Jacques

    2013-10-01

    HLA-DO (H2-O in mice) is an intracellular non-classical MHC class II molecule (MHCII). It forms a stable complex with HLA-DM (H2-M in mice) and shapes the MHC class II-associated peptide repertoire. Here, we tested the impact of HLA-DO and H2-O on the binding of superantigens (SAgs), which has been shown previously to be sensitive to the structural nature of the class II-bound peptides. We found that the binding of staphylococcal enterotoxin (SE) A and B, as well as toxic shock syndrome toxin 1 (TSST-1), was similar on the HLA-DO(+) human B cell lines 721.45 and its HLA-DO(-) counterpart. However, overexpressing HLA-DO in MHC class II(+) HeLa cells (HeLa-CIITA-DO) improved binding of SEA and TSST-1. Accordingly, knocking down HLA-DO expression using specific siRNAs decreased SEA and TSST-1 binding. We tested directly the impact of the class II-associated invariant chain peptide (CLIP), which dissociation from MHC class II molecules is inhibited by overexpressed HLA-DO. Loading of synthetic CLIP on HLA-DR(+) cells increased SEA and TSST-1 binding. Accordingly, knocking down HLA-DM had a similar effect. In mice, H2-O deficiency had no impact on SAgs binding to isolated splenocytes. Altogether, our results demonstrate that the sensitivity of SAgs to the MHCII-associated peptide has physiological basis and that the effect of HLA-DO on SEA and TSST-1 is mediated through the inhibition of CLIP release.

  2. Recombinant Lipoprotein Rv1016c Derived from Mycobacterium tuberculosis Is a TLR-2 Ligand that Induces Macrophages Apoptosis and Inhibits MHC II Antigen Processing

    PubMed Central

    Su, Haibo; Zhu, Shenglin; Zhu, Lin; Huang, Wei; Wang, Honghai; Zhang, Zhi; Xu, Ying

    2016-01-01

    TLR2-dependent cellular signaling in Mycobacterium tuberculosis-infected macrophages causes apoptosis and inhibits class II major histocompatibility complex (MHC-II) molecules antigen processing, leading to evasion of surveillance. Mycobacterium tuberculosis (MTB) lipoproteins are an important class of Toll-like receptor (TLR) ligand, and identified as specific components that mediate these effects. In this study, we identified and characterized MTB lipoprotein Rv1016c (lpqT) as a cell wall associated-protein that was exposed on the cell surface and enhanced the survival of recombinants M. smegmatis_Rv1016c under stress conditions. We found that Rv1016c lipoprotein was a novel TLR2 ligand and able to induce macrophage apoptosis in a both dose- and time-dependent manner. Additionally, apoptosis induced by Rv1016c was reserved in THP-1 cells blocked with anti-TLR-2 Abs or in TLR2−/− mouse macrophages, indicating that Rv1016c-induced apoptosis is dependent on TLR2. Moreover, we demonstrated that Rv1016c lipoprotein inhibited IFN-γ-induced MHC-II expression and processing of soluble antigens in a TLR2 dependent manner. Class II transactivator (CIITA) regulates MHC II expression. In this context, Rv1016c lipoprotein diminished IFN-γ-induced expression of CIITA IV through TLR2 and MAPK Signaling. TLR2-dependent apoptosis and inhibition of MHC-II Ag processing induced by Rv1016c during mycobacteria infection may promote the release of residual bacilli from apoptotic cells and decrease recognition by CD4+ T cells. These mechanisms may allow intracellular MTB to evade immune surveillance and maintain chronic infection. PMID:27917375

  3. FOXP1 suppresses immune response signatures and MHC class II expression in activated B-cell-like diffuse large B-cell lymphomas

    PubMed Central

    Brown, P J; Wong, K K; Felce, S L; Lyne, L; Spearman, H; Soilleux, E J; Pedersen, L M; Møller, M B; Green, T M; Gascoyne, D M; Banham, A H

    2016-01-01

    The FOXP1 (forkhead box P1) transcription factor is a marker of poor prognosis in diffuse large B-cell lymphoma (DLBCL). Here microarray analysis of FOXP1-silenced DLBCL cell lines identified differential regulation of immune response signatures and major histocompatibility complex class II (MHC II) genes as some of the most significant differences between germinal center B-cell (GCB)-like DLBCL with full-length FOXP1 protein expression versus activated B-cell (ABC)-like DLBCL expressing predominantly short FOXP1 isoforms. In an independent primary DLBCL microarray data set, multiple MHC II genes, including human leukocyte antigen DR alpha chain (HLA-DRA), were inversely correlated with FOXP1 transcript expression (P<0.05). FOXP1 knockdown in ABC-DLBCL cells led to increased cell-surface expression of HLA-DRA and CD74. In R-CHOP (rituximab, cyclophosphamide, doxorubicin, vincristine and prednisone)-treated DLBCL patients (n=150), reduced HLA-DRA (<90% frequency) expression correlated with inferior overall survival (P=0.0003) and progression-free survival (P=0.0012) and with non-GCB subtype stratified by the Hans, Choi or Visco–Young algorithms (all P<0.01). In non-GCB DLBCL cases with <90% HLA-DRA, there was an inverse correlation with the frequency (P=0.0456) and intensity (P=0.0349) of FOXP1 expression. We propose that FOXP1 represents a novel regulator of genes targeted by the class II MHC transactivator CIITA (MHC II and CD74) and therapeutically targeting the FOXP1 pathway may improve antigen presentation and immune surveillance in high-risk DLBCL patients. PMID:26500140

  4. HLA-G and MHC Class II Protein Expression in Diffuse Large B-Cell Lymphoma.

    PubMed

    Jesionek-Kupnicka, Dorota; Bojo, Marcin; Prochorec-Sobieszek, Monika; Szumera-Ciećkiewicz, Anna; Jabłońska, Joanna; Kalinka-Warzocha, Ewa; Kordek, Radzisław; Młynarski, Wojciech; Robak, Tadeusz; Warzocha, Krzysztof; Lech-Maranda, Ewa

    2016-06-01

    The expression of human leukocyte antigen-G (HLA-G) and HLA class II protein was studied by immunohistochemical staining of lymph nodes from 148 patients with diffuse large B-cell lymphoma (DLBCL) and related to the clinical course of the disease. Negative HLA-G expression was associated with a lower probability of achieving a complete remission (p = 0.04). Patients with negative HLA-G expression tended towards a lower 3-year overall survival (OS) rate compared to those with positive expression of HLA-G (p = 0.08). When restricting the analysis to patients receiving chemotherapy with rituximab, the estimated 3-year OS rate of patients with positive HLA-G expression was 73.3 % compared with 47.5 % (p = 0.03) in those with negative expression. Patients with negative HLA class II expression presented a lower 3-year OS rate compared to subjects with positive expression (p = 0.04). The loss of HLA class II expression (p = 0.05) and belonging to the intermediate high/high IPI risk group (p = 0.001) independently increased the risk of death. HLA class II expression also retained its prognostic value in patients receiving rituximab; the 3-year OS rate was 65.3 % in patients with positive HLA class II expression versus 29.6 % (p = 0.04) in subjects that had loss of HLA class II expression. To our knowledge, for the first time, the expression of HLA-G protein in DLBCL and its association with the clinical course of the disease was demonstrated. Moreover, the link between losing HLA class II protein expression and poor survival of patients treated with immunochemotherapy was confirmed.

  5. Multiple parasites mediate balancing selection at two MHC class II genes in the fossorial water vole: insights from multivariate analyses and population genetics.

    PubMed

    Tollenaere, C; Bryja, J; Galan, M; Cadet, P; Deter, J; Chaval, Y; Berthier, K; Ribas Salvador, A; Voutilainen, L; Laakkonen, J; Henttonen, H; Cosson, J-F; Charbonnel, N

    2008-09-01

    We investigated the factors mediating selection acting on two MHC class II genes (DQA and DRB) in water vole (Arvicola scherman) natural populations in the French Jura Mountains. Population genetics showed significant homogeneity in allelic frequencies at the DQA1 locus as opposed to neutral markers (nine microsatellites), indicating balancing selection acting on this gene. Moreover, almost exhaustive screening for parasites, including gastrointestinal helminths, brain coccidia and antibodies against viruses responsible for zoonoses, was carried out. We applied a co-inertia approach to the genetic and parasitological data sets to avoid statistical problems related to multiple testing. Two alleles, Arte-DRB-11 and Arte-DRB-15, displayed antagonistic associations with the nematode Trichuris arvicolae, revealing the potential parasite-mediated selection acting on DRB locus. Selection mechanisms acting on the two MHC class II genes thus appeared different. Moreover, overdominance as balancing selection mechanism was showed highly unlikely in this system.

  6. Complementary DNA sequences encoding the multimammate rat MHC class II DQ alpha and beta chains and cross-species sequence comparison in rodents.

    PubMed

    de Bellocq, J Goüy; Leirs, H

    2009-09-01

    Sequences of the complete open reading frame (ORF) for rodents major histocompatibility complex (MHC) class II genes are rare. Multimammate rat (Mastomys natalensis) complementary DNA (cDNA) encoding the alpha and beta chains of MHC class II DQ gene was cloned from a rapid amplifications of cDNA Emds (RACE) cDNA library. The ORFs consist of 801 and 771 bp encoding 266 and 256 amino acid residues for DQB and DQA, respectively. The genomic structure of Mana-DQ genes is globally analogous to that described for other rodents except for the insertion of a serine residue in the signal peptide of Mana-DQB, which is unique among known rodents.

  7. Developmental and cytokine-mediated regulation of MHC class II gene promoter occupancy in vivo.

    PubMed

    Kara, C J; Glimcher, L H

    1993-06-01

    The class II genes of the major histocompatibility complex are a family of genes whose expression is regulated developmentally in cells of the B lineage and by IFN-gamma in many other cell types. Using the approach of in vivo footprinting, which allows for the examination of protein-promoter interactions within intact cells, we demonstrated a transition from unoccupied to occupied to once again unoccupied class II promoters in cell lines representing the developmental pathway of B cells. IFN-gamma treatment of HeLa cells led to increased promoter occupancy of the DR alpha and DR beta promoters at the same sites that are constitutively bound in mature B cells. No IFN-gamma-specific binding site was induced. Additionally, an octamer element in the DR alpha gene displayed preferential binding in B cells. These results demonstrate that changes in the transcription of the class II genes are associated with changes in factor binding at the promoter in vivo. Moreover, given the ubiquity of class II promoter binding proteins, these results suggest that throughout B cell development and upon IFN-gamma stimulation, the accessibility of class II promoter DNA is subject to regulation.

  8. Genetic variation of the MHC class II DRB genes in the Japanese weasel, Mustela itatsi, endemic to Japan, compared with the Siberian weasel, Mustela sibirica.

    PubMed

    Nishita, Y; Abramov, A V; Kosintsev, P A; Lin, L-K; Watanabe, S; Yamazaki, K; Kaneko, Y; Masuda, R

    2015-12-01

    Major histocompatibility complex (MHC) genes encode proteins that play a critical role in vertebrate immune system and are highly polymorphic. To further understand the molecular evolution of the MHC genes, we compared MHC class II DRB genes between the Japanese weasel (Mustela itatsi), a species endemic to Japan, and the Siberian weasel (Mustela sibirica), a closely related species on the continent. We sequenced a 242-bp region of DRB exon 2, which encodes antigen-binding sites (ABS), and found 24 alleles from 31 M. itatsi individuals and 17 alleles from 21 M. sibirica individuals, including broadly distributed, species-specific and/or geographically restricted alleles. Our results suggest that pathogen-driven balancing selection have acted to maintain the diversity in the DRB genes. For predicted ABS, nonsynonymous substitutions exceeded synonymous substitutions, also indicating positive selection, which was not seen at non-ABS. In a Bayesian phylogenetic tree, two M. sibirica DRB alleles were basal to the rest of the sequences from mustelid species and may represent ancestral alleles. Trans-species polymorphism was evident between many mustelid DRB alleles, especially between M. itatsi and M. sibirica. These two Mustela species divided about 1.7 million years ago, but still share many MHC alleles, indicative of their close phylogenetic relationship.

  9. Drift Rather than Selection Dominates MHC Class II Allelic Diversity Patterns at the Biogeographical Range Scale in Natterjack Toads Bufo calamita

    PubMed Central

    Zeisset, Inga; Beebee, Trevor J. C.

    2014-01-01

    Study of major histocompatibility complex (MHC) loci has gained great popularity in recent years, partly due to their function in protecting vertebrates from infections. This is of particular interest in amphibians on account of major threats many species face from emergent diseases such as chytridiomycosis. In this study we compare levels of diversity in an expressed MHC class II locus with neutral genetic diversity at microsatellite loci in natterjack toad (Bufo (Epidalea) calamita) populations across the whole of the species’ biogeographical range. Variation at both classes of loci was high in the glacial refugium areas (REF) and much lower in postglacial expansion areas (PGE), especially in range edge populations. Although there was clear evidence that the MHC locus was influenced by positive selection in the past, congruence with the neutral markers suggested that historical demographic events were the main force shaping MHC variation in the PGE area. Both neutral and adaptive genetic variation declined with distance from glacial refugia. Nevertheless, there were also some indications from differential isolation by distance and allele abundance patterns that weak effects of selection have been superimposed on the main drift effect in the PGE zone. PMID:24937211

  10. Genetic Variation at Exon 2 of the MHC Class II DQB Locus in Blue Whale (Balaenoptera musculus) from the Gulf of California.

    PubMed

    Moreno-Santillán, Diana D; Lacey, Eileen A; Gendron, Diane; Ortega, Jorge

    2016-01-01

    The genes of the Major Histocompatibility Complex (MHC) play an important role in the vertebrate immune response and are among the most polymorphic genes known in vertebrates. In some marine mammals, MHC genes have been shown to be characterized by low levels of polymorphism compared to terrestrial taxa; this reduction in variation is often explained as a result of lower pathogen pressures in marine habitats. To determine if this same reduction in variation applies to the migratory population of blue whales (Balaenoptera musculus) that occurs in the Gulf of California, we genotyped a 172 bp fragment of exon 2 of the MHC Class II DQB locus for 80 members of this population. Twenty-two putatively functional DQB allotypes were identified, all of which were homologous with DQB sequences from other cetacean species. Up to 5 putative alleles per individual were identified, suggesting that gene duplication has occurred at this locus. Rates of non-synonymous to synonymous substitutions (ω) and maximum likelihood analyses of models of nucleotide variation provided potential evidence of ongoing positive selection at this exon. Phylogenetic analyses of DQB alleles from B. musculus and 16 other species of cetaceans revealed trans-specific conservation of MHC variants, suggesting that selection has acted on this locus over prolonged periods of time. Collectively our findings reveal that immunogenic variation in blue whales is comparable to that in terrestrial mammals, thereby providing no evidence that marine taxa are subject to reduced pathogen-induced selective pressures.

  11. Genetic Variation at Exon 2 of the MHC Class II DQB Locus in Blue Whale (Balaenoptera musculus) from the Gulf of California

    PubMed Central

    Moreno-Santillán, Diana D.; Lacey, Eileen A.; Gendron, Diane; Ortega, Jorge

    2016-01-01

    The genes of the Major Histocompatibility Complex (MHC) play an important role in the vertebrate immune response and are among the most polymorphic genes known in vertebrates. In some marine mammals, MHC genes have been shown to be characterized by low levels of polymorphism compared to terrestrial taxa; this reduction in variation is often explained as a result of lower pathogen pressures in marine habitats. To determine if this same reduction in variation applies to the migratory population of blue whales (Balaenoptera musculus) that occurs in the Gulf of California, we genotyped a 172 bp fragment of exon 2 of the MHC Class II DQB locus for 80 members of this population. Twenty-two putatively functional DQB allotypes were identified, all of which were homologous with DQB sequences from other cetacean species. Up to 5 putative alleles per individual were identified, suggesting that gene duplication has occurred at this locus. Rates of non-synonymous to synonymous substitutions (ω) and maximum likelihood analyses of models of nucleotide variation provided potential evidence of ongoing positive selection at this exon. Phylogenetic analyses of DQB alleles from B. musculus and 16 other species of cetaceans revealed trans-specific conservation of MHC variants, suggesting that selection has acted on this locus over prolonged periods of time. Collectively our findings reveal that immunogenic variation in blue whales is comparable to that in terrestrial mammals, thereby providing no evidence that marine taxa are subject to reduced pathogen-induced selective pressures. PMID:26761201

  12. Negative relationships between cellular immune response, Mhc class II heterozygosity and secondary sexual trait in the montane water vole.

    PubMed

    Charbonnel, Nathalie; Bryja, Josef; Galan, Maxime; Deter, Julie; Tollenaere, Charlotte; Chaval, Yannick; Morand, Serge; Cosson, Jean-François

    2010-05-01

    Heterogeneities in immune responsiveness may affect key epidemiological parameters and the dynamics of pathogens. The roles of immunogenetics in these variations remain poorly explored. We analysed the influence of Major histocompatibility complex (Mhc) genes and epigamic traits on the response to phytohaemagglutinin in males from cyclic populations of the montane water vole (Arvicola scherman). Besides, we tested the relevance of lateral scent glands as honest signals of male quality. Our results did not corroborate neither the hypotheses of genome-wide heterozygosity-fitness correlation nor the Mhc heterozygote advantage. We found a negative relationship between Mhc hetetozygosity and response to phytohaemagglutinin, mediated by a specific Mhc homozygous genotype. Our results therefore support the hypothesis of the Arte-Dqa-05 homozygous genotype being a 'good' Mhc variant in terms of immunogenetic quality. The development of the scent glands seems to be an honest signal for mate choice as it is negatively correlated with helminth load. The 'good gene' hypothesis was not validated as Arte-Dqa-05 homozygous males did not exhibit larger glands. Besides, the negative relationship observed between the size of these glands and the response to phytohaemagglutinin, mainly for Mhc homozygotes, corroborates the immunocompetence handicap hypothesis. The Mhc variants associated with larger glands remain yet to be determined.

  13. Negative relationships between cellular immune response, Mhc class II heterozygosity and secondary sexual trait in the montane water vole

    PubMed Central

    Charbonnel, Nathalie; Bryja, Josef; Galan, Maxime; Deter, Julie; Tollenaere, Charlotte; Chaval, Yannick; Morand, Serge; Cosson, Jean-François

    2010-01-01

    Heterogeneities in immune responsiveness may affect key epidemiological parameters and the dynamics of pathogens. The roles of immunogenetics in these variations remain poorly explored. We analysed the influence of Major histocompatibility complex (Mhc) genes and epigamic traits on the response to phytohaemagglutinin in males from cyclic populations of the montane water vole (Arvicola scherman). Besides, we tested the relevance of lateral scent glands as honest signals of male quality. Our results did not corroborate neither the hypotheses of genome-wide heterozygosity-fitness correlation nor the Mhc heterozygote advantage. We found a negative relationship between Mhc hetetozygosity and response to phytohaemagglutinin, mediated by a specific Mhc homozygous genotype. Our results therefore support the hypothesis of the Arte-Dqa-05 homozygous genotype being a ‘good’ Mhc variant in terms of immunogenetic quality. The development of the scent glands seems to be an honest signal for mate choice as it is negatively correlated with helminth load. The ‘good gene’ hypothesis was not validated as Arte-Dqa-05 homozygous males did not exhibit larger glands. Besides, the negative relationship observed between the size of these glands and the response to phytohaemagglutinin, mainly for Mhc homozygotes, corroborates the immunocompetence handicap hypothesis. The Mhc variants associated with larger glands remain yet to be determined. PMID:25567924

  14. A novel regulatory pathway for autoimmune disease: binding of partial MHC class II constructs to monocytes reduces CD74 expression and induces both specific and bystander T-cell tolerance.

    PubMed

    Vandenbark, Arthur A; Meza-Romero, Roberto; Benedek, Gil; Andrew, Shayne; Huan, Jianya; Chou, Yuan K; Buenafe, Abigail C; Dahan, Rony; Reiter, Yoram; Mooney, Jeffery L; Offner, Halina; Burrows, Gregory G

    2013-02-01

    Treatment with partial (p)MHC class II-β1α1 constructs (also referred to as recombinant T-cell receptor ligands - RTL) linked to antigenic peptides can induce T-cell tolerance, inhibit recruitment of inflammatory cells and reverse autoimmune diseases. Here we demonstrate a novel regulatory pathway that involves RTL binding to CD11b(+) mononuclear cells through a receptor comprised of MHC class II invariant chain (CD74), cell-surface histones and MHC class II itself for treatment of experimental autoimmune encephalomyelitis (EAE). Binding of RTL constructs with CD74 involved a previously unrecognized MHC class II-α1/CD74 interaction that inhibited CD74 expression, blocked activity of its ligand, macrophage migration inhibitory factor, and reduced EAE severity. These findings implicate binding of RTL constructs to CD74 as a key step in both antigen-driven and bystander T-cell tolerance important in treatment of inflammatory diseases.

  15. MHC class II transactivator is an in vivo regulator of osteoclast differentiation and bone homeostasis co-opted from adaptive immunity.

    PubMed

    Benasciutti, Elisa; Mariani, Elisabetta; Oliva, Laura; Scolari, Maria; Perilli, Egon; Barras, Emmanuele; Milan, Enrico; Orfanelli, Ugo; Fazzalari, Nicola L; Campana, Lara; Capobianco, Annalisa; Otten, Luc; Particelli, Francesca; Acha-Orbea, Hans; Baruffaldi, Fabio; Faccio, Roberta; Sitia, Roberto; Reith, Walter; Cenci, Simone

    2014-02-01

    The molecular networks controlling bone homeostasis are not fully understood. The common evolution of bone and adaptive immunity encourages the investigation of shared regulatory circuits. MHC Class II Transactivator (CIITA) is a master transcriptional co-activator believed to be exclusively dedicated for antigen presentation. CIITA is expressed in osteoclast precursors, and its expression is accentuated in osteoporotic mice. We thus asked whether CIITA plays a role in bone biology. To this aim, we fully characterized the bone phenotype of two mouse models of CIITA overexpression, respectively systemic and restricted to the monocyte-osteoclast lineage. Both CIITA-overexpressing mouse models revealed severe spontaneous osteoporosis, as assessed by micro-computed tomography and histomorphometry, associated with increased osteoclast numbers and enhanced in vivo bone resorption, whereas osteoblast numbers and in vivo bone-forming activity were unaffected. To understand the underlying cellular and molecular bases, we investigated ex vivo the differentiation of mutant bone marrow monocytes into osteoclasts and immune effectors, as well as osteoclastogenic signaling pathways. CIITA-overexpressing monocytes differentiated normally into effector macrophages or dendritic cells but showed enhanced osteoclastogenesis, whereas CIITA ablation suppressed osteoclast differentiation. Increased c-fms and receptor activator of NF-κB (RANK) signaling underlay enhanced osteoclast differentiation from CIITA-overexpressing precursors. Moreover, by extending selected phenotypic and cellular analyses to additional genetic mouse models, namely MHC Class II deficient mice and a transgenic mouse line lacking a specific CIITA promoter and re-expressing CIITA in the thymus, we excluded MHC Class II expression and T cells from contributing to the observed skeletal phenotype. Altogether, our study provides compelling genetic evidence that CIITA, the molecular switch of antigen presentation

  16. Specific Microbiota-Induced Intestinal Th17 Differentiation Requires MHC II but not GALT and Mesenteric Lymph Nodes

    PubMed Central

    Geem, Duke; Medina-Contreras, Oscar; McBride, Michelle; Newberry, Rodney D.; Koni, Pandelakis A.; Denning, Timothy L.

    2014-01-01

    Interleukin (IL)-17 expressing CD4+ T lymphocytes (Th17 cells) naturally reside in the intestine where specific cytokines and microbiota, such as segmented filamentous bacteria (SFB), promote their differentiation. Intestinal Th17 cells are believed to initially differentiate in the GALT and/or mesenteric lymph nodes (mLN) upon antigen encounter and subsequently home to the lamina propria (LP) where they mediate effector functions. However, whether GALT and/or mLN are required for intestinal Th17 differentiation, and how microbiota containing SFB regulate antigen-specific intestinal Th17 cells remain poorly defined. Here we observed that naïve CD4+ T cells were abundant in the intestinal LP prior to weaning and that the accumulation of Th17 cells in response to microbiota containing SFB occurred in the absence of lymphotoxin (LT)-dependent lymphoid structures and the spleen. Furthermore, the differentiation of intestinal Th17 cells in the presence of microbiota containing SFB was dependent on MHC II expression by CD11c+ cells. Lastly, the differentiation of antigen-specific Th17 cells required both the presence of cognate antigen and microbiota containing SFB. These findings suggest that microbiota containing SFB create an intestinal milieu that may induce antigen-specific Th17 differentiation against food and/or bacterial antigens directly in the intestinal LP. PMID:24899505

  17. Ex vivo analysis of human memory CD4 T cells specific for hepatitis C virus using MHC class II tetramers.

    PubMed

    Day, Cheryl L; Seth, Nilufer P; Lucas, Michaela; Appel, Heiner; Gauthier, Laurent; Lauer, Georg M; Robbins, Gregory K; Szczepiorkowski, Zbigniew M; Casson, Deborah R; Chung, Raymond T; Bell, Shannon; Harcourt, Gillian; Walker, Bruce D; Klenerman, Paul; Wucherpfennig, Kai W

    2003-09-01

    Containment of hepatitis C virus (HCV) and other chronic human viral infections is associated with persistence of virus-specific CD4 T cells, but ex vivo characterization of circulating CD4 T cells has not been achieved. To further define the phenotype and function of these cells, we developed a novel approach for the generation of tetrameric forms of MHC class II/peptide complexes that is based on the cellular peptide-exchange mechanism. HLA-DR molecules were expressed as precursors with a covalently linked CLIP peptide, which could be efficiently exchanged with viral peptides following linker cleavage. In subjects who spontaneously resolved HCV viremia, but not in those with chronic progressive infection, HCV tetramer-labeled cells could be isolated by magnetic bead capture despite very low frequencies (1:1,200 to 1:111,000) among circulating CD4 T cells. These T cells expressed a set of surface receptors (CCR7+CD45RA-CD27+) indicative of a surveillance function for secondary lymphoid structures and had undergone significant in vivo selection since they utilized a restricted Vbeta repertoire. These studies demonstrate a relationship between clinical outcome and the presence of circulating CD4 T cells directed against this virus. Moreover, they show that rare populations of memory CD4 T cells can be studied ex vivo in human diseases.

  18. Adrenomedullin 2 Improves Early Obesity-Induced Adipose Insulin Resistance by Inhibiting the Class II MHC in Adipocytes.

    PubMed

    Zhang, Song-Yang; Lv, Ying; Zhang, Heng; Gao, Song; Wang, Ting; Feng, Juan; Wang, Yuhui; Liu, George; Xu, Ming-Jiang; Wang, Xian; Jiang, Changtao

    2016-08-01

    MHC class II (MHCII) antigen presentation in adipocytes was reported to trigger early adipose inflammation and insulin resistance. However, the benefits of MHCII inhibition in adipocytes remain largely unknown. Here, we showed that human plasma polypeptide adrenomedullin 2 (ADM2) levels were negatively correlated with HOMA of insulin resistance in obese human. Adipose-specific human ADM2 transgenic (aADM2-tg) mice were generated. The aADM2-tg mice displayed improvements in high-fat diet-induced early adipose insulin resistance. This was associated with increased insulin signaling and decreased systemic inflammation. ADM2 dose-dependently inhibited CIITA-induced MHCII expression by increasing Blimp1 expression in a CRLR/RAMP1-cAMP-dependent manner in cultured adipocytes. Furthermore, ADM2 treatment restored the high-fat diet-induced early insulin resistance in adipose tissue, mainly via inhibition of adipocyte MHCII antigen presentation and CD4(+) T-cell activation. This study demonstrates that ADM2 is a promising candidate for the treatment of early obesity-induced insulin resistance. © 2016 by the American Diabetes Association. Readers may use this article as long as the work is properly cited, the use is educational and not for profit, and the work is not altered.

  19. Inhibition of HLA-DM Mediated MHC Class II Peptide Loading by HLA-DO Promotes Self Tolerance.

    PubMed

    Denzin, Lisa K

    2013-12-17

    Major histocompatibility class II (MHCII) molecules are loaded with peptides derived from foreign and self-proteins within the endosomes and lysosomes of antigen presenting cells (APCs). This process is mediated by interaction of MHCII with the conserved, non-polymorphic MHCII like molecule HLA-DM (DM). DM activity is directly opposed by HLA-DO (DO), another conserved, non-polymorphic MHCII like molecule. DO is an MHCII substrate mimic. Binding of DO to DM prevents MHCII from binding to DM, thereby inhibiting peptide loading. Inhibition of DM function enables low stability MHC complexes to survive and populate the surface of APCs. As a consequence, DO promotes the display of a broader pool of low abundance self-peptides. Broadening the peptide repertoire theoretically reduces the likelihood of inadvertently acquiring a density of self-ligands that is sufficient to activate self-reactive T cells. One function of DO, therefore, is to promote T cell tolerance by shaping the visible image of self. Recent data also shows that DO influences the adaptive immune response by controlling B cell entry into the germinal center reaction. This review explores the data supporting these concepts.

  20. Inhibition of HLA-DM Mediated MHC Class II Peptide Loading by HLA-DO Promotes Self Tolerance

    PubMed Central

    Denzin, Lisa K.

    2013-01-01

    Major histocompatibility class II (MHCII) molecules are loaded with peptides derived from foreign and self-proteins within the endosomes and lysosomes of antigen presenting cells (APCs). This process is mediated by interaction of MHCII with the conserved, non-polymorphic MHCII like molecule HLA-DM (DM). DM activity is directly opposed by HLA-DO (DO), another conserved, non-polymorphic MHCII like molecule. DO is an MHCII substrate mimic. Binding of DO to DM prevents MHCII from binding to DM, thereby inhibiting peptide loading. Inhibition of DM function enables low stability MHC complexes to survive and populate the surface of APCs. As a consequence, DO promotes the display of a broader pool of low abundance self-peptides. Broadening the peptide repertoire theoretically reduces the likelihood of inadvertently acquiring a density of self-ligands that is sufficient to activate self-reactive T cells. One function of DO, therefore, is to promote T cell tolerance by shaping the visible image of self. Recent data also shows that DO influences the adaptive immune response by controlling B cell entry into the germinal center reaction. This review explores the data supporting these concepts. PMID:24381574

  1. MHC class II/ESO tetramer-based generation of in vitro primed anti-tumor T-helper lines for adoptive cell therapy of cancer.

    PubMed

    Poli, Caroline; Raffin, Caroline; Dojcinovic, Danijel; Luescher, Immanuel; Ayyoub, Maha; Valmori, Danila

    2013-02-01

    Generation of tumor-antigen specific CD4(+) T-helper (T(H)) lines through in vitro priming is of interest for adoptive cell therapy of cancer, but the development of this approach has been limited by the lack of appropriate tools to identify and isolate low frequency tumor antigen-specific CD4(+) T cells. Here, we have used recently developed MHC class II/peptide tetramers incorporating an immunodominant peptide from NY-ESO-1 (ESO), a tumor antigen frequently expressed in different human solid and hematologic cancers, to implement an in vitro priming platform allowing the generation of ESO-specific T(H) lines. We isolated phenotypically defined CD4(+) T-cell subpopulations from circulating lymphocytes of DR52b(+) healthy donors by flow cytometry cell sorting and stimulated them in vitro with peptide ESO(119-143), autologous APC and IL-2. We assessed the frequency of ESO-specific cells in the cultures by staining with DR52b/ESO(119-143) tetramers (ESO-tetramers) and TCR repertoire of ESO-tetramer(+) cells by co-staining with TCR variable β chain (BV) specific antibodies. We isolated ESO-tetramer(+) cells by flow cytometry cell sorting and expanded them with PHA, APC and IL-2 to generate ESO-specific T(H) lines. We characterized the lines for antigen recognition, by stimulation with ESO peptide or recombinant protein, cytokine production, by intracellular staining using specific antibodies, and alloreactivity, by stimulation with allo-APC. Using this approach, we could consistently generate ESO-tetramer(+) T(H) lines from conventional CD4(+)CD25(-) naïve and central memory populations, but not from effector memory populations or CD4(+)CD25(+) Treg. In vitro primed T(H) lines recognized ESO with affinities comparable to ESO-tetramer(+) cells from patients immunized with an ESO vaccine and used a similar TCR repertoire. In this study, using MHC class II/ESO tetramers, we have implemented an in vitro priming platform allowing the generation of ESO

  2. MHC class II/ESO tetramer-based generation of in vitro primed anti-tumor T-helper lines for adoptive cell therapy of cancer

    PubMed Central

    Poli, Caroline; Raffin, Caroline; Dojcinovic, Danijel; Luescher, Immanuel; Ayyoub, Maha; Valmori, Danila

    2013-01-01

    Generation of tumor-antigen specific CD4+ T-helper (TH) lines through in vitro priming is of interest for adoptive cell therapy of cancer, but the development of this approach has been limited by the lack of appropriate tools to identify and isolate low frequency tumor antigen-specific CD4+ T cells. Here, we have used recently developed MHC class II/peptide tetramers incorporating an immunodominant peptide from NY-ESO-1 (ESO), a tumor antigen frequently expressed in different human solid and hematologic cancers, to implement an in vitro priming platform allowing the generation of ESO-specific TH lines. We isolated phenotypically defined CD4+ T-cell subpopulations from circulating lymphocytes of DR52b+ healthy donors by flow cytometry cell sorting and stimulated them in vitro with peptide ESO119-143, autologous APC and IL-2. We assessed the frequency of ESO-specific cells in the cultures by staining with DR52b/ESO119-143 tetramers (ESO-tetramers) and TCR repertoire of ESO-tetramer+ cells by co-staining with TCR variable β chain (BV) specific antibodies. We isolated ESO-tetramer+ cells by flow cytometry cell sorting and expanded them with PHA, APC and IL-2 to generate ESO-specific TH lines. We characterized the lines for antigen recognition, by stimulation with ESO peptide or recombinant protein, cytokine production, by intracellular staining using specific antibodies, and alloreactivity, by stimulation with allo-APC. Using this approach, we could consistently generate ESO-tetramer+ TH lines from conventional CD4+CD25− naïve and central memory populations, but not from effector memory populations or CD4+CD25+ Treg. In vitro primed TH lines recognized ESO with affinities comparable to ESO-tetramer+ cells from patients immunized with an ESO vaccine and used a similar TCR repertoire. In this study, using MHC class II/ESO tetramers, we have implemented an in vitro priming platform allowing the generation of ESO-monospecific polyclonal TH lines from non

  3. Patterns of MHC-G-Like and MHC-B Diversification in New World Monkeys

    PubMed Central

    Lugo, Juan S.; Cadavid, Luis F.

    2015-01-01

    The MHC class I (MHC-I) region in New World monkeys (Platyrrhini) has remained relatively understudied. To evaluate the diversification patterns and transcription behavior of MHC-I in Platyrrhini, we first analyzed public genomic sequences from the MHC-G-like subregion in Saimiri boliviensis, Ateles geoffroyi and Callicebus moloch, and from the MHC-B subregion in Saimiri boliviensis. While S. boliviensis showed multiple copies of both MHC-G-like (10) and –B (15) loci, A. geoffroyi and C. moloch had only three and four MHC-G-like genes, respectively, indicating that not all Platyrrhini species have expanded their MHC-I loci. We then sequenced MHC-G-like and -B cDNAs from nine Platyrrhini species, recovering two to five unique cDNAs per individual for both loci classes. In two Saguinus species, however, no MHC-B cDNAs were found. In phylogenetic trees, MHC-G-like cDNAs formed genus-specific clusters whereas the MHC-B cDNAs grouped by Platyrrhini families, suggesting a more rapid diversification of the former. Furthermore, cDNA sequencing in 12 capuchin monkeys showed that they transcribe at least four MHC-G-like and five MHC-B polymorphic genes, showing haplotypic diversity for gene copy number and signatures of positive natural selection at the peptide binding region. Finally, a quantitative index for MHC:KIR affinity was proposed and tested to predict putative interacting pairs. Altogether, our data indicate that i) MHC-I genes has expanded differentially among Platyrrhini species, ii) Callitrichinae (tamarins and marmosets) MHC-B loci have limited or tissue-specific expression, iii) MHC-G-like genes have diversified more rapidly than MHC-B genes, and iv) the MHC-I diversity is generated mainly by genetic polymorphism and gene copy number variation, likely promoted by natural selection for ligand binding. PMID:26121030

  4. CD45R, CD44 and MHC class II are signaling molecules for the cytoskeleton-dependent induction of dendrites and motility in activated B cells.

    PubMed

    Partida-Sánchez, S; Garibay-Escobar, A; Frixione, E; Parkhouse, R M; Santos-Argumedo, L

    2000-09-01

    Anti-CD44 or anti-MHC II antibodies bound to tissue culture plates have previously been shown to induce a dramatic generation of dendritic processes in activated murine B cells. In this study, we demonstrate a similar generation of dendrites and cell motility in activated B cells through CD45R. The dynamic formation of dendritic processes and associated induction of cell motility were analyzed by video microscopy and were characterized by a rapid, and multidirectional emission of dendrites with retractile behavior. The addition of cytochalasin E totally blocked dendrites formation and motility induced through either CD45R, CD44 or MHC II, suggesting that the necessary cytoskeletal rearrangements require active polymerization of actin. Confocal microscopy showed an accumulation of F-actin in the dendrites, as long as cells were elongating. In contrast, G-actin was localized in the perinuclear area and also accumulated in sites where dendrites originated. Preincubation of B cells with staurosporine (a PKC inhibitor) or BAPTA-AM (a calcium chelator) prevented these morphological changes, indicating additionally a requirement for a PKC-calcium-dependent activity. Dendrite formation and cellular motility, therefore, seem to be two manifestations of the same phenomenon, and CD44, CD45R and MHC II appear to be signaling molecules for the observed cytoskeleton-dependent morphological changes.

  5. Genome-wide association study identifies SNPs in the MHC class II loci that are associated with self-reported history of whooping cough.

    PubMed

    McMahon, George; Ring, Susan M; Davey-Smith, George; Timpson, Nicholas J

    2015-10-15

    Whooping cough is currently seeing resurgence in countries despite high vaccine coverage. There is considerable variation in subject-specific response to infection and vaccine efficacy, but little is known about the role of human genetics. We carried out a case-control genome-wide association study of adult or parent-reported history of whooping cough in two cohorts from the UK: the ALSPAC cohort and the 1958 British Birth Cohort (815/758 cases and 6341/4308 controls, respectively). We also imputed HLA alleles using dense SNP data in the MHC region and carried out gene-based and gene-set tests of association and estimated the amount of additive genetic variation explained by common SNPs. We observed a novel association at SNPs in the MHC class II region in both cohorts [lead SNP rs9271768 after meta-analysis, odds ratio [95% confidence intervals (CIs)] 1.47 (1.35, 1.6), P-value 1.21E - 18]. Multiple strong associations were also observed at alleles at the HLA class II loci. The majority of these associations were explained by the lead SNP rs9271768. Gene-based and gene-set tests and estimates of explainable common genetic variation could not establish the presence of additional associations in our sample. Genetic variation at the MHC class II region plays a role in susceptibility to whooping cough. These findings provide additional perspective on mechanisms of whooping cough infection and vaccine efficacy.

  6. Complement component 3 deficiency prolongs MHC-II disparate skin allograft survival by increasing the CD4+ CD25+ regulatory T cells population

    PubMed Central

    Zheng, Quan-you; Liang, Shen-ju; Li, Gui-qing; Lv, Yan-bo; Li, You; Tang, Ming; Zhang, Kun; Xu, Gui-lian; Zhang, Ke-qin

    2016-01-01

    Recent reports suggest that complement system contributes to allograft rejection. However, its underlying mechanism is poorly understood. Herein, we investigate the role of complement component 3 (C3) in a single MHC-II molecule mismatched murine model of allograft rejection using C3 deficient mice (C3−/−) as skin graft donors or recipients. Compared with C3+/+ B6 allografts, C3−/− B6 grafts dramatically prolonged survival in MHC-II molecule mismatched H-2bm12 B6 recipients, indicating that C3 plays a critical role in allograft rejection. Compared with C3+/+ allografts, both Th17 cell infiltration and Th1/Th17 associated cytokine mRNA levels were clearly reduced in C3−/− allografts. Moreover, C3−/− allografts caused attenuated Th1/Th17 responses, but increased CD4+CD25+Foxp3+ regulatory T (Treg) cell expression markedly in local intragraft and H-2bm12 recipients. Depletion of Treg cells by anti-CD25 monoclonal antibody (mAb) negated the survival advantages conferred by C3 deficiency. Our results indicate for the first time that C3 deficiency can prolong MHC-II molecule mismatched skin allograft survival, which is further confirmed to be associated with increased CD4+ CD25+ Treg cell population expansion and attenuated Th1/Th17 response. PMID:27641978

  7. Genome-wide association study identifies SNPs in the MHC class II loci that are associated with self-reported history of whooping cough

    PubMed Central

    McMahon, George; Ring, Susan M.; Davey-Smith, George; Timpson, Nicholas J.

    2015-01-01

    Whooping cough is currently seeing resurgence in countries despite high vaccine coverage. There is considerable variation in subject-specific response to infection and vaccine efficacy, but little is known about the role of human genetics. We carried out a case–control genome-wide association study of adult or parent-reported history of whooping cough in two cohorts from the UK: the ALSPAC cohort and the 1958 British Birth Cohort (815/758 cases and 6341/4308 controls, respectively). We also imputed HLA alleles using dense SNP data in the MHC region and carried out gene-based and gene-set tests of association and estimated the amount of additive genetic variation explained by common SNPs. We observed a novel association at SNPs in the MHC class II region in both cohorts [lead SNP rs9271768 after meta-analysis, odds ratio [95% confidence intervals (CIs)] 1.47 (1.35, 1.6), P-value 1.21E − 18]. Multiple strong associations were also observed at alleles at the HLA class II loci. The majority of these associations were explained by the lead SNP rs9271768. Gene-based and gene-set tests and estimates of explainable common genetic variation could not establish the presence of additional associations in our sample. Genetic variation at the MHC class II region plays a role in susceptibility to whooping cough. These findings provide additional perspective on mechanisms of whooping cough infection and vaccine efficacy. PMID:26231221

  8. DNA Vaccines: MHC II-Targeted Vaccine Protein Produced by Transfected Muscle Fibres Induces a Local Inflammatory Cell Infiltrate in Mice

    PubMed Central

    Løvås, Tom-Ole; Gundersen, Kristian; Bogen, Bjarne

    2014-01-01

    Vaccination with naked DNA holds great promise but immunogenicity needs to be improved. DNA constructs encoding bivalent proteins that bind antigen-presenting cells (APC) for delivery of antigen have been shown to enhance T and B cell responses and protection in tumour challenge experiments. However, the mechanism for the increased potency remains to be determined. Here we have constructed DNA vaccines that express the fluorescent protein mCherry, a strategy which allowed tracking of vaccine proteins. Transfected muscle fibres in mice were visualized, and their relationship to infiltrating mononuclear cells could be determined. Interestingly, muscle fibers that produced MHC class II-specific dimeric vaccine proteins with mCherry were for weeks surrounded by a localized intense cellular infiltrate composed of CD45+, MHC class II+ and CD11b+ cells. Increasing numbers of eosinophils were observed among the infiltrating cells from day 7 after immunization. The local infiltrate surrounding mCherry+ muscle fibers was dependent on the MHC II-specificity of the vaccine proteins since the control, a non-targeted vaccine protein, failed to induce similar infiltrates. Chemokines measured on day 3 in immunized muscle indicate both a DNA effect and an electroporation effect. No influence of targeting was observed. These results contribute to our understanding for why targeted DNA vaccines have an improved immunogenicity. PMID:25299691

  9. DNA vaccines: MHC II-targeted vaccine protein produced by transfected muscle fibres induces a local inflammatory cell infiltrate in mice.

    PubMed

    Løvås, Tom-Ole; Bruusgaard, Jo C; Øynebråten, Inger; Gundersen, Kristian; Bogen, Bjarne

    2014-01-01

    Vaccination with naked DNA holds great promise but immunogenicity needs to be improved. DNA constructs encoding bivalent proteins that bind antigen-presenting cells (APC) for delivery of antigen have been shown to enhance T and B cell responses and protection in tumour challenge experiments. However, the mechanism for the increased potency remains to be determined. Here we have constructed DNA vaccines that express the fluorescent protein mCherry, a strategy which allowed tracking of vaccine proteins. Transfected muscle fibres in mice were visualized, and their relationship to infiltrating mononuclear cells could be determined. Interestingly, muscle fibers that produced MHC class II-specific dimeric vaccine proteins with mCherry were for weeks surrounded by a localized intense cellular infiltrate composed of CD45+, MHC class II+ and CD11b+ cells. Increasing numbers of eosinophils were observed among the infiltrating cells from day 7 after immunization. The local infiltrate surrounding mCherry+ muscle fibers was dependent on the MHC II-specificity of the vaccine proteins since the control, a non-targeted vaccine protein, failed to induce similar infiltrates. Chemokines measured on day 3 in immunized muscle indicate both a DNA effect and an electroporation effect. No influence of targeting was observed. These results contribute to our understanding for why targeted DNA vaccines have an improved immunogenicity.

  10. MHC class II diversity of koala (Phascolarctos cinereus) populations across their range.

    PubMed

    Lau, Q; Jaratlerdsiri, W; Griffith, J E; Gongora, J; Higgins, D P

    2014-10-01

    Major histocompatibility complex class II (MHCII) genes code for proteins that bind and present antigenic peptides and trigger the adaptive immune response. We present a broad geographical study of MHCII DA β1 (DAB) and DB β1 (DBB) variants of the koala (Phascolarctos cinereus; n=191) from 12 populations across eastern Australia, with a total of 13 DAB and 7 DBB variants found. We identified greater MHCII variation and, possibly, additional gene copies in koala populations in the north (Queensland and New South Wales) relative to the south (Victoria), confirmed by STRUCTURE analyses and genetic differentiation using analysis of molecular variance. The higher MHCII diversity in the north relative to south could potentially be attributed to (i) significant founder effect in Victorian populations linked to historical translocation of bottlenecked koala populations and (ii) increased pathogen-driven balancing selection and/or local genetic drift in the north. Low MHCII genetic diversity in koalas from the south could reduce their potential response to disease, although the three DAB variants found in the south had substantial sequence divergence between variants. This study assessing MHCII diversity in the koala with historical translocations in some populations contributes to understanding the effects of population translocations on functional genetic diversity.

  11. Btn2a2, a T cell immunomodulatory molecule coregulated with MHC class II genes

    PubMed Central

    Sarter, Kerstin; Leimgruber, Elisa; Gobet, Florian; Agrawal, Vishal; Dunand-Sauthier, Isabelle; Barras, Emmanuèle; Mastelic-Gavillet, Béatris; Kamath, Arun; Fontannaz, Paola; Guéry, Leslie; Duraes, Fernanda do Valle; Lippens, Carla; Ravn, Ulla; Santiago-Raber, Marie-Laure; Magistrelli, Giovanni; Fischer, Nicolas; Siegrist, Claire-Anne; Hugues, Stéphanie

    2016-01-01

    Evidence has recently emerged that butyrophilins, which are members of the extended B7 family of co-stimulatory molecules, have diverse functions in the immune system. We found that the human and mouse genes encoding butyrophilin-2A2 (BTN2A2) are regulated by the class II trans-activator and regulatory factor X, two transcription factors dedicated to major histocompatibility complex class II expression, suggesting a role in T cell immunity. To address this, we generated Btn2a2-deficient mice. Btn2a2−/− mice exhibited enhanced effector CD4+ and CD8+ T cell responses, impaired CD4+ regulatory T cell induction, potentiated antitumor responses, and exacerbated experimental autoimmune encephalomyelitis. Altered immune responses were attributed to Btn2a2 deficiency in antigen-presenting cells rather than T cells or nonhematopoietic cells. These results provide the first genetic evidence that BTN2A2 is a co-inhibitory molecule that modulates T cell–mediated immunity. PMID:26809444

  12. MHC class II diversity of koala (Phascolarctos cinereus) populations across their range

    PubMed Central

    Lau, Q; Jaratlerdsiri, W; Griffith, J E; Gongora, J; Higgins, D P

    2014-01-01

    Major histocompatibility complex class II (MHCII) genes code for proteins that bind and present antigenic peptides and trigger the adaptive immune response. We present a broad geographical study of MHCII DA β1 (DAB) and DB β1 (DBB) variants of the koala (Phascolarctos cinereus; n=191) from 12 populations across eastern Australia, with a total of 13 DAB and 7 DBB variants found. We identified greater MHCII variation and, possibly, additional gene copies in koala populations in the north (Queensland and New South Wales) relative to the south (Victoria), confirmed by STRUCTURE analyses and genetic differentiation using analysis of molecular variance. The higher MHCII diversity in the north relative to south could potentially be attributed to (i) significant founder effect in Victorian populations linked to historical translocation of bottlenecked koala populations and (ii) increased pathogen-driven balancing selection and/or local genetic drift in the north. Low MHCII genetic diversity in koalas from the south could reduce their potential response to disease, although the three DAB variants found in the south had substantial sequence divergence between variants. This study assessing MHCII diversity in the koala with historical translocations in some populations contributes to understanding the effects of population translocations on functional genetic diversity. PMID:24690756

  13. Characterization of MHC class II B polymorphism in multiple populations of wild gorillas using non-invasive samples and next-generation sequencing.

    PubMed

    Hans, Jörg B; Haubner, Anne; Arandjelovic, Mimi; Bergl, Richard A; Fünfstück, Tillmann; Gray, Maryke; Morgan, David B; Robbins, Martha M; Sanz, Crickette; Vigilant, Linda

    2015-11-01

    Genes encoded by the major histocompatibility complex (MHC) are crucial for the recognition and presentation of antigens to the immune system. In contrast to their closest relatives, chimpanzees and humans, much less is known about variation in gorillas at these loci. This study explored the exon 2 variation of -DPB1, -DQB1, and -DRB genes in 46 gorillas from four populations while simultaneously evaluating the feasibility of using fecal samples for high-throughput MHC genotyping. By applying strict similarity- and frequency-based analysis, we found, despite our modest sample size, a total of 18 alleles that have not been described previously, thereby illustrating the potential for efficient and highly accurate MHC genotyping from non-invasive DNA samples. We emphasize the importance of controlling for multiple potential sources of error when applying this massively parallel short-read sequencing technology to PCR products generated from low concentration DNA extracts. We observed pronounced differences in MHC variation between species, subspecies and populations that are consistent with both the ancient and recent demographic histories experienced by gorillas.

  14. Crystal structure of the human CD4 N-terminal two-domain fragment complexed to a class II MHC molecule.

    SciTech Connect

    Wang, J.-H.; Meijers, R.; Xiong, Y.; Liu, J.-H.; Sakihama, T.; Zhang, R.-G.; Joachimiak, A.; Reinherz, E. L.; Biosciences Division; Dana-Farber Cancer Inst.; Harvard Medical School

    2001-09-11

    The structural basis of the interaction between the CD4 coreceptor and a class II major histocompatibility complex (MHC) is described. The crystal structure of a complex containing the human CD4 N-terminal two-domain fragment and the murine I-A{sup k }class II MHC molecule with associated peptide (pMHCII) shows that only the 'top corner' of the CD4 molecule directly contacts pMHCII. The CD4 Phe-43 side chain extends into a hydrophobic concavity formed by MHC residues from both {alpha}2 and {beta}2 domains. A ternary model of the CD4-pMHCII-T-cell receptor (TCR) reveals that the complex appears V-shaped with the membrane-proximal pMHCII at the apex. This configuration excludes a direct TCR-CD4 interaction and suggests how TCR and CD4 signaling is coordinated around the antigenic pMHCII complex. Human CD4 binds to HIV gp120 in a manner strikingly similar to the way in which CD4 interacts with pMHCII. Additional contacts between gp120 and CD4 give the CD4-gp120 complex a greater affinity. Thus, ligation of the viral envelope glycoprotein to CD4 occludes the pMHCII-binding site on CD4, contributing to immunodeficiency.

  15. Association of major histocompatibility complex II with cholesterol- and sphingolipid-rich membranes precedes peptide loading.

    PubMed

    Karacsonyi, Claudia; Knorr, Ruth; Fülbier, Angela; Lindner, Robert

    2004-08-13

    Major histocompatibility complex class II protein (MHC II) molecules present antigenic peptides to CD4-positive T-cells. Efficient T cell stimulation requires association of MHC II with membrane microdomains organized by cholesterol and glycosphingolipids or by tetraspanins. Using detergent extraction at 37 degrees C combined with a modified flotation assay, we investigated the sequence of events leading to the association of MHC II with cholesterol- and glycosphingolipid-rich membranes (DRMs) that are distinct from tetraspanins. We find two stages of association of MHC II with DRMs. In stage one, complexes of MHC II and invariant chain, a chaperone involved in MHC II transport, enter DRMs in the Golgi stack. In early endosomes, these complexes are almost quantitatively associated with DRMs. Upon transport to late endocytic compartments, MHC II-bound invariant chain is stepwise proteolyzed to the MHC class II-associated invariant chain peptide (CLIP) that remains MHC II-bound and retains a preference for DRMs. At the transition between the two stages, CLIP is exchanged against processed antigens, and the resulting MHC II-peptide complexes are transported to the cell surface. In the second stage, MHC II shows a lower overall association with DRMs. However, surface MHC II molecules occupied with peptides that induce resistance to denaturation by SDS are enriched in DRMs relative to SDS-sensitive MHC II-peptide complexes. Likewise, MHC II molecules loaded with long-lived processing products of hen-egg lysozyme containing the immunodominant epitope 48-61 show a very high preference for DRMs. Thus after an initial mainly intracellular stage of high DRM association, MHC II moves to a second stage in which its preference for DRMs is modulated by bound peptides.

  16. Toward a Network Model of MHC Class II-Restricted Antigen Processing

    PubMed Central

    Miller, Michael A.; Ganesan, Asha Purnima V.; Eisenlohr, Laurence C.

    2013-01-01

    The standard model of Major Histocompatibility Complex class II (MHCII)-restricted antigen processing depicts a straightforward, linear pathway: internalized antigens are converted into peptides that load in a chaperone dependent manner onto nascent MHCII in the late endosome, the complexes subsequently trafficking to the cell surface for recognition by CD4+ T cells (TCD4+). Several variations on this theme, both moderate and radical, have come to light but these alternatives have remained peripheral, the conventional pathway generally presumed to be the primary driver of TCD4+ responses. Here we continue to press for the conceptual repositioning of these alternatives toward the center while proposing that MHCII processing be thought of less in terms of discrete pathways and more in terms of a network whose major and minor conduits are variable depending upon many factors, including the epitope, the nature of the antigen, the source of the antigen, and the identity of the antigen-presenting cell. PMID:24379819

  17. Linkage relationships and haplotype polymorphism among cichlid Mhc class II B loci.

    PubMed Central

    Málaga-Trillo, E; Zaleska-Rutczynska, Z; McAndrew, B; Vincek, V; Figueroa, F; Sültmann, H; Klein, J

    1998-01-01

    The species flocks of cichlid fishes in the Great East African Lakes are paradigms of adaptive radiation and hence, of great interest to evolutionary biologists. Phylogenetic studies of these fishes have, however, been hampered by the lack of suitable polymorphic markers. The genes of the major histocompatibility complex hold the promise to provide, through their extensive polymorphism, a large number of such markers, but their use has been hampered by the complexity of the genetic system and the lack of definition of the individual loci. In this study we take the first substantial step to alleviate this problem. Using a combination of methods, including the typing of single sperm cells, gyno- or androgenetic individuals, and haploid embryos, as well as sequencing of class II B restriction fragments isolated from gels for Southern blots, we identify the previously characterized homology groups as distinct loci. At least 17 polymorphic class II B loci, all of which are presumably transcribed, have been found among the different species studied. Most of these loci are shared across the various cichlid species and genera. The number of loci per haplotype varies from individual to individual, ranging from 1 to 13. A total of 21 distinct haplotypes differing in the number of loci they carry has thus far been identified. All the polymorphic loci are part of the same cluster in which, however, distances between at least some of the loci (as indicated by recombination frequencies) are relatively large. Both the individual loci and the haplotypes can now be used to study phylogenetic relationships among the members of the species flocks and the mode in which speciation occurs during adaptive radiation. PMID:9649539

  18. MHC class I and class II genes in Tunisian patients with reactive and undifferentiated arthritis.

    PubMed

    Siala, M; Mahfoudh, N; Fourati, H; Gdoura, R; Younes, M; Kammoun, A; Chour, I; Meddeb, N; Gaddour, L; Hakim, F; Baklouti, S; Bargaoui, N; Sellami, S; Hammami, A; Makni, H

    2009-01-01

    To study HLA class I and class II association in Tunisian patients with reactive (ReA) and undifferentiated arthritis (UA). The study included 17 patients with ReA defined according to the European Spondylarthropathy Study Group criteria for spondylarthropathy (SpA), 11 patients classified as having undifferentiated arthritis and 100 unrelated healthy controls. HLA class I antigens were typed serologically and HLA class II alleles were genotyped molecularly by the polymerase chain reaction with sequence-specific primers technique. There was a major difference between HLA alleles in ReA and UA patients when compared separately with controls. Increased frequencies of HLA-B27 (p=7.76 10-12, OR=59.30), HLA-B51 (p=0.015, OR=4.91) and HLA-DRB1*04 (p=0.033, OR=2.90) alleles were found in patients with ReA but not in patients with UA. HLA-B27 was not expressed totally in our cohort of UA patients. A significant increase of HLA-B15 (p=0.002, OR=18.40) and a moderate increase of HLA-B7 (p=0.043, OR=5.15) was found in patients with UA, but not in patients with ReA. In the B27 negative patients, HLA-DRB1*04 association with ReA was found independently of B27. Our data confirmed a significant association of HLA-B27 with ReA in the Tunisian population. Our results also suggested that some of the additional HLA antigens were associated with ReA including HLA-B51 and HLA-DRB1*04 alleles. UA seemed to have a genetic background different from ReA in Tunisian patients.

  19. MHC expression and chemokine production in the murine vagina following intra-vaginal administration of ligands to toll-like receptors 3, 7 and 9.

    PubMed

    Nurkkala, Merja; Nordström, Inger; Telemo, Esbjörn; Eriksson, Kristina

    2007-04-01

    The expression of MHC class I, MHC class II and the chemokines IP-10, MIP-1alpha, RANTES, fractalkine and I-TAC has been analyzed after intra-vaginal treatment with three synthetic toll-like receptors (TLR) agonists-double-stranded RNA (poly I:C), imiquimod and CpG-rich oligonucleotides (CpG-ODN). These compounds act mainly through TLR3, TLR7 and TLR9, respectively. CpG-ODN induced an accumulation of leucocytes in the vagina, and a strong up-regulation of MHC class I expression on both leucocytes and epithelial cells. Imiquimod and poly I:C induced a weak MHC class I up-regulation in the epithelium but not in the lamina propria. Neither treatment had any profound effect on expression of MHC class II on epithelial cells but poly I:C and to a lesser extent CpG-ODN, up-regulated MHC class II staining intensity which, in the case of CpG-ODN, treatment, was associated with a strong accumulation of CD11c-positive dendritic cells. All three treatments induced an early (8h) but transient IP-10 response. Imiquimod and CpG-ODN, but not poly I:C induced an early MIP-1alpha response which remained for at least 7 days in CpG-ODN-treated animals but not in imiquimod-treated mice. Poly I:C and CpG-ODN, but not imiquimod, induced significant levels of RANTES at different time-points post-treatment. None of the treatments induced any significant changes in the levels of fractalkine, I-TAC or IFN-alpha. These studies have implications for the manipulation of the genital immune response and also improving the outcome of vaginal immunotherapy.

  20. Comprehensive analysis of MHC class II genes in teleost fish genomes reveals dispensability of the peptide-loading DM system in a large part of vertebrates

    PubMed Central

    2013-01-01

    Background Classical major histocompatibility complex (MHC) class II molecules play an essential role in presenting peptide antigens to CD4+ T lymphocytes in the acquired immune system. The non-classical class II DM molecule, HLA-DM in the case of humans, possesses critical function in assisting the classical MHC class II molecules for proper peptide loading and is highly conserved in tetrapod species. Although the absence of DM-like genes in teleost fish has been speculated based on the results of homology searches, it has not been definitively clear whether the DM system is truly specific for tetrapods or not. To obtain a clear answer, we comprehensively searched class II genes in representative teleost fish genomes and analyzed those genes regarding the critical functional features required for the DM system. Results We discovered a novel ancient class II group (DE) in teleost fish and classified teleost fish class II genes into three major groups (DA, DB and DE). Based on several criteria, we investigated the classical/non-classical nature of various class II genes and showed that only one of three groups (DA) exhibits classical-type characteristics. Analyses of predicted class II molecules revealed that the critical tryptophan residue required for a classical class II molecule in the DM system could be found only in some non-classical but not in classical-type class II molecules of teleost fish. Conclusions Teleost fish, a major group of vertebrates, do not possess the DM system for the classical class II peptide-loading and this sophisticated system has specially evolved in the tetrapod lineage. PMID:24279922

  1. Association of SNP variants of MHC Class II DRB gene with thermo-physiological traits in tropical goats.

    PubMed

    Yakubu, Abdulmojeed; Salako, Adebowale E; De Donato, Marcos; Peters, Sunday O; Takeet, Michael I; Wheto, Mathew; Okpeku, Moses; Imumorin, Ikhide G

    2017-02-01

    Host defense in vertebrates depend on many secreted regulatory proteins such as major histocompatibility complex (MHC) class II which provide important regulatory and effector functions of T cells. Gene polymorphism in the second exon of Capra-DRB gene in three major Nigerian goat breeds [West African Dwarf (WAD), Red Sokoto (RS), and Sahel (SH)] was analyzed by restriction fragment length polymorphisms (RFLP). Four restriction enzymes, BsaHI, AluI, HaeIII, and SacII, were utilized. The association between the polymorphic sites and some heat tolerance traits were also investigated in a total of 70 WAD, 90 RS, and 50 SH goats. Fourteen different types of alleles identified in the Nigerian goats, four of which were found in the peptide coding region (A57G, Q89R, G104D, and T112I), indicate a high degree of polymorphism at the DRB locus in this species. An obvious excess (P < 0.01) of non-synonymous substitutions than synonymous (dN/dS) in this locus is a reflection of adaptive evolution and positive selection. The phylogenetic trees revealed largely species-wise clustering in DRB gene. BsaHI, AluI, HaeIII, and SacII genotype frequencies were in Hardy-Weinberg equilibrium (P > 0.05), except AluI in RS goats and HaeIII in WAD goats (P < 0.05). The expected heterozygosity (H), which is a measure of gene diversity in the goat populations, ranged from 0.16 to 0.50. Genotypes AA (BsaHI), GG, GC and CC (AluI) and GG, GA, AA (HaeIII) appeared better in terms of heat tolerance. The heat-tolerant ability of SH and RS goats to the hot and humid tropical environment of Nigeria seemed better than that of the WAD goats. Sex effect (P < 0.05) was mainly on pulse rate and heat stress index, while there were varying interaction effects on heat tolerance. Variation at the DRB locus may prove to be important in possible selection and breeding for genetic resistance to heat stress in the tropics.

  2. Allergen-specific MHC class II tetramer+ cells are detectable in allergic, but not in nonallergic, individuals.

    PubMed

    Macaubas, Claudia; Wahlstrom, Jan; Galvão da Silva, Ana Paula; Forsthuber, Thomas G; Sønderstrup, Grete; Kwok, William W; DeKruyff, Rosemarie H; Umetsu, Dale T

    2006-04-15

    Allergen-specific cells are present in very low frequency in peripheral blood of humans, and differ in function in allergic and nonallergic individuals. We report in this study that soluble class II MHC tetramers can be used to directly identify and study such allergen epitope-specific CD4+ T cells in humans. We identified the major antigenic epitope of rye grass allergen Lol p 1 in HLA-DRB1*0401 individuals using HLA-DR*0401 transgenic mice and peripheral blood cells from HLA-DR*0401 individuals. Using DRB1*0401 tetramers loaded with this major epitope of Lol p 1, we detected allergen-specific CD4+ T cells in the peripheral blood of DRB1*0401 rye grass allergic individuals after ex vivo expansion with allergen. These tetramer-positive cells produced IL-4, but little IFN-gamma. In contrast, we were unable to detect rye grass tetramer-positive cells in cultures from HLA-DR*0401 nonallergic individuals, even after expansion with IL-2. Thus, our results suggest that rye grass allergen-specific T cells in DR*0401 nonallergic subjects are present at very low levels (e.g., because of deletion or suppression), differ in a fundamental way in their requirement for ex vivo expansion (e.g., they may be anergic), or use TCRs distinct from those of allergic individuals. Thus, analysis using DRB1*0401 tetramers loaded with a major epitope of Lol p 1 indicates that allergen-specific CD4+ T cells in nonallergic individuals are distinct from those in allergic subjects.

  3. Endosomally stored MHC class II does not contribute to antigen presentation by dendritic cells at inflammatory conditions.

    PubMed

    ten Broeke, Toine; van Niel, Guillaume; Wauben, Marca H M; Wubbolts, Richard; Stoorvogel, Willem

    2011-08-01

    Major histocompatibility complex (MHC) class II (MHCII) is constitutively expressed by immature dendritic cells (DC), but has a short half-life as a consequence of its transport to and degradation in lysosomes. For its transfer to lysosomes, MHCII is actively sorted to the intraluminal vesicles (ILV) of multivesicular bodies (MVB), a process driven by its ubiquitination. ILV have, besides their role as an intermediate compartment in lysosomal transfer, also been proposed to function as a site for MHCII antigen loading and temporal storage. In that scenario, DC would recruit antigen-loaded MHCII to the cell surface in response to a maturation stimulus by allowing ILV to fuse back with the MVB delimiting membrane. Other studies, however, explained the increase in cell surface expression during DC maturation by transient upregulation of MHCII synthesis and reduced sorting of newly synthesized MHCII to lysosomes. Here, we have characterized the relative contributions from the biosynthetic and endocytic pathways and found that the vast majority of antigen-loaded MHCII that is stably expressed at the plasma membrane by mature DC is synthesized after exposure to inflammatory stimuli. Pre-existing endosomal MHCII contributed only when it was not yet sorted to ILV at the moment of DC activation. Together with previous records, our current data are consistent with a model in which passage of MHCII through ILV is not required for antigen loading in maturing DC and in which sorting to ILV in immature DC provides a one-way ticket for lysosomal degradation. © 2011 John Wiley & Sons A/S.

  4. LAMP-2C Inhibits MHC Class II Presentation of Cytoplasmic Antigens by Disrupting Chaperone-Mediated Autophagy.

    PubMed

    Pérez, Liliana; McLetchie, Shawna; Gardiner, Gail J; Deffit, Sarah N; Zhou, Delu; Blum, Janice S

    2016-03-15

    Cells use multiple autophagy pathways to sequester macromolecules, senescent organelles, and pathogens. Several conserved isoforms of the lysosome-associated membrane protein-2 (LAMP-2) regulate these pathways influencing immune recognition and responses. LAMP-2A is required for chaperone-mediated autophagy (CMA), which promotes Ag capture and MHC class II (MHCII) presentation in B cells and signaling in T cells. LAMP-2B regulates lysosome maturation to impact macroautophagy and phagocytosis. Yet, far less is known about LAMP-2C function. Whereas LAMP2A and LAMP2B mRNA were broadly detected in human tissues, LAMP2C expression was more limited. Transcripts for the three LAMP2 isoforms increased with B cell activation, although specific gene induction varied depending on TLR versus BCR engagement. To examine LAMP-2C function in human B cells and specifically its role in Ag presentation, we used ectopic gene expression. Increased LAMP-2C expression in B cells did not alter MHCII expression or invariant chain processing, but did perturb cytoplasmic Ag presentation via CMA. MHCII presentation of epitopes from exogenous and membrane Ags was not affected by LAMP-2C expression in B cells. Similarly, changes in B cell LAMP-2C expression did not impact macroautophagy. The gene expression of other LAMP2 isoforms and proteasome and lysosomal proteases activities were unperturbed by LAMP-2C ectopic expression. LAMP-2C levels modulated the steady-state expression of several cytoplasmic proteins that are targeted for degradation by CMA and diminished peptide translocation via this pathway. Thus, LAMP-2C serves as a natural inhibitor of CMA that can selectively skew MHCII presentation of cytoplasmic Ags.

  5. Up-regulated MHC-class II expression and gamma-IFN and soluble IL-2R in lupus nephritis.

    PubMed

    Yokoyama, H; Takabatake, T; Takaeda, M; Wada, T; Naito, T; Ikeda, K; Goshima, S; Takasawa, K; Tomosugi, N; Kobayashi, K

    1992-09-01

    Expression of MHC-class II molecules (HLA-DR and -DQ), serum gamma-interferon (gamma-IFN) and soluble interleukin-2 receptor (sIL-2R) levels were studied in 35 Japanese patients with lupus nephritis (LN) to clarify intraglomerular cellular activation and cytokine involvement in human LN. In 11 normal kidney specimens, HLA-DR(Ia1) was noted in glomerular tufts, but HLA-DQ was either not or was faintly detected in glomeruli by the indirect immunofluorescence technique. HLA-DR and -DQ were observed mainly on the surface of glomerular endothelial cells in 100% and 50% of 28 lupus kidney specimens except for necrotic or sclerotic lesions. HLA-DQ was expressed in a high incidence of 67%, 86% in patients with proliferative LN (WHO Class III-IV) and active lesions, respectively. Serum gamma-IFN and sIL-2R levels were 1.2 +/- 0.2 U/ml and 190 +/- 24 U/ml (mean +/- SEM; N = 30) in normal controls, and elevated in patients with proliferative LN (4.1 +/- 1.0 U/ml, 383 +/- 81 U/ml, N = 25), especially with active lesions (6.2 +/- 1.5 U/ml, 500 +/- 110 U/ml, N = 14). Overall, glomerular lesions such as HLA-DQ expression, the activity index and leukocyte infiltration correlated positively with serum gamma-IFN levels (r = 0.55; P less than 0.01 for HLA-DQ, r = 0.68; P less than 0.001 for activity index, r = 0.38; P less than 0.05 for leukocyte infiltration), but not with serum sIL-2R levels, anti-DNA antibody titers and CH50 titers.(ABSTRACT TRUNCATED AT 250 WORDS)

  6. Both IFN-γ and IL-4 Induce MHC Class II Expression at the Surface of Mouse Pro-B Cells

    PubMed Central

    Lombard-Platet, Suzanne; Meyer, Valerie

    1997-01-01

    Pro-B cells are early B-cell progenitors that retain macrophage potential. We have studied MHC class II molecules and invariant chain inducibility on four class II negative mouse pro- B-cell clones. We analyzed the effects of IL-4 and IFN-γ, which represent the major inducers of class II in the B-lymphoid and monocytic/macrophage lineages, respectively. After 48 h of treatment with either cytokine, three pro-B-cell clones (C2.13, A1.5, and F2.2) expressed intracellular invariant chain and cell-surface class II molecules. One clone (D2.1) remained negative. As already reported, more differentiated 70Z/3 pre-B cells were inducible by IL-4 only. These data suggest that the induction of class II and invariant-chain genes are subject to regulation throughout B-cell differentiation. PMID:9587711

  7. Functional expression of a cattle MHC class II DR-like antigen on mouse L cells

    SciTech Connect

    Fraser, D.C.; Craigmile, S.; Campbell, J.D.M.

    1996-09-01

    Cattle DRA and DRB genes, cloned by reverse-transcription polymerase chain reaction, were transfected into mouse L cells. The cattle DR-expressing L-cell transfectant generated was analyzed serologically, biochemically, and functionally. Sequence analysis of the transfected DRB gene clearly showed showed that it was DRB3 allele DRB3*0101, which corresponds to the 1D-IEF-determined allele DRBF3. 1D-IEF analysis of the tranfectant confirmed that the expressed DR product was DRBF3. Functional integrity of the transfected gene products was demonstrated by the ability of the transfectant cell line to present two antigens (the foot-and-mouth disease virus-derived peptide FMDV15, and ovalbumin) to antigen-specific CD4{sup +} T cells from both the original animal used to obtain the genes, and also from an unrelated DRBF3{sup +} heterozygous animal. Such transfectants will be invaluable tools, allowing us to dissect the precise contributions each locus product makes to the overall immune response in heterozygous animals, information essential for rational vaccine design. 45 refs., 5 figs., 1 tab.

  8. MHC-mismatched mixed chimerism augments thymic regulatory T-cell production and prevents relapse of EAE in mice

    PubMed Central

    Wu, Limin; Li, Nainong; Zhang, Mingfeng; Xue, Sheng-Li; Cassady, Kaniel; Lin, Qing; Riggs, Arthur D.; Zeng, Defu

    2015-01-01

    Multiple sclerosis (MS) is an autoimmune inflammatory disease of the central nervous system with demyelination, axon damage, and paralysis. Induction of mixed chimerism with allogeneic donors has been shown to not cause graft-versus-host disease (GVHD) in animal models and humans. We have reported that induction of MHC-mismatched mixed chimerism can cure autoimmunity in autoimmune NOD mice, but this approach has not yet been tested in animal models of MS, such as experimental autoimmune encephalomyelitis (EAE). Here, we report that MHC-mismatched mixed chimerism with C57BL/6 (H-2b) donor in SJL/J (H-2s) EAE recipients eliminates clinical symptoms and prevents relapse. This cure is demonstrated by not only disappearance of clinical signs but also reversal of autoimmunity; elimination of infiltrating T, B, and macrophage cells in the spinal cord; and regeneration of myelin sheath. The reversal of autoimmunity is associated with a marked reduction of autoreactivity of CD4+ T cells and significant increase in the percentage of Foxp3+ Treg among host-type CD4+ T cells in the spleen and lymph nodes. The latter is associated with a marked reduction of the percentage of host-type CD4+CD8+ thymocytes and an increase of Treg percentage among the CD4+CD8+ and CD4+CD8− thymocytes. Thymectomy leads to loss of prevention of EAE relapse by induction of mixed chimerism, although there is a dramatic expansion of host-type Treg cells in the lymph nodes. These results indicate that induction of MHC-mismatched mixed chimerism can restore thymic negative selection of autoreactive CD4+ T cells, augment production of Foxp3+ Treg, and cure EAE. PMID:26647186

  9. Design of a predicted MHC restricted short peptide immunodiagnostic and vaccine candidate for Fowl adenovirus C in chicken infection.

    PubMed

    Valdivia-Olarte, Hugo; Requena, David; Ramirez, Manuel; Saravia, Luis E; Izquierdo, Ray; Falconi-Agapito, Francesca; Zavaleta, Milagros; Best, Iván; Fernández-Díaz, Manolo; Zimic, Mirko

    2015-01-01

    Fowl adenoviruses (FAdVs) are the ethiologic agents of multiple pathologies in chicken. There are five different species of FAdVs grouped as FAdV-A, FAdV-B, FAdV-C, FAdV-D, and FAdV-E. It is of interest to develop immunodiagnostics and vaccine candidate for Peruvian FAdV-C in chicken infection using MHC restricted short peptide candidates. We sequenced the complete genome of one FAdV strain isolated from a chicken of a local farm. A total of 44 protein coding genes were identified in each genome. We sequenced twelve Cobb chicken MHC alleles from animals of different farms in the central coast of Peru, and subsequently determined three optimal human MHC-I and four optimal human MHC-II substitute alleles for MHC-peptide prediction. The potential MHC restricted short peptide epitope-like candidates were predicted using human specific (with determined suitable chicken substitutes) NetMHC MHC-peptide prediction model with web server features from all the FAdV genomes available. FAdV specific peptides with calculated binding values to known substituted chicken MHC-I and MHC-II were further filtered for diagnostics and potential vaccine epitopes. Promiscuity to the 3/4 optimal human MHC-I/II alleles and conservation among the available FAdV genomes was considered in this analysis. The localization on the surface of the protein was considered for class II predicted peptides. Thus, a set of class I and class II specific peptides from FAdV were reported in this study. Hence, a multiepitopic protein was built with these peptides, and subsequently tested to confirm the production of specific antibodies in chicken.

  10. Design of a predicted MHC restricted short peptide immunodiagnostic and vaccine candidate for Fowl adenovirus C in chicken infection

    PubMed Central

    Valdivia-Olarte, Hugo; Requena, David; Ramirez, Manuel; Saravia, Luis E; Izquierdo, Ray; Falconi-Agapito, Francesca; Zavaleta, Milagros; Best, Iván; Fernández-Díaz, Manolo; Zimic, Mirko

    2015-01-01

    Fowl adenoviruses (FAdVs) are the ethiologic agents of multiple pathologies in chicken. There are five different species of FAdVs grouped as FAdV-A, FAdV-B, FAdV-C, FAdV-D, and FAdV-E. It is of interest to develop immunodiagnostics and vaccine candidate for Peruvian FAdV-C in chicken infection using MHC restricted short peptide candidates. We sequenced the complete genome of one FAdV strain isolated from a chicken of a local farm. A total of 44 protein coding genes were identified in each genome. We sequenced twelve Cobb chicken MHC alleles from animals of different farms in the central coast of Peru, and subsequently determined three optimal human MHC-I and four optimal human MHC-II substitute alleles for MHC-peptide prediction. The potential MHC restricted short peptide epitope-like candidates were predicted using human specific (with determined suitable chicken substitutes) NetMHC MHC-peptide prediction model with web server features from all the FAdV genomes available. FAdV specific peptides with calculated binding values to known substituted chicken MHC-I and MHC-II were further filtered for diagnostics and potential vaccine epitopes. Promiscuity to the 3/4 optimal human MHC-I/II alleles and conservation among the available FAdV genomes was considered in this analysis. The localization on the surface of the protein was considered for class II predicted peptides. Thus, a set of class I and class II specific peptides from FAdV were reported in this study. Hence, a multiepitopic protein was built with these peptides, and subsequently tested to confirm the production of specific antibodies in chicken. PMID:26664030

  11. Expression of various MHC class II molecules and of intracellular adhesion molecule-1 (ICAM-1) on focal clusters of dendritic cells in iodine deficiency goitres

    PubMed Central

    Dimal, P.; Wilders-Truschnig, M.; Mooij, P.; Leb, G.; Eber, O.; Langsteger, W.; Hebenstreit, J.; Beham, A.; Stiegler, C.; Dohr, G.; Drexhage, H. A.

    1993-01-01

    Thyroid sections from 18 consecutive euthyroid patients undergoing surgery for iodine deficiency goitre were investigated by means of immunohistochemistry and immunofluorescence, evaluating the expression of MHC class II antigens (HLA-DR, -DP, -DQ, and RFD1) and intercellular adhesion molecule-1 on the formerly described clusters of dendritic cells, as well as on thyrocytes. Eleven of 18 iodine deficiency goitres contained clusters of dendritic cells. These clusters appeared to express only HLA-DR in two cases; in nine of 12 cases they showed a differential expression of class II molecules in the following frequency: HLA-DR>DQ and/or -DP>RFD1. These dendritic cells also were ICAM-1+. In four of 18 iodine deficiency goitres, thyroid epithelial cells showed MHC class II expression in several combinations, but were ICAM-1-. In normal thyroids and in nodular goitres from inhabitants of the endemic area not having an actual iodine deficiency, only sparse clusters of dendritic cells were found; these cells were only HLA-DR+. Follicle lining cells were negative for the MHC class II molecules. In normal thyroids from an area with sufficient iodine supply, no clusters of dendritic cells were seen. The few dendritic cells observed were lying isolated in the interstitium and only positive for HLA-DR and ICAM-1; epithelial cells were negative for the studied markers. These data show clusters of dendritic cells in thyroids of inhabitants of an endemic area. When goitre is accompanied by iodine deficiency at the moment of operation, there appears to be activation of these dendritic cells and of thyroid epithelial cells. ImagesFig. 1Fig. 2 PMID:8099855

  12. Processing and MHC class II presentation of exogenous soluble antigen involving a proteasome-dependent cytosolic pathway in CD40-activated B cells.

    PubMed

    Becker, Hans Jiro; Kondo, Eisei; Shimabukuro-Vornhagen, Alexander; Theurich, Sebastian; von Bergwelt-Baildon, Michael S

    2016-08-01

    Activated B cells have the capacity to present antigen and induce immune responses as potent antigen-presenting cells (APCs). As in other APCs, antigen presentation by B cells involves antigen internalization, antigen processing, and peptide loading onto MHC molecules. However, while the mechanism of antigen processing has been studied extensively in other APCs, this pathway remains elusive in B cells. The aim of this study was to investigate the MHC class II processing pathway in CD40-activated B cells (CD40Bs), as a model for activated, antigen-presenting B cells. Using CMV pp65 as a model antigen, we evaluated processing and presentation of the CD4 + T-cell epitope 509-523 (K509) by human CD40Bs in ELISPOT assays. As expected, stimulation of specific CD4 + T-cell clones was attenuated after pretreatment of CD40Bs with inhibitors of classic class II pathway components. However, proteasome inhibitors such as epoxomicin limited antigen presentation as well. This suggests that the antigen is processed in a non-classical, cytosolic MHC class II pathway. Further experiments with truncated protein variants revealed involvement of the proteasome in processing of the N and C extensions of the epitope. Access to the cytosol was shown to be size dependent. Epoxomicin sensitivity exclusively in CD40B cells, but not in dendritic cells, suggests a novel processing mechanism unique to this APC. Our data suggest that B cells process antigen using a distinct, non-classical class II pathway.

  13. Anti-TNF monoclonal antibodies prevent haemorrhage-induced suppression of Kupffer cell antigen presentation and MHC class II antigen expression.

    PubMed Central

    Ertel, W; Morrison, M H; Ayala, A; Perrin, M M; Chaudry, I H

    1991-01-01

    Kupffer cells (KC), by virtue of their ability to present antigen (AP) and express major histocompatibility complex (MHC) class II antigen (Ia), play a pivotal role in the host defence system against invading micro-organisms. Although haemorrhagic shock depresses the above KC functions, it is not known whether increased KC tumour necrosis factor (TNF) production and elevated TNF plasma levels following haemorrhage are responsible for it. To study this, C3H/HeN mice were pretreated intraperitoneally with either anti-murine TNF antibody (anti-TNF Ab) or saline. Twenty hours later mice were bled and maintained at a mean blood pressure of 35 mmHg for 60 min followed by adequate fluid resuscitation. Two and 24 hr later, plasma was collected and KC were isolated. AP was measured by co-culturing KC with the D10.G4.1 Th cell clone. Ia expression was determined by direct immunofluorescence. Interleukin (IL)-1, IL-6 and TNF levels in KC supernatants and plasma were measured with bioassays or ELISA. Haemorrhage increased circulating TNF levels by 215% at 2 hr and by 76% at 24 hr (P less than 0.05), which was prevented by pretreatment with anti-TNF Ab. Haemorrhage-induced increase of circulating IL-6 was abolished (P less than 0.05) at 2 hr but not at 24 hr in the anti-TNF Ab group. The suppression of KC AP (P less than 0.05) and Ia expression (P less than 0.05) due to haemorrhage was attenuated (P less than 0.05) in anti-TNF Ab-treated mice at 2 and 24 hr and KC IL-1 and TNF synthesis was further (P less than 0.01) increased. These results indicate that TNF plays a critical role in the initiation and regulation of KC AP, Ia expression, and cytokine production following haemorrhage. PMID:1748476

  14. Duplication, balancing selection and trans-species evolution explain the high levels of polymorphism of the DQA MHC class II gene in voles (Arvicolinae).

    PubMed

    Bryja, J; Galan, M; Charbonnel, N; Cosson, J F

    2006-04-01

    Major histocompatibility complex (MHC) genes play important role in host-parasite interactions and parasites are crucial factors influencing the population dynamics of hosts. We described the structure and diversity of exon 2 of the MHC class II DQA gene in three species of voles (Arvicolinae) exhibiting regular multi-annual fluctuations of population density and analysed the processes leading to the observed MHC polymorphism. By using cloning-sequencing methodology and capillary electrophoresis-single strand conformation polymorphism, we described seven sequences in the water, eight in the common, and seven in the bank voles coming from an area of 70 km(2) around the Nozeroy canton in the Jura Mountains (Franche Comté, France). All exon 2 sequences translate to give unique amino acid sequences and positive selection was found to act very intensively on antigen binding sites. We documented the presence of recombination at vole DQA region but its importance in generating allelic polymorphism seems to be relatively limited. For the first time within rodents, we documented the duplication of the DQA gene in all three species with both copies being transcriptionally active. Phylogenetic analysis of allelic sequences revealed extensive trans-species polymorphism within the subfamily although no alleles were shared between species in our data set. We discuss possible role of parasites in forming the recent polymorphism pattern of the DQA locus in voles.

  15. Genome-Wide Analysis Identifies a Quantitative Trait Locus in the MHC Class II Region Associated with Generalized Vitiligo Age of Onset

    PubMed Central

    Jin, Ying; Birlea, Stanca A.; Fain, Pamela R.; Gowan, Katherine; Riccardi, Sheri L.; Holland, Paulene J.; Bennett, Dorothy C.; Herbstman, Deborah M.; Wallace, Margaret R.; McCormack, Wayne T.; Kemp, E. Helen; Gawkrodger, David J.; Weetman, Anthony P.; Picardo, Mauro; Leone, Giovanni; Taïeb, Alain; Jouary, Thomas; Ezzedine, Khaled; van Geel, Nanny; Lambert, Jo; Overbeck, Andreas; Spritz, Richard A.

    2011-01-01

    Generalized vitiligo is a common autoimmune disease in which acquired patchy depigmentation of skin, hair, and mucous membranes results from loss of melanocytes from involved areas. Previous genetic analyses have focused on vitiligo susceptibility, and have identified a number of genes involved in disease risk. Age of onset of generalized vitiligo also involves a substantial genetic component, but has not previously been studied systematically. In this study, we report a genome-wide association study of vitiligo age of onset in 1,339 generalized vitiligo patients, with replication in an independent cohort of 677 cases. We identified a quantitative trait locus for vitiligo age of onset in the major histocompatibility complex (MHC) class II region, located near c6orf10-BTNL2 (rs7758128; P = 8.14×10−11), a region that is also associated with generalized vitiligo susceptibility. In contrast, there was no association of vitiligo age of onset with any other MHC or non-MHC loci that are associated with vitiligo susceptibility. These findings highlight the differing roles played by genes involved in vitiligo susceptibility versus vitiligo age of onset, and illustrate that genome-wide analyses can be used to identify genes involved in quantitative aspects of disease natural history, as well as disease susceptibility per se. PMID:21326295

  16. Sorting signals in the MHC class II invariant chain cytoplasmic tail and transmembrane region determine trafficking to an endocytic processing compartment

    PubMed Central

    1994-01-01

    Targeting of MHC class II molecules to the endocytic compartment where they encounter processed antigen is determined by the invariant chain (Ii). By analysis of Ii-transferrin receptor (TR) chimera trafficking, we have identified sorting signals in the Ii cytoplasmic tail and transmembrane region that mediate this process. Two non-tyrosine-based sorting signals in the Ii cytoplasmic tail were identified that mediate localization to plasma membrane clathrin-coated pits and promote rapid endocytosis. Leu7 and Ile8 were required for the activity of the signal most distal to the cell membrane whereas Pro15 Met16 Leu17 were important for the membrane-proximal signal. The same or overlapping non- tyrosine-based sorting signals are essential for delivery of Ii-TR chimeras, either by an intracellular route or via the plasma membrane, to an endocytic compartment where they are rapidly degraded. The Ii transmembrane region is also required for efficient delivery to this endocytic processing compartment and contains a signal distinct from the Ii cytoplasmic tail. More than 80% of the Ii-TR chimera containing the Ii cytoplasmic tail and transmembrane region is delivered directly to the endocytic pathway by an intracellular route, implying that the Ii sorting signals are efficiently recognized by sorting machinery located in the trans-Golgi. PMID:8034737

  17. A four-step model for the IL-6 amplifier, a regulator of chronic inflammations in tissue-specific MHC class II-associated autoimmune diseases.

    PubMed

    Murakami, Masaaki; Hirano, Toshio

    2011-01-01

    It is commonly thought that autoimmune diseases are caused by the breakdown of self-tolerance, which suggests the recognition of specific antigens by autoreactive CD4+ T cells contribute to the specificity of autoimmune diseases (Marrack et al., 2001; Mathis and Benoist, 2004). In several cases, however, even for diseases associated with class II major histocompatibility complex (MHC) alleles, the causative tissue-specific antigens recognized by memory/activated CD4+ T cells have not been established (Mocci et al., 2000; Skapenko et al., 2005). Rheumatoid arthritis (RA) and arthritis in F759 knock-in mice (F759 mice) are such examples (Atsumi et al., 2002; Brennan et al., 2002; Falgarone et al., 2009). These include associations with class II MHC and CD4 molecules; increased numbers of memory/activated CD4+ T cells; and improved outcomes in response to suppressions and/or deficiencies in class II MHC molecules, CD4+ T cells, and the T cell survival cytokine IL-7. Regarding the development of arthritis in F759 mice, it is not only the immune system, but also non-immune tissue that are involved, indicating that the importance of their interactions (Sawa et al., 2006, 2009; Ogura et al., 2008; Hirano, 2010; Murakami et al., 2011). Furthermore, we have shown that local events such as microbleeding together with an accumulation of activated CD4+ T cells in a manner independent of tissue antigen-recognitions induces arthritis in the joints of F759 mice (Murakami et al., 2011). For example, local microbleeding-mediated CCL20 expression induce such an accumulation, causing arthritis development via chronic activation of an IL-17A-dependent IL-6 signaling amplification loop in type 1 collagen+ cells that is triggered by CD4+ T cell-derived cytokine(s) such as IL-17A, which leads to the synergistic activation of STAT3 and NFκB in non-hematopoietic cells in the joint (Murakami et al., 2011). We named this loop the IL-6-mediated inflammation amplifier, or IL-6 amplifier for

  18. RNA-seq liver transcriptome analysis reveals an activated MHC-I pathway and an inhibited MHC-II pathway at the early stage of vaccine immunization in zebrafish

    PubMed Central

    2012-01-01

    Background Zebrafish (Danio rerio) is a prominent vertebrate model of human development and pathogenic disease and has recently been utilized to study teleost immune responses to infectious agents threatening the aquaculture industry. In this work, to clarify the host immune mechanisms underlying the protective effects of a putative vaccine and improve its immunogenicity in the future efforts, high-throughput RNA sequencing technology was used to investigate the immunization-related gene expression patterns of zebrafish immunized with Edwardsiella tarda live attenuated vaccine. Results Average reads of 18.13 million and 14.27 million were obtained from livers of zebrafish immunized with phosphate buffered saline (mock) and E. tarda vaccine (WED), respectively. The reads were annotated with the Ensembl zebrafish database before differential expressed genes sequencing (DESeq) comparative analysis, which identified 4565 significantly differentially expressed genes (2186 up-regulated and 2379 down-regulated in WED; p<0.05). Among those, functional classifications were found in the Gene Ontology database for 3891 and in the Kyoto Encyclopedia of Genes and Genomes database for 3467. Several pathways involved in acute phase response, complement activation, immune/defense response, and antigen processing and presentation were remarkably affected at the early stage of WED immunization. Further qPCR analysis confirmed that the genes encoding the factors involved in major histocompatibility complex (MHC)-I processing pathway were up-regulated, while those involved in MHC-II pathway were down-regulated. Conclusion These data provided insights into the molecular mechanisms underlying zebrafish immune response to WED immunization and might aid future studies to develop a highly immunogenic vaccine against gram-negative bacteria in teleosts. PMID:22805612

  19. RNA-seq liver transcriptome analysis reveals an activated MHC-I pathway and an inhibited MHC-II pathway at the early stage of vaccine immunization in zebrafish.

    PubMed

    Yang, Dahai; Liu, Qin; Yang, Minjun; Wu, Haizhen; Wang, Qiyao; Xiao, Jingfan; Zhang, Yuanxing

    2012-07-17

    Zebrafish (Danio rerio) is a prominent vertebrate model of human development and pathogenic disease and has recently been utilized to study teleost immune responses to infectious agents threatening the aquaculture industry. In this work, to clarify the host immune mechanisms underlying the protective effects of a putative vaccine and improve its immunogenicity in the future efforts, high-throughput RNA sequencing technology was used to investigate the immunization-related gene expression patterns of zebrafish immunized with Edwardsiella tarda live attenuated vaccine. Average reads of 18.13 million and 14.27 million were obtained from livers of zebrafish immunized with phosphate buffered saline (mock) and E. tarda vaccine (WED), respectively. The reads were annotated with the Ensembl zebrafish database before differential expressed genes sequencing (DESeq) comparative analysis, which identified 4565 significantly differentially expressed genes (2186 up-regulated and 2379 down-regulated in WED; p<0.05). Among those, functional classifications were found in the Gene Ontology database for 3891 and in the Kyoto Encyclopedia of Genes and Genomes database for 3467. Several pathways involved in acute phase response, complement activation, immune/defense response, and antigen processing and presentation were remarkably affected at the early stage of WED immunization. Further qPCR analysis confirmed that the genes encoding the factors involved in major histocompatibility complex (MHC)-I processing pathway were up-regulated, while those involved in MHC-II pathway were down-regulated. These data provided insights into the molecular mechanisms underlying zebrafish immune response to WED immunization and might aid future studies to develop a highly immunogenic vaccine against gram-negative bacteria in teleosts.

  20. Changes in variation at the MHC class II DQA locus during the final demise of the woolly mammoth.

    PubMed

    Pečnerová, Patrícia; Díez-Del-Molino, David; Vartanyan, Sergey; Dalén, Love

    2016-05-04

    According to the nearly-neutral theory of evolution, the relative strengths of selection and drift shift in favour of drift at small population sizes. Numerous studies have analysed the effect of bottlenecks and small population sizes on genetic diversity in the MHC, which plays a central role in pathogen recognition and immune defense and is thus considered a model example for the study of adaptive evolution. However, to understand changes in genetic diversity at loci under selection, it is necessary to compare the genetic diversity of a population before and after the bottleneck. In this study, we analyse three fragments of the MHC DQA gene in woolly mammoth samples radiocarbon dated to before and after a well-documented bottleneck that took place about ten thousand years ago. Our results indicate a decrease in observed heterozygosity and number of alleles, suggesting that genetic drift had an impact on the variation on MHC. Based on coalescent simulations, we found no evidence of balancing selection maintaining MHC diversity during the Holocene. However, strong trans-species polymorphism among mammoths and elephants points to historical effects of balancing selection on the woolly mammoth lineage.

  1. Changes in variation at the MHC class II DQA locus during the final demise of the woolly mammoth

    NASA Astrophysics Data System (ADS)

    Pečnerová, Patrícia; Díez-Del-Molino, David; Vartanyan, Sergey; Dalén, Love

    2016-05-01

    According to the nearly-neutral theory of evolution, the relative strengths of selection and drift shift in favour of drift at small population sizes. Numerous studies have analysed the effect of bottlenecks and small population sizes on genetic diversity in the MHC, which plays a central role in pathogen recognition and immune defense and is thus considered a model example for the study of adaptive evolution. However, to understand changes in genetic diversity at loci under selection, it is necessary to compare the genetic diversity of a population before and after the bottleneck. In this study, we analyse three fragments of the MHC DQA gene in woolly mammoth samples radiocarbon dated to before and after a well-documented bottleneck that took place about ten thousand years ago. Our results indicate a decrease in observed heterozygosity and number of alleles, suggesting that genetic drift had an impact on the variation on MHC. Based on coalescent simulations, we found no evidence of balancing selection maintaining MHC diversity during the Holocene. However, strong trans-species polymorphism among mammoths and elephants points to historical effects of balancing selection on the woolly mammoth lineage.

  2. Changes in variation at the MHC class II DQA locus during the final demise of the woolly mammoth

    PubMed Central

    Pečnerová, Patrícia; Díez-del-Molino, David; Vartanyan, Sergey; Dalén, Love

    2016-01-01

    According to the nearly-neutral theory of evolution, the relative strengths of selection and drift shift in favour of drift at small population sizes. Numerous studies have analysed the effect of bottlenecks and small population sizes on genetic diversity in the MHC, which plays a central role in pathogen recognition and immune defense and is thus considered a model example for the study of adaptive evolution. However, to understand changes in genetic diversity at loci under selection, it is necessary to compare the genetic diversity of a population before and after the bottleneck. In this study, we analyse three fragments of the MHC DQA gene in woolly mammoth samples radiocarbon dated to before and after a well-documented bottleneck that took place about ten thousand years ago. Our results indicate a decrease in observed heterozygosity and number of alleles, suggesting that genetic drift had an impact on the variation on MHC. Based on coalescent simulations, we found no evidence of balancing selection maintaining MHC diversity during the Holocene. However, strong trans-species polymorphism among mammoths and elephants points to historical effects of balancing selection on the woolly mammoth lineage. PMID:27143688

  3. Novel classical MHC class I alleles identified in horses by sequencing clones of reverse transcription-PCR products.

    PubMed

    Chung, C; Leib, S R; Fraser, D G; Ellis, S A; McGuire, T C

    2003-12-01

    Improved typing of horse classical MHC class I is required to more accurately define these molecules and to extend the number identified further than current serological assays. Defining classical MHC class I alleleic polymorphism is important in evaluating cytotoxic T lymphocyte (CTL) responses in horses. In this study, horse classical MHC class I genes were analyzed based on reverse transcription (RT)-PCR amplification of sequences encoding the polymorphic peptide binding region and the more conserved alpha 3, transmembrane and cytoplasmic regions followed by cloning and sequencing. Primer sets included a horse classical MHC class I-specific reverse primer and a forward primer conserved in all known horse MHC class I genes. Sequencing at least 25 clones containing MHC class I sequences from each of 13 horses identified 25 novel sequences and three others which had been described. Of these, nine alleles were identified from different horses or different RT-PCR and 19 putative alleles were identified in multiple clones from the same RT-PCR. The primer pairs did not amplify putative non-classical MHC class I genes as only classical MHC class I and related pseudogenes were found in 462 clones. This method also identified classical MHC class I alleles shared between horses by descent, and defined differences in alleles between horses varying in equine leukocyte antigen (ELA)-A haplotype as determined by serology. However, horses sharing ELA-A haplotypes defined by serotyping did not always share cDNA sequences, suggesting subhaplotypic variations within serologically defined ELA-A haplotypes. The 13 horses in this study had two to five classical MHC class I sequences, indicating that multiple loci code for these genes. Sequencing clones from RT-PCR with classical MHC class I-specific primers should be useful for selection of haplotype matched and mismatched horses for CTL studies, and provides sequence information needed to develop easier and more discriminating

  4. Identification of cathepsin B from large yellow croaker (Pseudosciaena crocea) and its role in the processing of MHC class II-associated invariant chain.

    PubMed

    Li, Mingyu; Li, Qiuhua; Yang, Zhijun; Hu, Guohai; Li, Ting; Chen, Xinhua; Ao, Jingqun

    2014-08-01

    In teleost, cathepsin B has been identified from several species and shown to play roles in the host immune response during pathogen challenge. However, the mechanism of how cathepsin B modulates the immune response in teleosts remains poorly understood. In this study, we identified and characterized cathepsin B (LycCatB) and invariant chain (LycIi) from the large yellow croaker (Pseudosciaena crocea). Sequence comparison and phylogenetic analysis indicated that LycCatB and LycIi are highly conserved within teleosts. Quantitative RT-PCR analysis showed that LycCatB mRNA was widely expressed in all examined tissues. We then recombinantly expressed LycCatB and Lyc-TR-Ii (transmembrane domain removed Ii chain) in Pichia pastoris and Escherichiacoli, respectively. The recombinant LycCatB (rLycCatB) can hydrolyze the substrate Z-FR-AMC with a Km value of 40.68μM. Furthermore, co-incubation of rLycCatB with rLyc-TR-Ii led to an efficient cleavage of rLyc-TR-Ii in a time-dependant manner. These results indicated that cathepsin B may be involved in MHC class II-associated Ii processing in large yellow croaker, and provide new information helping to elucidate the immunological functions of teleost cathepsin B. Copyright © 2014 Elsevier Ltd. All rights reserved.

  5. Rejection versus escape: the tumor MHC dilemma.

    PubMed

    Garrido, Federico; Ruiz-Cabello, Francisco; Aptsiauri, Natalia

    2017-02-01

    Most tumor cells derive from MHC-I-positive normal counterparts and remain positive at early stages of tumor development. T lymphocytes can infiltrate tumor tissue, recognize and destroy MHC class I (MHC-I)-positive cancer cells ("permissive" phase I). Later, MHC-I-negative tumor cell variants resistant to T-cell killing emerge. During this process, tumors first acquire a heterogeneous MHC-I expression pattern and finally become uniformly MHC-I-negative. This stage (phase II) represents a "non-permissive" encapsulated structure with tumor nodes surrounded by fibrous tissue containing different elements including leukocytes, macrophages, fibroblasts, etc. Molecular mechanisms responsible for total or partial MHC-I downregulation play a crucial role in determining and predicting the antigen-presenting capacity of cancer cells. MHC-I downregulation caused by reversible ("soft") lesions can be upregulated by TH1-type cytokines released into the tumor microenvironment in response to different types of immunotherapy. In contrast, when the molecular mechanism of the tumor MHC-I loss is irreversible ("hard") due to a genetic defect in the gene/s coding for MHC-I heavy chains (chromosome 6) or beta-2-microglobulin (B2M) (chromosome 15), malignant cells are unable to upregulate MHC-I, remain undetectable by cytotoxic T-cells, and continue to grow and metastasize. Based on the tumor MHC-I molecular analysis, it might be possible to define MHC-I phenotypes present in cancer patients in order to distinguish between non-responders, partial/short-term responders, and likely durable responders. This highlights the need for designing strategies to enhance tumor MHC-I expression that would allow CTL-mediated tumor rejection.

  6. Crystallographic and Computational Studies of a Class II MHC Complex with a Nonconforming Peptide: HLA-DRA/DRB3*0101

    NASA Astrophysics Data System (ADS)

    Parry, Christian S.; Gorski, Jack; Stern, Lawrence J.

    2003-03-01

    The stable binding of processed foreign peptide to a class II major histocompatibility (MHC) molecule and subsequent presentation to a T cell receptor is a central event in immune recognition and regulation. Polymorphic residues on the floor of the peptide binding site form pockets that anchor peptide side chains. These and other residues in the helical wall of the groove determine the specificity of each allele and define a motif. Allele specific motifs allow the prediction of epitopes from the sequence of pathogens. There are, however, known epitopes that do not satisfy these motifs: anchor motifs are not adequate for predicting epitopes as there are apparently major and minor motifs. We present crystallographic studies into the nature of the interactions that govern the binding of these so called nonconforming peptides. We would like to understand the role of the P10 pocket and find out whether the peptides that do not obey the consensus anchor motif bind in the canonical conformation observed in in prior structures of class II MHC-peptide complexes. HLA-DRB3*0101 complexed with peptide crystallized in unit cell 92.10 x 92.10 x 248.30 (90, 90, 90), P41212, and the diffraction data is reliable to 2.2ÅWe are complementing our studies with dynamical long time simulations to answer these questions, particularly the interplay of the anchor motifs in peptide binding, the range of protein and ligand conformations, and water hydration structures.

  7. Forced expression of HLA-DM at the surface of dendritic cells increases loading of synthetic peptides on MHC class II molecules and modulates T cell responses.

    PubMed

    Pezeshki, Abdul Mohammad; Côté, Marie-Hélène; Azar, Georges A; Routy, Jean-Pierre; Boulassel, Mohamed-Rachid; Thibodeau, Jacques

    2011-07-01

    Adoptive transfer of autologous dendritic cells (DCs) loaded with tumor-associated CD4 and CD8 T cell epitopes represents a promising avenue for the immunotherapy of cancer. In an effort to increase the loading of therapeutic synthetic peptides on MHC II molecules, we used a mutant of HLA-DM (DMY) devoid of its lysosomal sorting motif and that accumulates at the cell surface. Transfection of DMY into HLA-DR(+) cells resulted in increased loading of the exogenously supplied HA(307-318) peptide, as well as increased stimulation of HA-specific T cells. Also, on transduction in mouse and human DCs, DMY increased loading of HEL(48-61) and of the tumor Ag-derived gp100(174-190) peptides, respectively. Interestingly, expression of DMY at the surface of APCs favored Th1 differentiation over Th2. Finally, we found that DMY(-) and DMY(+) mouse APCs differentially stimulated T cell hybridomas sensitive to the fine conformation of peptide-MHC II complexes. Taken together, our results suggest that the overexpression of HLA-DMY at the plasma membrane of DCs may improve quantitatively, but also qualitatively, the presentation of CD4 T cell epitopes in cellular vaccine therapies for cancer.

  8. MHC class II-transduced tumor cells originating in the immune-privileged eye prime and boost CD4(+) T lymphocytes that cross-react with primary and metastatic uveal melanoma cells.

    PubMed

    Bosch, Jacobus J; Thompson, James A; Srivastava, Minu K; Iheagwara, Uzoma K; Murray, Timothy G; Lotem, Michal; Ksander, Bruce R; Ostrand-Rosenberg, Suzanne

    2007-05-01

    Uveal melanoma, the most common malignancy of the eye, has a 50% rate of liver metastases among patients with large primary tumors. Several therapies prolong survival of metastatic patients; however, none are curative and no patients survive. Therefore, we are exploring immunotherapy as an alternative or adjunctive treatment. Uveal melanoma may be particularly appropriate for immunotherapy because primary tumors arise in an immune-privileged site and may express antigens to which the host is not tolerized. We are developing MHC class II (MHC II)-matched allogeneic, cell-based uveal melanoma vaccines that activate CD4(+) T lymphocytes, which are key cells for optimizing CD8(+) T-cell immunity, facilitating immune memory, and preventing tolerance. Our previous studies showed that tumor cells genetically modified to express costimulatory and MHC II molecules syngeneic to the recipient are potent inducers of antitumor immunity. Because the MHC II-matched allogeneic vaccines do not express the accessory molecule, Invariant chain, they present MHC II-restricted peptides derived from endogenously encoded tumor antigens. We now report that MHC II-matched allogeneic vaccines, prepared from primary uveal melanomas that arise in the immune-privileged eye, prime and boost IFNgamma-secreting CD4(+) T cells from the peripheral blood of either healthy donors or uveal melanoma patients that cross-react with primary uveal melanomas from other patients and metastatic tumors. In contrast, vaccines prepared from metastatic cells in the liver are less effective at activating CD4(+) T cells, suggesting that tumor cells originating in immune-privileged sites may have enhanced capacity for inducing antitumor immunity and for serving as immunotherapeutic agents.

  9. Differential regulation of expression of the MHC class II molecules RT1.B and RT1.D on rat B lymphocytes: effects of interleukin-4, interleukin-13 and interferon-gamma.

    PubMed Central

    Roos, A; Schilder-Tol, E J; Chand, M A; Claessen, N; Lakkis, F G; Pascual, D W; Weening, J J; Aten, J

    1998-01-01

    Susceptibility to induction of both T helper 1- (Th1) and Th2-mediated autoimmunity is multifactorial and involves genetic linkage to the major histocompatibility complex (MHC) class II haplotype. Brown Norway (BN) rats exposed to mercuric chloride develop a Th2-dependent systemic autoimmunity, whereas Lewis rats, which are highly susceptible to Th1-mediated autoimmunity, develop immune suppression after mercuric chloride exposure. Exposure to mercuric chloride is known to enhance B-lymphocyte expression of the MHC class II molecule RT1.B, predominantly in BN rats. We demonstrate that, in contrast, expression of RT1.D was unmodified on these B cells, whereas both RT1.B and RT1.D were up-regulated on epithelial cells. Regulation of B-cell MHC class II isotype expression was further studied in vitro, using BN rat lymph node (LN) cells. Interleukin-4 (IL-4) strongly enhanced B-cell expression of RT1.B (2.8-fold), whereas RT1.D expression was only slightly, although significantly, modified (1.2-fold). B cells from Lewis rats showed a similar IL-4-induced enhancement of RT1.B expression (2.5-fold), whereas, in contrast, RT1.D expression was unmodified. Exposure of LN cells from BN rats to interferon-gamma induced a moderate increase of B-cell MHC class II expression, predominantly of RT1.B. Strong and rapid enhancement of B-cell RT1.D expression was observed after stimulation by phorbol 12-myristate 13-acetate and ionomycin. Rat IL-13 did not modify B-cell MHC class II expression; however, it induced typical morphological changes in peritoneal macrophages. These experiments demonstrate isotype-specific and strain-dependent regulation of MHC class II expression on rat B lymphocytes, which may be of pathophysiological relevance for the strain-dependent susceptibility for Th1- or Th2-mediated autoimmunity. Images Figure 1 Figure 5 PMID:9536116

  10. Polymorphisms in the F8 Gene and MHC-II Variants as Risk Factors for the Development of Inhibitory Anti-Factor VIII Antibodies during the Treatment of Hemophilia A: A Computational Assessment

    PubMed Central

    Pandey, Gouri Shankar; Yanover, Chen; Howard, Tom E.; Sauna, Zuben E.

    2013-01-01

    The development of neutralizing anti-drug-antibodies to the Factor VIII protein-therapeutic is currently the most significant impediment to the effective management of hemophilia A. Common non-synonymous single nucleotide polymorphisms (ns-SNPs) in the F8 gene occur as six haplotypes in the human population (denoted H1 to H6) of which H3 and H4 have been associated with an increased risk of developing anti-drug antibodies. There is evidence that CD4+ T-cell response is essential for the development of anti-drug antibodies and such a response requires the presentation of the peptides by the MHC-class-II (MHC-II) molecules of the patient. We measured the binding and half-life of peptide-MHC-II complexes using synthetic peptides from regions of the Factor VIII protein where ns-SNPs occur and showed that these wild type peptides form stable complexes with six common MHC-II alleles, representing 46.5% of the North American population. Next, we compared the affinities computed by NetMHCIIpan, a neural network-based algorithm for MHC-II peptide binding prediction, to the experimentally measured values and concluded that these are in good agreement (area under the ROC-curve of 0.778 to 0.972 for the six MHC-II variants). Using a computational binding predictor, we were able to expand our analysis to (a) include all wild type peptides spanning each polymorphic position; and (b) consider more MHC-II variants, thus allowing for a better estimation of the risk for clinical manifestation of anti-drug antibodies in the entire population (or a specific sub-population). Analysis of these computational data confirmed that peptides which have the wild type sequence at positions where the polymorphisms associated with haplotypes H3, H4 and H5 occur bind MHC-II proteins significantly more than a negative control. Taken together, the experimental and computational results suggest that wild type peptides from polymorphic regions of FVIII constitute potential T-cell epitopes and thus

  11. Identification of the expressed MHC class II DQB gene of the Asiatic black bear, Ursus thibetanus, in Japan.

    PubMed

    Yasukochi, Yoshiki; Kurosaki, Toshifumi; Yoneda, Masaaki; Koike, Hiroko

    2010-04-01

    Genetic diversity estimation of the major histocompatibility complex (MHC) gene may be an important tool in the assessment of immune response ability against infectious disease. We were able to identify a near full-length expressed DQB sequence by RACE-PCR method from the Asiatic black bear, Ursus thibetanus in Japan. This is the first such full length expression in the Ursidae. The bear had at least one functional DQB locus. In phylogenetic tree analysis its DQB amino acid sequence formed a monophyletic group with DQB sequences from members of the order Carnivora and had a 90% nucleotide sequence similarity with the DQB allele of the California sea lion, Zalophus californianus. We compared the DQB amino acid composition of U. thibetanus with those of several other mammalian species including Homo sapiens. Amino acid residues known to be functionally important for human MHC genes, tended to be also conserved among other mammalian species while PBRs in the beta1 domain were heterogeneous among mammalian species. The DQB sequence obtained from the bear had not only no putative frameshifts or deletions but also no abnormal amino acid mutations such as had been observed in human DQB molecules. This suggests that the bear DQB sequence was an apparently functional DQB allele. As a preliminary study, we sequenced the exon 2 region of DQB alleles from genomic DNA, and succeeded to amplify the exon 2 of DQB loci. Our study will provide useful information for conservation genetics of the U. thibetanus as well as more generally regarding the mammalian MHC region.

  12. Lung cancer patients’ CD4+ T cells are activated in vitro by MHC II cell-based vaccines despite the presence of myeloid-derived suppressor cells

    PubMed Central

    Srivastava, Minu K.; Bosch, Jacobus J.; Thompson, James A.; Ksander, Bruce R.; Edelman, Martin J.

    2009-01-01

    Background Advanced non-small cell lung cancer (NSCLC) remains an incurable disease. Immunotherapies that activate patients’ T cells against resident tumor cells are being developed; however, these approaches may not be effective in NSCLC patients due to tumor-induced immune suppression. A major cause of immune suppression is myeloid-derived suppressor cells (MDSC). Because of the strategic role of CD4+ T lymphocytes in the activation of cytotoxic CD8+ T cells and immune memory, we are developing cell-based vaccines that activate tumor-specific CD4+ T cells in the presence of MDSC. The vaccines are NSCLC cell lines transfected with costimulatory (CD80) plus major histocompatibility complex class II (MHC II) genes that are syngeneic to the recipient. The absence of invariant chain promotes the presentation of endogenously synthesized tumor antigens, and the activation of MHC II-restricted, tumor-antigen-specific CD4+ T cells. Methods Potential vaccine efficacy was tested in vitro by priming and boosting peripheral blood mononuclear cells from ten NSCLC patients who had varying levels of MDSC. CD4+ T cell activation was quantified by measuring Type 1 and Type 2 cytokine release. Results The vaccines activated CD4+ T cells from all ten patients, despite the presence of CD33+CD11b+ MDSC. Activated CD4+ T cells were specific for NSCLC and did not cross-react with tumor cells derived from non-lung tissue or normal lung fibroblasts. Conclusions The NSCLC vaccines activate tumor-specific CD4+ T cells in the presence of potent immune suppression, and may be useful for the treatment of patients with NSCLC. PMID:18322683

  13. Lung cancer patients' CD4(+) T cells are activated in vitro by MHC II cell-based vaccines despite the presence of myeloid-derived suppressor cells.

    PubMed

    Srivastava, Minu K; Bosch, Jacobus J; Thompson, James A; Ksander, Bruce R; Edelman, Martin J; Ostrand-Rosenberg, Suzanne

    2008-10-01

    Advanced non-small cell lung cancer (NSCLC) remains an incurable disease. Immunotherapies that activate patients' T cells against resident tumor cells are being developed; however, these approaches may not be effective in NSCLC patients due to tumor-induced immune suppression. A major cause of immune suppression is myeloid-derived suppressor cells (MDSC). Because of the strategic role of CD4(+) T lymphocytes in the activation of cytotoxic CD8(+) T cells and immune memory, we are developing cell-based vaccines that activate tumor-specific CD4(+) T cells in the presence of MDSC. The vaccines are NSCLC cell lines transfected with costimulatory (CD80) plus major histocompatibility complex class II (MHC II) genes that are syngeneic to the recipient. The absence of invariant chain promotes the presentation of endogenously synthesized tumor antigens, and the activation of MHC II-restricted, tumor-antigen-specific CD4(+) T cells. Potential vaccine efficacy was tested in vitro by priming and boosting peripheral blood mononuclear cells from ten NSCLC patients who had varying levels of MDSC. CD4(+) T cell activation was quantified by measuring Type 1 and Type 2 cytokine release. The vaccines activated CD4(+) T cells from all ten patients, despite the presence of CD33(+)CD11b(+) MDSC. Activated CD4(+) T cells were specific for NSCLC and did not cross-react with tumor cells derived from non-lung tissue or normal lung fibroblasts. The NSCLC vaccines activate tumor-specific CD4(+) T cells in the presence of potent immune suppression, and may be useful for the treatment of patients with NSCLC.

  14. Rafting MHC-II domains in the APC (presynaptic) plasma membrane and the thresholds for T-cell activation and immunological synapse formation.

    PubMed

    Gombos, Imre; Detre, Cynthia; Vámosi, György; Matkó, János

    2004-03-29

    Glycosphingolipid- and cholesterol-rich membrane microdomains (rafts) in T-cells are important in triggering and regulation of T(H)-cell activation in immunological synapses (IS), which in turn may control the T-cell repertoire in lymph nodes and at the periphery. It is less known, however, how the "presynaptic side" controls formation and function of IS. We investigated here activation signals and synapse formation frequency of murine IP12-7 T(H) hybridoma cell specific to influenza virus HA-peptide upon stimulation with two B-lymphoma cells, A20 and 2PK3, pulsed with peptide antigen. Confocal microscopic colocalization and FRET data consonantly revealed clustered distribution and constitutive raft-association of a major fraction of MHC-II molecules in both APCs. Costimulatory molecules (CD80 and CD86), not associated constitutively with rafts, were expressed at much lower level in A20 cells. T-cells responded to 2PK3 APC with much higher signal strength than to A20 cells, in good correlation with the frequency of IS formation, as assessed by microscopic conjugation assay. Disruption of rafts by cholesterol depletion in 2PK3 cells largely decreased the magnitude of T(H) cell activation signals, especially at low peptide antigen doses, similarly to masking CD4 with mAb on T-cells. The frequency of IS formation was reduced by blocking LFA-1 on T-cells and CD80 on APCs, by lowering the temperature below the phase transition of the membrane or by disrupting actin cytoskeleton. These data together suggest that the surface density and affinity/stability of peptide-MHC-II complexes and the costimulatory level are primary determinants for an efficient TCR recognition and the strength of the subsequent T-cell signals, as well as of the IS formation, which additionally requires a cytoskeleton-dependent remodeling of APC surface after the initial TCR signal. The threshold of T-cell activation can be further set by rafting MHC-II domains via concentrating high affinity

  15. Lacking prognostic significance of beta 2-microglobulin, MHC class I and class II antigen expression in breast carcinomas.

    PubMed Central

    Wintzer, H. O.; Benzing, M.; von Kleist, S.

    1990-01-01

    To evaluate the impact of MHC antigen expression on the survival of patients with cancer, 77 human breast carcinomas were investigated for the expression of beta 2-microglobulin (beta 2m), HLA-A,B,C and HLA-DR. Thirty-one benign breast tumours were stained for comparison. The results for the carcinomas were related to the survival data of the cancer patients. The expression of beta 2m, HLA-A,B,C and HLA-DR was significantly lower in malignant tumours compared to the benign lesions. Whereas all benign tumours were positive for beta 2m and HLA-A,B,C and 28/31 positive for HLA-DR the following positivity rates were found in carcinomas: 74/77 for beta 2m, 57/77 for HLA-A,B,C and 10/77 for HLA-DR. The follow-up (median 45 months) of 66 cancer patients for overall survival and of 65 patients for disease-free survival revealed no influence of beta 2m, HLA-A,B,C or HLA-DR expression on the prognosis of this cancer. In conclusion, experimental data indicating the importance of MHC antigens in anti-tumour responses are not confirmed by the analysis of cancer patient survival data. Images Figure 1 Figure 2 Figure 3 Figure 4 PMID:2201398

  16. Innate lymphoid cells and the MHC.

    PubMed

    Robinette, M L; Colonna, M

    2016-01-01

    Innate lymphoid cells (ILCs) are a new class of immune cells that include natural killer (NK) cells and appear to be the innate counterparts to CD4(+) helper T cells and CD8(+) cytotoxic T cells based on developmental and functional similarities. Like T cells, both NK cells and other ILCs also show connections to the major histocompatibility complex (MHC). In human and mouse, NK cells recognize and respond to classical and nonclassical MHC I molecules as well as structural homologues, whereas mouse ILCs have recently been shown to express MHC II. We describe the history of MHC I recognition by NK cells and discuss emerging roles for MHC II expression by ILC subsets, making comparisons between both mouse and human when possible. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  17. The Dendritic Cell Major Histocompatibility Complex II (MHC II) Peptidome Derives from a Variety of Processing Pathways and Includes Peptides with a Broad Spectrum of HLA-DM Sensitivity*

    PubMed Central

    Clement, Cristina C.; Becerra, Aniuska; Yin, Liusong; Zolla, Valerio; Huang, Liling; Merlin, Simone; Follenzi, Antonia; Shaffer, Scott A.; Stern, Lawrence J.; Santambrogio, Laura

    2016-01-01

    The repertoire of peptides displayed in vivo by MHC II molecules derives from a wide spectrum of proteins produced by different cell types. Although intracellular endosomal processing in dendritic cells and B cells has been characterized for a few antigens, the overall range of processing pathways responsible for generating the MHC II peptidome are currently unclear. To determine the contribution of non-endosomal processing pathways, we eluted and sequenced over 3000 HLA-DR1-bound peptides presented in vivo by dendritic cells. The processing enzymes were identified by reference to a database of experimentally determined cleavage sites and experimentally validated for four epitopes derived from complement 3, collagen II, thymosin β4, and gelsolin. We determined that self-antigens processed by tissue-specific proteases, including complement, matrix metalloproteases, caspases, and granzymes, and carried by lymph, contribute significantly to the MHC II self-peptidome presented by conventional dendritic cells in vivo. Additionally, the presented peptides exhibited a wide spectrum of binding affinity and HLA-DM susceptibility. The results indicate that the HLA-DR1-restricted self-peptidome presented under physiological conditions derives from a variety of processing pathways. Non-endosomal processing enzymes add to the number of epitopes cleaved by cathepsins, altogether generating a wider peptide repertoire. Taken together with HLA-DM-dependent and-independent loading pathways, this ensures that a broad self-peptidome is presented by dendritic cells. This work brings attention to the role of “self-recognition” as a dynamic interaction between dendritic cells and the metabolic/catabolic activities ongoing in every parenchymal organ as part of tissue growth, remodeling, and physiological apoptosis. PMID:26740625

  18. The Dendritic Cell Major Histocompatibility Complex II (MHC II) Peptidome Derives from a Variety of Processing Pathways and Includes Peptides with a Broad Spectrum of HLA-DM Sensitivity.

    PubMed

    Clement, Cristina C; Becerra, Aniuska; Yin, Liusong; Zolla, Valerio; Huang, Liling; Merlin, Simone; Follenzi, Antonia; Shaffer, Scott A; Stern, Lawrence J; Santambrogio, Laura

    2016-03-11

    The repertoire of peptides displayed in vivo by MHC II molecules derives from a wide spectrum of proteins produced by different cell types. Although intracellular endosomal processing in dendritic cells and B cells has been characterized for a few antigens, the overall range of processing pathways responsible for generating the MHC II peptidome are currently unclear. To determine the contribution of non-endosomal processing pathways, we eluted and sequenced over 3000 HLA-DR1-bound peptides presented in vivo by dendritic cells. The processing enzymes were identified by reference to a database of experimentally determined cleavage sites and experimentally validated for four epitopes derived from complement 3, collagen II, thymosin β4, and gelsolin. We determined that self-antigens processed by tissue-specific proteases, including complement, matrix metalloproteases, caspases, and granzymes, and carried by lymph, contribute significantly to the MHC II self-peptidome presented by conventional dendritic cells in vivo. Additionally, the presented peptides exhibited a wide spectrum of binding affinity and HLA-DM susceptibility. The results indicate that the HLA-DR1-restricted self-peptidome presented under physiological conditions derives from a variety of processing pathways. Non-endosomal processing enzymes add to the number of epitopes cleaved by cathepsins, altogether generating a wider peptide repertoire. Taken together with HLA-DM-dependent and-independent loading pathways, this ensures that a broad self-peptidome is presented by dendritic cells. This work brings attention to the role of "self-recognition" as a dynamic interaction between dendritic cells and the metabolic/catabolic activities ongoing in every parenchymal organ as part of tissue growth, remodeling, and physiological apoptosis. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  19. IL-17A and IL-2-expanded regulatory T cells cooperate to inhibit Th1-mediated rejection of MHC II disparate skin grafts.

    PubMed

    Vokaer, Benoît; Charbonnier, Louis-Marie; Lemaître, Philippe H; Spilleboudt, Chloé; Le Moine, Alain

    2013-01-01

    Several evidences suggest that regulatory T cells (Treg) promote Th17 differentiation. Based on this hypothesis, we tested the effect of IL-17A neutralization in a model of skin transplantation in which long-term graft survival depends on a strong in vivo Treg expansion induced by transient exogenous IL-2 administration. As expected, IL-2 supplementation prevented rejection of MHC class II disparate skin allografts but, surprisingly, not in IL-17A-deficient recipients. We attested that IL-17A was not required for IL-2-mediated Treg expansion, intragraft recruitment or suppressive capacities. Instead, IL-17A prevented allograft rejection by inhibiting Th1 alloreactivity independently of Tregs. Indeed, T-bet expression of naive alloreactive CD4+ T cells and the subsequent Th1 immune response was significantly enhanced in IL-17A deficient mice. Our results illustrate for the first time a protective role of IL-17A in CD4+-mediated allograft rejection process.

  20. Negative selection and peptide chemistry determine the size of naive foreign peptide-MHC class II-specific CD4+ T cell populations.

    PubMed

    Chu, H Hamlet; Moon, James J; Kruse, Andrew C; Pepper, Marion; Jenkins, Marc K

    2010-10-15

    Naive CD4(+) T cell populations that express TCRs specific for different foreign peptide-MHC class II complex (pMHCII) ligands can vary in size over several orders of magnitude. This variation may explain why immune responses to some peptides are stronger than others. In this study, we used a sensitive pMHCII-tetramer-based cell enrichment method to study the derivation of two naive foreign pMHCII-specific naive CD4(+) T cell populations that differed in size by 8-fold in normal mice. Analysis of mice in which thymic negative selection was impaired revealed that the smaller population underwent more clonal deletion than the larger population. In addition, large naive cell populations tended to recognize peptides with tryptophan residues as TCR contacts. Thus, the foreign pMHCII that tend to be recognized by large naive populations induce minimal clonal deletion and contain certain amino acids with the capacity to interact favorably with TCRs.

  1. HLA-DRB1*11 and variants of the MHC class II locus are strong risk factors for systemic juvenile idiopathic arthritis.

    PubMed

    Ombrello, Michael J; Remmers, Elaine F; Tachmazidou, Ioanna; Grom, Alexei; Foell, Dirk; Haas, Johannes-Peter; Martini, Alberto; Gattorno, Marco; Özen, Seza; Prahalad, Sampath; Zeft, Andrew S; Bohnsack, John F; Mellins, Elizabeth D; Ilowite, Norman T; Russo, Ricardo; Len, Claudio; Hilario, Maria Odete E; Oliveira, Sheila; Yeung, Rae S M; Rosenberg, Alan; Wedderburn, Lucy R; Anton, Jordi; Schwarz, Tobias; Hinks, Anne; Bilginer, Yelda; Park, Jane; Cobb, Joanna; Satorius, Colleen L; Han, Buhm; Baskin, Elizabeth; Signa, Sara; Duerr, Richard H; Achkar, J P; Kamboh, M Ilyas; Kaufman, Kenneth M; Kottyan, Leah C; Pinto, Dalila; Scherer, Stephen W; Alarcón-Riquelme, Marta E; Docampo, Elisa; Estivill, Xavier; Gül, Ahmet; de Bakker, Paul I W; Raychaudhuri, Soumya; Langefeld, Carl D; Thompson, Susan; Zeggini, Eleftheria; Thomson, Wendy; Kastner, Daniel L; Woo, Patricia

    2015-12-29

    Systemic juvenile idiopathic arthritis (sJIA) is an often severe, potentially life-threatening childhood inflammatory disease, the pathophysiology of which is poorly understood. To determine whether genetic variation within the MHC locus on chromosome 6 influences sJIA susceptibility, we performed an association study of 982 children with sJIA and 8,010 healthy control subjects from nine countries. Using meta-analysis of directly observed and imputed SNP genotypes and imputed classic HLA types, we identified the MHC locus as a bona fide susceptibility locus with effects on sJIA risk that transcended geographically defined strata. The strongest sJIA-associated SNP, rs151043342 [P = 2.8 × 10(-17), odds ratio (OR) 2.6 (2.1, 3.3)], was part of a cluster of 482 sJIA-associated SNPs that spanned a 400-kb region and included the class II HLA region. Conditional analysis controlling for the effect of rs151043342 found that rs12722051 independently influenced sJIA risk [P = 1.0 × 10(-5), OR 0.7 (0.6, 0.8)]. Meta-analysis of imputed classic HLA-type associations in six study populations of Western European ancestry revealed that HLA-DRB1*11 and its defining amino acid residue, glutamate 58, were strongly associated with sJIA [P = 2.7 × 10(-16), OR 2.3 (1.9, 2.8)], as was the HLA-DRB1*11-HLA-DQA1*05-HLA-DQB1*03 haplotype [6.4 × 10(-17), OR 2.3 (1.9, 2.9)]. By examining the MHC locus in the largest collection of sJIA patients assembled to date, this study solidifies the relationship between the class II HLA region and sJIA, implicating adaptive immune molecules in the pathogenesis of sJIA.

  2. Peripheral blood CD8αα+CD11c+MHC-II+CD3- cells attenuate autoimmune glomerulonephritis in rats.

    PubMed

    Wu, Jean; Zhou, Cindy; Robertson, Julie; Carlock, Colin; Lou, Ya-Huan

    2014-05-01

    In an anti-glomerular basement membrane (GBM) glomerulonephritis (GN) model, GN-resistant Lewis rats naturally recover from early glomerular inflammation. Here we investigated recovery mechanisms for development of a potential immunotherapy for autoimmune GN. Our previous studies suggested that glomeruli-infiltrating leukocytes with a phenotype of CD8αα+CD11c+MHC-II+CD3- (GIL CD8αα+ cells) were responsible for recovery through induction of T-cell apoptosis. Now, we identified peripheral blood CD8αα+CD11c+MHC-II+CD3- cells (PBMC CD8αα+CD3- cells), which shared 9 markers with GIL CD8αα+ cells. Upon incubation, PBMC CD8αα+CD3- cells displayed a morphology resembling that of dendritic cells. Similar to GIL CD8αα+ cells, PBMC CD8αα+CD3- cells were capable of inducing T-cell apoptosis in vitro. Hence, PBMC CD8αα+CD3- cells were likely the precursor of GIL CD8αα+ cells. We next tested their potential in vivo function. PBMC CD8αα+CD3- cells were able to infiltrate inflamed but not normal glomeruli. Isolated PBMC CD8αα+CD3- cells of Lewis rats were transferred into GN-prone Wistar-Kyoto rats at early inflammatory stage (days 17-25). When examined at day 45, both histopathology and blood urea nitrogen/serum creatinine level showed significantly attenuated GN in 80% of cell recipient Wistar-Kyoto rats. Separate experiments verified infiltration of transferred Lewis PBMC CD8αα+CD3- into the glomeruli, accompanied with apoptotic CD4+ T cells in the glomeruli of the recipient Wistar-Kyoto rats. Thus, PBMC CD8αα+CD3- cells of Lewis rats were able to terminate ongoing autoimmune inflammation in the glomeruli.

  3. Reversal of type 1 diabetes by a new MHC II-peptide chimera: "Single-epitope-mediated suppression" to stabilize a polyclonal autoimmune T-cell process.

    PubMed

    Lin, Marvin; Stoica-Nazarov, Cristina; Surls, Jacqueline; Kehl, Margaret; Bona, Constantin; Olsen, Cara; Brumeanu, Teodor D; Casares, Sofia

    2010-08-01

    Polyclonality of self-reactive CD4(+) T cells is the hallmark of several autoimmune diseases like type 1 diabetes. We have previously reported that a soluble dimeric MHC II-peptide chimera prevents and reverses type 1 diabetes induced by a monoclonal diabetogenic T-cell population in double Tg mice [Casares, S. et al., Nat. Immunol. 2002. 3: 383-391]. Since most of the glutamic acid decarboxylase 65 (GAD65)-specific CD4(+) T cells in the NOD mouse are tolerogenic but unable to function in an autoimmune environment, we have activated a silent, monoclonal T-regulatory cell population (GAD65(217-230)-specific CD4(+) T cells) using a soluble I-A(αβ) (g7)/GAD65(217-230)/Fcγ2a dimer, and measured the effect on the ongoing polyclonal diabetogenic T-cell process. Activated GAD65(217-230)-specific T cells and a fraction of the diabetogenic (B(9-23)-specific) T cells were polarized toward the IL-10-secreting T-regulatory type 1-like function in the pancreas of diabetic NOD mice. More importantly, this led to the reversal of hyperglycemia for more than 2 months post-therapy in 80% of mice in the context of stabilization of pancreatic insulitis and improved insulin secretion by the β cells. These findings argue for the stabilization of a polyclonal self-reactive T-cell process by a single epitope-mediated bystander suppression. Dimeric MHC class II-peptide chimeras-like approach may provide rational grounds for the development of more efficient antigen-specific therapies in type 1 diabetes.

  4. Deficient Peptide Loading and MHC Class II Endosomal Sorting in a Human Genetic Immunodeficiency Disease: the Chediak-Higashi Syndrome

    PubMed Central

    Faigle, Wolfgang; Raposo, Graça; Tenza, Daniele; Pinet, Valérie; Vogt, Anne B.; Kropshofer, Harald; Fischer, Alain; de Saint-Basile, Geneviève; Amigorena, Sebastian

    1998-01-01

    The Chediak-Higashi syndrome (CHS) is a human recessive autosomal disease caused by mutations in a single gene encoding a protein of unknown function, called lysosomal-trafficking regulator. All cells in CHS patients bear enlarged lysosomes. In addition, T- and natural killer cell cytotoxicity is defective in these patients, causing severe immunodeficiencies. We have analyzed major histocompatibility complex class II functions and intracellular transport in Epstein Barr Virus–transformed B cells from CHS patients. Peptide loading onto major histocompatibility complex class II molecules and antigen presentation are strongly delayed these cells. A detailed electron microscopy analysis of endocytic compartments revealed that only lysosomal multilaminar compartments are enlarged (reaching 1–2 μm), whereas late multivesicular endosomes have normal size and morphology. In contrast to giant multilaminar compartments that bear most of the usual lysosomal markers in these cells (HLA-DR, HLA-DM, Lamp-1, CD63, etc.), multivesicular late endosomes displayed reduced levels of all these molecules, suggesting a defect in transport from the trans-Golgi network and/or early endosomes into late multivesicular endosomes. Further insight into a possible mechanism of this transport defect came from immunolocalizing the lysosomal trafficking regulator protein, as antibodies directed to a peptide from its COOH terminal domain decorated punctated structures partially aligned along microtubules. These results suggest that the product of the Lyst gene is required for sorting endosomal resident proteins into late multivesicular endosomes by a mechanism involving microtubules. PMID:9606205

  5. Deficient peptide loading and MHC class II endosomal sorting in a human genetic immunodeficiency disease: the Chediak-Higashi syndrome.

    PubMed

    Faigle, W; Raposo, G; Tenza, D; Pinet, V; Vogt, A B; Kropshofer, H; Fischer, A; de Saint-Basile, G; Amigorena, S

    1998-06-01

    The Chediak-Higashi syndrome (CHS) is a human recessive autosomal disease caused by mutations in a single gene encoding a protein of unknown function, called lysosomal-trafficking regulator. All cells in CHS patients bear enlarged lysosomes. In addition, T- and natural killer cell cytotoxicity is defective in these patients, causing severe immunodeficiencies. We have analyzed major histocompatibility complex class II functions and intracellular transport in Epstein Barr Virus-transformed B cells from CHS patients. Peptide loading onto major histocompatibility complex class II molecules and antigen presentation are strongly delayed these cells. A detailed electron microscopy analysis of endocytic compartments revealed that only lysosomal multilaminar compartments are enlarged (reaching 1-2 micron), whereas late multivesicular endosomes have normal size and morphology. In contrast to giant multilaminar compartments that bear most of the usual lysosomal markers in these cells (HLA-DR, HLA-DM, Lamp-1, CD63, etc.), multivesicular late endosomes displayed reduced levels of all these molecules, suggesting a defect in transport from the trans-Golgi network and/or early endosomes into late multivesicular endosomes. Further insight into a possible mechanism of this transport defect came from immunolocalizing the lysosomal trafficking regulator protein, as antibodies directed to a peptide from its COOH terminal domain decorated punctated structures partially aligned along microtubules. These results suggest that the product of the Lyst gene is required for sorting endosomal resident proteins into late multivesicular endosomes by a mechanism involving microtubules.

  6. NLRC5 controls basal MHC class I gene expression in an MHC enhanceosome-dependent manner.

    PubMed

    Neerincx, Andreas; Rodriguez, Galaxia M; Steimle, Viktor; Kufer, Thomas A

    2012-05-15

    Nucleotide-binding domain and leucine-rich repeat (NLR) proteins play important roles in innate immune responses as pattern-recognition receptors. Although most NLR proteins act in cell autonomous immune pathways, some do not function as classical pattern-recognition receptors. One such NLR protein is the MHC class II transactivator, the master regulator of MHC class II gene transcription. In this article, we report that human NLRC5, which we recently showed to be involved in viral-mediated type I IFN responses, shuttles to the nucleus and activates MHC class I gene expression. Knockdown of NLRC5 in different human cell lines and primary dermal fibroblasts leads to reduced MHC class I expression, whereas introduction of NLRC5 into cell types with very low expression of MHC class I augments MHC class I expression to levels comparable to those found in lymphocytes. Expression of NLRC5 positively correlates with MHC class I expression in human tissues. Functionally, we show that both the N-terminal effector domain of NLRC5 and its C-terminal leucine-rich repeat domain are needed for activation of MHC class I expression. Moreover, nuclear shuttling and function depend on a functional Walker A motif. Finally, we identified a promoter sequence in the MHC class I promoter, the X1 box, to be involved in NLRC5-mediated MHC class I gene activation. Taken together, this suggested that NLRC5 acts in a manner similar to class II transactivator to drive MHC expression and revealed NLRC5 as an important regulator of basal MHC class I expression.

  7. Enhanced and sustained CD8+ T cell responses with an adenoviral vector-based hepatitis C virus vaccine encoding NS3 linked to the MHC class II chaperone protein invariant chain.

    PubMed

    Mikkelsen, Marianne; Holst, Peter Johannes; Bukh, Jens; Thomsen, Allan Randrup; Christensen, Jan Pravsgaard

    2011-02-15

    Potent and broad cellular immune responses against the nonstructural (NS) proteins of hepatitis C virus (HCV) are associated with spontaneous viral clearance. In this study, we have improved the immunogenicity of an adenovirus (Ad)-based HCV vaccine by fusing NS3 from HCV (Strain J4; Genotype 1b) to the MHC class II chaperone protein invariant chain (Ii). We found that, after a single vaccination of C57BL/6 or BALB/c mice with Ad-IiNS3, the HCV NS3-specific CD8(+) T cell responses were significantly enhanced, accelerated, and prolonged compared with the vaccine encoding NS3 alone. The AdIiNS3 vaccination induced polyfunctional CD8(+) T cells characterized by coproduction of IFN-γ, TNF-α and IL-2, and this cell phenotype is associated with good viral control. The memory CD8(+) T cells also expressed high levels of CD27 and CD127, which are markers of long-term survival and maintenance of T cell memory. Functionally, the AdIiNS3-vaccinated mice had a significantly increased cytotoxic capacity compared with the AdNS3 group. The AdIiNS3-induced CD8(+) T cells protected mice from infection with recombinant vaccinia virus expressing HCV NS3 of heterologous 1b strains, and studies in knockout mice demonstrated that this protection was mediated primarily through IFN-γ production. On the basis of these promising results, we suggest that this vaccination technology should be evaluated further in the chimpanzee HCV challenge model.

  8. Polymorphism in the major histocompatibility complex (MHC class II B) genes of the Rufous-backed Bunting (Emberiza jankowskii)

    PubMed Central

    Li, Dan; Zhao, Yunjiao; Lin, Aiqing; Li, Shi; Feng, Jiang

    2017-01-01

    Genetic diversity is one of the pillars of conservation biology research. High genetic diversity and abundant genetic variation in an organism may be suggestive of capacity to adapt to various environmental changes. The major histocompatibility complex (MHC) is known to be highly polymorphic and plays an important role in immune function. It is also considered an ideal model system to investigate genetic diversity in wildlife populations. The Rufous-backed Bunting (Emberiza jankowskii) is an endangered species that has experienced a sharp decline in both population and habitat size. Many historically significant populations are no longer present in previously populated regions, with only three breeding populations present in Inner Mongolia (i.e., the Aolunhua, Gahaitu and Lubei557 populations). Efforts focused on facilitating the conservation of the Rufous-backed Bunting (Emberiza jankowskii) are becoming increasingly important. However, the genetic diversity of E. jankowskii has not been investigated. In the present study, polymorphism in exon 2 of the MHCIIB of E. jankowskii was investigated. This polymorphism was subsequently compared with a related species, the Meadow Bunting (Emberiza cioides). A total of 1.59 alleles/individual were detected in E. jankowskii and 1.73 alleles/individual were identified in E. cioides. The maximum number of alleles per individual from the three E. jankowskii populations suggest the existence of at least three functional loci, while the maximum number of alleles per individual from the three E. cioides populations suggest the presence of at least four functional loci. Two of the alleles were shared between the E. jankowskii and E. cioides. Among the 12 unique alleles identified in E. jankowskii, 10.17 segregating sites per allele were detected, and the nucleotide diversity was 0.1865. Among the 17 unique alleles identified in E. cioides, eight segregating sites per allele were detected, and the nucleotide diversity was 0

  9. The diversity of bovine MHC class II DRB3 genes in Japanese Black, Japanese Shorthorn, Jersey and Holstein cattle in Japan.

    PubMed

    Takeshima, S; Saitou, N; Morita, M; Inoko, H; Aida, Y

    2003-10-16

    We sequenced exon 2 of the major histocompatibility complex (MHC) class II DRB3 gene from 471 individuals in four different Japanese populations of cattle (201 Japanese Black, 101 Holstein, 100 Japanese Shorthorn, and 69 Jersey cattle) using a new method for sequence-based typing (SBT). We identified the 34 previously reported alleles and four novel alleles. These alleles were 80.0-100.0% identical at the nucleotide level and 77.9-100.0% identical at the amino acid level to the bovine MHC (BoLA)-DRB3 cDNA clone NR1. Among the 38 alleles, eight alleles were found in only one breed in this study. However, these alleles did not form specific clusters on a phylogenetic tree of 236-base pairs (bp) nucleotide sequences. Furthermore, these breeds exhibited similar variations with respect to average frequencies of nucleotides and amino acids, as well as synonymous and non-synonymous substitutions, in all pairwise comparisons of the alleles found in this study. By contrast, analysis of the frequencies of the various BoLA-DRB3 alleles in each breed indicated that DRB3*1101 was the most frequent allele in Holstein cattle (16.8%), DRB3*4501 was the most frequent allele in Jersey cattle (18.1%), DRB3*1201 was the most frequent allele in Japanese Shorthorn cattle (16.0%) and DRB3*1001 was the most frequent allele in Japanese Black cattle (17.4%), indicating that the frequencies of alleles were differed in each breed. In addition, a population tree based on the frequency of BoLA-DRB3 alleles in each breed suggested that Holstein and Japanese Black cattle were the most closely related, and that Jersey cattle were more different from both these breeds than Japanese Shorthorns.

  10. Chemical biology of antigen presentation by MHC molecules.

    PubMed

    van Kasteren, Sander I; Overkleeft, Hermen; Ovaa, Huib; Neefjes, Jacques

    2014-02-01

    MHC class I and MHC class II molecules present peptides to the immune system to drive proper T cell responses. Pharmacological modulation of T-cell responses can offer treatment options for a range of immune-related diseases. Pharmacological downregulation of MHC molecules may find application in treatment of auto-immunity and transplantation rejection while pharmacological activation of antigen presentation would support immune responses to infection and cancer. Since the cell biology of MHC class I and MHC class II antigen presentation is understood in great detail, many potential targets for manipulation have been defined over the years. Here, we discuss how antigen presentation by MHC molecules can be modulated by pharmacological agents and how chemistry can further support the study of antigen presentation in general. The chemical biology of antigen presentation by MHC molecules shows surprising options for immune modulation and the development of future therapies. Copyright © 2013 Elsevier Ltd. All rights reserved.

  11. Antigen Presentation by MHC-Dressed Cells

    PubMed Central

    Nakayama, Masafumi

    2015-01-01

    Professional antigen-presenting cells (APCs) such as conventional dendritic cells (DCs) process protein antigens to MHC-bound peptides and then present the peptide–MHC complexes to T cells. In addition to this canonical antigen presentation pathway, recent studies have revealed that DCs and non-APCs can acquire MHC class I (MHCI) and/or MHC class II (MHCII) from neighboring cells through a process of cell–cell contact-dependent membrane transfer called trogocytosis. These MHC-dressed cells subsequently activate or regulate T cells via the preformed antigen peptide–MHC complexes without requiring any further processing. In addition to trogocytosis, intercellular transfer of MHCI and MHCII can be mediated by secretion of membrane vesicles such as exosomes from APCs, generating MHC-dressed cells. This review focuses on the physiological role of antigen presentation by MHCI- or MHCII-dressed cells, and also discusses differences and similarities between trogocytosis and exosome-mediated transfer of MHC. PMID:25601867

  12. Inhibitory effects of thymus-independent type 2 antigens on MHC class II-restricted antigen presentation: comparative analysis of carbohydrate structures and the antigen presenting cell.

    PubMed

    González-Fernández, M; Carrasco-Marín, E; Alvarez-Domínguez, C; Outschoorn, I M; Leyva-Cobián, F

    1997-02-25

    The role of thymus-independent type 2 (TI-2) antigens (polysaccharides) on the MHC-II-restricted processing of protein antigens was studied in vitro. In general, antigen presentation is inhibited when both peritoneal and splenic macrophages (M phi) as well as Küpffer cells (KC) are preincubated with acidic polysaccharides or branched dextrans. However, the inhibitory effect of neutral polysaccharides was minimal when KC were used as antigen presenting cells (APC). Morphological evaluation of the uptake of fluoresceinated polysaccharides clearly correlates with this selective and differential interference. Polysaccharides do not block MHC-I-restricted antigen presentation. Some chemical characteristics shared by different saccharides seem to be specially related to their potential inhibitory abilities: (i) those where two anomeric carbon atoms of two interlinked sugars and (ii) those containing several sulfate groups per disaccharide repeating unit. No polysaccharide being inhibitory in M phi abrogated antigen processing in other APC: lipopolysaccharide-activated B cells, B lymphoma cells, or dendritic cells (DC). Using radiolabeled polysaccharides it was observed that DC and B cells incorporated less radioactivity as a function of time than M phi. Morphological evaluation of these different APC incubated for extended periods of time with inhibitory concentrations of polysaccharides revealed intense cytoplasmic vacuolization in M phi but not in B cells or DC. The large majority of M phi lysosomes containing polysaccharides fail to fuse with incoming endocytic vesicles and delivery of fluid-phase tracers was reduced, suggesting that indigestible carbohydrates reduced the fusion of these loaded lysosomes with endosomes containing recently internalized tracers. It is suggested that the main causes of this antigen presentation blockade are (i) the chemical characteristics of certain carbohydrates and whether the specific enzymatic machinery for their intracellular

  13. CITA/NLRC5: A critical transcriptional regulator of MHC class I gene expression.

    PubMed

    Downs, Isaac; Vijayan, Saptha; Sidiq, Tabasum; Kobayashi, Koichi S

    2016-07-08

    Major histocompatibility complex (MHC) class I and class II molecules play essential roles in the development and activation of the human adaptive immune system. An NLR protein, CIITA (MHC class II transactivator) has been recognized as a master regulator of MHC class II gene expression, albeit knowledge about the regulatory mechanism of MHC class I gene expression had been limited. Recently identified MHC class I transactivator (CITA), or NLRC5, also belongs to the NLR protein family and constitutes a critical regulator for the transcriptional activation of MHC class I genes. In addition to MHC class I genes, CITA/NLRC5 induces the expression of β2 -microglobulin, TAP1 and LMP2, essential components of the MHC class I antigen presentation pathway. Therefore, CITA/NLRC5 and CIITA are transcriptional regulators that orchestrate the concerted expression of critical components in the MHC class I and class II pathways, respectively. © 2016 BioFactors, 42(4):349-357, 2016.

  14. Influence of pre-ovulatory insemination and early pregnancy on the distribution of CD2, CD4, CD8 and MHC class II expressing cells in the sow endometrium.

    PubMed

    Kaeoket, K; Persson, E; Dalin, A-M

    2003-04-15

    The aim was to investigate the distribution of CD2(+), CD4(+) and CD8(+) lymphocyte subpopulations and MHC class II expressing cells in the sow endometrium following pre-ovulatory insemination and during early pregnancy. Crossbred multiparous sows (Swedish Landrace x Swedish Yorkshire) were inseminated once at 15-20 h before ovulation. The sows were slaughtered at 5-6h (group I, n=4) after AI or at 20-25 h (group II, n=4) and 70 h (group III, n=4) after ovulation, day 11 (group IV, day 1=first day of standing oestrus, n=3) and day 19 (group V, n=3). Uterine horns were flushed to control for the presence of spermatozoa and neutrophils (groups I-IV) and/or for recovery of oocytes and/or embryos (groups II-IV, control of pregnancy). Cryofixed mesometrial uterine samples were analysed by immunohistochemistry with an avidin-biotin-peroxidase method using monoclonal antibodies to lymphocyte subpopulations and MHC class II molecules. The surface (SE) and glandular (GE) epithelia as well as connective tissue layers in subepithelial (SL) and glandular (GL) areas were examined by light microscopy. Taking all groups and different tissue layers together, the most commonly observed positive cells were CD2(+) cells (PII, and the smallest number in group V. In the SE and GE, more CD8(+) (T cytotoxic) cells were observed than CD4(+) (T helper) cells. In the SL and GL, the largest numbers of CD2(+), CD4(+) and CD8(+) cells were found in group V. Taking all groups together, a larger number of CD4(+) cells compared with CD8(+) cells were found. For the proportion of (CD4(+)+CD8(+))/CD2(+) cells, there were significantly (PMHC class II expressing cells in the SE was observed in groups I, II and III compared with the other groups. In the SL, a larger number of MHC class II expressing cells was observed in groups

  15. CONSTRUCTION AND EXPRESSION OF DERMATOPHAGOIDES PTERONYSSINUS GROUP 1 MAJOR ALLERGEN T CELL FUSION EPITOPE PEPTIDE VACCINE VECTOR BASED ON THE MHC II PATHWAY.

    PubMed

    Li, Chaopin; Zhao, Beibei; Jiang, Yuxin; Diao, Jidong; Li, Na; Lu, Wei

    2015-11-01

    Antecedentes y objetivo: el Dermatophagoides peteronyssinus es uno de los principales ácaros del polvo doméstico responsables del asma alérgica que se pueden administrar provisionalmente para una inmunoterapia específica. El presente estudio busca construir un vector que codifique epítopos de células T del grupo de alérgenos principal, el Grupo 1 de Dermatophagoides pteronyssinus como una vacuna suministrada mediante la vía MHC de clase II. Métodos: se sintetizaron las secuencias de nucleótidos de los 3 genes objetivo, incluyendo TAT, IhC y el fragmento recombinante de Der p 1 encargado de codificar 3 epítopos de célula T. Después de la amplificación de los 3 fragmentos objetivo por PCR y digestión con endonucleasas de restricción correspondientes, el gen recombinante TAT-IhC-Der p 1-3T se ligó usando T4 DNA ligasa y se insertó en el vector de expresión procariota pET28a (+) para construir el plásmido recombinante pET 28a (+)-TAT-IHC-Der p 1-3T, que se confirmó por digestión con endonucleasas de restricción y secuenciación. El vector recombinante se transformó en E. coli cepa BL21 (DE3) y se indujo con IPTG, y la proteína inducida TATIHC- Der p1-3T se detectó mediante SDS-PAGE. Después de la purificación, la proteina recombinante se confirmó por análisis de inmunotransferencia (Western blot) y se probó su alergenicidad usando el ensayo de unión a IgE. Resultados: el plásmido recombinante pET-28a-TATIHCDer p1-3T se construyó con éxito, se confirmó por digestión con endonucleasas de restricción y la secuenciación y la expresión de la proteína recombinante TAT-IHCDer p1-3T fue inducida en E. coli. Purificación con éxito verificada mediante Western blot de la proteína objetivo, que mostró una capacidad de unión a IgE más fuerte que Der p1. Conclusión: hemos construido con éxito el vector de expresión recombinante pET-28a-TAT-IHC-Der p1-3T que expresa una vacuna de epítopo de células T administrada por vía MHC II con

  16. B Cells Induce Tolerance by Presenting Endogenous Peptide-IgG on MHC Class II Molecules via an IFN-γ-Inducible Lysosomal Thiol Reductase-Dependent Pathway1

    PubMed Central

    Su, Yan; Carey, Gregory; Marić, Maja; Scott, David W.

    2008-01-01

    We have previously demonstrated that splenic B cells, transduced with peptide-IgG fusion proteins, are efficient tolerogenic APCs in vivo. Specific hyporesponsiveness to epitopes encoded in the peptide-IgG fusion protein has been achieved to over one dozen Ags, and clinical efficacy has been established in animal models for several autoimmune diseases and hemophilia. Previous studies also demonstrated that tolerance in this system requires MHC class II expression by the transduced B cells. Yet, the mechanisms of this B cell tolerogenic processing pathway remain unclear. In this study, we show that MHC class II molecules on tolerogenic B cells present epitopes derived from endogenous, but not exogenous (secreted), peptide-IgG fusion protein. These class II epitopes from the IgG fusion protein are processed in lysosomes/endosomes in an IFN-γ-inducible lysosomal thiol reductase-dependent manner. We suggest that the MHC class II presentation of endogenously produced fusion protein epitopes represents a novel mechanism for tolerance induced by peptide-IgG-transduced B cells. An understanding of this process might provide insights into central and peripheral tolerance induced by other professional and nonprofessional APCs. PMID:18606668

  17. Generation of CD4+ blastoid T cells showing marked upregulation of CD4, class I and II MHC, and IL2 receptor molecules is required for the expression of potent encephalitogenicity.

    PubMed

    Kira, J; Itoyama, Y; Goto, I

    1989-10-15

    The relationship between surface molecule expression and encephalitogenicity of myelin basic protein (BP)-sensitized cells induced by three different sensitization protocols was studied using adoptive transfer in Lewis rats. (i) In BP/CFA sensitization, CD4+ blastoid T cells showing marked upregulation of CD4, class I and II MHC, and IL2 receptor molecules, but not CD5, CD8, or CD45, were generated after culture with BP. In this case, BP-cultured cells were strongly encephalitogenic in the recipients. (ii) In the case of BP/IFA sensitization, CD4+ T cells showed no remarkable change of cell size or surface molecule expression after culture with BP and were weakly encephalitogenic in the recipients. Vigorous proliferation of the cells induced by addition of recombinant IL2 to the culture with BP neither enhanced the encephalitogenicity nor produced CD4+ blastoid T cells showing marked upregulation of CD4, class I and II MHC, and IL2 receptor molecules. (iii) The sequentially transferred naive T cells showed no remarkable change of cell size or surface molecule expression, even after a second culture with BP, and were the least encephalitogenic. These data suggest that the generation of CD4+ blastoid T cells showing marked upregulation of CD4, class I and II MHC, and IL2 receptor molecules but not vigorous proliferation correlates closely with the potent encephalitogenicity in vivo.

  18. Genomic organization of the crested ibis MHC provides new insight into ancestral avian MHC structure

    PubMed Central

    Chen, Li-Cheng; Lan, Hong; Sun, Li; Deng, Yan-Li; Tang, Ke-Yi; Wan, Qiu-Hong

    2015-01-01

    The major histocompatibility complex (MHC) plays an important role in immune response. Avian MHCs are not well characterized, only reporting highly compact Galliformes MHCs and extensively fragmented zebra finch MHC. We report the first genomic structure of an endangered Pelecaniformes (crested ibis) MHC containing 54 genes in three regions spanning ~500 kb. In contrast to the loose BG (26 loci within 265 kb) and Class I (11 within 150) genomic structures, the Core Region is condensed (17 within 85). Furthermore, this Region exhibits a COL11A2 gene, followed by four tandem MHC class II αβ dyads retaining two suites of anciently duplicated “αβ” lineages. Thus, the crested ibis MHC structure is entirely different from the known avian MHC architectures but similar to that of mammalian MHCs, suggesting that the fundamental structure of ancestral avian class II MHCs should be “COL11A2-IIαβ1-IIαβ2.” The gene structures, residue characteristics, and expression levels of the five class I genes reveal inter-locus functional divergence. However, phylogenetic analysis indicates that these five genes generate a well-supported intra-species clade, showing evidence for recent duplications. Our analyses suggest dramatic structural variation among avian MHC lineages, help elucidate avian MHC evolution, and provide a foundation for future conservation studies. PMID:25608659

  19. Mhc-linked survival and lifetime reproductive success in a wild population of great tits.

    PubMed

    Sepil, Irem; Lachish, Shelly; Sheldon, Ben C

    2013-01-01

    Major histocompatibility complex (Mhc) genes are frequently used as a model for adaptive genetic diversity. Although associations between Mhc and disease resistance are frequently documented, little is known about the fitness consequences of Mhc variation in wild populations. Further, most work to date has involved testing associations between Mhc genotypes and fitness components. However, the functional diversity of the Mhc, and hence the mechanism by which selection on Mhc acts, depends on how genotypes map to the functional properties of Mhc molecules. Here, we test three hypotheses that relate Mhc diversity to fitness: (i) the maximal diversity hypothesis, (ii) the optimal diversity hypothesis and (iii) effect of specific Mhc types. We combine mark-recapture methods with analysis of long-term breeding data to investigate the effects of Mhc class I functional diversity (Mhc supertypes) on individual fitness in a wild great tit (Parus major) population. We found that the presence of three different Mhc supertypes was associated with three different components of individual fitness: survival, annual recruitment and lifetime reproductive success (LRS). Great tits possessing Mhc supertype 3 experienced higher survival rates than those that did not, whereas individuals with Mhc supertype 6 experienced higher LRS and were more likely to recruit offspring each year. Conversely, great tits that possessed Mhc supertype 5 had reduced LRS. We found no evidence for a selective advantage of Mhc diversity, in terms of either maximal or optimal supertype diversity. Our results support the suggestion that specific Mhc types are an important determinant of individual fitness.

  20. Decreased expression of MHC class II and cathepsin E in dendritic cells might contribute to impaired induction of antigen-specific T cell response in NC/Nga mice.

    PubMed

    Sakai, Tohru; Shuto, Emi; Taki, Tomoyo; Imamura, Honami; Kioka, Miku; Nakamoto, Akiko; Mizoguchi, Seiji; Amano, Shouko; Kawano, Yukari; Nakamoto, Mariko; Tsutumi, Rie; Hosaka, Toshio

    2012-01-01

    NC/Nga (NC) mice are an animal model for human atopic dermatitis. We found that induction of antigen (Ag)-specific T cell response is diminished in ovalbumin (OVA)-immunized NC mice. Ability of Ag presentation in NC mouse dendritic cells (DCs) was significantly weaker than that in BALB/c and DBA/2 mouse DCs. Expression levels of MHC class II molecules and cathepsin E in NC mouse DCs were significantly lower that those in BALB/c and DBA/2 mouse DCs. These results indicate that low expression levels of MHC class II and cathepsin E might contribute to the defect in induction of Ag-specific T cells in NC mice.

  1. Genotypic characterization of an MHC class II locus in lake trout ( Salvelinus namaycush) from Lake Superior by single-stranded conformational polymorphism analysis and reference strand-mediated conformational analysis.

    PubMed

    Noakes, Marc A; Reimer, Tara; Phillips, Ruth B

    2003-01-01

    This study compares the genotypic information provided by reference strand-mediated conformational analysis and single-stranded confirmational polymorphism (SSCP) analysis for the major histocompatibility complex (MHC) II locus in lake trout. For this study 80 wild-caught animals from the Apostle Islands of Lake Superior were genotyped using both RSCA and SSCP analysis. Their genotypes were recorded using both methods and compared. The genotypic information provided by the 2 methods was essentially the same although some inconsistencies were observed. Both methods detected approximately 65 genotypes, and both were able to distinguish heterozygous and homozygous animals. The analyses determined that only approximately 20% of alleles were shared between 2 morphologically different populations within the sample set, and identified the dominant alleles. SSCP analysis was quicker, simple, and more robust than RSCA. SSCP analysis using fluorescence technologies could be the method of choice for future genotypic analysis of the MHC II locus in salmonids.

  2. MHC II lung cancer vaccines prime and boost tumor-specific CD4+ T cells that cross-react with multiple histologic subtypes of nonsmall cell lung cancer cells.

    PubMed

    Srivastava, Minu K; Bosch, Jacobus J; Wilson, Ashley L; Edelman, Martin J; Ostrand-Rosenberg, Suzanne

    2010-12-01

    Nonsmall cell lung cancer (NSCLC) is the major cause of lung cancer-related deaths in the United States. We are developing cell-based vaccines as a new approach for the treatment of NSCLC. NSCLC is broadly divided into 3 histologic subtypes: adenocarcinoma, squamous cell carcinoma and large cell carcinoma. Since these subtypes are derived from the same progenitor cells, we hypothesized that they share common tumor antigens, and vaccines that induce immune reactivity against 1 subtype may also induce immunity against other subtypes. Our vaccine strategy has focused on activating tumor-specific CD4(+) T cells, a population of lymphocytes that facilitates the optimal activation of effector and memory cytotoxic CD8(+) T cells. We now report that our NSCLC MHC II vaccines prepared from adeno, squamous or large cell carcinomas each activate CD4(+) T cells that cross-react with the other NSCLC subtypes and do not react with HLA-DR-matched normal lung fibroblasts or other HLA-DR-matched nonlung tumor cells. Using MHC II NSCLC vaccines expressing the DR1, DR4, DR7 or DR15 alleles, we also demonstrate that antigens shared among the different subtypes are presented by multiple HLA-DR alleles. Therefore, MHC II NSCLC vaccines expressing a single HLA-DR allele activate NSCLC-specific CD4(+) T cells that react with the 3 major classes of NSCLC, and the antigens recognized by the activated T cells are presented by several common HLA-DR alleles, suggesting that the MHC II NSCLC vaccines are potential immunotherapeutics for a range of NSCLC patients.

  3. MHC II Lung Cancer Vaccines Prime and Boost Tumor-specific CD4+ T Cells that Cross-React with Multiple Histologic Subtypes of Non-small Cell Lung Cancer Cells

    PubMed Central

    Srivastava, Minu K.; Bosch, Jacobus J.; Wilson, Ashley L.; Edelman, Martin J.; Ostrand-Rosenberg, Suzanne

    2010-01-01

    Non-small cell lung cancer (NSCLC) is the major cause of lung cancer-related deaths in the United States. We are developing cell-based vaccines as a new approach for the treatment of NSCLC. NSCLC is broadly divided into three histologic subtypes: adenocarcinoma, squamous cell carcinoma, and large cell carcinoma. Since these subtypes are derived from the same progenitor cells, we hypothesized that they share common tumor antigens and vaccines that induce immune reactivity against one subtype may also induce immunity against other subtypes. Our vaccine strategy has focused on activating tumor-specific CD4+ T cells, a population of lymphocytes that facilitates the optimal activation of effector and memory cytotoxic CD8+ T cells. We now report that our NSCLC MHC II vaccines prepared from adeno, squamous, or large cell carcinomas each activate CD4+ T cells that cross-react with the other NSCLC subtypes and do not react with HLA-DR-matched normal lung fibroblasts or other HLA-DR-matched non-lung tumor cells. Using MHC II NSCLC vaccines expressing the DR1, DR4, DR7, or DR15 alleles, we also demonstrate that antigens shared among the different subtypes are presented by multiple HLA-DR alleles. Therefore, MHC II NSCLC vaccines expressing a single HLA-DR allele activate NSCLC-specific CD4+ T cells that react with the three major classes of NSCLC and the antigens recognized by the activated T cells are presented by several common HLA-DR alleles, suggesting that the MHC II NSCLC vaccines are potential immunotherapeutics for a range of NSCLC patients. PMID:20473949

  4. Prevention of soya-induced enteritis in Atlantic salmon (Salmo salar) by bacteria grown on natural gas is dose dependent and related to epithelial MHC II reactivity and CD8α+ intraepithelial lymphocytes.

    PubMed

    Romarheim, Odd H; Hetland, Dyveke L; Skrede, Anders; Øverland, Margareth; Mydland, Liv T; Landsverk, Thor

    2013-03-28

    An experiment was carried out to study the preventive effect of bacterial meal (BM) produced from natural gas against plant-induced enteropathy in Atlantic salmon (Salmo salar). Salmon were fed a diet based on fish meal (FM) or seven diets with 200 g/kg solvent-extracted soyabean meal (SBM) to induce enteritis in combination with increasing levels of BM from 0 to 300 g/kg. Salmon fed a SBM-containing diet without BM developed typical SBM-induced enteritis. The enteritis gradually disappeared with increasing inclusion of BM. By morphometry, no significant (P>0.05) differences in the size of stretches stained for proliferating cell nuclear antigen were found with 150 g/kg BM compared with the FM diet. Increasing BM inclusion caused a gradual decline in the number of cluster of differentiation 8 α positive (CD8α+) intraepithelial lymphocytes, and fish fed BM at 200 g/kg or higher revealed no significant difference from the FM diet. Histological sections stained with antibody for MHC class II (MHC II) showed that fish with intestinal inflammation had more MHC II-reactive cells in the lamina propria and submucosa, but less in the epithelium and brush border, compared with fish without inflammation. There were no significant (P>0.05) differences in growth among the diets, but the highest levels of BM slightly reduced protein digestibility and increased the weight of the distal intestine. In conclusion, the prevention of SBM-induced enteritis by BM is dose dependent and related to intestinal levels of MHC II- and CD8α-reactive cells.

  5. Host DNA released in response to aluminum adjuvant enhances MHC class II-mediated antigen presentation and prolongs CD4 T-cell interactions with dendritic cells

    PubMed Central

    McKee, Amy S.; Burchill, Matthew A.; Munks, Michael W.; Jin, Lei; Kappler, John W.; Friedman, Rachel S.; Jacobelli, Jordan; Marrack, Philippa

    2013-01-01

    Many vaccines include aluminum salts (alum) as adjuvants despite little knowledge of alum’s functions. Host DNA rapidly coats injected alum. Here, we further investigated the mechanism of alum and DNA’s adjuvant function. Our data show that DNase coinjection reduces CD4 T-cell priming by i.m. injected antigen + alum. This effect is partially replicated in mice lacking stimulator of IFN genes, a mediator of cellular responses to cytoplasmic DNA. Others have shown that DNase treatment impairs dendritic cell (DC) migration from the peritoneal cavity to the draining lymph node in mice immunized i.p. with alum. However, our data show that DNase does not affect accumulation of, or expression of costimulatory proteins on, antigen-loaded DCs in lymph nodes draining injected muscles, the site by which most human vaccines are administered. DNase does inhibit prolonged T-cell–DC conjugate formation and antigen presentation between antigen-positive DCs and antigen-specific CD4 T cells following i.m. injection. Thus, from the muscle, an immunization site that does not require host DNA to promote migration of inflammatory DCs, alum acts as an adjuvant by introducing host DNA into the cytoplasm of antigen-bearing DCs, where it engages receptors that promote MHC class II presentation and better DC–T-cell interactions. PMID:23447566

  6. Down-regulation of poison ivy/oak-induced contact sensitivity by treatment with a class II MHC binding peptide:hapten conjugate.

    PubMed

    Gelber, C; Gemmell, L; McAteer, D; Homola, M; Swain, P; Liu, A; Wilson, K J; Gefter, M

    1997-03-01

    Immune regulation of contact sensitivity to the poison ivy/oak catechol was studied at the level of class II MHC-restricted T cell recognition of hapten:peptide conjugates. In this study we have shown that 1) T cells from C3H/HeN (H-2k) mice, immunized with a synthetic I-Ak binding peptide coupled to 3-pentadecyl-catechol (PDC; a representative catechol in urushiol), recognized peptides derived from syngeneic cells linked to the same catechol; 2) T cells from draining lymph nodes of C3H/HeN mice skin-painted with PDC proliferated in response to a peptide carrier:PDC conjugate only when it was linked at the 7th, but not the 4th or the 10th, position on the peptide carrier; and 3) tolerization studies confirmed down-regulation of PDC-induced delayed-type hypersensitivity following treatment with a single I-Ak binding peptide carrying PDC covalently bound to a lysine residue at the middle (7th) TCR contact position. Tolerization with peptide:PDC conjugate resulted in abrogation of hapten-specific T cell proliferative responses that correlated with diminished IL-2 secretion. On the basis of these data we propose that it may be sufficient to couple the hapten at a single, well-chosen position on a carrier peptide to target a relevant population of T cells involved in contact sensitivity.

  7. H2-O, a MHC class II-like protein, sets a threshold for B-cell entry into germinal centers.

    PubMed

    Draghi, Nicole A; Denzin, Lisa K

    2010-09-21

    Upon antigen (Ag) encounter, B cells require T-cell help to enter the germinal center (GC). They obtain this help by presenting Ag-derived peptides on MHC class II (MHCII) for recognition by the T-cell receptor (TCR) of CD4(+) T cells. Peptides are loaded onto MHCII in endosomal compartments in a process catalyzed by the MHCII-like protein H2-M (HLA-DM in humans). This process is modulated by another MHCII-like protein, H2-O (HLA-DO in humans). H2-O is a biochemical inhibitor of peptide loading onto MHCII; however, on the cellular level, it has been shown to have varying effects on Ag presentation. Thus, the function of H2-O in the adaptive immune response remains unclear. Here, we examine the effect of H2-O expression on the ability of Ag-specific B cells to enter the GC. We show that when Ag specific WT and H2-O(-/-) B cells are placed in direct competition, H2-O(-/-) B cells preferentially populate the GC. This advantage is confined to Ag-specific B cells and is due to their superior ability to obtain Ag-specific T-cell help when T-cell help is limiting. Overall, our work shows that H2-O expression reduces the ability of B cells to gain T-cell help and participate in the GC reaction.

  8. Pandemic 2009 H1N1 Influenza Venus reporter virus reveals broad diversity of MHC class II-positive antigen-bearing cells following infection in vivo.

    PubMed

    DiPiazza, Anthony; Nogales, Aitor; Poulton, Nicholas; Wilson, Patrick C; Martínez-Sobrido, Luis; Sant, Andrea J

    2017-09-07

    Although it is well established that Influenza A virus infection is initiated in the respiratory tract, the sequence of events and the cell types that become infected or access viral antigens remains incompletely understood. In this report, we used a novel Influenza A/California/04/09 (H1N1) reporter virus that stably expresses the Venus fluorescent protein to identify antigen-bearing cells over time in a mouse model of infection using flow cytometry. These studies revealed that many hematopoietic cells, including subsets of monocytes, macrophages, dendritic cells, neutrophils and eosinophils acquire influenza antigen in the lungs early post-infection. Surface staining of the viral HA revealed that most cell populations become infected, most prominently CD45(neg) cells, alveolar macrophages and neutrophils. Finally, differences in infection status, cell lineage and MHC class II expression by antigen-bearing cells correlated with differences in their ability to re-stimulate influenza-specific CD4 T cells ex vivo. Collectively, these studies have revealed the cellular heterogeneity and complexity of antigen-bearing cells within the lung and their potential as targets of antigen recognition by CD4 T cells.

  9. MHC II tetramers visualize human CD4+ T cell responses to Epstein-Barr virus infection and demonstrate atypical kinetics of the nuclear antigen EBNA1 response.

    PubMed

    Long, Heather M; Chagoury, Odette L; Leese, Alison M; Ryan, Gordon B; James, Eddie; Morton, Laura T; Abbott, Rachel J M; Sabbah, Shereen; Kwok, William; Rickinson, Alan B

    2013-05-06

    Virus-specific CD4(+) T cells are key orchestrators of host responses to viral infection yet, compared with their CD8(+) T cell counterparts, remain poorly characterized at the single cell level. Here we use nine MHC II-epitope peptide tetramers to visualize human CD4(+) T cell responses to Epstein-Barr virus (EBV), the causative agent of infectious mononucleosis (IM), a disease associated with large virus-specific CD8(+) T cell responses. We find that, while not approaching virus-specific CD8(+) T cell expansions in magnitude, activated CD4(+) T cells specific for epitopes in the latent antigen EBNA2 and four lytic cycle antigens are detected at high frequencies in acute IM blood. They then fall rapidly to values typical of life-long virus carriage where most tetramer-positive cells display conventional memory markers but some, unexpectedly, revert to a naive-like phenotype. In contrast CD4(+) T cell responses to EBNA1 epitopes are greatly delayed in IM patients, in line with the well-known but hitherto unexplained delay in EBNA1 IgG antibody responses. We present evidence from an in vitro system that may explain these unusual kinetics. Unlike other EBNAs and lytic cycle proteins, EBNA1 is not naturally released from EBV-infected cells as a source of antigen for CD4(+) T cell priming.

  10. Evidence for the ‘Good Genes’ Model: Association of MHC Class II DRB Alleles with Ectoparasitism and Reproductive State in the Neotropical Lesser Bulldog Bat, Noctilio albiventris

    PubMed Central

    Schad, Julia; Dechmann, Dina K. N.; Voigt, Christian C.; Sommer, Simone

    2012-01-01

    The adaptive immune system has a major impact on parasite resistance and life history strategies. Immunological defence is costly both in terms of immediate activation and long-term maintenance. The ‘good genes’ model predicts that males with genotypes that promote a good disease resistance have the ability to allocate more resources to reproductive effort which favours the transmission of good alleles into future generations. Our study shows a correlation between immune gene constitution (Major Histocompatibility Complex, MHC class II DRB), ectoparasite loads (ticks and bat flies) and the reproductive state in a neotropical bat, Noctilio albiventris. Infestation rates with ectoparasites were linked to specific Noal-DRB alleles, differed among roosts, increased with body size and co-varied with reproductive state particularly in males. Non-reproductive adult males were more infested with ectoparasites than reproductively active males, and they had more often an allele (Noal-DRB*02) associated with a higher tick infestation than reproductively active males or subadults. We conclude that the individual immune gene constitution affects ectoparasite susceptibility, and contributes to fitness relevant trade-offs in male N. albiventris as suggested by the ‘good genes’ model. PMID:22615910

  11. Fc receptor density, MHC antigen expression and superoxide production are increased in interferon-gamma-treated microglia isolated from adult rat brain.

    PubMed Central

    Woodroofe, M N; Hayes, G M; Cuzner, M L

    1989-01-01

    Treatment of microglia isolated from adult rat brain with interferon-gamma (IFN-gamma) at a concentration of 1 U/ml resulted in enhanced expression of Fc receptors and major histocompatibility complex (MHC) antigens and increased production of superoxide anions. Neonatal microglia and peritoneal macrophages, isolated and cultured in the same manner, displayed functional properties very similar to those of adult microglia, indicating a common origin for different macrophage populations. The Fc binding capacity of microglia was found to be significantly greater than that of peritoneal cells, thus underlining the potential role of microglia in immune-mediated demyelination. PMID:2556346

  12. An immunocytochemical study of pulpal responses to cavity preparation by laser ablation in rat molars by using antibodies to heat shock protein (Hsp) 25 and class II MHC antigen.

    PubMed

    Suzuki, Takeshi; Nomura, Shuichi; Maeda, Takeyasu; Ohshima, Hayato

    2004-03-01

    Initial responses of odontoblasts and immunocompetent cells to cavity preparation by laser ablation were investigated in rat molars. In untreated control teeth, intense heat shock protein (Hsp) 25 immunoreactivity was found in the cell bodies of odontoblasts, whereas cells immunopositive for the class II major histocompatibility complex (MHC) antigen were predominantly located beneath the odontoblast layer in the dental pulp. Cavity preparation caused the destruction of the odontoblast layer and the shift of most class-II-MHC-positive cells from the pulp-dentin border toward the pulp core at the affected site. Twelve hours after cavity preparation, numerous class-II-MHC-positive cells appeared along the pulp-dentin border and extended their processes deep into the exposed dentinal tubules, but subsequently disappeared from the pulp-dentin border together with Hsp-25-immunopositive cells by 24 h after the operation. By 3-5 days postoperation, distinct abscess formation consisting of polymorphonuclear leukocytes was found in the dental pulp. The penetration of masses of oral bacteria was recognizable in the dentinal tubules beneath the prepared cavity. These findings indicate that cavity preparation by laser ablation induces remarkable inflammation by continuous bacterial infections via dentinal tubules in this experimental model, thereby delaying pulpal regeneration.

  13. A recombinant single-chain human class II MHC molecule (HLA-DR1) as a covalently linked heterotrimer of alpha chain, beta chain, and antigenic peptide, with immunogenicity in vitro and reduced affinity for bacterial superantigens.

    PubMed

    Zhu, X; Bavari, S; Ulrich, R; Sadegh-Nasseri, S; Ferrone, S; McHugh, L; Mage, M

    1997-08-01

    Major histocompatibility complex (MHC) class II molecules bind to numerous peptides and display these on the cell surface for T cell recognition. In a given immune response, receptors on T cells recognize antigenic peptides that are a minor population of MHC class II-bound peptides. To control which peptides are presented to T cells, it may be desirable to use recombinant MHC molecules with covalently bound antigenic peptides. To study T cell responses to such homogeneous peptide-MHC complexes, we engineered an HLA-DR1 cDNA coding for influenza hemagglutinin, influenza matrix, or HIV p24 gag peptides covalently attached via a peptide spacer to the N terminus of the DR1 beta chain. Co-transfection with DR alpha cDNA into mouse L cells resulted in surface expression of HLA-DR1 molecules that reacted with monoclonal antibodies (mAb) specific for correctly folded HLA-DR epitopes. This suggested that the spacer and peptide did not alter expression or folding of the molecule. We then engineered an additional peptide spacer between the C terminus of a truncated beta chain (without transmembrane or cytoplasmic domains) and the N terminus of full-length DR alpha chain. Transfection of this cDNA into mouse L cells resulted in surface expression of the entire covalently linked heterotrimer of peptide, beta chain, and alpha chain with the expected molecular mass of approximately 66 kDa. These single-chain HLA-DR1 molecules reacted with mAb specific for correctly folded HLA-DR epitopes, and identified one mAb with [MHC + peptide] specificity. Affinity-purified soluble secreted single-chain molecules with truncated alpha chain moved in electrophoresis as compact class II MHC dimers. Cell surface two-chain or single-chain HLA-DR1 molecules with a covalent HA peptide stimulated HLA-DR1-restricted HA-specific T cells. They were immunogenic in vitro for peripheral blood mononuclear cells. The two-chain and single-chain HLA-DR1 molecules with covalent HA peptide had reduced binding

  14. Novel MHC Class II Breast Cancer Vaccine Using RNA Interference (RNAi) to Down Regulate Invariant Chain (li)

    DTIC Science & Technology

    2006-05-01

    patients, and may provide a powerful tool for activation of the immune system against primary tumor and metastatic disease . Body: Statement of...cells with IL-12 reduces established metastatic disease and stimulates immune effectors and monokine-induced by interferon-gamma. Canc. Immunol...concomitant down-regulation of Ii via RNAi may further improve vaccine efficacy and protect and/or treat tumor recurrence and/ or metastatic disease

  15. NLRC5: a key regulator of MHC class I-dependent immune responses.

    PubMed

    Kobayashi, Koichi S; van den Elsen, Peter J

    2012-12-01

    The expression of MHC class I molecules is crucial for the initiation and regulation of adaptive immune responses against pathogens. NOD-, LRR- and CARD-containing 5 (NLRC5) was recently identified as a specific transactivator of MHC class I genes (CITA). NLRC5 and the master regulator for MHC class II genes, class II transactivator (CIITA), interact with similar MHC promoter-bound factors. Here, we provide a broad overview of the molecular mechanisms behind MHC class I transcription and the role of the class I transactivator NLRC5 in MHC class I-dependent immune responses.

  16. Characterization of the major histocompatibility complex class II DQB (MhcMamu-DQB1) alleles in a cohort of Chinese rhesus macaques (Macaca mulatta).

    PubMed

    Qiu, Chen-Li; Yang, Gui-Bo; Yu, Kai; Li, Yue; Li, Xiao-Li; Liu, Qiang; Zhao, Hui; Xing, Hui; Shao, Yiming

    2008-08-01

    Rhesus macaques have long been used in animal models for various human diseases, the susceptibility and/or resistance to some of which have been associated with the major histocompatibilty complex (MHC). To gain insight into the MHC background and to facilitate the experimental use of Chinese rhesus macaques, the second exon of MhcMamu-DQB1 genes in 105 rhesus macaques were characterized by cloning and sequencing. A total of 37 MhcMamu-DQB1 alleles were identified, illustrating a marked allelic polymorphism at DQB1 in these monkeys. In addition to 10 alleles were novel sequences that had not been documented in earlier reports, at least 14 alleles reported in earlier studies were not detected in this study. Most of the sequences (73%) observed in this study belong to DQB1 06 (13 alleles) and DQB1 18 (14 alleles) lineages, and the rest (27%) belong to DQB1 15, DQB1 16 and DQB1 17 lineages. The most frequent allele detected among these monkeys was MhcMamu-DQB1 06111 (22%), followed by DQB1 1503 (19%); and most of the novel alleles were present at a frequency of less than 2.5%. As for individual animals, 24 of 105 (23%) were homozygous whereas 81 of 105 (77%) were heterozygous at the MhcMamu-DQB1 locus. These data indicated significant differences in MhcMamu-DQB1 allele distribution between the Chinese rhesus macaques and the previously reported rhesus macaques, which were mostly of Indian origin. This information will not only promote the understanding of rhesus macaque MHC diversity and polymorphism but will also facilitate the use of Chinese rhesus macaques in human disease studies, especially those that may be associated with HLA-DQB genes.

  17. Conserved MHC gene orthologs genetically map to the turkey MHC- B.

    PubMed

    Reed, Kent M; Benoit, Benjamin; Wang, Xiong; Greenshields, Molly A; Hughes, Camilla H K; Mendoza, Kristelle M

    2014-01-01

    The avian MHC-associated gene set includes orthologs to genes found throughout the human major histocompatibility complex (MHC), including some loci of the evolutionarily conserved class III region. In the turkey and other Galliformes, genes linked to the MHC have been identified because they are closely associated with class I or class II genes. This study was designed to evaluate additional class III genes for linkage to the avian MHC to further determine conservation of these loci in birds. BLAST searches were used to locate sequences in the turkey genome with similarity to genes shared between the MHC of Xenopus and humans. Primers were designed to target 25 genes, and putative orthologs were amplified by PCR and sequenced. Sequence polymorphisms were identified for 15 genes in turkey reference mapping families, and 8 genes showed significant genetic linkage to the turkey MHC-B locus. These new genetic markers and linkage relationships broaden our understanding of the composition of the avian MHC and expand the gene content for the turkey MHC-B.

  18. The MHC-II transactivator CIITA inhibits Tat function and HIV-1 replication in human myeloid cells.

    PubMed

    Forlani, Greta; Turrini, Filippo; Ghezzi, Silvia; Tedeschi, Alessandra; Poli, Guido; Accolla, Roberto S; Tosi, Giovanna

    2016-04-18

    We previously demonstrated that the HLA class II transactivator CIITA inhibits HIV-1 replication in T cells by competing with the viral transactivator Tat for the binding to Cyclin T1 subunit of the P-TEFb complex. Here, we analyzed the anti-viral function of CIITA in myeloid cells, another relevant HIV-1 target cell type. We sinvestigated clones of the U937 promonocytic cell line, either permissive (Plus) or non-permissive (Minus) to HIV-1 replication. This different phenotype has been associated with the expression of TRIM22 in U937 Minus but not in Plus cells. U937 Plus cells stably expressing CIITA were generated and HLA-II positive clones were selected by cell sorting and cloning. HLA and CIITA proteins were analyzed by cytofluorometry and western blotting, respectively. HLA-II DR and CIITA mRNAs were quantified by qRT-PCR. Tat-dependent transactivation was assessed by performing the HIV-1 LTR luciferase gene reporter assay. Cells were infected with HIV-1 and viral replication was evaluated by measuring the RT activity in culture supernatants. CIITA was expressed only in HLA-II-positive U937 Minus cells, and this was strictly correlated with inhibition of Tat-dependent HIV-1 LTR transactivation in Minus but not in Plus cells. Overexpression of CIITA in Plus cells restored the suppression of Tat transactivation, confirming the inhibitory role of CIITA. Importantly, HIV-1 replication was significantly reduced in Plus-CIITA cells with respect to Plus parental cells. This effect was independent of TRIM22 as CIITA did not induce TRIM22 expression in Plus-CIITA cells. U937 Plus and Minus cells represent an interesting model to study the role of CIITA in HIV-1 restriction in the monocytic/macrophage cell lineage. The differential expression of CIITA in CIITA-negative Plus and CIITA-positive Minus cells correlated with their capacity to support or not HIV-1 replication, respectively. In Minus cells CIITA targeted the viral transactivator Tat to inhibit HIV-1

  19. Attenuation of thioacetamide-induced hepatocellular injury by short-term repeated injections associated with down-regulation of metabolic enzymes and relationship with MHC class II-presenting cells.

    PubMed

    Kuramochi, Mizuki; Izawa, Takeshi; Pervin, Munmun; Bondoc, Alexandra; Kuwamura, Mitsuru; LaMarre, Jonathan; Yamate, Jyoji

    2017-10-02

    The liver is the primary organ participating in the metabolism of xenobiotics and is therefore an important target in the safety assessment of drugs, chemicals and environmental toxins. Drug-induced liver injury (DILI) has recently become widely recognized in human medicine as an adverse event. The progression of DILI often involves "damage-associated molecular patterns" (DAMPs) of gene and protein expression such as high-mobility group boxes (HMGBs), S100 proteins and heat shock proteins (Hsp). DAMPs are released from injured or necrotic cells and are bound to Toll-like receptors (TLRs) and modulate inflammatory reactions. Previously, in thioacetamide (TAA; 300mg/kg body weight, single injection)-induced rat liver, we demonstrated that the expressions of DAMPs, TLR4 and major histocompatibility complex (MHC) class II were simultaneously increased, accompanied with progression of hepatocellular injury and inflammation. Here we investigated the association of DILI and DAMPs, TLRs and MHC class II by using rat livers repeated injections with TAA (100mg/kg body weight, once, three times). Two days after TAA single injection, centrilobular hepatocellular necrosis with infiltration of mononuclear cells was observed, being paralleled with increase in serum levels of aspartate transaminase (AST), alanine transaminase (ALT) and alkaline phosphatase (ALP). However, two days after duplicate and triplicate injections, only mild degenerative change of hepatocytes and slight infiltration of mononuclear cells were seen in the affected centrilobular area. Serum levels of AST, ALT and ALP were also decreased to the same levels of control. mRNA expressions of DAMPs (HMGBs, S100A4 and Hsp 70-2), TLR4 and MHC class II tended to be increased only on single injection, although the number of MHC class II-positive cells in the centrilobular area was still increased on each examination point. The analysis of enzymes (CYP2E1 and Flavin monooxygenase (FMO) 3), which metabolize TAA in

  20. Monoclonal antibodies to NF-Y define its function in MHC class II and albumin gene transcription.

    PubMed Central

    Mantovani, R; Pessara, U; Tronche, F; Li, X Y; Knapp, A M; Pasquali, J L; Benoist, C; Mathis, D

    1992-01-01

    NF-Y is a sequence-specific DNA-binding protein which, as a heterodimer, recognizes CCAAT motifs in a variety of transcriptional promoters. We have generated a panel of monoclonal and affinity-purified polyclonal antibodies directed against various epitopes of NF-Y. These reagents are highly specific for either of the A or B subunits; we have mapped the epitopes recognized by the monoclonal antibodies to the glutamine-rich activation domain of NF-YA. The antibodies inhibit in vitro transcription from the promoters of the albumin gene and of Ea, a class II gene of the major histocompatibility complex. These data definitively demonstrate the role of NF-Y in regulating the transcription of two tissue-specific genes whose expression patterns do not overlap. Interestingly, the antibodies cannot inhibit a formed pre-initiation complex, but do block reinitiation of subsequent rounds of transcription from the same templates. Images PMID:1380453

  1. Acidosis increases MHC class II-restricted presentation of a protein endowed with a pH-dependent heparan sulfate-binding ability.

    PubMed

    Knittel, Delphine; Savatier, Alexandra; Upert, Grégory; Lortat-Jacob, Hugues; Léonetti, Michel

    2015-04-15

    Heparan sulfate proteoglycans (HSPGs) are ubiquitously expressed molecules that participate in numerous biological processes. We previously showed that HSPGs expressed on the surface of APCs can serve as receptors for a hybrid protein containing an HS ligand and an Ag, which leads to more efficient stimulation of Th cells. To investigate whether such behavior is shared by proteins with inherent HS-binding ability, we looked for proteins endowed with this characteristic. We found that diphtheria toxin and its nontoxic mutant, called CRM197, can interact with HS. However, we observed that their binding ability is higher at pH 6 than at pH 7.4. Therefore, as extracellular acidosis occurs during infection by various micro-organisms, we assessed whether HS-binding capacity affects MHC class II-restricted presentation at different pHs. We first observed that pH decrease allows CRM197 binding to HSPG-expressing cells, including APCs. Then, we showed that this interaction enhances Ag uptake and presentation to Th cells. Lastly, we observed that pH decrease does not affect processing and presentation abilities of the APCs. Our findings show that acidic pH causes an HSPG-mediated uptake and an enhancement of T cell stimulation of Ags with the inherent ability to bind HSPGs pH-dependently. Furthermore, they suggest that proteins from micro-organisms with this binding characteristic might be supported more efficiently by the adaptive immune system when acidosis is triggered during infection. Copyright © 2015 by The American Association of Immunologists, Inc.

  2. Identification of Streptococcus mutans PAc peptide motif binding with human MHC class II molecules (DRB1*0802, *1101, *1401 and *1405).

    PubMed Central

    Senpuku, H; Yanagi, K; Nisizawa, T

    1998-01-01

    A surface protein antigen (PAc) of Streptococcus mutans, in particular the A-region of this PAc molecule, has been noted as a possible target in research for an effective dental caries vaccine. To identify the antigenic peptide binding to major histocompatibility complex (MHC) class II (HLA-DR) molecules in the A-region, we prepared a panel of overlapping synthetic peptides in the second unit of the A-region, and established that a simple enzyme-linked immunosorbent assay (ELISA) binding assay could be achieved by incubating the DR-crude. Binding to DR molecules of these peptides from nine donors was investigated by using the ELISA binding assay. It was revealed that the PAc(316-334) peptide bound more strongly to the HLA-DR molecule in seven out of nine subjects. In particular, DR8 (DRB1*0802), DR5 (DRB1*1101) and DR6 (DRB1*1402 and *1405), which bound strongly to PAc(316-334) peptide, were identified. Moreover, we synthesized glycine-substituted peptide analogues of the peptide and examined the binding motif of the binding region. As a result, the multiple binding motif in DR8, DR5 and DR6 was found in L-RV-K-A. It is suggested that a peptide vaccine for dental caries that is more effective for humans, with fewer adverse side-effects, could be designed by combining the multiple binding motif with the B-cell epitope to produce only the inhibiting antibody against dental caries. The peptide could therefore be useful for peptide vaccine development in the general human population. PMID:9824493

  3. Identification of Streptococcus mutans PAc peptide motif binding with human MHC class II molecules (DRB1*0802, *1101, *1401 and *1405).

    PubMed

    Senpuku, H; Yanagi, K; Nisizawa, T

    1998-11-01

    A surface protein antigen (PAc) of Streptococcus mutans, in particular the A-region of this PAc molecule, has been noted as a possible target in research for an effective dental caries vaccine. To identify the antigenic peptide binding to major histocompatibility complex (MHC) class II (HLA-DR) molecules in the A-region, we prepared a panel of overlapping synthetic peptides in the second unit of the A-region, and established that a simple enzyme-linked immunosorbent assay (ELISA) binding assay could be achieved by incubating the DR-crude. Binding to DR molecules of these peptides from nine donors was investigated by using the ELISA binding assay. It was revealed that the PAc(316-334) peptide bound more strongly to the HLA-DR molecule in seven out of nine subjects. In particular, DR8 (DRB1*0802), DR5 (DRB1*1101) and DR6 (DRB1*1402 and *1405), which bound strongly to PAc(316-334) peptide, were identified. Moreover, we synthesized glycine-substituted peptide analogues of the peptide and examined the binding motif of the binding region. As a result, the multiple binding motif in DR8, DR5 and DR6 was found in L-RV-K-A. It is suggested that a peptide vaccine for dental caries that is more effective for humans, with fewer adverse side-effects, could be designed by combining the multiple binding motif with the B-cell epitope to produce only the inhibiting antibody against dental caries. The peptide could therefore be useful for peptide vaccine development in the general human population.

  4. Comparative Genome Analyses Reveal Distinct Structure in the Saltwater Crocodile MHC

    PubMed Central

    Jaratlerdsiri, Weerachai; Deakin, Janine; Godinez, Ricardo M.; Shan, Xueyan; Peterson, Daniel G.; Marthey, Sylvain; Lyons, Eric; McCarthy, Fiona M.; Isberg, Sally R.; Higgins, Damien P.; Chong, Amanda Y.; John, John St; Glenn, Travis C.; Ray, David A.; Gongora, Jaime

    2014-01-01

    The major histocompatibility complex (MHC) is a dynamic genome region with an essential role in the adaptive immunity of vertebrates, especially antigen presentation. The MHC is generally divided into subregions (classes I, II and III) containing genes of similar function across species, but with different gene number and organisation. Crocodylia (crocodilians) are widely distributed and represent an evolutionary distinct group among higher vertebrates, but the genomic organisation of MHC within this lineage has been largely unexplored. Here, we studied the MHC region of the saltwater crocodile (Crocodylus porosus) and compared it with that of other taxa. We characterised genomic clusters encompassing MHC class I and class II genes in the saltwater crocodile based on sequencing of bacterial artificial chromosomes. Six gene clusters spanning ∼452 kb were identified to contain nine MHC class I genes, six MHC class II genes, three TAP genes, and a TRIM gene. These MHC class I and class II genes were in separate scaffold regions and were greater in length (2–6 times longer) than their counterparts in well-studied fowl B loci, suggesting that the compaction of avian MHC occurred after the crocodilian-avian split. Comparative analyses between the saltwater crocodile MHC and that from the alligator and gharial showed large syntenic areas (>80% identity) with similar gene order. Comparisons with other vertebrates showed that the saltwater crocodile had MHC class I genes located along with TAP, consistent with birds studied. Linkage between MHC class I and TRIM39 observed in the saltwater crocodile resembled MHC in eutherians compared, but absent in avian MHC, suggesting that the saltwater crocodile MHC appears to have gene organisation intermediate between these two lineages. These observations suggest that the structure of the saltwater crocodile MHC, and other crocodilians, can help determine the MHC that was present in the ancestors of archosaurs. PMID:25503521

  5. Comparative genome analyses reveal distinct structure in the saltwater crocodile MHC.

    PubMed

    Jaratlerdsiri, Weerachai; Deakin, Janine; Godinez, Ricardo M; Shan, Xueyan; Peterson, Daniel G; Marthey, Sylvain; Lyons, Eric; McCarthy, Fiona M; Isberg, Sally R; Higgins, Damien P; Chong, Amanda Y; John, John St; Glenn, Travis C; Ray, David A; Gongora, Jaime

    2014-01-01

    The major histocompatibility complex (MHC) is a dynamic genome region with an essential role in the adaptive immunity of vertebrates, especially antigen presentation. The MHC is generally divided into subregions (classes I, II and III) containing genes of similar function across species, but with different gene number and organisation. Crocodylia (crocodilians) are widely distributed and represent an evolutionary distinct group among higher vertebrates, but the genomic organisation of MHC within this lineage has been largely unexplored. Here, we studied the MHC region of the saltwater crocodile (Crocodylus porosus) and compared it with that of other taxa. We characterised genomic clusters encompassing MHC class I and class II genes in the saltwater crocodile based on sequencing of bacterial artificial chromosomes. Six gene clusters spanning ∼452 kb were identified to contain nine MHC class I genes, six MHC class II genes, three TAP genes, and a TRIM gene. These MHC class I and class II genes were in separate scaffold regions and were greater in length (2-6 times longer) than their counterparts in well-studied fowl B loci, suggesting that the compaction of avian MHC occurred after the crocodilian-avian split. Comparative analyses between the saltwater crocodile MHC and that from the alligator and gharial showed large syntenic areas (>80% identity) with similar gene order. Comparisons with other vertebrates showed that the saltwater crocodile had MHC class I genes located along with TAP, consistent with birds studied. Linkage between MHC class I and TRIM39 observed in the saltwater crocodile resembled MHC in eutherians compared, but absent in avian MHC, suggesting that the saltwater crocodile MHC appears to have gene organisation intermediate between these two lineages. These observations suggest that the structure of the saltwater crocodile MHC, and other crocodilians, can help determine the MHC that was present in the ancestors of archosaurs.

  6. MHC-linked susceptibility to a bacterial infection, but no MHC-linked cryptic female choice in whitefish.

    PubMed

    Wedekind, C; Walker, M; Portmann, J; Cenni, B; Müller, R; Binz, T

    2004-01-01

    Non-random gamete fusion is one of several potential cryptic female choice mechanisms that have been postulated and that may enhance the survival probability of the offspring. Previous studies have found that gamete fusion in mice is influenced by genes of the major histocompatibility complex (MHC) region. Here we test (i) whether there is MHC-dependent gamete fusion in whitefish (Coregonus sp.) and (ii) whether there is a link between the MHC and embryo susceptibility to an infection by the bacterium Pseudomonas fuorescens. We experimentally bred whitefish and reared sibships in several batches that either experienced or did not experience strong selection by P. fluorescens. We then determined the MHC class II B1 genotype of 1016 surviving larvae of several full sibships. We found no evidence for MHC-linked gamete fusion. However, in one of seven sibships we found a strong connection between the MHC class II genotype and embryo susceptibility to P. fluorescens. This connection was still significant after correcting for multiple testing. Hence, the MHC class II genotype can considerably influence embryo survival in whitefish, but gamete fusion seems to be random with respect to the MHC.

  7. Double loading of dendritic cell MHC class I and MHC class II with an AML antigen repertoire enhances correlates of T-cell immunity in vitro via amplification of T-cell help.

    PubMed

    Decker, William K; Xing, Dongxia; Li, Sufang; Robinson, Simon N; Yang, Hong; Yao, Xin; Segall, Harry; McMannis, John D; Komanduri, Krishna V; Champlin, Richard E; Shpall, Elizabeth J

    2006-04-12

    Therapeutic vaccination with dendritic cells presenting tumor-specific antigens is now recognized as an important investigational therapy for the treatment of neoplastic disease. Dendritic cell cross-presentation is credited with the ability of tumor lysate-loaded dendritic cells to prime both CD4 and CD8-specific T-lymphocyte responses, enabling the generation of cancer specific CTL activity without the loading of the classical MHC class I compartment. Recently, however, several reports have raised doubts as to the efficiency of cross-presentation as a mechanism for CTL priming in vivo. To examine this issue, we have doubly-loaded human dendritic cells with both AML-specific tumor lysate and AML-specific tumor mRNA. Our results show that these doubly-loaded dendritic cells can mediate superior primary, recall, and effector lytic responses in vitro in comparison to those of dendritic cells loaded with either tumor lysate or tumor mRNA alone. Enhanced recall responses appeared to be influenced by CD40/CD40L signaling, underscoring the importance of T-cell help in the generation and perpetuation of the adaptive immune response.

  8. Natural selection of the major histocompatibility complex (Mhc) in Hawaiian honeycreepers (Drepanidinae)

    USGS Publications Warehouse

    Jarvi, S.I.; Tarr, C.L.; Mcintosh, C.E.; Atkinson, C.T.; Fleischer, R.C.

    2004-01-01

    The native Hawaiian honeycreepers represent a classic example of adaptive radiation and speciation, but currently face one the highest extinction rates in the world. Although multiple factors have likely influenced the fate of Hawaiian birds, the relatively recent introduction of avian malaria is thought to be a major factor limiting honeycreeper distribution and abundance. We have initiated genetic analyses of class II ?? chain Mhc genes in four species of honeycreepers using methods that eliminate the possibility of sequencing mosaic variants formed by cloning heteroduplexed polymerase chain reaction products. Phylogenetic analyses group the honeycreeper Mhc sequences into two distinct clusters. Variation within one cluster is high, with dN > d S and levels of diversity similar to other studies of Mhc (B system) genes in birds. The second cluster is nearly invariant and includes sequences from honeycreepers (Fringillidae), a sparrow (Emberizidae) and a blackbird (Emberizidae). This highly conserved cluster appears reminiscent of the independently segregating Rfp-Y system of genes defined in chickens. The notion that balancing selection operates at the Mhc in the honeycreepers is supported by transpecies polymorphism and strikingly high dN/dS ratios at codons putatively involved in peptide interaction. Mitochondrial DNA control region sequences were invariant in the i'iwi, but were highly variable in the 'amakihi. By contrast, levels of variability of class II ?? chain Mhc sequence codons that are hypothesized to be directly involved in peptide interactions appear comparable between i'iwi and 'amakihi. In the i'iwi, natural selection may have maintained variation within the Mhc, even in the face of what appears to a genetic bottleneck.

  9. CDF II production farm project

    SciTech Connect

    Baranovski, A.; Benjamin, D.; Cooper, G.; Farrington, S.; Genser, K.; Hou, S.; Hsieh, T.; Kotwal, A.; Lipeles, E.; Murat, P.; Norman, M.; /Fermilab /Duke U. /Taiwan, Inst. Phys. /UC, San Diego /Glasgow U. /Frascati

    2006-12-01

    We describe the architecture and discuss our operational experience in running the off-line reconstruction farm of the CDFII experiment. The Linux PC-based farm performs a wide set of tasks,ranging from producing calibrations and primary event reconstruction to large scale ntuple production.The farm control software uses a standard Condor toolkit and the data handling part is based on SAM (Sequential Access via Metadata)software.During its lifetime,the CDFII experiment will integrate a large amount of data (several petabytes)and the data processing chain is one of the key components of the successful physics program of the experiment.

  10. Immunization with a peptide containing MHC class I and II epitopes derived from the tumor antigen SIM2 induces an effective CD4 and CD8 T-cell response.

    PubMed

    Kissick, Haydn T; Sanda, Martin G; Dunn, Laura K; Arredouani, Mohamed S

    2014-01-01

    Here, we sought to determine whether peptide vaccines designed harbor both class I as well as class II restricted antigenic motifs could concurrently induce CD4 and CD8 T cell activation against autologous tumor antigens. Based on our prior genome-wide interrogation of human prostate cancer tissues to identify genes over-expressed in cancer and absent in the periphery, we targeted SIM2 as a prototype autologous tumor antigen for these studies. Using humanized transgenic mice we found that the 9aa HLA-A*0201 epitope, SIM2(237-245), was effective at inducing an antigen specific response against SIM2-expressing prostate cancer cell line, PC3. Immunization with a multi-epitope peptide harboring both MHC-I and MHC-II restricted epitopes induced an IFN-γ response in CD8 T cells to the HLA-A*0201-restricted SIM2(237-245) epitope, and an IL-2 response by CD4 T cells to the SIM2(240-254) epitope. This peptide was also effective at inducing CD8+ T-cells that responded specifically to SIM2-expressing tumor cells. Collectively, the data presented in this study suggest that a single peptide containing multiple SIM2 epitopes can be used to induce both a CD4 and CD8 T cell response, providing a peptide-based vaccine formulation for potential use in immunotherapy of various cancers.

  11. Heavy flavor production in CDF II detector

    SciTech Connect

    Gorelov, Igor V.; /New Mexico U.

    2006-01-01

    For data collected with the CDF Run II detector, measurements of the charm and bottom production cross-sections are presented. The results are based both on large samples of fully reconstructed hadron decay products of charm and bottom made available by the tracking triggers and on a calorimeter jet triggered sample tagged by the presence of a secondary vertex. The experimental data are compared with theoretical predictions from recent next-to-leading order (NLO) QCD calculations.

  12. Shared evolutionary origin of MHC polymorphism in sympatric lemurs.

    PubMed

    Kaesler, Eva; Kappeler, Peter M; Brameier, Markus; Demeler, Janina; Kraus, Cornelia; Rakotoniaina, Josué H; Hämäläinen, Anni M; Huchard, Elise

    2017-08-21

    Genes of the Major Histocompatibility Complex (MHC) play a central role in adaptive immune responses of vertebrates. They exhibit remarkable polymorphism, often crossing species boundaries with similar alleles or allelic motifs shared across species. This pattern may reflect parallel parasite-mediated selective pressures, either favouring the long maintenance of ancestral MHC allelic lineages across successive speciation events by balancing selection ('trans-species polymorphism'), or alternatively favouring the independent emergence of functionally similar alleles post-speciation via convergent evolution. Here we investigate the origins of MHC similarity across several species of dwarf and mouse lemurs (Cheirogaleidae). We examined MHC class II variation in two highly polymorphic loci (DRB, DQB) and evaluated the overlap of gut-parasite communities in four sympatric lemurs. We tested for parasite-MHC associations across species to determine whether similar parasite pressures may select for similar MHC alleles in different species. Next, we integrated our MHC data with those previously obtained from other Cheirogaleidae to investigate the relative contribution of convergent evolution and co-ancestry to shared MHC polymorphism by contrasting patterns of codon usage at functional versus neutral sites. Our results indicate that parasites shared across species may select for functionally similar MHC alleles, implying that the dynamics of MHC-parasite co-evolution should be envisaged at the community level. We further show that balancing selection maintaining trans-species polymorphism, rather than convergent evolution, is the primary mechanism explaining shared MHC sequence motifs between species that diverged up to 30 million years ago. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

  13. Human Megakaryocyte Progenitors Derived from Hematopoietic Stem Cells of Normal Individuals are MHC class II-Expressing Professional APC that Enhance Th17 and Th1/Th17 Responses

    PubMed Central

    Finkielsztein, Ariel; Schlinker, Alaina C.; Zhang, Li; Miller, William M.; Datta, Syamal K.

    2014-01-01

    Platelets, like stromal cells, present antigen only via MHC class I, but the immune potential of their progenitors has not been explored in humans. We derived CD34+CD117+CD41+CD151+ megakaryocyte progenitors (MKp) in vitro from mobilized peripheral blood hematopoietic stem and progenitor cells (HSPC) of normal subjects using culture conditions akin to bone marrow niche, or organs that support extramedullary hematopoiesis. The MKp expressed MHC Class II in contrast to platelets and functioned as professional APC before they matured further. Moreover, MKp constitutively expressed mRNA encoding mediators for human Th17 expansion, including IL-1, IL-18, IL-6, TGFβ, IL-23, BAFF, and COX2. MKp also expressed high levels of type I interferon and IRF5 mRNA. In contrast to platelets, MKp augmented the expansion of Th17, Th1, and potent Th17/Th1 double-positive cells in normal PBMC and CD4 line T cells from normal subjects or lupus patients. The Th cell augmentation involved pre-committed memory cells, and was significant although modest, because only non-cognate MKp-T cell interactions could be studied, under non-polarizing conditions. Importantly, the MKp-mediated expansion was observed in the presence or absence of direct MKp-T cell contact. Furthermore, MKp augmented Th17 responses against Candida albicans, a serious opportunistic pathogen. These results indicate an immunologic role of MKp in situations associated with extramedullary hematopoiesis and mobilization of HSPC. PMID:25454068

  14. The production, purification and crystallization of a soluble form of the nonclassical MHC HLA-G: the essential role of cobalt

    SciTech Connect

    Clements, Craig S.; Kjer-Nielsen, Lars; Kostenko, Lyudmila; McCluskey, James; Rossjohn, Jamie

    2006-01-01

    X-ray diffraction data were collected to 1.9 Å from crystals of HLA-G. Cobalt ions were found to be essential for the production of diffracting crystals. HLA-G is a nonclassical class I major histocompatibility complex (MHC) molecule that is primarily expressed at the foetal–maternal interface. Although the role of HLA-G has not been fully elucidated, current evidence suggests it protects the foetus from the maternal immune response. In this report, HLA-G (44 kDa) is characterized by expression in Escherichia coli. The inclusion bodies were refolded in complex with a peptide derived from histone H2A (RIIPRHLQL), purified and subsequently crystallized. Correct refolding was determined using two conformation-dependent antibodies. Cobalt ions were shown to be an essential ingredient for obtaining diffraction-quality crystals. The crystals, which diffracted to 1.9 Å resolution, belonged to space group P3{sub 2}2{sub 1}, with unit-cell parameters a = b = 77.15, c = 151.72 Å.

  15. Productive association between MHC class I and tapasin requires the tapasin transmembrane/cytosolic region and the tapasin C-terminal Ig-like domain

    PubMed Central

    Simone, Laura C.; Georgesen, Corey J.; Simone, Peter D.; Wang, Xiaojian; Solheim, Joyce C.

    2011-01-01

    The current model of antigen assembly with major histocompatibility complex (MHC) class I molecules posits that interactions between the tapasin N-terminal immunoglobulin (Ig)-like domain and the MHC class I peptide-binding groove permit tapasin to regulate antigen selection. Much less is known regarding interactions that might involve the tapasin C-terminal Ig-like domain. Additionally, the tapasin transmembrane/cytoplasmic region enables tapasin to bridge the MHC class I molecule to the transporter associated with antigen processing (TAP). In this investigation, we made use of two tapasin mutants to determine the relative contribution of the tapasin C-terminal Ig-like domain and the tapasin transmembrane/cytoplasmic region to the assembly of MHC class I molecules. Deletion of a loop within the tapasin C-terminal Ig-like domain (Δ334-342) prevented tapasin association with the MHC class I molecule Kd. Although tapasin Δ334-342 did not increase the efficiency of Kd folding, Kd surface expression was enhanced on cells expressing this mutant relative to tapasin-deficient cells. In contrast to tapasin Δ334-342, a soluble tapasin mutant lacking the transmembrane/cytoplasmic region retained the ability to bind to Kd molecules, but did not facilitate Kd surface expression. Furthermore, when soluble tapasin and tapasin Δ334-342 were co-expressed, soluble tapasin had a dominant negative effect on the folding and surface expression of not only Kd, but also Db and Kb. In addition, our molecular modeling of the MHC class I-tapasin interface revealed novel potential interactions involving tapasin residues 334-342. Together, these findings demonstrate that the tapasin C-terminal and transmembrane/cytoplasmic regions are critical to tapasin's capacity to associate effectively with the MHC class I molecule. PMID:22169163

  16. Sympatric and Allopatric Divergence of MHC Genes in Threespine Stickleback

    PubMed Central

    Matthews, Blake; Harmon, Luke J.; M'Gonigle, Leithen; Marchinko, Kerry B.; Schaschl, Helmut

    2010-01-01

    Parasites can strongly affect the evolution of their hosts, but their effects on host diversification are less clear. In theory, contrasting parasite communities in different foraging habitats could generate divergent selection on hosts and promote ecological speciation. Immune systems are costly to maintain, adaptable, and an important component of individual fitness. As a result, immune system genes, such as those of the Major Histocompatability Complex (MHC), can change rapidly in response to parasite-mediated selection. In threespine stickleback (Gasterosteus aculeatus), as well as in other vertebrates, MHC genes have been linked with female mating preference, suggesting that divergent selection acting on MHC genes might influence speciation. Here, we examined genetic variation at MHC Class II loci of sticklebacks from two lakes with a limnetic and benthic species pair, and two lakes with a single species. In both lakes with species pairs, limnetics and benthics differed in their composition of MHC alleles, and limnetics had fewer MHC alleles per individual than benthics. Similar to the limnetics, the allopatric population with a pelagic phenotype had few MHC alleles per individual, suggesting a correlation between MHC genotype and foraging habitat. Using a simulation model we show that the diversity and composition of MHC alleles in a sympatric species pair depends on the amount of assortative mating and on the strength of parasite-mediated selection in adjacent foraging habitats. Our results indicate parallel divergence in the number of MHC alleles between sympatric stickleback species, possibly resulting from the contrasting parasite communities in littoral and pelagic habitats of lakes. PMID:20585386

  17. Differential scanning fluorimetry based assessments of the thermal and kinetic stability of peptide-MHC complexes.

    PubMed

    Hellman, Lance M; Yin, Liusong; Wang, Yuan; Blevins, Sydney J; Riley, Timothy P; Belden, Orrin S; Spear, Timothy T; Nishimura, Michael I; Stern, Lawrence J; Baker, Brian M

    2016-05-01

    Measurements of thermal stability by circular dichroism (CD) spectroscopy have been widely used to assess the binding of peptides to MHC proteins, particularly within the structural immunology community. Although thermal stability assays offer advantages over other approaches such as IC50 measurements, CD-based stability measurements are hindered by large sample requirements and low throughput. Here we demonstrate that an alternative approach based on differential scanning fluorimetry (DSF) yields results comparable to those based on CD for both class I and class II complexes. As they require much less sample, DSF-based measurements reduce demands on protein production strategies and are amenable for high throughput studies. DSF can thus not only replace CD as a means to assess peptide/MHC thermal stability, but can complement other peptide-MHC binding assays used in screening, epitope discovery, and vaccine design. Due to the physical process probed, DSF can also uncover complexities not observed with other techniques. Lastly, we show that DSF can also be used to assess peptide/MHC kinetic stability, allowing for a single experimental setup to probe both binding equilibria and kinetics. Copyright © 2016 Elsevier B.V. All rights reserved.

  18. Sex-associated expression of co-stimulatory molecules CD80, CD86, and accessory molecules, PDL-1, PDL-2 and MHC-II, in F480+ macrophages during murine cysticercosis.

    PubMed

    Togno-Peirce, Cristián; Nava-Castro, Karen; Terrazas, Luis Ignacio; Morales-Montor, Jorge

    2013-01-01

    Macrophages are critically involved in the interaction between T. crassiceps and the murine host immune system. Also, a strong gender-associated susceptibility to murine cysticercosis has been reported. Here, we examined the sex-associated expression of molecules MHC-II, CD80, CD86, PD-L1, and PD-L2 on peritoneal F4/80(hi) macrophages of BALB/c mice infected with Taenia crassiceps. Peritoneal macrophages from both sexes of mice were exposed to T. crassiceps total extract (TcEx). BALB/c Females mice recruit higher number of macrophages to the peritoneum. Macrophages from infected animals show increased expression of PDL2 and CD80 that was dependent from the sex of the host. These findings suggest that macrophage recruitment at early time points during T. crassiceps infection is a possible mechanism that underlies the differential sex-associated susceptibility displayed by the mouse gender.

  19. Definition of MHC and T cell receptor contacts in the HLA-DR4restricted immunodominant epitope in type II collagen and characterization of collagen-induced arthritis in HLA-DR4 and human CD4 transgenic mice

    PubMed Central

    Andersson, Ellen Christina; Hansen, Bjarke Endel; Jacobsen, Helle; Madsen, Lars S.; Andersen, Claus B.; Engberg, Jan; Rothbard, Jonathan B.; McDevitt, Grete Sønderstrup; Malmström, Vivianne; Holmdahl, Rikard; Svejgaard, Arne; Fugger, Lars

    1998-01-01

    Rheumatoid arthritis (RA) is an autoimmune disease associated with the HLA-DR4 and DR1 alleles. The target autoantigen(s) in RA is unknown, but type II collagen (CII) is a candidate, and the DR4- and DR1-restricted immunodominant T cell epitope in this protein corresponds to amino acids 261–273 (CII 261–273). We have defined MHC and T cell receptor contacts in CII 261–273 and provide strong evidence that this peptide corresponds to the peptide binding specificity previously found for RA-associated DR molecules. Moreover, we demonstrate that HLA-DR4 and human CD4 transgenic mice homozygous for the I-Abβ0 mutation are highly susceptible to collagen-induced arthritis and describe the clinical course and histopathological changes in the affected joints. PMID:9636191

  20. Both man & bird & beast: Comparative organization of MHC genes

    SciTech Connect

    Trowsdale, J.

    1995-01-01

    The major histocompatibility complex (MHC) is the center of the immune universe. Genes in the MHC determine which antigens are processed and presented. Not surprisingly, the MHC contributes the major genetic component to important autoimmune diseases and will no doubt, although evidence is limited, contribute to resistance to infectious disorders. Vertebrates all seem to have MHC genes and it should be possible to determine, within the next few years, whether the clustering of antigen processing and presenting genes in this region is a conserved feature. One could imagine an evolutionary advantage to maintaining the MHC as a unit, either to coordinate expression of the genes in different tissues, or to coordinate T-cell selection during thymic ontogeny, since inheriting a linked set of polymorphic gene products may help to avoid conflicts during positive and negative selection. 153 refs., 9 figs., 3 tabs.