Major histocompatibility class II genes in rainbow trout (Oncorhynchus mykiss) exhibit temperature dependent downregulation

Major histocompatibility (MH) class II receptors are expressed on the surface of specialized antigen-presenting cells in vertebrate immune systems. Their function is to present peptides derived from exogenous pathogens to CD4+ T cells. Variation in the level of expression of these genes has been linked to pathogenesis in various diseases. Very little has been published on the function of MH class II receptors in teleost fish to date. In this study, we have produced polyclonal antibodies recognizing MH class II alpha and beta proteins of rainbow trout and employed them to characterize the expression pattern of these genes. Deglycosylation using N-glycosidase F and endoglycosidase H showed that MH class II alpha is glycosylated in rainbow trout. MH class II beta was also found to be glycosylated as reported previously. Results from Northern blotting revealed that the expression of these genes was not affected by exposure of rainbow trout to temperature of 5°C. However, at 2°C, downregulation of MH class II alpha and beta genes was evident at both the mRNA and protein levels as assessed by Northern and Western blotting, respectively. Because MH class II antigens play an important role in generating an immune response to bacterial and fungal pathogens, downregulation of these genes at low temperature could account for the susceptibility of fish to low temperature-related diseases such as bacterial cold-water disease and winter saprolegniosis.     

The beta 2-microglobulin locus of rainbow trout (Oncorhynchus mykiss) contains three polymorphic genes

Beta2-microglobulin (beta2m) associates with MHC and related class I H chains to form cell surface glycoproteins that mediate a variety of functions in defense. In humans, monomorphism of a single beta2m gene contrasts with the diversity and polymorphism of the class I H chain genes, and a similar picture was seen in almost all other species examined. In this regard, rainbow trout (Oncorhynchus mykiss) appeared unusual: trout beta2m genes gave a complicated and polymorphic pattern in Southern blots, and a minimum of 10 different mRNA encoding two distinct types of beta2m were expressed by a single fish. Characterization of genomic clones from the same fish now shows that the rainbow trout beta2m locus consists of two expressed genes and one partial gene that are closely linked. Four copies of the locus were identified and allelic variants of each gene defined, largely through comparison of the noncoding regions. A dramatic variation in the lengths of introns is caused by variable repetitive elements and accounts for the complex pattern seen in Southern blots. By comparison to noncoding sequences, the coding regions are conserved but the three loci differ within a cluster of codons that encode residues of beta2m that do not interact with class I H chains. Additional diversity in the trout beta2m genes appears to be due to somatic mutation that might be facilitated by the abundance of repetitive DNA elements within the 12 beta2m genes of an individual rainbow trout.     

Stable surface expression of invariant chain prevents peptide presentation by HLA-DR.

Class II major histocompatibility complex (MHC) molecules are cell surface glycoproteins that bind and present immunogenic peptides to T cells. Intracellularly, class II molecules associate with a polypeptide referred to as the invariant (Ii) chain. Ii is proteolytically degraded and dissociates from the class II complex prior to cell surface expression of the mature class II alpha beta heterodimer. Using human fibroblasts transfected with HLA-DR1 and Ii cDNAs, we now demonstrate that truncation of the cytoplasmic domain of Ii results in the failure of Ii to dissociate from the alpha beta Ii complex and leads to stable expression of class II alpha beta Ii complexes on the cell surface. Furthermore, biochemical analysis and peptide presentation assays demonstrated that transfectants with stable surface alpha beta Ii complexes expressed very few free alpha beta heterodimers at the surface and were very inefficient in their ability to present immunogenic peptides to T cells. These results support the hypothesis that the cytoplasmic domain of Ii is responsible for endosomal targeting of alpha beta Ii and directly demonstrate that association with Ii interferes with the antigen presentation function of class II molecules.     

The rainbow trout classical MHC class I molecule Onmy-UBA*501 is expressed in similar cell types as mammalian classical MHC class I molecules

Onmy-UBA is a polymorphic classical major histocompatibility (MHC) class I locus in rainbow trout (Oncorhynchus mykiss). A common allomorph is Onmy-UBA*501, which has been detected in several wildtype strains, in the clonal homozygous rainbow trout C25 and, in the current study, in the rainbow trout gonad cell line RTG-2. The extracellular domain of this allomorph was expressed in E. coli and a murine monoclonal antibody designated H9 was generated against the recombinant protein. In Western blot analysis Mab H9 specifically recognised an n-glycosylated protein of 45 kDa in leucocytes and erythrocytes of C25 fish and in RTG-2 cells. The level of Onmy-UBA*501 expression in erythrocytes was very low. Immunocytochemistry of isolated cells indicated expression in lymphocytes, macrophages, neutrophils, erythrocytes, RTG-2 cells and Onmy-UBA *501 transfected CHO cells, but not in untransfected CHO cells. Immunohistochemistry using frozen sections of C25 fish indicated that Onmy-UBA*501 expression is strong in the lymphoid organs (thymus, head kidney and spleen) and in the epithelia and endothelia of several organs. No significant expression was observed in muscle fibres, hepatocytes or neurons. These observations demonstrate that in jawed fish, the lowest phylogenetic group possessing an MHC system, the classical MHC class I molecules are expressed in similar cell types as in higher vertebrates.     

Classical MHC class I genes composed of highly divergent sequence lineages share a single locus in rainbow trout (Oncorhynchus mykiss).

The classical MHC class I genes have been known to be highly polymorphic in various vertebrates. To date, putative allelic sequences of the classical MHC class I genes in teleost fish have been reported in several studies. However, the establishment of their allelic status has been hampered in most cases by the lack of appropriate genomic information. In the present study, using heterozygous and homozygous fish, we obtained classical-type MHC class I sequences of rainbow trout (Oncorhynchus mykiss) and investigated their allelic relationship by gene amplification and Southern and Northern hybridization analyses. The results indicated that all MHC class I sequences we obtained were derived from a single locus. Based on this, a unique polymorphic nature of the MHC class I locus of rainbow trout has been revealed. The mosaic combination of highly divergent ancient sequences in the peptide-binding domains is notable, and the variable nature around the boundary between the alpha3 and transmembrane domains is unprecedented.     

Genetic modulation of tumor antigen presentation

An effective cancer-cell vaccine is created by expressing major-histocompatibility-complex (MHC) class II molecules without the invariant chain protein (Ii) that normally blocks the antigenic-peptide-binding site of MHC class II molecules at their synthesis in the endoplasmic reticulum. Such tumor-cell constructs are created either by the transfer of genes for MHC class IIalpha and beta chains, or by the induction of MHC class II molecules and Ii protein with a transacting factor, followed by Ii suppression using antisense methods. Preclinical validation of this approach is reviewed with the goal of using this immunotherapy for metastatic human cancers.     

Modes of salmonid MHC class I and II evolution differ from the primate paradigm

Rainbow trout (Oncorhynchus mykiss) and brown trout (Salmo trutta) represent two salmonid genera separated for 15--20 million years. cDNA sequences were determined for the classical MHC class I heavy chain gene UBA and the MHC class II beta-chain gene DAB from 15 rainbow and 10 brown trout. Both genes are highly polymorphic in both species and diploid in expression. The MHC class I alleles comprise several highly divergent lineages that are represented in both species and predate genera separation. The class II alleles are less divergent, highly species specific, and probably arose after genera separation. The striking difference in salmonid MHC class I and class II evolution contrasts with the situation in primates, where lineages of class II alleles have been sustained over longer periods of time relative to class I lineages. The difference may arise because salmonid MHC class I and II genes are not linked, whereas in mammals they are closely linked. A prevalent mechanism for evolving new MHC class I alleles in salmonids is recombination in intron II that shuffles alpha 1 and alpha 2 domains into different combinations.     

Effect of decreasing the affinity of the class II-associated invariant chain peptide on the MHC class II peptide repertoire in the presence or absence of H-2M

The class II-associated invariant chain peptide (CLIP) region of the invariant chain (Ii) directly influences MHC class II presentation by occupying the MHC class II peptide-binding groove, thereby preventing premature loading of peptides. Different MHC class II alleles exhibit distinct affinities for CLIP, and a low affinity interaction has been associated with decreased dependence upon H-2M and increased susceptibility to rheumatoid arthritis, suggesting that decreased CLIP affinity alters the MHC class II-bound peptide repertoire, thereby promoting autoimmunity. To examine the role of CLIP affinity in determining the MHC class II peptide repertoire, we generated transgenic mice expressing either wild-type human Ii or human Ii containing a CLIP region of low affinity for MHC class II. Our data indicate that although degradation intermediates of Ii containing a CLIP region with decreased affinity for MHC class II do not remain associated with I-A(b), this does not substantially alter the peptide repertoire bound by MHC class II or increase autoimmune susceptibility in the mice. This implies that the affinity of the CLIP:MHC class II interaction is not a strong contributory factor in determining the probability of developing autoimmunity. In contrast, in the absence of H-2M, MHC class II peptide repertoire diversity is enhanced by decreasing the affinity of CLIP for MHC class II, although MHC class II cell surface expression is reduced. Thus, we show clearly, in vivo, the critical chaperone function of H-2M, which preserves MHC class II molecules for high affinity peptide binding upon dissociation of Ii degradation intermediates.     

Dissection of the interferon gamma-MHC class II signal transduction pathway reveals that type I and type II interferon systems share common signalling component(s).

We have used a herpes virus thymidine kinase (HSV-TK) based metabolic selection system to isolate mutants defective in the interferon gamma mediated induction of the MHC class II promoter. All the mutations act in trans and result in no detectable induction of MHC and invariant chain (Ii) gene expression. Scatchard analysis indicates that the mutants have a normal number of surface IFN gamma receptors with the same affinity constant. The mutants fall into two broad categories. One class of mutants is still able to induce MHC class I, IRF-1, 9-27, 1-8 and GBP genes by IFN gamma. A second class of mutants is defective for the IFN gamma induction of all the genes tested; surprisingly, the IFN alpha/beta induction of MHC class I, 9-27, ISG54 and ISG15 genes is also defective in these mutants, although different members of this class can be discriminated by the response of the GBP and IRF-1 genes to type I interferons. These data demonstrate that the signalling pathways of both type I and type II interferon systems share common signal transduction component(s). These mutants will be useful for the study of IFN gamma regulation of class II genes and Ii chain, and to elucidate molecular components of type I and type II interferon signal transduction.     

How MHC class II molecules reach the endocytic pathway.

We have examined trafficking of major histocompatibility complex (MHC) class II molecules in human B cells exposed to concanamycin B, a highly specific inhibitor of the vacuolar H(+)-ATPases required for acidification of the vacuolar system and for early to late endosomal transport. Neutralization of vacuolar compartments prevents breakdown of the invariant chain (Ii) and blocks conversion of MHC class II molecules to peptide-loaded, SDS-stable alpha beta dimers. Ii remains associated with alpha beta and this complex accumulates internally, as ascertained biochemically and by morphological methods. In concanamycin B-treated cells, a slow increase (> 20-fold) in surface expression of Ii, mostly complexed with alpha beta, is detected. This surface-disposed fraction of alpha beta Ii is nevertheless a minor population that reaches the cell surface directly, or is routed via early endosomes as intermediary stations. In inhibitor-treated cells, the bulk of newly synthesized alpha beta Ii is no longer accessible to fluid phase endocytic markers. It is concluded that the majority of alpha beta Ii is targeted directly from the trans-Golgi network to the compartment for peptide loading, bypassing the cell surface and early endosomes en route to the endocytic pathway.     

Association with BiP and aggregation of class II MHC molecules synthesized in the absence of invariant chain.

Class II molecules of the major histocompatibility complex (MHC) are composed of two polymorphic glycoprotein chains (alpha and beta), that associate in the ER with a third, non-polymorphic glycoprotein known as the invariant chain (Ii). We have examined the relationship between the intracellular transport and physico-chemical characteristics of various combinations of murine alpha, beta and Ii chains. Biochemical and morphological analyses of transfected fibroblasts expressing class II MHC chains show that both unassembled alpha and beta chains, as well as a large fraction of alpha+beta complexes synthesized in the absence of Ii chain, are retained in the ER in association with the immunoglobulin heavy chain binding protein, BiP. Analyses by sedimentation velocity on sucrose gradients show that most incompletely assembled class II MHC species exist as high molecular weight aggregates in both transfected fibroblasts and spleen cells from mice carrying a disruption of the Ii chain gene. This is in contrast to the sedimentation properties of alpha beta Ii complexes from normal mice, which migrate as discrete, stoichiometric complexes of M(r) approximately 200,000-300,000. These observations suggest that assembly with the Ii chain prevents accumulation of aggregated alpha and beta chains in the ER, which might relate to the known ability of the Ii chain to promote exit of class II MHC molecules from the ER.     

Cathepsin B cleavage of Ii from class II MHC alpha- and beta-chains

Class II MHC-associated invariant chain (Ii) might regulate binding of digested peptides to the Ag binding site (desetope) of class II MHC proteins by directly or allosterically blocking that site until cleavage and release of Ii from MHC alpha- and beta-chains at the time of peptide charging. We examined the cleavage and release of Ii from class II MHC alpha/beta Ii trimers by cathepsin B, which has been shown by others to colocalize with class II MHC molecules in intracellular compartments and to generate antigenic peptide fragments. Cathepsin B at pH 5.0 cleaved and released Ii from class II MHC alpha- and beta-chains. Cathepsin B digested Ii from alpha- and beta-chains in a dose-dependent fashion, yielding 23-, 21-, and 10-kDa fragments. Blockage of cathepsin B activity with leupeptin restored the 2D(nonequilibrium pH gradient gel electrophoresis/SDS) PAGE patterns of Ii and sialic acid-derivatized forms of Ii seen without the protease. The fragmentation pattern of cathepsin D treatment was different from that of cathepsin B, yielding 25-kDa intermediates.     

Expression of invariant chain (CD 74) and major histocompatibility complex (MHC) class II antigens in the human fetus.

During the initiation of an immune response, antigen-presenting cells employ MHC class II antigens as key molecules to present small peptides to CD4-positive lymphocytes. The invariant chain (Ii; CD74) plays a critical role in this process by influencing the expression and peptide loading of the MHC class II molecules. Therefore, coordinate expression of these molecules is believed to play an important role in antigen presentation. This study explores the expression of these molecules in fetal tissues. Formalin-fixed, paraffin-embedded multi-organ tissue blocks from aborted fetuses (age range 7-22 weeks) were immunostained for Ii/CD74 and MHC class II antigens using commercially available monoclonal antibodies for Ii/CD74 (LN2) and MHC class II antigens (LN3), respectively. Coordinate staining for Ii/CD74 and MHC class II antigens was seen in the skin, proximal renal tubules, tips of small intestinal mucosa, and cells of the reticuloendothelial system, including the spleen and thymus. Expression of Ii/CD74, but not of MHC class II antigens, was seen in pulmonary alveolar epithelium in all cases and in testicular Leydig cells (11 of 11 testes examined). The distribution and intensity of staining did not change significantly with age. In conclusion, this study describes distribution of Ii/CD74 and MHC class II antigens in human fetal tissues. Coordinate expression of Ii/CD74 and MHC class II antigens was identified in most fetal tissues, but there were also notable exceptions. In all cases this took the form of expression of Ii/CD74 in the absence of MHC class II expression. Discordance was particularly striking in pulmonary alveolar epithelium and testicular Leydig cells. This suggests that the Ii/CD74 molecule has functional roles in addition to its role in antigen presentation.     

The MHC class I linkage group is a major determinant in the in vivo rejection of allogeneic erythrocytes in rainbow trout (<Emphasis Type="Italic">Oncorhynchus mykiss</Emphasis>)

Despite accumulating sequence data, information on the function of major histocompatibility complex (MHC) genes in fish is scarce. In contrast to the genome organization in higher vertebrates, the polymorphic MHC class I and II genes are not linked in the teleost genome. A previous study found an MHC class II linkage group to be a major determinant in the rejection of allogeneic scales by a teleost species (Cardwell et al. 2001). The present study investigated whether the teleost MHC class I linkage group can be involved in allograft rejection. Erythrocytes were chosen as grafts since they express MHC class I, but do not express class II. Rainbow trout erythrocytes expressing different MHC class I alleles were differentially stained, mixed and injected into recipients that were of the same sibling group as the donors. The MHC class I linkage group was the major determinant for in vivo graft rejection.     

MHC class II molecules on the move for successful antigen presentation

Major histocompatibility complex class II (MHC II) molecules are targeted to endocytic compartments, known as MIIC, by the invariant chain (Ii) that is degraded upon arrival in these compartments. MHC II acquire antigenic fragments from endocytosed proteins for presentation at the cell surface. In a unique and complex series of reactions, MHC II succeed in exchanging a remaining fragment of Ii for other protein fragments in subdomains of MIIC before transport to the cell surface. Here, the mechanisms regulating loading and intracellular trafficking of MHC II are discussed.     

Invariant chain protects class II histocompatibility antigens from binding intact polypeptides in the endoplasmic reticulum.

Unlike class I histocompatibility (MHC) antigens, most newly synthesized MHC class II molecules fail to be loaded with peptides in the endoplasmic reticulum (ER), binding instead to the invariant chain glycoprotein (Ii). Ii blocks the class II peptide binding groove until the class II:Ii complexes are transported to endosomes where Ii is removed by proteolysis, thus permitting loading with endosomal short peptides (approximately 12-25 amino acids). Ligands from which the groove is protected by Ii have not yet been identified; theoretically they could be short peptides or longer polypeptides (or both), because the class II groove is open at both ends. Here we show that in Ii- deficient cells, but not in cells expressing large amounts of Ii, a substantial fraction of class II alpha beta dimers forms specific, SDS-resistant 1:1 complexes with a variety of polypeptides. Different sets of polypeptides bound to H-2Ak, Ek, Ed and HLA-DR1 class II molecules; for Ak, a major species of Mr 50 kDa (p50) and further distinct 20 and 130 kDa polypeptides were detectable. Class II binding of p50 was characterized in detail. Point mutations within the Ak antigen binding groove destabilized the p50:class II complexes; a mutation outside the groove had no effect. A short segment of p50 was sufficient for association with Ak. The p50 polypeptide was synthesized endogenously, bound to Ak in a pre-Golgi compartment, and was transported to the cell surface in association with Ak. Thus, Ii protects the class II groove from binding endogenous, possibly misfolded polypeptides in the ER. The possibility is discussed that polypeptide binding is an ancestral function of the MHC antigen binding domain.     

Chromosome mapping of MHC class I in rainbow trout (Oncorhynchus mykiss)

The major histocompatibility complex (MHC) is well-studied in mammals. Much research has addressed the genomic organisation of MHC genes and it is well established that human MHC class I genes are located on chromosome 6. However, information on the organisation of the MHC complex in rainbow trout is only beginning to become available. In the present study it was determined that rainbow trout MHC class I sequences are located on chromosome 18. This is the first reported use of fluorescence in situ hybridisation (FISH) to identify the chromosomal location of genes involved in the immune system of fish.