Molecular analysis of chicken immune response genes

We have recently isolated immune response genes of the major histocompatibility B complex of the chicken (the B-L beta genes) by cross-hybridization in low stringency with an HLA class II beta chain probe. After reviewing the main results obtained, we present a detailed analysis of the region flanking the first gene characterized, B-L beta III. By Southern blot analysis with exon-specific probes, we demonstrate the presence of another related B-L beta gene 10 kb on the 3' side of B-L beta III, the B-L beta V gene. Moreover, retrospective analysis of the phage clones initially isolated with the HLA-DQ beta probe, using a chicken class I probe that we isolated by chromosome walking from the B-L beta genes, indicates that the B-L beta III gene is closely linked on its 5' side to a class I gene, B-FVI.     

Isolation and characterization of cDNA clones for chicken major histocompatibility complex class II molecules

The chicken major histocompatibility complex (MHC), the B complex, is being intensively analysed at the DNA level. To further probe the molecular structure of chicken MHC class II genes, cDNA clones coding for chicken MHC class II (B-L) p chain molecules were isolated from an inbred G-B2 Leghorn chicken spleen and liver. Twenty-nine cDNA clones were isolated from the spleen and eight cDNA clones were isolated from the liver. Based on restriction maps, most clones could be clustered into one family of genes. Four cDNA clones were sequenced (S7, S10 and S19 from the spleen and L1, which was identical to S19, from the liver). Complete amino acid sequences of B-Lβ chain molecules were predicted from the nucleotide sequences of the cDNA clones. Although both the nature and the location of the conserved residues were similar in chicken and mammalian sequences, some species-specific differences were found, suggesting that the structures of the B-L molecules of this haplotype are similar, but not identical, to their mammalian counterparts.     

The major histocompatibility complex in the chicken

The chicken B complex is the first non-mammalian MHC characterized at the molecular level. It differs from the human HLA and murine H-2 complexes in the small size of the class I (B-F) and class II (B-L) genes and their close proximity. This proximity accounts for the absence of recombination between B-F and B-L genes and leaves no space for class III genes. Moreover the B-F and B-L genes are tightly linked to unrelated genes absent from mammalian MHCs, such as the polymorphic B-G genes and a member of the G protein beta subunit family. This linkage could form the basis for resistance to viral-induced tumors associated with some B complex haplotypes.     

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

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.     

The simple chicken major histocompatibility complex: life and death in the face of pathogens and vaccines

In contrast to the major histocompatibility complex (MHC) of well-studied mammals such as humans and mice, the particular haplotype of the B-F/B-L region of the chicken B locus determines life and death in response to certain infectious pathogens as well as to certain vaccines. We found that the B-F/B-L region is much smaller and simpler than the typical mammalian MHC, with an important difference being the expression of a single class I gene at a high level of RNA and protein. The peptide-binding specificity of this dominantly expressed class I molecule in different haplotypes correlates with resistance to tumours caused by Rous sarcoma virus, while the cell-surface expression level correlates with susceptibility to tumours caused by Marek's disease virus. A similar story is developing with class II beta genes and response to killed viral vaccines. This apparently suicidal strategy of single dominantly expressed class I and class II molecules may be due to coevolution between genes within the compact chicken MHC.     

A PCR method for typing B-LβII family (class II MHC) alleles in broiler chickens

Certain haplotypes of the major histocompatibility (B) complex are strongly associated with resistance or susceptibility to several infectious diseases in Leghorn chickens. Identification of chicken haplotypes based on the nucleotide sequence of B complex loci could provide more precise identification of haplotypes than traditional serological methods. We report the development and application of polymerase chain reaction with sequence specific primers (PCR-SSP) to type broiler chicken B haplotypes based on the DNA sequence of B-L beta II family genes. Five well-defined standard B haplotypes from White Leghorns and 12 recently characterized B haplotypes from a broiler breeder line were used to develop the test system. The B-L beta II family loci were amplified from genomic DNA by B-L beta II family specific primers and then characterized by PCR-SSP. In total, ten pairs of primers, derived from the sequences of expressed B-L beta II family alleles, were used in the PCR typing test to discriminate the chicken B haplotypes identified previously by serological means. The PCR-SSP showed that each haplotype had a different amplification pattern, except those haplotypes known or suspected to have the same B-L beta alleles. Cloning and sequencing of the family specific PCR products indicated that two loci in the B-L beta II family, presumably B-L beta I and B-L beta II, were amplified. Finally, B-L beta PCR-SSP typing was used in combination with B-G RFLP analyses to characterize unusual (variant) B serotypes; the results indicate that some of these are natural recombinants within the B complex.     

Structure, biosynthesis, and polymorphism of chicken MHC class II (B-L) antigens and associated molecules

Chicken MHC class II (B-L) antigens were immunoprecipitated by the monoclonal antibody TaP1 from inbred chicken splenic leukocytes and a lymphoblastoid B cell line (RP9), and were studied by two dimensional gel electrophoresis. B-L antigens are composed of one alpha and one beta chain that are noncovalently bound at the cell surface. In all haplotypes studied, a single acidic 34,000 dalton non-polymorphic chain was observed, whereas two polymorphic chains could be distinguished, differing in both pH and m.w. The alpha-beta heterodimer is associated during its maturation in the cytoplasm with several basic invariant molecules with m.w. ranging from 30,000 to 42,000 daltons. Treatment of cells with tunicamycin and treatment of immunoprecipitated molecules with several glycosidases revealed a complex process of maturation for all of these molecules. The alpha and beta chains undergo a N-glycosylation of complex type, whereas the invariant molecules bear N-linked high mannose glycans, and perhaps also O-linked glycans in the RP9 lymphoblastoid line. Overall, the B-L antigens appear very similar to the HLA-DR and I-E antigens.     

Identification of peptides associated with chicken major histocompatibility complex class II molecules of <Emphasis Type="Italic">B21</Emphasis> and <Emphasis Type="Italic">B19</Emphasis> haplotypes

Chicken major histocompatibility complex (MHC) molecules present peptides to T cells to initiate immune response. Some variants of the chicken MHC, such as B19 and B21 haplotypes, are strongly associated with susceptibility and resistance to Mareks disease, respectively. The objective of the present study was to characterize the repertoire and origin of self-peptides presented by chicken MHC class II (B-L) molecules of B19 and B21 haplotypes. Following immunoaffinity purification of B21 and B19 B-L molecules from transformed B cell lines, their associated peptides were eluted, high performance liquid chromatography-fractionated, and sequenced by tandem mass spectrometry. Four peptides were identified associated with B21 B-L molecules. These ranged from 16 to 21 residues in length and had originated from membrane-bound, cytosolic, and mitochondrial proteins. Two of these peptides were present in form of an overlapping set, which is a common characteristic of MHC II-associated peptides. The single B19-associated peptide was 17 residues long and had originated from a cytosolic source. Presentation of endogenous peptides, such as those derived from cytosolic and mitochondrial proteins, by B-L molecules is indicative of cross-sampling between MHC class I and II antigen presentation pathways. These findings facilitate future studies aimed at elucidating mechanisms of chicken MHC association with disease resistance.     

Evolutionary relationships of class II major-histocompatibility-complex genes in mammals

The major histocompatibility complex (MHC) class II molecule consists of noncovalently associated alpha and beta chains. In mammals studied so far, the class II MHC can be divided into a number of regions, each containing one or more alpha-chain genes (A genes) and beta-chain genes (B genes), and it has been known for some time that orthologous relationships exist between genes in corresponding regions from different mammalian species. A phylogenetic analysis of DNA sequences of class II A and B genes confirmed these relationships; but no such orthologous relationship was observed between the B genes of mammals and those of birds. Thus, the class II regions have diverged since the separation of birds and mammals (approximately 300 Mya) but before the radiation of the placental mammalian orders (60-80 Mya). Comparison of the phylogenetic trees for A and B genes revealed an unexpected characteristic of DP-region genes: DPB genes are most closely related to DQB genes, whereas DPA chain genes are most closely related to DRA-chain genes. Thus, the DP region seems to have originated through a recombinational event which brought together a DQB gene and a DRA gene (perhaps approximately 120 Mya). The 5' untranslated region of all class II genes includes sequences which are believed to be important in regulating class II gene expression but which are not conserved in known pseudogenes. These sequences are conserved to an extraordinary degree in the human DQB1 gene and its mouse homologue A beta 1, suggesting that regulation of expression of this locus may play a key role in expression of the entire class II MHC.     

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

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.     

Marsupial MHC class II beta genes are not orthologous to the eutherian beta gene families

The major histocompatibility complex (MHC) class II DRB, DQB, DPB, and DOB gene clusters are shared by different eutherian orders. Such an orthologous relationship is not seen between the beta genes of birds and eutherians. A high degree of uncertainty surrounds the evolutionary relationship of marsupial class II beta sequences with eutherian beta gene families. In particular, it has been suggested that marsupials utilize the DRB gene cluster. A cDNA encoding an MHC class II beta molecule was isolated from a brushtail possum mesenteric lymph node cDNA library. This clone is most similar to Macropus rufogriseus DBB. Our analysis suggests that all known marsupial beta-chain genes, excluding DMB, fall into two separate clades, which are distinct from the eutherian DRB, DQB, DPB, or DOB gene clusters. We recommend that the DAB and DBB nomenclature be reinstated. DAB and DBB orthologs are not present in eutherians. It appears that the marsupial and eutherian lineages have retained different gene clusters following gene duplication events early in mammalian evolution.     

Beta 57-Asp plays an essential role in the unique SDS stability of HLA-DQA1*0102/DQB1*0602 alpha beta protein dimer, the class II MHC allele associated with protection from insulin-dependent diabetes mellitus

Studies of the stability of HLA-DQ have revealed a correlation between SDS stability of MHC class II alphabeta dimers and insulin-dependent diabetes mellitus (IDDM) susceptibility. The MHC class II alphabeta dimer encoded by HLA-DQA1*0102/DQB1*0602 (DQ0602), which is a dominant protective allele in IDDM, exhibits the greatest SDS stability among HLA-DQ molecules in EBV-transformed B-lymphoblastoid cells and PBLs. DQ0602 is also uniquely SDS stable in the HLA-DM-deficient cell line, BLS-1. We addressed the molecular mechanism of the stability of DQ0602 in BLS-1. A panel of mutants based on the polymorphic differences between HLA-DQA1*0102/DQB1*0602 and HLA-DQA1*0102/DQB1*0604 were generated and expressed in BLS-1. An Asp at beta57 was found to be critical for SDS stability, whereas Tyr at beta30, Gly at beta70, and Ala at beta86 played secondary roles. Furthermore, the level of class II-associated invariant chain peptide bound to HLA-DQ did not correlate with SDS stability, suggesting that class II-associated invariant chain peptide does not play a direct role in the unique SDS stability of DQ0602. These results support a role for DQB1 codon 57 in HLA-DQ alphabeta dimer stability and IDDM susceptibility.     

B-L antigens (Class II) of the chicken major histocompatibility complex control T-B cell interaction

The detailed study of the genetic control of T-B cell interactions in the chicken has been hampered by the lack of defined major histocompatibility complex (MHC) recombinant chicken lines. In the present study we have used some recently described MHC recombinant chicken lines separating regions encoding antigens that are homologous to class I and class II antigens of mammals in adoptive bursa cell transfer experiments, in which bursa cells from newly hatched chicks were transplanted into cyclophosphamide (Cy)-treated chicks. Subsequent immunizations of the recipients with a thymus-dependent antigen (SRBC) and a thymus-independent antigen (Brucella abortus) showed that the generation of germinal centers in the spleen and the production of antibodies to SRBC required identity between donor and recipient class II antigens (B-L antigens), whereas response to Brucella antigen did not require identity at any of the known MHC loci of the chicken. The results thus reveal that also in the chicken class II (B-L) region genes encode cell-surface glycoproteins that serve as restriction elements in T-B cell cooperation.This work has been presented in part at the Ninth International Congress of the Transplantation Society (Vainio et al. 1983a).