Single locus typing of MHC class I and class II B loci in a population of red jungle fowl

In species with duplicated major histocompatibility complex (MHC) genes, estimates of genetic variation often rely on multilocus measures of diversity. It is possible that such measures might not always detect more detailed patterns of selection at individual loci. Here, we describe a method that allows us to investigate classical MHC diversity in red jungle fowl (Gallus gallus), the wild ancestor of the domestic chicken, using a single locus approach. This is possible due to the well-characterised gene organisation of the ‘minimal essential’ MHC (BF/BL region) of the domestic chicken, which comprises two differentially expressed duplicated class I (BF) and two class II B (BLB) genes. Using a combination of reference strand-mediated conformation analysis, cloning and sequencing, we identify nine BF and ten BLB alleles in a captive population of jungle fowl. We show that six BF and five BLB alleles are from the more highly expressed locus of each gene, BF2 and BLB2, respectively. An excess of non-synonymous substitutions across the jungle fowl BF/BL region suggests that diversifying selection has acted on this population. Importantly, single locus screening reveals that the strength of selection is greatest on the highly expressed BF2 locus. This is the first time that a population of red jungle fowl has been typed at the MHC region, laying the basis for further research into the underlying processes acting to maintain MHC diversity in this and other species.     

Structural analysis of MHC alleles in an RSV tumour regression chicken using a BAC library

The chicken major histocompatibility complex (MHC-B locus) has a strong association with resistance and susceptibility to numerous diseases. We have found a B haplotype designated WLA that associated with the regression of tumours caused by Rous sarcoma virus J strain (RSV-J). Haplotype WLA was identical to the regressive B6 haplotype when partial genotyping was performed (Poultry Science, 89, 2010, 651). We then constructed a bacterial artificial chromosome (BAC) library from a WLA homozygote chicken to evaluate the structure of this regression haplotype and compared it to those of the B6 haplotype. Comparison between WLA and B6 above 59 kb within the 167 kb, including 14 genes from BG1 to BF2, revealed 75 SNPs and 14 indels. However, several genes were identical between WLA and B6, including the BF1 and BF2 genes, which encode a class I molecule previously suggested to be related to the regression phenotype. The BLB2 gene encoding the MHC class II beta chain showed the greatest diversity, with 19 non-synonymous SNPs. A comparison of WLA and B6 haplotpyes that are associated with tumour regression and RIRa and B24 haplotypes associated with tumour progression suggests that DMA1, DMA2, BRD2, TAPBP and BLB2 genes are not involved in the intensity of RSV J tumour regression.     

High-resolution typing for chicken BF2 (MHC class I) alleles by automated sequencing

Sequence-based typing (SBT) was developed for major histocompatibility complex (MHC) class I and class II alleles in humans. We report here the development and application of a SBT method for alleles of the chicken BF2 locus (the more polymorphic of the two MHC class I loci in chickens). Exon 2 of the BF2 gene was selectively amplified from genomic DNA using a BF2 locus-specific PCR primer. Exon 2 sequences were sufficient to identify the 21 distinct BF2 alleles described in standard B haplotypes of Leghorns and in commercial broiler-breeder lines. Sixty-six samples from MHC typed, pedigreed chickens were tested, including 50 different heterozygous combinations. BF2 sequences from all B homozygotes were successfully amplified, and all combinations of BF2 alleles in heterozygotes were co-amplified equally. The two different BF2 alleles in heterozygotes could be identified unambiguously by distinct sequence motif patterns. In tests of samples of unknown B genotype in commercial broiler-breeder flocks, we identified expected BF2 alleles as well as an allele not previously encountered in one of the lines.     

The peptide motif of the single dominantly expressed class I molecule of the chicken MHC can explain the response to a molecular defined vaccine of infectious bursal disease virus (IBDV)

In contrast to typical mammals, the chicken MHC (the BF-BL region of the B locus) has strong genetic associations with resistance and susceptibility to infectious pathogens as well as responses to vaccines. We have shown that the chicken MHC encodes a single dominantly expressed class I molecule whose peptide-binding motifs can determine resistance to viral pathogens, such as Rous sarcoma virus and Marek’s disease virus. In this report, we examine the response to a molecular defined vaccine, fp-IBD1, which consists of a fowlpox virus vector carrying the VP2 gene of infectious bursal disease virus (IBDV) fused with β-galactosidase. We vaccinated parental lines and two backcross families with fp-IBD1, challenged with the virulent IBDV strain F52/70, and measured damage to the bursa. We found that the MHC haplotype B15 from line 15I confers no protection, whereas B2 from line 6₁ and B12 from line C determine protection, although another locus from line 6₁ was also important. Using our peptide motifs, we found that many more peptides from VP2 were predicted to bind to the dominantly expressed class I molecule BF2*1201 than BF2*1501. Moreover, most of the peptides predicted to bind BF2*1201 did in fact bind, while none bound BF2*1501. Using peptide vaccination, we identified one B12 peptide that conferred protection to challenge, as assessed by bursal damage and viremia. Thus, we show the strong genetic association of the chicken MHC to a T cell vaccine can be explained by peptide presentation by the single dominantly expressed class I molecule.     

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.     

The dominant MHC class I gene is adjacent to the polymorphic<Emphasis Type="Italic"> TAP2</Emphasis> gene in the duck,<Emphasis Type="Italic"> Anas platyrhynchos</Emphasis>

We are investigating the expression and linkage of major histocompatibility complex (MHC) class I genes in the duck (Anas platyrhynchos) with a view toward understanding the susceptibility of ducks to two medically important viruses: influenza A and hepatitis B. In mammals, there are multiple MHC class I loci, and alleles at a locus are polymorphic and co-dominantly expressed. In contrast, in lower vertebrates the expression of one locus predominates. Southern-blot analysis and amplification of genomic sequences suggested that ducks have at least four loci encoding MHC class I. To identify expressed MHC genes, we constructed an unamplified cDNA library from the spleen of a single duck and screened for MHC class I. We sequenced 44 positive clones and identified four MHC class I sequences, each sharing approximately 85% nucleotide identity. Allele-specific oligonucleotide hybridization to a Northern blot indicated that only two of these sequences were abundantly expressed. In chickens, the dominantly expressed MHC class I gene lies adjacent to the transporter of antigen processing (TAP2) gene. To investigate whether this organization is also found in ducks, we cloned the gene encoding TAP2 from the cDNA library. PCR amplification from genomic DNA allowed us to determine that the dominantly expressed MHC class I gene was adjacent to TAP2. Furthermore, we amplified two alleles of the TAP2 gene from this duck that have significant and clustered amino acid differences that may influence the peptides transported. This organization has implications for the ability of ducks to eliminate viral pathogens.The nucleotide sequence data reported in this paper have been submitted to the GenBank nucleotide sequence database and have been assigned the accession numbers AY294416–22     

Expression, linkage, and polymorphism of MHC-related genes in rainbow trout, Oncorhynchus mykiss.

The architecture of the MHC in teleost fish, which display a lack of linkage between class I and II genes, differs from all other vertebrates. Because rainbow trout have been examined for a variety of immunologically relevant genes, they present a good teleost model for examining both the expression and organization of MHC-related genes. Full-length cDNA and partial gDNA clones for proteasome delta, low molecular mass polypeptide (LMP) 2, TAP1, TAP2A, TAP2B, class Ia, and class IIB were isolated for this study. Aside from the expected polymorphisms associated with class I genes, LMP2 and TAP2 are polygenic. More specifically, we found a unique lineage of LMP2 (LMP2/delta) that shares identity to both LMP2 and delta but is expressed like the standard LMP2. Additionally, two very different TAP2 loci were found, one of which encodes polymorphic alleles. In general, the class I pathway genes are expressed in most tissues, with highest levels in lymphoid tissue. We then analyzed the basic genomic organization of the trout MHC in an isogenic backcross. The main class Ia region does not cosegregate with the class IIB locus, but LMP2, LMP2/delta, TAP1A, and TAP2B are linked to the class Ia locus. Interestingly, TAP2A (second TAP2 locus) is a unique lineage in sequence composition that appears not to be linked to this cluster or to class IIB. These results support and extend the recent findings of nonlinkage between class I and II in a different teleost order (cyprinids), suggesting that this unique arrangement is common to all teleosts.     

Proteasome, transporter associated with antigen processing, and class I genes in the nurse shark Ginglymostoma cirratum: evidence for a stable class I region and MHC haplotype lineages.

Cartilaginous fish (e.g., sharks) are derived from the oldest vertebrate ancestor having an adaptive immune system, and thus are key models for examining MHC evolution. Previously, family studies in two shark species showed that classical class I (UAA) and class II genes are genetically linked. In this study, we show that proteasome genes LMP2 and LMP7, shark-specific LMP7-like, and the TAP1/2 genes are linked to class I/II. Functional LMP7 and LMP7-like genes, as well as multiple LMP2 genes or gene fragments, are found only in some sharks, suggesting that different sets of peptides might be generated depending upon inherited MHC haplotypes. Cosmid clones bearing the MHC-linked classical class I genes were isolated and shown to contain proteasome gene fragments. A non-MHC-linked LMP7 gene also was identified on another cosmid, but only two exons of this gene were detected, closely linked to a class I pseudogene (UAA-NC2); this region probably resulted from a recent duplication and translocation from the functional MHC. Tight linkage of proteasome and class I genes, in comparison with gene organizations of other vertebrates, suggests a primordial MHC organization. Another nonclassical class I gene (UAA-NC1) was detected that is linked neither to MHC nor to UAA-NC2; its high level of sequence similarity to UAA suggests that UAA-NC1 also was recently derived from UAA and translocated from MHC. These data further support the principle of a primordial class I region with few class I genes. Finally, multiple paternities in one family were demonstrated, with potential segregation distortions.     

Mass spectral data for 64 eluted peptides and structural modeling define peptide binding preferences for class I alleles in two chicken MHC-<Emphasis Type="Italic">B</Emphasis> haplotypes associated with opposite responses to Marek's disease

In the chicken, resistance to lymphomas that form following infection with oncogenic strains of Marek's herpesvirus is strongly linked to the major histocompatibility complex (MHC)-B complex. MHC-B21 haplotype is associated with lower tumor-related mortality compared to other haplotypes including MHC-B13. The single, dominantly expressed class I gene (BF2) is postulated as responsible for the MHC-B haplotype association. We used mass spectrometry to identify peptides and structural modeling to define the peptide binding preferences of BF2*2101 and BF2*1301 proteins. Endogenous peptides (8-12 residues long) were eluted from affinity-purified BF2*2101 and BF2*1301 proteins obtained from transduced cDNA expressed in RP9 cells, hence expressed in the presence of heterologous TAP. Sequences of individual peptides were identified by mass spectrometry. BF2*2101 peptides appear to be tethered at the binding groove margins with longer peptides arching out but selected by preferred residues at positions P3, P5, and P8: X-X-[AVILFP]-X((1-5))-[AVLFWP]-X((2-3))-[VILFM]. BF2*1301 peptides appear selected for residues at P2, P3, P5, and P8: X-[DE]-[AVILFW]-X((1-2))-[DE]-X-X-[ED]-X((0-4)). Some longer BF2*1301 peptides likely also arch out, but others are apparently accommodated by repositioning of Arg83 so that peptides extend beyond the last preferred residue at P8. Comparisons of these peptides with earlier peptides derived in the presence of homologous TAP transport revealed the same side chain preferences. Scanning of Marek's and other viral proteins with the BF2*2101 motif identified many matches, as did the control human leukocyte antigen A*0201 motif. The BF2*1301 motif is more restricting suggesting that this allele may confer a selective advantage only in infections with a subset of viral pathogens.     

A novel, nonclassical MHC class I molecule specific to the common chimpanzee

All expressed human MHC class I genes (HLA-A, -B, -C, -E, -F, and -G) have functional orthologues in the MHC of the common chimpanzee (Pan troglodytes). In contrast, a nonclassical MHC class I gene discovered in the chimpanzee is not present in humans or the other African ape species. In exons and more so in introns, this Patr-AL gene is similar to the expressed A locus in the orangutan, Popy-A, suggesting they are orthologous. Patr-AL/Popy-A last shared a common ancestor with the classical MHC-A locus >20 million years ago. Population analysis revealed little Patr-AL polymorphism: just three allotypes differing only at residues 52 and 91. Patr-AL is expressed in PBMC and B cell lines, but at low level compared with classical MHC class I. The Patr-AL polypeptide is unusually basic, but its glycosylation, association with beta(2)-microglobulin, and antigenicity at the cell surface are like other MHC class I. No Patr-AL-mediated inhibition of polyclonal chimpanzee NK cells was detected. The Patr-AL gene is present in 50% of chimpanzee MHC haplotypes, correlating with presence of a 9.8-kb band in Southern blots. The flanking regions of Patr-AL contain repetitive/retroviral elements not flanking other class I genes. In sequenced HLA class I haplotypes, a similar element is present in the A*2901 haplotype but not the A*0201 or A*0301 haplotypes. This element, 6 kb downstream of A*2901, appears to be the relic of a human gene related to Patr-AL. Patr-AL has characteristics of a class I molecule of innate immunity with potential to provide common chimpanzees with responses unavailable to humans.     

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.     

Multiple divergent haplotypes express completely distinct sets of class I MHC genes in zebrafish

The zebrafish is an important animal model for stem cell biology, cancer, and immunology research. Histocompatibility represents a key intersection of these disciplines; however, histocompatibility in zebrafish remains poorly understood. We examined a set of diverse zebrafish class I major histocompatibility complex (MHC) genes that segregate with specific haplotypes at chromosome 19, and for which donor-recipient matching has been shown to improve engraftment after hematopoietic transplantation. Using flanking gene polymorphisms, we identified six distinct chromosome 19 haplotypes. We describe several novel class I U lineage genes and characterize their sequence properties, expression, and haplotype distribution. Altogether, ten full-length zebrafish class I genes were analyzed, mhc1uba through mhc1uka. Expression data and sequence properties indicate that most are candidate classical genes. Several substitutions in putative peptide anchor residues, often shared with deduced MHC molecules from additional teleost species, suggest flexibility in antigen binding. All ten zebrafish class I genes were uniquely assigned among the six haplotypes, with dominant or codominant expression of one to three genes per haplotype. Interestingly, while the divergent MHC haplotypes display variable gene copy number and content, the different genes appear to have ancient origin, with extremely high levels of sequence diversity. Furthermore, haplotype variability extends beyond the MHC genes to include divergent forms of psmb8. The many disparate haplotypes at this locus therefore represent a remarkable form of genomic region configuration polymorphism. Defining the functional MHC genes within these divergent class I haplotypes in zebrafish will provide an important foundation for future studies in immunology and transplantation.     

Critical role for the tapasin-docking site of TAP2 in the functional integrity of the MHC class I-peptide-loading complex

The transporter associated with Ag processing (TAP) translocates antigenic peptides into the endoplasmic reticulum for binding onto MHC class I (MHC I) molecules. Tapasin organizes a peptide-loading complex (PLC) by recruiting MHC I and accessory chaperones to the N-terminal regions (N domains) of the TAP subunits TAP1 and TAP2. To investigate the function of the tapasin-docking sites of TAP in MHC I processing, we expressed N-terminally truncated variants of TAP1 and TAP2 in combination with wild-type chains, as fusion proteins or as single subunits. Strikingly, TAP variants lacking the N domain in TAP2, but not in TAP1, build PLCs that fail to generate stable MHC I-peptide complexes. This correlates with a substantially reduced recruitment of accessory chaperones into the PLC demonstrating their important role in the quality control of MHC I loading. However, stable surface expression of MHC I can be rescued in post-endoplasmic reticulum compartments by a proprotein convertase-dependent mechanism.     

A novel instance of class I modification (cim) affecting two of three rat class I RT1-A molecules within one MHC haplotype

MHC class I expression by rats of the RT1(o), RT1(d), and RT1(m) MHC haplotypes was investigated. Identical, functional cDNAs were obtained from RT1(o) and BDIX (RT1(dv1)) rats for three MHC class I molecules. RT1-A1(o/d) and -A2(o/d) are closely related in sequence to other cloned rat class Ia genes that have been shown to map to the RT1-A region, while RT1-A3 degrees is highly homologous to a class I gene identified by sequencing an RT1-A(n) genomic contig and is named A3(n). Detailed analysis of the three molecules was undertaken using serology with mAbs, two-dimensional gel analysis of immunoprecipitates, and killing assays using cytotoxic T cells. Arguments are presented suggesting that A1 degrees is the principal MHC class Ia (classical) restricting element of this haplotype. A2 degrees, which is highly cross-reactive with A1 degrees, and A3 degrees probably play more minor or distinct roles in Ag presentation. Unexpectedly, cDNAs encoding exactly the same three molecules were cloned from rats of the RT1(m) haplotype, an MHC that until now was thought to possess unique class Ia genes. RT1(m) contains the TAP-B allele of the TAP transporter, and we present evidence that functional polymorphism in rat TAP has an even greater impact on the expression of RT1-A1 degrees and -A2 degrees than it does on RT1-A(a) in the established case of class I modification (cim). Historically, this led to the misclassification of RT1(m) class Ia molecules as separate and distinct.