The Diverse Major Histocompatibility Complex Haplotypes of a Common Commercial Chicken Line and Their Effect on Marek's Disease Virus Pathogenesis and Tumorigenesis.

The major histocompatibility complex (MHC) is crucial for appropriate immune responses against invading pathogens. Chickens possess a single predominantly-expressed class I molecule with strong associations between disease resistance and MHC haplotype. For Marek's disease virus (MDV) infections of chickens, the MHC haplotype is one of the major determinants of genetic resistance and susceptibility. VALO specific pathogen free (SPF) chickens are widely used in biomedical research and vaccine production. While valuable findings originate from MDV infections of VALO SPF chickens, their MHC haplotypes and associated disease resistance remained elusive. In this study, we used several typing systems to show that VALO SPF chickens possess MHC haplotypes that include B9, B9:02, B15, B19 and B21 at various frequencies. Moreover, we associate the MHC haplotypes to MDV-induced disease and lymphoma formation and found that B15 homozygotes had the lowest tumor incidence while B21 homozygotes had the lowest number of organs with tumors. Finally, we found transmission at variable levels to all contact birds except B15/B21 heterozygotes. These data have immediate implications for the use of VALO SPF chickens and eggs in the life sciences and add another piece to the puzzle of the chicken MHC complex and its role in infections with this oncogenic herpesvirus.

Chickens as a simple system for scientific discovery: The example of the MHC.

Chickens have played many roles in human societies over thousands of years, most recently as an important model species for scientific discovery, particularly for embryology, virology and immunology. In the last few decades, biomedical models like mice have become the most important model organism for understanding the mechanisms of disease, but for the study of outbred populations, they have many limitations. Research on humans directly addresses many questions about disease, but frank experiments into mechanisms are limited by practicality and ethics. For research into all levels of disease simultaneously, chickens combine many of the advantages of humans and of mice, and could provide an independent, integrated and overarching system to validate and/or challenge the dogmas that have arisen from current biomedical research. Moreover, some important systems are simpler in chickens than in typical mammals. An example is the major histocompatibility complex (MHC) that encodes the classical MHC molecules, which play crucial roles in the innate and adaptive immune systems. Compared to the large and complex MHCs of typical mammals, the chicken MHC is compact and simple, with single dominantly-expressed MHC molecules that can determine the response to infectious pathogens. As a result, some fundamental principles have been easier to discover in chickens, with the importance of generalist and specialist MHC alleles being the latest example.

A potential nomenclature for the Immuno Polymorphism Database (IPD) of chicken MHC genes: progress and problems.

Among the genes with the highest allelic polymorphism and sequence diversity are those encoding the classical class I and class II molecules of the major histocompatibility complex (MHC). Although many thousands of MHC sequences have been deposited in general sequence databases like GenBank, the availability of curated MHC sequences with agreed nomenclature has been enormously beneficial. Along with the Immuno Polymorphism Database-IMunoGeneTics/human leukocyte antigen (IPD-IMGT/HLA) database, a collection of databases for curated sequences of immune importance has been developed. A recent addition is an IPD-MHC database for chickens. For many years, the nomenclature system for chicken MHC genes has been based on a list of standard, presumed to be stable, haplotypes. However, these standard haplotypes give different names to identical sequences. Moreover, the discovery of new recombinants between haplotypes and a rapid increase in newly discovered alleles leaves the old system untenable. In this review, a new nomenclature is considered, for which alleles of different loci are given names based on the system used for other MHCs, and then haplotypes are named according to the alleles present. The new nomenclature system is trialled, first with standard haplotypes and then with validated sequences from the scientific literature. In the trial, some class II B sequences were found in both class II loci, presumably by gene conversion or inversion, so that identical sequences would receive different names. This situation prompts further suggestions to the new nomenclature system. In summary, there has been progress, but also problems, with the new IPD-MHC system for chickens.

Surface expression, peptide repertoire, and thermostability of chicken class I molecules correlate with peptide transporter specificity.

The chicken major histocompatibility complex (MHC) has strong genetic associations with resistance and susceptibility to certain infectious pathogens. The cell surface expression level of MHC class I molecules varies as much as 10-fold between chicken haplotypes and is inversely correlated with diversity of peptide repertoire and with resistance to Marek's disease caused by an oncogenic herpesvirus. Here we show that the average thermostability of class I molecules isolated from cells also varies, being higher for high-expressing MHC haplotypes. However, we find roughly the same amount of class I protein synthesized by high- and low-expressing MHC haplotypes, with movement to the cell surface responsible for the difference in expression. Previous data show that chicken TAP genes have high allelic polymorphism, with peptide translocation specific for each MHC haplotype. Here we use assembly assays with peptide libraries to show that high-expressing B15 class I molecules can bind a much wider variety of peptides than are found on the cell surface, with the B15 TAPs restricting the peptides available. In contrast, the translocation specificity of TAPs from the low-expressing B21 haplotype is even more permissive than the promiscuous binding shown by the dominantly expressed class I molecule. B15/B21 heterozygote cells show much greater expression of B15 class I molecules than B15/B15 homozygote cells, presumably as a result of receiving additional peptides from the B21 TAPs. Thus, chicken MHC haplotypes vary in several correlated attributes, with the most obvious candidate linking all these properties being molecular interactions within the peptide-loading complex (PLC).

Structure of the chicken CD3εδ/γ heterodimer and its assembly with the αßT cell receptor.

In mammals, the αßT cell receptor (TCR) signaling complex is composed of a TCRαß heterodimer that is noncovalently coupled to three dimeric signaling molecules, CD3εδ, CD3εγ, and CD3ζζ. The nature of the TCR signaling complex and subunit arrangement in different species remains unclear however. Here we present a structural and biochemical analysis of the more primitive ancestral form of the TCR signaling complex found in chickens. In contrast to mammals, chickens do not express separate CD3δ and CD3γ chains but instead encode a single hybrid chain, termed CD3δ/γ, that is capable of pairing with CD3ε. The NMR structure of the chicken CD3εδ/γ heterodimer revealed a unique dimer interface that results in a heterodimer with considerable deviation from the distinct side-by-side architecture found in human and murine CD3εδ and CD3εγ. The chicken CD3εδ/γ heterodimer also contains a unique molecular surface, with the vast majority of surface-exposed, nonconserved residues being clustered to a single face of the heterodimer. Using an in vitro biochemical assay, we demonstrate that CD3εδ/γ can assemble with both chicken TCRα and TCRß via conserved polar transmembrane sites. Moreover, analogous to the human TCR signaling complex, the presence of two copies of CD3εδ/γ is required for ζζ assembly. These data provide insight into the evolution of this critical receptor signaling apparatus.

CD40 ligand supports the long-term maintenance and differentiation of chicken B cells in culture.

TNF family members play crucial roles in mammalian B-cell differentiation and function, many of which have not been demonstrated in other species. To investigate the avian CD40/CD40L system, a chicken CD40 cDNA, obtained by expression screening, was used to raise monoclonal antibodies showing that CD40 was expressed on chicken B cells, monocytes and macrophages, like mammalian CD40. CD40 ligand fusion protein supported the proliferation of B cells in culture for up to 3 weeks, during which they differentiated towards a plasma cell phenotype. CD40L-activated B cells from immunised birds secreted antigen-specific IgM and IgG. These results showed important conserved functions of CD40 and its ligand in mammals and birds. CD40L provides a means for maintenance and differentiation of untransformed chicken B cells in culture, for the first time, allowing new approaches to study of post-bursal B cell biology and host-pathogen interactions with B cell tropic viruses.

Conservation of biological properties of the CD40 ligand, CD154 in a non-mammalian vertebrate.

Signals delivered by the CD40 ligand, CD154, have crucial roles in immune responses in mammals, being required for development of germinal centres, maturation of T-dependent antibody responses, and generation of B-cell memory. To determine whether these functions were conserved in a non-mammalian species, a putative chicken CD 154 cDNA was used to make an oligomeric fusion protein, and to raise monoclonal antibodies. The antibodies detected surface expression on activated T-cells. The fusion protein detected expression of a receptor on B-cells, thrombocytes and macrophages. Biological effects of the fusion protein included induction of NO synthesis in a macrophage cell line, enhancement of splenic B-cell survival, and induction of apoptosis in a bursal lymphoma cell line. These observations demonstrated substantial functional equivalence with mammalian CD 154 and thus provided evidence for the early evolutionary emergence of the set of functions associated with this molecule, and its central role in the vertebrate immune system.

The chicken CD4 gene has remained conserved in evolution.

CD4 has a central role in thymocyte differentiation and cell-mediated immunity. We isolated and analyzed chicken CD4. The gene spans 11.5 kb and is composed of ten exons. The promoter is TATA-less and similar to the mouse and human CD4 promoters, with two transcription start sites as determined by 5'RACE analysis. In general the introns are short, although the 5'untranslated region includes a large intron of 5.6 kb containing binding sites of the putative CD4 silencer. The single-strand conformation polymorphism technique was used to identify a polymorphism to map the gene, which lies on chicken Chromosome 1 in a position showing conserved synteny to mouse and human. This is the first report describing the organization of CD4 from a non-mammalian species. The structure and localization of chicken CD4 and many sequence motifs important in its regulation have remained conserved during evolution.