A chromosome-level genome assembly for the Silkie chicken resolves complete sequences for key chicken metabolic, reproductive, and immunity genes.

A set of high-quality pan-genomes would help identify important genes that are still hidden/incomplete in bird reference genomes. In an attempt to address these issues, we have assembled a de novo chromosome-level reference genome of the Silkie (Gallus gallus domesticus), which is an important avian model for unique traits, like fibromelanosis, with unclear genetic foundation. This Silkie genome includes the complete genomic sequences of well-known, but unresolved, evolutionarily, endocrinologically, and immunologically important genes, including leptin, ovocleidin-17, and tumor-necrosis factor-α. The gap-less and manually annotated MHC (major histocompatibility complex) region possesses 38 recently identified genes, with differentially regulated genes recovered in response to pathogen challenges. We also provide whole-genome methylation and genetic variation maps, and resolve a complex genetic region that may contribute to fibromelanosis in these animals. Finally, we experimentally show leptin binding to the identified leptin receptor in chicken, confirming an active leptin ligand-receptor system. The Silkie genome assembly not only provides a rich data resource for avian genome studies, but also lays a foundation for further functional validation of resolved genes.

The minor chicken class I gene BF1 is deleted between short imperfect direct repeats in the B14 and typical B15 major histocompatibility complex (MHC) haplotypes.

The chicken major histocompatibility complex (MHC, also known as the BF-BL region of the B locus) is notably small and simple with few genes, most of which are involved in antigen processing and presentation. There are two classical class I genes, of which only BF2 is well and systemically expressed as the major ligand for cytotoxic T lymphocytes (CTLs). The other class I gene, BF1, is believed to be primarily a natural killer (NK) cell ligand. Among most standard chicken MHC haplotypes examined in detail, BF1 is expressed tenfold less than BF2 at the RNA level due to defects in the promoter or in a splice site. However, in the B14 and typical B15 haplotypes, BF1 RNA was not detected, and here, we show that a deletion between imperfect 32 nucleotide direct repeats has removed the BF1 gene entirely. The phenotypic effects of not having a BF1 gene (particularly on resistance to infectious pathogens) have not been systematically explored, but such deletions between short direct repeats are also found in some BF1 promoters and in the 5' untranslated region (5'UTR) of some BG genes found in the BG region of the B locus. Despite the opposite transcriptional orientation of homologous genes in the chicken MHC, which might prevent the loss of key genes from a minimal essential MHC, it appears that small direct repeats can still lead to deletion.

Understanding the evolution of immune genes in jawed vertebrates.

Driven by co-evolution with pathogens, host immunity continuously adapts to optimize defence against pathogens within a given environment. Recent advances in genetics, genomics and transcriptomics have enabled a more detailed investigation into how immunogenetic variation shapes the diversity of immune responses seen across domestic and wild animal species. However, a deeper understanding of the diverse molecular mechanisms that shape immunity within and among species is still needed to gain insight into-and generate evolutionary hypotheses on-the ultimate drivers of immunological differences. Here, we discuss current advances in our understanding of molecular evolution underpinning jawed vertebrate immunity. First, we introduce the immunome concept, a framework for characterizing genes involved in immune defence from a comparative perspective, then we outline how immune genes of interest can be identified. Second, we focus on how different selection modes are observed acting across groups of immune genes and propose hypotheses to explain these differences. We then provide an overview of the approaches used so far to study the evolutionary heterogeneity of immune genes on macro and microevolutionary scales. Finally, we discuss some of the current evidence as to how specific pathogens affect the evolution of different groups of immune genes. This review results from the collective discussion on the current key challenges in evolutionary immunology conducted at the ESEB 2021 Online Satellite Symposium: Molecular evolution of the vertebrate immune system, from the lab to natural populations.

A Wider and Deeper Peptide-Binding Groove for the Class I Molecules from B15 Compared with B19 Chickens Correlates with Relative Resistance to Marek's Disease.

The chicken MHC is known to confer decisive resistance or susceptibility to various economically important pathogens, including the iconic oncogenic herpesvirus that causes Marek's disease (MD). Only one classical class I gene, BF2, is expressed at a high level in chickens, so it was relatively easy to discern a hierarchy from well-expressed thermostable fastidious specialist alleles to promiscuous generalist alleles that are less stable and expressed less on the cell surface. The class I molecule BF2*1901 is better expressed and more thermostable than the closely related BF2*1501, but the peptide motif was not simpler as expected. In this study, we confirm for newly developed chicken lines that the chicken MHC haplotype B15 confers resistance to MD compared with B19. Using gas phase sequencing and immunopeptidomics, we find that BF2*1901 binds a greater variety of amino acids in some anchor positions than does BF2*1501. However, by x-ray crystallography, we find that the peptide-binding groove of BF2*1901 is narrower and shallower. Although the self-peptides that bound to BF2*1901 may appear more various than those of BF2*1501, the structures show that the wider and deeper peptide-binding groove of BF2*1501 allows stronger binding and thus more peptides overall, correlating with the expected hierarchies for expression level, thermostability, and MD resistance. Our study provides a reasonable explanation for greater promiscuity for BF2*1501 compared with BF2*1901, corresponding to the difference in resistance to MD.

New vistas unfold: Chicken MHC molecules reveal unexpected ways to present peptides to the immune system.

The functions of a wide variety of molecules with structures similar to the classical class I and class II molecules encoded by the major histocompatibility complex (MHC) have been studied by biochemical and structural studies over decades, with many aspects for humans and mice now enshrined in textbooks as dogma. However, there is much variation of the MHC and MHC molecules among the other jawed vertebrates, understood in the most detail for the domestic chicken. Among the many unexpected features in chickens is the co-evolution between polymorphic TAP and tapasin genes with a dominantly-expressed class I gene based on a different genomic arrangement compared to typical mammals. Another important discovery was the hierarchy of class I alleles for a suite of properties including size of peptide repertoire, stability and cell surface expression level, which is also found in humans although not as extreme, and which led to the concept of generalists and specialists in response to infectious pathogens. Structural studies of chicken class I molecules have provided molecular explanations for the differences in peptide binding compared to typical mammals. These unexpected phenomena include the stringent binding with three anchor residues and acidic residues at the peptide C-terminus for fastidious alleles, and the remodelling binding sites, relaxed binding of anchor residues in broad hydrophobic pockets and extension at the peptide C-terminus for promiscuous alleles. The first few studies for chicken class II molecules have already uncovered unanticipated structural features, including an allele that binds peptides by a decamer core. It seems likely that the understanding of how MHC molecules bind and present peptides to lymphocytes will broaden considerably with further unexpected discoveries through biochemical and structural studies for chickens and other non-mammalian vertebrates.

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.

Some thoughts about what non-mammalian jawed vertebrates are telling us about antigen processing and peptide loading of MHC molecules.

The major histocompatibility complex (MHC) of mammals encodes highly polymorphic classical class I and class II molecules with crucial roles in immune responses, as well as various nonclassical molecules encoded by the MHC and elsewhere in the genome that have a variety of functions. These MHC molecules are supported by antigen processing and peptide loading pathways which are well-understood in mammals. This review considers what has been learned about the MHC, MHC molecules and the supporting pathways in non-mammalian jawed vertebrates. From the initial understanding from work with the chicken MHC, a great deal of diversity in the structure and function has been found. Are there underlying principles?

Innate immune genes of the chicken MHC and related regions.

Compared to the major histocompatibility complex (MHC) of typical mammals, the chicken BF/BL region is small and simple, with most of the genes playing central roles in the adaptive immune response. However, some genes of the chicken MHC are almost certainly involved in innate immunity, such as the complement component C4 and the lectin-like receptor/ligand gene pair BNK and Blec. The poorly expressed classical class I molecule BF1 is known to be recognised by natural killer (NK) cells and, analogous to mammalian immune responses, the classical class I molecules BF1 and BF2, the CD1 homologs and the butyrophilin homologs called BG may be recognised by adaptive immune lymphocytes with semi-invariant receptors in a so-called adaptate manner. Moreover, the TRIM and BG regions next to the chicken MHC, along with the genetically unlinked Y and olfactory/scavenger receptor regions on the same chromosome, have multigene families almost certainly involved in innate and adaptate responses. On this chicken microchromosome, the simplicity of the adaptive immune gene systems contrasts with the complexity of the gene systems potentially involved in innate immunity.

The dominantly expressed class II molecule from a resistant MHC haplotype presents only a few Marek's disease virus peptides by using an unprecedented binding motif.

Viral diseases pose major threats to humans and other animals, including the billions of chickens that are an important food source as well as a public health concern due to zoonotic pathogens. Unlike humans and other typical mammals, the major histocompatibility complex (MHC) of chickens can confer decisive resistance or susceptibility to many viral diseases. An iconic example is Marek's disease, caused by an oncogenic herpesvirus with over 100 genes. Classical MHC class I and class II molecules present antigenic peptides to T lymphocytes, and it has been hard to understand how such MHC molecules could be involved in susceptibility to Marek's disease, given the potential number of peptides from over 100 genes. We used a new in vitro infection system and immunopeptidomics to determine peptide motifs for the 2 class II molecules expressed by the MHC haplotype B2, which is known to confer resistance to Marek's disease. Surprisingly, we found that the vast majority of viral peptide epitopes presented by chicken class II molecules arise from only 4 viral genes, nearly all having the peptide motif for BL2*02, the dominantly expressed class II molecule in chickens. We expressed BL2*02 linked to several Marek's disease virus (MDV) peptides and determined one X-ray crystal structure, showing how a single small amino acid in the binding site causes a crinkle in the peptide, leading to a core binding peptide of 10 amino acids, compared to the 9 amino acids in all other reported class II molecules. The limited number of potential T cell epitopes from such a complex virus can explain the differential MHC-determined resistance to MDV, but raises questions of mechanism and opportunities for vaccine targets in this important food species, as well as providing a basis for understanding class II molecules in other species including humans.

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.

Cas9-expressing chickens and pigs as resources for genome editing in livestock.

Genetically modified animals continue to provide important insights into the molecular basis of health and disease. Research has focused mostly on genetically modified mice, although other species like pigs resemble the human physiology more closely. In addition, cross-species comparisons with phylogenetically distant species such as chickens provide powerful insights into fundamental biological and biomedical processes. One of the most versatile genetic methods applicable across species is CRISPR-Cas9. Here, we report the generation of transgenic chickens and pigs that constitutively express Cas9 in all organs. These animals are healthy and fertile. Functionality of Cas9 was confirmed in both species for a number of different target genes, for a variety of cell types and in vivo by targeted gene disruption in lymphocytes and the developing brain, and by precise excision of a 12.7-kb DNA fragment in the heart. The Cas9 transgenic animals will provide a powerful resource for in vivo genome editing for both agricultural and translational biomedical research, and will facilitate reverse genetics as well as cross-species comparisons.

Quantitative trait loci and transcriptome signatures associated with avian heritable resistance to Campylobacter.

Campylobacter is the leading cause of bacterial foodborne gastroenteritis worldwide. Handling or consumption of contaminated poultry meat is a key risk factor for human campylobacteriosis. One potential control strategy is to select poultry with increased resistance to Campylobacter. We associated high-density genome-wide genotypes (600K single nucleotide polymorphisms) of 3000 commercial broilers with Campylobacter load in their caeca. Trait heritability was modest but significant (h2 = 0.11 ± 0.03). Results confirmed quantitative trait loci (QTL) on chromosomes 14 and 16 previously identified in inbred chicken lines, and detected two additional QTLs on chromosomes 19 and 26. RNA-Seq analysis of broilers at the extremes of colonisation phenotype identified differentially transcribed genes within the QTL on chromosome 16 and proximal to the major histocompatibility complex (MHC) locus. We identified strong cis-QTLs located within MHC suggesting the presence of cis-acting variation in MHC class I and II and BG genes. Pathway and network analyses implicated cooperative functional pathways and networks in colonisation, including those related to antigen presentation, innate and adaptive immune responses, calcium, and renin-angiotensin signalling. While co-selection for enhanced resistance and other breeding goals is feasible, the frequency of resistance-associated alleles was high in the population studied and non-genetic factors significantly influenced Campylobacter colonisation.

The Combination of CD8αα and Peptide-MHC-I in a Face-to-Face Mode Promotes Chicken γδT Cells Response.

The CD8αα homodimer is crucial to both thymic T cell selection and the antigen recognition of cytotoxic T cells. The CD8-pMHC-I interaction can enhance CTL immunity via stabilizing the TCR-pMHC-I interaction and optimizing the cross-reactivity and Ag sensitivity of CD8+ T cells at various stages of development. To date, only human and mouse CD8-pMHC-I complexes have been determined. Here, we resolved the pBF2*1501 complex and the cCD8αα/pBF2*1501 and cCD8αα/pBF2*0401 complexes in nonmammals for the first time. Remarkably, cCD8αα/pBF2*1501 and the cCD8αα/pBF2*0401 complex both exhibited two binding modes, including an "antibody-like" mode similar to that of the known mammal CD8/pMHC-I complexes and a "face-to-face" mode that has been observed only in chickens to date. Compared to the "antibody-like" mode, the "face-to-face" binding mode changes the binding orientation of the cCD8αα homodimer to pMHC-I, which might facilitate abundant γδT cells to bind diverse peptides presented by limited BF2 alleles in chicken. Moreover, the forces involving in the interaction of cCD8αα/pBF2*1501 and the cCD8αα/pBF2*0401 are different in this two binding model, which might change the strength of the CD8-pMHC-I interaction, amplifying T cell cross-reactivity in chickens. The coreceptor CD8αα of TCR has evolved two peptide-MHC-I binding patterns in chickens, which might enhance the T cell response to major or emerging pathogens, including chicken-derived pathogens that are relevant to human health, such as high-pathogenicity influenza viruses.

From Chickens to Humans: The Importance of Peptide Repertoires for MHC Class I Alleles.

In humans, killer immunoglobulin-like receptors (KIRs), expressed on natural killer (NK) and thymus-derived (T) cells, and their ligands, primarily the classical class I molecules of the major histocompatibility complex (MHC) expressed on nearly all cells, are both polymorphic. The variation of this receptor-ligand interaction, based on which alleles have been inherited, is known to play crucial roles in resistance to infectious disease, autoimmunity, and reproduction in humans. However, not all the variation in response is inherited, since KIR binding can be affected by a portion of the peptide bound to the class I molecules, with the particular peptide presented affecting the NK response. The extent to which the large multigene family of chicken immunoglobulin-like receptors (ChIRs) is involved in functions similar to KIRs is suspected but not proven. However, much is understood about the two MHC-I molecules encoded in the chicken MHC. The BF2 molecule is expressed at a high level and is thought to be the predominant ligand of cytotoxic T lymphocytes (CTLs), while the BF1 molecule is expressed at a much lower level if at all and is thought to be primarily a ligand for NK cells. Recently, a hierarchy of BF2 alleles with a suite of correlated properties has been defined, from those expressed at a high level on the cell surface but with a narrow range of bound peptides to those expressed at a lower level on the cell surface but with a very wide repertoire of bound peptides. Interestingly, there is a similar hierarchy for human class I alleles, although the hierarchy is not as wide. It is a question whether KIRs and ChIRs recognize class I molecules with bound peptide in a similar way, and whether fastidious to promiscuous hierarchy of class I molecules affect both T and NK cell function. Such effects might be different from those predicted by the similarities of peptide-binding based on peptide motifs, as enshrined in the idea of supertypes. Since the size of peptide repertoire can be very different for alleles with similar peptide motifs from the same supertype, the relative importance of these two properties may be testable.

Origins of the RAG Transposome and the MHC.

How innate immunity gave rise to adaptive immunity in vertebrates remains unknown. We propose an evolutionary scenario beginning with pathogen-associated molecular pattern(s) (PAMPs) being presented by molecule(s) on one cell to specific receptor(s) on other cells, much like MHC molecules and T cell receptors (TCRs). In this model, mutations in MHC-like molecule(s) that bound new PAMP(s) would not be recognized by original TCR-like molecule(s), and new MHC-like gene(s) would be lost by neutral drift. Integrating recombination activating gene (RAG) transposon(s) in a TCR-like gene would result in greater recognition diversity, with new MHC-like variants recognized and selected, along with a new RAG/TCR-like system. MHC genes would be selected to present many peptides, through multigene families, allelic polymorphism, and peptide-binding promiscuity.

Advances in the Evolutionary Understanding of MHC Polymorphism.

Proteins encoded by the classical major histocompatibility complex (MHC) genes incite the vertebrate adaptive immune response by presenting peptide antigens on the cell surface. Here, we review mechanisms explaining landmark features of these genes: extreme polymorphism, excess of nonsynonymous changes in peptide-binding domains, and long gene genealogies. Recent studies provide evidence that these features may arise due to pathogens evolving ways to evade immune response guided by the locally common MHC alleles. However, complexities of selection on MHC genes are simultaneously being revealed that need to be incorporated into existing theory. These include pathogen-driven selection for antigen-binding breadth and expansion of the MHC gene family, associated autoimmunity trade-offs, hitchhiking of deleterious mutations linked to the MHC, geographic subdivision, and adaptive introgression.

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.