Jim Kaufman

The chicken B locus is a minimal essential major histocompatibility complex.

Here we report the sequence of the region that determines rapid allograft rejection in chickens, the chicken major histocompatibility complex (MHC). This 92-kilobase region of the B locus contains only 19 genes, making the chicken MHC roughly 20-fold smaller than the human MHC. Virtually all the genes have counterparts in the human MHC, defining a minimal essential set of MHC genes conserved over 200 million years of divergence between birds and mammals. They are organized differently, with the class III region genes located outside the class II and class I region genes. The absence of proteasome genes is unexpected and might explain unusual peptide-binding specificities of chicken class I molecules. The presence of putative natural killer receptor gene(s) is unprecedented and might explain the importance of the B locus in the response to the herpes virus responsible for Mareks disease. The small size and simplicity of the chicken MHC allows co-evolution of genes as haplotypes over considerable periods of time, and makes it possible to study the striking MHC-determined pathogen-specific disease resistance at the molecular level.

A "minimal essential Mhc" and an "unrecognized Mhc": two extremes in selection for polymorphism.

The high polymorphism of classical Mhc molecules found in mammals is not simply the result of strong selection for pathogen resistance in the recent past, since there are virtually no examples of diseases caused by infectious pathogens for which resistance is determined by particular Mhc haplotypes, and in the best-studied case, a particular aspect of malaria in humans, the selection is remarkably weak. We discuss three possibilities to explain high polymorphism in mammals: accumulating, merging and boosting. The mammalian Mhc is complicated and redundant, so that every Mhc haplotype may give some level of resistance due to multiple classical Mhc genes as well as other disease resistance genes; this frustrates the attempts to demonstrate selection for disease resistance. We have looked at two vertebrate groups that may represent two extreme examples of selection for Mhc polymorphism. Birds, like mammals, have highly a polymorphic Mhc that determines strong allograft rejection. However, chickens have a much smaller, compact and simpler Mhc than mammals, as though the Mhc has been stripped down to the essentials during evolution. The selection on a single Mhc gene should be much stronger than on a large multigene family and, in fact, there are a number of viral diseases for which resistance and susceptibility are determined by particular chicken Mhc haplotypes. We have determined the peptide motifs for the chicken class I molecules from a number of haplotypes, which may explain some disease associations quite simply. On the other hand, salamanders have very low Mhc polymorphism and slow allograft rejection. We have isolated axolotl Mhc molecules and shown that they cosegregate with the locus that determines graft rejection in the axolotl, have only a few alleles and only weakly stimulate axolotl T lymphocytes in mixed lymphocyte culture. We believe that salamanders have classical Mhc molecules but most T cells do not recognize them, so that there is no strong selection for polymorphism.

Peptide motifs of the single dominantly expressed class I molecule explain the striking MHC-determined response to Rous sarcoma virus in chickens

Compared with the MHC of typical mammals, the chicken MHC is smaller and simpler, with only two class I genes found in the B12 haplotype. We make five points to show that there is a single-dominantly expressed class I molecule that can have a strong effect on MHC function. First, we find only one cDNA for two MHC haplotypes (B14 and B15) and cDNAs corresponding to two genes for the other six (B2, B4, B6, B12, B19, and B21). Second, we find, for the B4, B12, and B15 haplotypes, that one cDNA is at least 10-fold more abundant than the other. Third, we use 2D gel electrophoresis of class I molecules from pulse-labeled cells to show that there is only one heavy chain spot for the B4 and B15 haplotypes, and one major spot for the B12 haplotype. Fourth, we determine the peptide motifs for B4, B12, and B15 cells in detail, including pool sequences and individual peptides, and show that the motifs are consistent with the peptides binding to models of the class I molecule encoded by the abundant cDNA. Finally, having shown for three haplotypes that there is a single dominantly expressed class I molecule at the level of RNA, protein, and antigenic peptide, we show that the motifs can explain the striking MHC-determined resistance and susceptibility to Rous sarcoma virus. These results are consistent with the concept of a “minimal essential MHC” for chickens, in strong contrast to typical mammals.

Gene organisation determines evolution of function in the chicken MHC

Summary: Some years ago, we used our data for class I genes, proteins and peptide‐binding specificities to develop the hypothesis that the chicken B‐F/B‐L region represents a “minimal essential MHC”, In this view, the B locus contains the classical (highly expressed and polymorphic) class I α and class II β multigene families, which are reduced to one or two members, with many other genes moved away or deleted from the chicken genome altogether. We found that a single dominantly expressed class I gene determines the immune response to certain infectious pathogens, due to peptide‐binding specificity and cell‐surface expression level. This stands in stark contrast to well‐studied mammals like humans and mice, in which every haplotype is more‐or‐less responsive to every pathogen and vaccine, presumably due to the multigene family of MHC molecules present. In order to approach the basis for a single dominantly expressed class I molecule, we have sequenced a portion of the B complex and examined the location and polymorphism of the class I (B‐F)α, TAP and class II (B‐L) β genes. The region is remarkably compact and simple, with many of the genes expected from the MHC of mammals absent, including LMP, class II α and DO genes as well as most class III region genes. However, unexpected genes were present, including tapasin and putative natural killer receptor genes. The region is also organised differently from mammals, with the TAPs in between the class I genes, the tapasin gene in between the class II (B‐L) β genes, and the C4 gene outside of the class I α and class II β genes. The close proximity of TAP and class I α genes leads to the possibility of co‐evolution, which can drive the use of a single dominantly expressed class I molecule with peptide‐binding specificity like the TAP molecule. There is also a single dominantly expressed class II β gene, but the reason for this is not yet clear. Finally, the presence of the C4 gene outside of the classical class I α and class II β genes suggests the possibility that this organisation was ancestral, although a number of models of organization and evolution are still possible, given the presence of the Rfp‐γ region with non‐classical class I α and class II β genes as well as the presence of multigene families of B‐G and rRNA genes.

2004 nomenclature for the chicken major histocompatibility (B and Y) complex

The first standard nomenclature for the chicken (Gallus gallus) major histocompatibility (B) complex published in 1982 describing chicken major histocompatibility complex (MHC) variability is being revised to include subsequent findings. Considerable progress has been made in identifying the genes that define this polymorphic region. Allelic sequences for MHC genes are accumulating at an increasing rate without a standard system of nomenclature in place. The recommendations presented here were derived in workshops held during International Society of Animal Genetics and Avian Immunology Research Group meetings. A nomenclature for B and Y (Rfp-Y) loci and alleles has been developed that can be applied to existing and newly defined haplotypes including recombinants. A list of the current standard B haplotypes is provided with reference stock, allele designations, and GenBank numbers for corresponding MHC class I and class IIβ sequences. An updated list of proposed names for B recombinant haplotypes is included, as well as a list of over 17 Y haplotypes designated to date.

Reversible epigenetic down-regulation of MHC molecules by devil facial tumour disease illustrates immune escape by a contagious cancer

Contagious cancers that pass between individuals as an infectious cell line are highly unusual pathogens. Devil facial tumor disease (DFTD) is one such contagious cancer that emerged 16 y ago and is driving the Tasmanian devil to extinction. As both a pathogen and an allograft, DFTD cells should be rejected by the host–immune response, yet DFTD causes 100% mortality among infected devils with no apparent rejection of tumor cells. Why DFTD cells are not rejected has been a question of considerable confusion. Here, we show that DFTD cells do not express cell surface MHC molecules in vitro or in vivo, due to down-regulation of genes essential to the antigen-processing pathway, such as β2-microglobulin and transporters associated with antigen processing. Loss of gene expression is not due to structural mutations, but to regulatory changes including epigenetic deacetylation of histones. Consequently, MHC class I molecules can be restored to the surface of DFTD cells in vitro by using recombinant devil IFN-γ, which is associated with up-regulation of the MHC class II transactivator, a key transcription factor with deacetylase activity. Further, expression of MHC class I molecules by DFTD cells can occur in vivo during lymphocyte infiltration. These results explain why T cells do not target DFTD cells. We propose that MHC-positive or epigenetically modified DFTD cells may provide a vaccine to DFTD. In addition, we suggest that down-regulation of MHC molecules using regulatory mechanisms allows evolvability of transmissible cancers and could affect the evolutionary trajectory of DFTD.

ISAG/IUIS-VIC Comparative MHC Nomenclature Committee report, 2005

Nomenclature for Major Histocompatibility Complex (MHC) genes and alleles in species other than humans and mice has historically been overseen either informally by groups generating sequences, or by formal nomenclature committees set up by the International Society for Animal Genetics (ISAG). The suggestion for a Comparative MHC Nomenclature Committee was made at the ISAG meeting held in Göttingen, Germany (2002), and the committee met for the first time at the Institute for Animal Health, Compton, UK in January 2003. To publicize its activity and extend its scope, the committee organized a workshop at the International Veterinary Immunology Symposium (IVIS) in Quebec (2004) where it was decided to affiliate with the Veterinary Immunology Committee (VIC) of the International Union of Immunological Societies (IUIS). The goals of the committee are to establish a common framework and guidelines for MHC nomenclature in any species; to demonstrate this in the form of a database that will ensure that in the future, researchers can easily access a source of validated MHC sequences for any species; to facilitate discussion on this area between existing groups and nomenclature committees. A further meeting of the committee was held in September 2005 in Glasgow, UK. This was attended by most of the existing committee members with some additional invited participants (Table 1). The aims of this meeting were to facilitate the inclusion of new species onto the database, to discuss extension, improvement and funding of the database, and to address a number of nomenclature issues raised at the previous workshop.

Generation and characterization of chicken bone marrow‐derived dendritic cells

Dendritic cells (DCs) are bone marrow‐derived professional antigen‐presenting cells. The in vitro generation of DCs from either bone marrow or blood is routine in mammals. Their distinct morphology and phenotype and their unique ability to stimulate naïve T cells are used to define DCs. In this study, chicken bone marrow cells were cultured in the presence of recombinant chicken granulocyte–macrophage colony‐stimulating factor (GM‐CSF) and recombinant chicken interleukin‐4 (IL‐4) for 7 days. The cultured population showed the typical morphology of DCs, with the surface phenotype of major histocompatibility complex (MHC) class II+ (high), CD11c+ (high), CD40+ (moderate), CD1·1+ (moderate), CD86+ (low), CD83− and DEC‐205−. Upon maturation with lipopolysaccharide (LPS) or CD40L, surface expression of CD40, CD1·1, CD86, CD83 and DEC‐205 was greatly increased. Endocytosis and phagocytosis were assessed by fluorescein isothiocyanate (FITC)‐dextran uptake and fluorescent bead uptake, respectively, and both decreased after stimulation. Non‐stimulated chicken bone marrow‐derived DCs (chBM‐DCs) stimulated both allogeneic and syngeneic peripheral blood lymphocytes (PBLs) to proliferate in a mixed lymphocyte reaction (MLR). LPS‐ or CD40L‐stimulated chBM‐DCs were more effective T‐cell stimulators in MLR than non‐stimulated chBM‐DCs. Cultured chBM‐DCs could be matured to a T helper type 1 (Th1)‐promoting phenotype by LPS or CD40L stimulation, as determined by mRNA expression levels of Th1 and Th2 cytokines. We have therefore cultured functional chBM‐DCs in a non‐mammalian species for the first time.

Characterization of the Chicken C-Type Lectin-Like Receptors B-NK and B-lec Suggests That the NK Complex and the MHC Share a Common Ancestral Region

The sequencing of the chicken MHC led to the identification of two open reading frames, designated B-NK and B-lec, that were predicted to encode C-type lectin domains. C-type lectin domains are not encoded in the MHC of any animal described to date; therefore, this observation was completely unexpected, particularly given that the chicken has a “minimal essential MHC.” In this study, we describe the initial characterization of the B-NK and B-lec genes, and show that they share greatest homology with C-type lectin-like receptors encoded in the human NK complex (NKC), in particular NKR-P1 and lectin-like transcript 1 (LLT1), respectively. In common with NKR-P1 and LLT1, B-NK and B-lec are located next to each other and transcribed in opposite orientation. Like human NKR-P1, B-NK has a functional inhibitory signaling motif in the cytoplasmic tail and is expressed in NK cells. In contrast, B-lec contains an endocytosis motif in the cytoplasmic tail, and like LLT1, is an early activation Ag. Further analysis leads us to propose that there are four subgroups of C-type lectin-like receptors in the NKC, which arose as a result of duplication events. Moreover, this analysis suggests that the NKC may be considered a fifth paralogous region, and therefore shares an ancient common origin with the MHC. This provides evidence that C-type lectin-like receptors were present in the preduplication, primordial MHC region, and suggests that an original function of MHC molecules was for recognition by NK cell receptors encoded nearby.

The dominantly expressed class I molecule of the chicken MHC is explained by coevolution with the polymorphic peptide transporter (TAP) genes

In most mammals, the MHC class I molecules are polymorphic and determine the specificity of peptide presentation, whereas the transporter associated with antigen presentation (TAP) heterodimers are functionally monomorphic. In chickens, there are two classical class I genes but only one is expressed at a high level, which can result in strong MHC associations with resistance to particular infectious pathogens. However, the basis for having a single dominantly expressed class I molecule has been unclear. Here we report TAP1 and TAP2 sequences from 16 chicken lines, and show that both genes have high allelic polymorphism and moderate sequence diversity, with variation in positions expected for peptide binding. We analyze peptide translocation in two MHC haplotypes, showing that chicken TAPs specify translocation at three peptide positions, matching the peptide motif of the single dominantly expressed class I molecule. These results show that coevolution between class I and TAP genes can explain the presence of a single dominantly expressed class I molecule in common chicken MHC haplotypes. Moreover, such coevolution in the primordial MHC may have been responsible for the appearance of the antigen presentation pathways at the birth of the adaptive immune system.