Bi-Functional Chicken Immunoglobulin-Like Receptors With a Single Extracellular Domain (ChIR-AB1): Potential Framework Genes Among a Relatively Stable Number of Genes Per Haplotype.

The leukocyte receptor complex (LRC) in humans encodes many receptors with immunoglobulin-like (Ig-like) extracellular domains, including the killer Ig-like receptors (KIRs) expressed on natural killer (NK) cells among others, the leukocyte Ig-like receptors (LILRs) expressed on myeloid and B cells, and an Fc receptor (FcR), all of which have important roles in the immune response. These highly-related genes encode activating receptors with positively-charged residues in the transmembrane region, inhibitory receptors with immuno-tyrosine based motifs (ITIMs) in the cytoplasmic tail, and bi-functional receptors with both. The related chicken Ig-like receptors (ChIRs) are almost all found together on a microchromosome, with over 100 activating (A), inhibitory (B), and bi-functional (AB) genes, bearing either one or two extracellular Ig-like domains, interspersed over 500-1,000 kB in the genome of an individual chicken. Sequencing studies have suggested rapid divergence and little overlap between ChIR haplotypes, so we wished to begin to understand their genetics. We chose to use a hybridization technique, reference strand-mediated conformational analysis (RSCA), to examine the ChIR-AB1 family, with a moderate number of genes dispersed across the microchromosome. Using fluorescently-labeled references (FLR), we found that RSCA and sequencing of ChIR-AB1 extracellular exon gave two groups of peaks with mobility correlated with sequence relationship to the FLR. We used this system to examine widely-used and well-characterized experimental chicken lines, finding only one or a few simple ChIR haplotypes for each line, with similar numbers of peaks overall. We found much more complicated patterns from a broiler line from a commercial breeder and a flock of red junglefowl, but trios of parents and offspring from another commercial chicken line show that the complicated patterns are due to heterozygosity, indicating a relatively stable number of peaks within haplotypes of these birds. Some ChIR-AB1 peaks were found in all individuals from the commercial lines, and some of these were shared with red junglefowl and the experimental lines derived originally from egg-laying chickens. Overall, this analysis suggests that there are some simple features underlying the apparent complexity of the ChIR locus.

Identification of an Activating Chicken Ig-like Receptor Recognizing Avian Influenza Viruses.

Chicken Ig-like receptors (CHIRs) represent a multigene family encoded by the leukocyte receptor complex that encodes a variety of receptors that are subdivided into activating CHIR-A, inhibitory CHIR-B, and bifunctional CHIR-AB. Apart from CHIR-AB, which functions as an Fc receptor, CHIR ligands are unknown. In the current study, we used a panel of different BWZ.36 CHIR reporter cells to identify an interaction between specific CHIRs and avian influenza virus (AIV). The specificity of the CHIR-AIV interaction was further demonstrated using CHIR fusion proteins that bound to AIV-coated plates and were able to reduce the interaction of reporter cells with AIV. There was no difference in binding of CHIR to different AIV strains. Furthermore, CHIR fusion proteins reduced AIV-induced in vitro activation of NK cells obtained from lungs of AIV-infected animals, as judged by the lower frequency of CD107+ cells. Because the original CHIR reporter lines were generated based on sequence information about extracellular CHIR domains, we next identified a full-length CHIR that displayed similar binding to AIV. The sequence analysis identified this CHIR as a CHIR-A. Neuraminidase treatment of coated CHIR-human Ig proteins reduced binding of trimeric H5 proteins to CHIR. This suggests that the interaction is dependent on sialic acid moieties on the receptor. In conclusion, this article identifies AIV as a ligand of CHIR-A and describes the functional consequences of this interaction.

Chicken TREM-B1, an Inhibitory Ig-Like Receptor Expressed on Chicken Thrombocytes.

Triggering receptors expressed on myeloid cells (TREM) form a multigene family of immunoregulatory Ig-like receptors and play important roles in the regulation of innate and adaptive immunity. In chickens, three members of the TREM family have been identified on chromosome 26. One of them is TREM-B1 which possesses two V-set Ig-domains, an uncharged transmembrane region and a long cytoplasmic tail with one ITSM and two ITIMs indicating an inhibitory function. We generated specific monoclonal antibodies by immunizing a Balb/c mouse with a TREM-B1-FLAG transfected BWZ.36 cell line and tested the hybridoma supernatants on TREM-B1-FLAG transfected 2D8 cells. We obtained two different antibodies specific for TREM-B1, mab 7E8 (mouse IgG1) and mab 1E9 (mouse IgG2a) which were used for cell surface staining. Single and double staining of different tissues, including whole blood preparations, revealed expression on thrombocytes. Next we investigated the biochemical properties of TREM-B1 by using the specific mab 1E9 for immunoprecipitation of either lysates of surface biotinylated peripheral blood cells or stably transfected 2D8 cells. Staining with streptavidin coupled horse radish peroxidase revealed a glycosylated monomeric protein of about 50 kDa. Furthermore we used the stably transfected 2D8 cell line for analyzing the cytoplasmic tyrosine based signaling motifs. After pervanadate treatment, we detected phosphorylation of the tyrosine residues and subsequent recruitment of the tyrosine specific protein phosphatase SHP-2, indicating an inhibitory potential for TREM-B1. We also showed the inhibitory effect of TREM-B1 in chicken thrombocytes using a CD107 degranulation assay. Crosslinking of TREM-B1 on activated primary thrombocytes resulted in decreased CD107 surface expression of about 50-70%.

Chicken CD300a homolog is found on B lymphocytes, various leukocytes populations and binds to phospholipids.

The chicken CD300 cluster contains three genes that encode inhibitory, activating and soluble forms. In the present study, we have generated a monoclonal antibody against the inhibitory CD300L-B1 molecule. The mab 1D4 was specific for the CD300L-B1 form and showed no crossreactivity with the related CD300L-X1. Virtually all bursal cells expressed CD300L-B1, whereas only a small positive subset was found in thymus that was identified as thymic B cell subpopulation. In peripheral tissues, CD300L-B1 was found to be expressed on lymphocyte subpopulations in blood and spleen. Double immunofluorescence analysis with B- and T-cell specific markers identified these subsets as B lymphocytes. In addition, analysis of PBMC revealed that CD300L-B1 was also present on monocytes, heterophils, blood NK cells and in vitro differentiated macrophages. We utilized a reporter cell line in order to identify potential ligands of CD300L-B1. When several phospholipids were tested, only phosphatidylserine and phosphatidylethanolamine were found to trigger strong reaction of the reporter cells. The two phospholipids elicited a response only in CD300L-B1 reporter cells, but not in CD300L-X1 reporter cells. Moreover the interaction could be blocked with the specific mab. In conclusion, we provide evidence for the expression of chicken CD300L-B1 on immature and mature B cells, monocytes, heterophils, macrophages and NK cells and identify phosphatidylserine and phosphatidylethanolamine as CD300L-B1 ligands.

Identification of novel chicken CD4⁺ CD3⁻ blood population with NK cell like features.

Chicken NK cells have been defined in embryonic spleen and intestinal epithelium as CD8(+) lymphoid cells that lack BCR and TCR, whereas blood NK cells have not been phenotypically defined. Here we employed the mab, 8D12 directed against CHIR-AB1, a chicken Fc receptor, to define a previously uncharacterized lymphoid cell population in the blood. Although CHIR-AB1 expression was found on several cell populations, cells with extraordinary high CHIR-AB1 levels ranged between 0.4 and 2.8% in five different chicken lines. The widespread applicability of the CHIR-AB1 mab was unexpected, since CHIR-AB1-like genes form a polygenic and polymorphic subfamily. Surprisingly the CHIR-AB1 high cells coexpressed low MHCII, low CD4 and CD5, while other T cell markers CD3 and CD8, the B cell marker Bu1, the macrophage marker KUL01 were absent. Moreover, they stained with the mab 28-4, 20E5 and 1G7, which define chicken NK cells and they also expressed CD25, CD57, CD244 and the vitronectin receptor (αVß3 integrin). In functional assays, PMA stimulation led to high levels of IFNγ release, while spontaneous cytotoxicity was not detectable. The expression of typical NK cell markers in the absence of characteristic B- or T-cell markers, and their IFNγ release is suggestive of a yet unidentified NK like population.

Chicken SLAMF4 (CD244, 2B4), a receptor expressed on thrombocytes, monocytes, NK cells, and subsets of αß-, γδ- T cells and B cells binds to SLAMF2.

The SLAM family of membrane receptors is involved in the regulation of immune responses by controlling cytokines production, cytotoxicity as well as cell development, differentiation and proliferation, but has only been described in chickens, recently. The aim of this study was to characterize the avian homologue to mammalian SLAMF4 (CD244, 2B4), a cell surface molecule which belongs to the SLAM family of membrane receptors. We generated a SLAMF4 specific monoclonal antibody (mab) designated 8C7 and analyzed the SLAMF4 expression on cells isolated from various lymphoid organs. Subsets of αß and γδ T cells found in peripheral blood lymphocytes (PBL) and spleen coexpressed SLAMF4. The expression was restricted to CD8α(+) T cells, whereas CD4(+) T cells and all thymocytes showed little or no reactivity upon staining with the 8C7 mab. Blood and splenic γδ T cells could be further differentiated according to their expression levels of SLAMF4 into two and three subsets, respectively. SLAMF4 was absent from bursal and splenic B cells, however, it was expressed by a distinct fraction of circulating B cells that were characterized by high level expression of Bu1, Ig, and CD40. SLAMF4 was also present on NK cells isolated from intestine of adult chickens or embryonic splenocytes identified by their coexpression of the 28-4 NK cell marker. Moreover, SLAMF4 expression was found on thrombocytes and monocytes. The interaction of SLAMF4 with SLAMF2 was proven by a reporter assay and could be blocked with the 8C7 mab. In conclusion, the avian SLAMF4 expression markedly differs from mammals; it binds to SLAMF2 and will be an important tool to discriminate several γδ T cell subsets.

Chicken immunoregulatory Ig-like receptor families: an overview and expression details on ggTREM-A1.

Paired immunoregulatory receptors facilitate the coordination of the immune response at the cellular level. In recent years, our group characterized chicken homologues to mammalian immunoregulatory Ig-like receptor families. The first part of this review focuses on the current progress on chicken immunoregulatory Ig-like receptor families. One of these receptors is gallus gallus TREM-A1, which was described as the only member of the chicken TREM family with activating potential. The second part of this review presents a study initiated to further characterize ggTREM-A1 expression. For this purpose we established real-time RT-PCR and generated a specific mab to analyze the expression profile of ggTREM-A1 on mRNA and protein level, respectively. GgTREM-A1 mRNA was predominantly expressed in macrophages, but was also detected in brain, bone marrow, bursa, thymus, spleen and PBMC. Analyzing ggTREM-A1 surface expression by mab staining validated the expression on macrophages. Additionally, we showed high expression on blood monocytes, heterophils and NK cells and on monocytes isolated from bone marrow. Moreover, we detected ggTREM-A1 protein also on thrombocytes, B and T cell subsets, but antigen expression seemed to be lower and more variable in these cells. Immunohistochemistry of chicken brain tissue, combining ggTREM-A1 mab and various markers specific for various brain cell subsets showed expression of ggTREM-A1 on microglial cells, but also on neurons, astrocytes and oligodendrocytes. In conclusion, ggTREM-A1 is expressed on a variety of cells, relevant for the immune system, possibly combining physiological function of different mammalian TREM.

Chicken NK cell receptors.

Natural killer cells are innate immune cells that destroy virally infected or transformed cells. They recognize these altered cells by a plethora of diverse receptors and thereby differ from other lymphocytes that use clonally distributed antigen receptors. To date, several receptor families that play a role in either activating or inhibiting NK cells have been identified in mammals. In the chicken, NK cells have been functionally and morphologically defined, however, a conclusive analysis of receptors involved in NK cell mediated functions has not been available. This is partly due to the low frequencies of NK cells in blood or spleen that has hampered their intensive characterization. Here we will review recent progress regarding the diverse NK cell receptor families, with special emphasis on novel families identified in the chicken genome with potential as chicken NK cell receptors.

The Turkey Ig-like receptor family: identification, expression and function.

The chicken leukocyte receptor complex located on microchromosome 31 encodes the chicken Ig-like receptors (CHIR), a vastly expanded gene family which can be further divided into three subgroups: activating CHIR-A, bifunctional CHIR-AB and inhibitory CHIR-B. Here, we investigated the presence of CHIR homologues in other bird species. The available genome databases of turkey, duck and zebra finch were screened with different strategies including BLAST searches employing various CHIR sequences, and keyword searches. We could not identify CHIR homologues in the distantly related zebra finch and duck, however, several partial and complete sequences of CHIR homologues were identified on chromosome 3 of the turkey genome. They were designated as turkey Ig-like receptors (TILR). Using cDNA derived from turkey blood and spleen RNA, six full length TILR could be amplified and further divided according to the typical sequence features into one activating TILR-A, one inhibitory TILR-B and four bifunctional TILR-AB. Since the TILR-AB sequences all displayed the critical residues shown to be involved in binding to IgY, we next confirmed the IgY binding using a soluble TILR-AB1-huIg fusion protein. This fusion protein reacted with IgY derived from various gallinaceous birds, but not with IgY from other bird species. Finally, we tested various mab directed against CHIR for their crossreactivity with either turkey or duck leukocytes. Whereas no staining was detectable with duck cells, the CHIR-AB1 specific mab 8D12 and the CHIR-A2 specific mab 13E2 both reacted with a leukocyte subpopulation that was further identified as thrombocytes by double immunofluorescence employing B-cell, T-cell and thrombocyte specific reagents. In summary, although the turkey harbors similar LRC genes as the chicken, their distribution seems to be distinct with predominance on thrombocytes rather than lymphocytes.

The chicken SLAM family.

The signaling lymphocytic activation molecule (SLAM) family of receptors is critically involved in the immune regulation of lymphocytes but has only been detected in mammals, with one member being present in Xenopus. Here, we describe the identification, cloning, and analysis of the chicken homologues to the mammalian SLAMF1 (CD150), SLAMF2 (CD48), and SLAMF4 (CD244, 2B4). Two additional chicken SLAM genes were identified and designated SLAMF3like and SLAM5like in order to stress that those two receptors have no clear mammalian counterpart but share some features with mammalian SLAMF3 and SLAMF5, respectively. Three of the chicken SLAM genes are located on chromosome 25, whereas two are currently not yet assigned. The mammalian and chicken receptors share a common structure with a V-like domain that lacks conserved cysteine residues and a C2-type Ig domain with four cysteines forming two disulfide bonds. Chicken SLAMF2, like its mammalian counterpart, lacks a transmembrane and cytoplasmic domain and thus represents a glycosyl-phosphatidyl-inositol-anchored protein. The cytoplasmic tails of SLAMF1 and SLAMF4 display two and four conserved immunoreceptor tyrosine-based switch motifs (ITSMs), respectively, whereas both chicken SLAMF3like and SLAMF5like have only a single ITSM. We have also identified the chicken homologues of the SLAM-associated protein family of adaptors (SAP), SAP and EAT-2. Chicken SAP shares about 70 % identity with mammalian SAP, and chicken EAT-2 is homologous to mouse EAT-2, whereas human EAT-2 is much shorter. The characterization of the chicken SLAM family of receptors and the SAP adaptors demonstrates the phylogenetic conservation of this family, in particular, its signaling capacities.

A striking example of convergent evolution observed for the ggFcR:IgY interaction closely resembling that of mammalian FcR:IgG.

We have recently identified a novel IgY specific chicken FcR (ggFcR) on chromosome 20, a region where no FcR gene is present in mammals. Serially deleted IgY fusion proteins were tested in a reporter assay to identify C(H) domains involved in ggFcR binding. Single C(H) domains did not bind to ggFcR, whereas Fcυ2 to Fcυ4 induced good and the Fcυ3 to Fcυ4 domains moderate activity. When IgY from diverse birds were assayed, only IgY from gallinaceous birds showed binding, which enabled us to pinpoint several potential contact sites by a sequence comparison and molecular modelling. Point mutations of critical residues at these sites revealed the Fcυ2 and Fcυ3 domains as major ggFcR:IgY binding sites similar to mammalian IgG. These results demonstrate that ggFcR has a contact site to IgY which closely resembles that of human IgG bound to FcR.

The chicken leukocyte receptor cluster.

Receptors of the immunoglobulin-like superfamily are critically involved in virtually every aspect of immune responses. One large chromosomal area encoding such immunoregulatory receptors is the leukocyte receptor cluster. Here we review various aspects of the chicken Ig-like receptor (CHIR) family, located on microchromosome 31, an orthologous position to the mammalian leukocyte receptor cluster. The CHIR family has been massively expanded with over hundred CHIR genes that are further distinguished into activating, inhibitory and bifunctional receptors. Comparisons of various features such as amino acid motifs, genomic structure, expression and associated adaptor molecules reveal the homology of CHIR to both the killer Ig-like and the leukocyte Ig-like receptor families, with most pronounced correlation of certain CHIR to the NK cell receptor KIR2DL4. To date the CHIR ligands remain largely obscure with the exception of CHIR-AB1 that binds to chicken IgY. Detailed analyses of CHIR-AB1, its crystal structure, the interaction to IgY and functional capabilities allow us to draw conclusions regarding Fc receptor phylogeny and function.

The red jungle fowl leukocyte receptor complex contains a large, highly diverse number of chicken immunoglobulin-like receptor (CHIR) genes.

The chicken Ig-like receptor (CHIR) gene family is located on microchromosome 31, the orthologous region to the mammalian leukocyte receptor complex. CHIR are equally related to the mammalian killer Ig-like receptors and leukocyte Ig-like transcripts, but they occur in a much higher number and diversity. The chicken microchromosome 31 has been neglected in the genome sequence analysis. Here, we provide a first analysis of this region. For this purpose bacterial artificial chromosome (BAC) sequences originating from a single inbred red jungle fowl that served as basis for the chicken genome project were screened for the presence of CHIR sequences and eight BACs were identified as major CHIR containing regions. Since the sequences of these BACs that were available in the database were not complete, sequence gaps were further closed by novel data from the chicken genome project. The entire sequence was aligned into 26 contigs covering 875kbp that contained 84 functional CHIR and 46 CHIR pseudogenes that were hampered by different reasons such as premature stop codons. The 84 functional CHIR were further categorized into 35 activating (CHIRA), 26 inhibitory (CHIRB) and 23 bifunctional (CHIRAB) genes. A detailed comparison of the annotated sequence taking also into account the previously published CHIR BAC sequence originating from an Lohman selected leghorn chicken revealed that the CHIR locus seems to be a very active region with a high degree of gene reorganization that resembles a constant birth and death evolution. The present report provides a framework for the future completion of the CHIR locus. It further suggests that the entire microchromosome 31 may resemble a locus of extraordinary genomic diversity that is beneficial for the development of a large CHIR repertoire, but that has therefore lost all other genes, where such a diversification would be fatal.

Complexity of expressed CHIR genes.

The chicken leukocyte receptor complex (LRC) encodes an unprecedented number of chicken Ig-like receptor (CHIR) genes compared to the mammalian LRC. Although there are at least 100 CHIR genes in the LRC, only little information is available about the number and variability of expressed CHIR. Recently, we showed that CHIR with one Ig domain encode a variety of different affinity IgY receptors, which are highly variable in different chicken strains. The current report focused on expressed CHIR with two Ig domains. Oligonucleotides specific for conserved regions at the 5' end of Ig1 and 3' end of Ig2 were used on PBMC mRNA obtained from two individual chickens with different MHC haplotypes (M11, R11). Sequencing of 142 colonies of M11 and 117 of R11 yielded 98 and 70 different CHIR2D amino acid sequences, respectively. Comparing a total of 219 CHIR sequences, including also a genomic dataset from an LSL chicken, revealed a single amino acid sequence identical between all three chicken strains, and four sequence pairs either shared between M11 and R11 or between M11 and LSL. Calculating Wu-Kabat variability revealed three amino acid positions, which were highly variable and the analysis of synonymous/non-synonymous ratio indicated positive selection. This analysis of expressed CHIR genes in different chickens demonstrates an unusual polymorphism of expressed receptors, where only few are conserved between chickens.

Chicken IgY binds its receptor at the CH3/CH4 interface similarly as the human IgA: Fc alpha RI interaction.

Chicken IgY, the ancestral form of mammalian IgE and IgG, is recognized by the high-affinity FcY receptor CHIR-AB1, a member of the leukocyte receptor family. In this study, we have characterized the receptor ligand interaction site by consecutive truncations of the Fcv IgY domains and mutational analyses of selected residues. Using several fusion proteins that linked the human Cgamma2 and Cgamma3 domains with the Fcv IgY domains, a binding assay revealed that both the Fcv3 and Fcv4 domains were essential for the IgY CHIR-AB1 interaction. Sequence comparisons of chicken IgY with human IgA demonstrated that 11 of the 19 contact residues important for the IgA FcalphaRI interaction have been conserved in chicken IgY, although the overall amino acid identity is only 34%. Among the 19 amino acids at respective positions in IgY, the mutation of two residues in the Fcv3 and two in the Fcv4 domain completely abolished the IgY to CHIR-AB1 binding revealed by two independent assays. Three further mutations substantially altered the interaction. Molecular modeling on the Cv3 to Cv4 crystal structure revealed that all critical residues, although on two domains, are in close proximity. The importance of N-linked carbohydrates was demonstrated by the failure of the CHIR-AB1 interaction after mutation of the glycosylation site. The identification of the IgY Cv3/Cv4 interdomain region as critical for binding to CHIR-AB1 significantly enhances our understanding of the IgY receptor interaction and allows further conclusions regarding the FcR phylogeny.

The chicken leukocyte receptor complex encodes a family of different affinity FcY receptors.

Chicken Ig-like receptors (CHIR) form a large family in the leukocyte receptor complex on microchromosome 31 with inhibitory, activating, and bifunctional receptors. Recently, we characterized CHIR-AB1 as a high-affinity, primordial FcY receptor. Given that the CHIR family represents a multigene family, it is plausible that more than a single receptor binds to IgY. Therefore, after comparing CHIR-AB1-like sequences in databases, we cloned CHIR-AB1 homologues from two individual chickens representing the lines M11 and R11 with primers binding to highly conserved regions. In both lines this approach yielded 18 different CHIR-AB amino acid versions, with one sequence out of each line that was identical with the previously characterized B19 CHIR-AB1 Ig domain and two additional R11-M11 identical sequence pairs. All M11-derived CHIR-AB homologues were then expressed as soluble human Ig fusion proteins. Following standardization of the fusion protein concentration with an ELISA, the IgY, IgM, and IgA binding activities were determined by ELISA. Six fusion proteins recognized IgY, whereas none bound to IgM and IgA. The affinities of selected fusion proteins were determined using surface plasmon resonance yielding an equilibrium binding constant between 25 nM for high binders and 260 nM for low binders. Sequence comparisons and subsequent mutational analysis of selected residues identified five amino acids that are potentially involved in IgY binding. These results imply that multiple FcY receptors of variable affinity are encoded by the CHIR locus and that different chicken lines may express both unique as well as highly conserved FcY receptors.

A novel activating chicken IgY FcR is related to leukocyte receptor complex (LRC) genes but is located on a chromosomal region distinct from the LRC and FcR gene clusters.

FcRs have multifaceted roles in the immune system. Chicken FcRs were demonstrated on macrophages decades ago; however, only recently the chicken Ig-like receptor AB1, encoded in the leukocyte receptor complex, was molecularly identified as a high-affinity FcR. The present study was initiated to identify additional receptors with the capability to bind chicken immunoglobulins. Based on database searches, we cloned a novel chicken FcR, designated gallus gallus FcR (ggFcR), which was shown to bind selectively chicken IgY. The receptor consists of four extracellular C2-set Ig domains, followed by a transmembrane region containing arginine as a positively charged amino acid and a short cytoplasmic tail. ggFcR associates with the common gamma-chain, indicative for an activating receptor, and real-time RT-PCR revealed high expression on PBMC, thrombocytes, and macrophages. The genomic organization is similar to most Ig-like receptor genes, where each Ig domain is encoded by a separate exon. Additionally, the ggFcR signal peptide is encoded by two exons, the second of which is 36 bp, a hallmark for genes encoded in the leukocyte receptor complex. Phylogenetic analysis also showed a relationship to genes encoded in the leukocyte receptor complex. Surprisingly, ggFcR is not encoded in the leukocyte receptor complex, but it is located as a single isolated gene at the extremity of chicken chromosome 20.

Avian NK activities, cells and receptors.

Natural killer (NK) activity has been examined in birds for over 30 years, but evidence that avian NK activity plays crucial roles in disease is only suggestive. In chickens, NK activity is mediated by TCR0 cells in the intestinal epithelium, but elsewhere subsets of alphabeta and gammadelta T cells (NKT cells) may be more important. There are few lectin-like NK receptor genes, located in the genomic region syntenic with the natural killer complex (NKC) as well as the major histocompatibility complex (MHC). In contrast, a huge number of Ig-like receptor genes are located in a region syntenic with the leukocyte receptor complex (LRC).

The crystal structure of CHIR-AB1: a primordial avian classical Fc receptor.

CHIR-AB1 is a newly identified avian immunoglobulin (Ig) receptor that includes both activating and inhibitory motifs and was therefore classified as a potentially bifunctional receptor. Recently, CHIR-AB1 was shown to bind the Fc region of chicken IgY and to induce calcium mobilization via association with the common gamma-chain, a subunit that transmits signals upon ligation of many different immunoreceptors. Here we describe the 1.8-A-resolution crystal structure of the CHIR-AB1 ectodomain. The receptor ectodomain consists of a single C2-type Ig domain resembling the Ig-like domains found in mammalian Fc receptors such as FcgammaRs and FcalphaRI. Unlike these receptors and other monomeric Ig superfamily members, CHIR-AB1 crystallized as a 2-fold symmetrical homodimer that bears no resemblance to variable or constant region dimers in an antibody. Analytical ultracentrifugation demonstrated that CHIR-AB1 exists as a mixture of monomers and dimers in solution, and equilibrium gel filtration revealed a 2:1 receptor/ligand binding stoichiometry. Measurement of the 1:1 CHIR-AB1/IgY interaction affinity indicates a relatively low affinity complex, but a 2:1 CHIR-AB1/IgY interaction allows an increase in apparent affinity due to avidity effects when the receptor is tethered to a surface. Taken together, these results add to the structural understanding of Fc receptors and their functional mechanisms.

Characterization of the chicken CD200 receptor family.

The modulation of myeloid cells via inhibitory and activating immunoglobulin superfamily members has been a subject of intense study in mammals. One such example is the inhibitory receptor for CD200, which is shown to regulate the activation threshold of myeloid cells by interaction with the broadly distributed CD200 molecule. By looking at sequence homology and synteny conservation in the chicken genome, we identified two members of the CD200 receptor family in chicken on chromosome one. Cloning and further characterization of the protein sequence yielded a potentially inhibitory ggCD200R-B1 with a splice variant lacking a transmembrane region and a potentially soluble ggCD200R-S1. Both showed a typical V/C2-set Ig domain arrangement and we present evidence that these two genes have evolved by gene duplication. The inhibitory receptor displayed an uncharged transmembrane region and a long cytoplasmic tail encoding four tyrosine residues, one of them embedded in a motif similar to the mammalian NPxY motif. Further characterization of ggCD200R-B1 showed that it is expressed as a highly glycosylated protein and that its cytoplasmic tyrosine residues can be phosphorylated. Real-time RT-PCR analysis of various tissues and primary cells showed that ggCD200R-B1 is predominantly expressed in macrophages, whereas ggCD200R-S1 is highly expressed in peripheral blood mononuclear cells, but not macrophages. In summary, we showed that there is a homologue of mammalian CD200R conserved in chicken suggesting a similar function in avian species. Furthermore, the presence of potentially soluble CD200R molecules implies an important role for these in the regulation of myeloid cells in chicken.