NK and T cells constitute two major, functionally distinct intestinal epithelial lymphocyte subsets in the chicken

Non-mammalian NK cells have not been characterized in detail; however, their analysis is essential for the understanding of the NK cell receptor phylogeny. As a first step towards defining chicken NK cells, several tissues were screened for the presence of NK cells, phenotypically defined as CD8(+) cells lacking T- or B-lineage specific markers. By this criteria, approximately 30% of CD8(+) intestinal intraepithelial lymphocytes (IEL), but <1% of splenocytes or peripheral blood lymphocytes were defined as NK cells. These CD8(+)CD3(-) IEL were used for the generation of the 28-4 mAb, immunoprecipitating a 35-kDa glycoprotein with a 28-kDa protein core. The CD3 and 28-4 mAb were used to separate IEL into CD3(+) IEL T cells and 28-4(+) cells, both co-expressing the CD8 antigen. During ontogeny, 28-4(+) cells were abundant in the IEL and in the embryonic spleen, where two subsets could be distinguished according to their CD8 and c-kit expression. Most importantly, 28-4(+) IEL lysed NK-sensitive targets, whereas intestinal T cells did not have any spontaneous cytolytic activity. These results define two major, phenotypically and functionally distinct IEL subpopulations, and imply an important role of NK cells in the mucosal immune system

Cytokines of Birds: Conserved Functions

Targeted disruptions of the mouse genes for cytokines, cytokine receptors, or components of cytokine signaling cascades convincingly revealed the important roles of these molecules in immunologic processes. Cytokines are used at present as drugs to fight chronic microbial infections and cancer in humans, and they are being evaluated as immune response modifiers to improve vaccines. Until recently, only a few avian cytokines have been characterized, and potential applications thus have remained limited to mammals. Classic approaches to identify cytokine genes in birds proved difficult because sequence conservation is generally low. As new technology and high throughput sequencing became available, this situation changed quickly. We review here recent work that led to the identification of genes for the avian homologs of interferon-a/b (IFNa/b) and IFN-g, various interleukins (IL), and several chemokines. From the initial data on the biochemical properties of these molecules, a picture is emerging that shows that avian and mammalian cytokines may perform similar tasks, although their primary structures in most cases are remarkably different

Chicken BAFF

Members of the tumor necrosis factor (TNF) family play key roles in the regulation of inflammation, immune responses and tissue homeostasis. Here we describe the identification of the chicken homologue of mammalian B cell activating factor of the TNF family (BAFF/BLyS). By searching a chicken EST database we identified two overlapping cDNA clones that code for the entire open reading frame of chicken BAFF (chBAFF), which contains a predicted transmembrane domain and a putative furin protease cleavage site like its mammalian counterparts. The amino acid identity between soluble chicken and human BAFF is 76%, considerably higher than for most other known cytokines. The chBAFF gene is most strongly expressed in the bursa of Fabricius. Soluble recombinant chBAFF produced by human 293T cells interacted with the mammalian cell-surface receptors TACI, BCMA and BAFF-R. It bound to chicken B cells, but not to other lymphocytes, and it promoted the survival of splenic chicken B cells in culture. Furthermore, bacterially expressed chBAFF induced the selective expansion of B cells in the spleen and cecal tonsils when administered to young chicks. Our results suggest that like its mammalian counterpart, chBAFF plays an important role in survival and/or proliferation of chicken B cells

Chicken γδ T cells proliferate upon IL-2 and IL-12 treatment and show a restricted receptor repertoire in cell culture

In chickens, γδ T cells represent a large fraction of peripheral T cells; however, their function remains largely unknown. Here, we describe the selective in vitro expansion of γδ T cells from total splenocytes by stimulation with the cytokines IL-2 and IL-12. Under these conditions, γδ T cells proliferated preferentially and reached frequencies of >95% within three weeks. Although IL-2 alone also triggered proliferation, an increased proliferation rate was observed in combination with IL-12. Most of the expanded cells were γδ TCR and CD8 double-positive. Splenocytes sorted into TCR1+CD8+, TCR1highCD8−, and TCR1lowCD8− subsets proliferated well upon dual stimulation with IL-2/IL-12, indicating that none of the three γδ T cell subsets require bystander activation for proliferation. TCR1+CD8+ cells maintained CD8 surface expression during stimulation, whereas CD8− subpopulations showed varied levels of CD8 upregulation, with the highest upregulation observed in the TCR1high subset. Changes in the γδ T-cell receptor repertoire during cell culture from day 0 to day 21 were analyzed by next-generation sequencing of the γδ variable regions. Overall, long-term culture led to a restricted γ and δ chain repertoire, characterized by a reduced number of unique variable region clonotypes, and specific V genes were enriched at day 21. On day 0, the δ chain repertoire was highly diverse, and the predominant clonotypes differed between animals, while the most frequent γ-chain clonotypes were shared between animals. However, on day 21, the most frequent clonotypes in both the γ and δ chain repertoires were different between animals, indicating that selective expansion of dominant clonotypes during stimulation seems to be an individual outcome. In conclusion, IL-2 and IL-12 were sufficient to stimulate the in vitro outgrowth of γδ T cells. Analyses of the TCR repertoire indicate that the culture leads to an expansion of individual T cell clones, which may reflect previous in vivo activation. This system will be instrumental in studying γδ T cell function

Identification of a Candidate CD5 Homologue in the Amphibian Xenopus laevis

We identified a novel T cell Ag in the South African clawed toad (Xenopus laevis) by a mAb designated 2B1. This Ag is present in relatively high levels on most thymocytes, approximately 65% of splenocytes, 55% of PBL, and 65% of intestinal lymphocytes, but is rarely seen on IgM+ B cells in any of these tissues. Lymphocytes bearing the 2B1 Ag proliferate in response to stimulation with Con A or PHA, whereas the 2B1- lymphocytes are reactive to LPS. Biochemical analysis indicates that this Ag is a differentially phosphorylated glycoprotein of 71 to 82 kDa. The protein core of 64 kDa bears both N- and O-linked carbohydrate side chains. The amino-terminal protein sequence of the 2B1 Ag shares significant homology with both the macrophage scavenger receptor type 1 motif and the mammalian CD5/CD6 family. The biochemical characteristics and cellular distribution of the 2B1 Ag suggest that it represents the CD5 homologue in X. laevis. While T cells constitutively express this highly conserved molecule, Xenopus B cells acquire the CD5 homologue only when they are stimulated in the presence of T cell

Identification of T-cell receptor a-chain genes in the chicken

T-cell receptor (TCR) -chain (TCR) and ß-chain (TCRß) genes are well characterized in mammals, while only TCRß genes have been identified in other vertebrates. To identify avian TCR genes, we used monoclonal anti-CD3 antibodies to isolate chicken TCR for peptide sequence analysis. Degenerate oligonucleotide probes were then used to isolate a candidate TCR cDNA clone that hybridized with a 1.7-kb mRNA species present only in ß T cells and in tissues populated by these cells. Southern blot analysis revealed gene rearrangement in thymocytes and ß T-cell lines. The TCR cDNA candidate encoded an openreading frame of 275 amino acids, the predicted variable (V)-, joining (J)-, and constant (C)-region amino acid sequences of which shared 40%, 60%, and 25% homology with corresponding mammalian sequences. A single C gene and 25 V genes were identified by using region-specific probes. The V cDNA probe isolated from a Vß1+ cell line reacted with transcripts from one of five Vß2+ cell lines, suggesting shared use of V genes by Vß1+ and Vß2+ T cells and the existence of other V gene families. A genomic V sequence was flanked by classical recombination signal sequences but, unlike previously defined V genes, the leader and V region were encoded by a single exon. The data indicate evolutionary conservation of the basic TCR gene structure in birds and mammal

Unraveling the chicken T cell repertoire with enhanced genome annotation

T cell receptor (TCR) repertoire sequencing has emerged as a powerful tool for understanding the diversity and functionality of T cells within the host immune system. Yet, the chicken TCR repertoire remains poorly understood due to incomplete genome annotation of the TCR loci, despite the importance of chickens in agriculture and as an immunological model. Here, we addressed this critical issue by employing 5’ rapid amplification of complementary DNA ends (5’RACE) TCR repertoire sequencing with molecular barcoding of complementary DNA (cDNA) molecules. Simultaneously, we enhanced the genome annotation of TCR Variable (V), Diversity (D, only present in β and δ loci) and Joining (J) genes in the chicken genome. To enhance the efficiency of TCR annotations, we developed VJ-gene-finder, an algorithm designed to extract VJ gene candidates from deoxyribonucleic acid (DNA) sequences. Using this tool, we achieved a comprehensive annotation of all known chicken TCR loci, including the α/δ locus on chromosome 27. Evolutionary analysis revealed that each locus evolved separately by duplication of long homology units. To define the baseline TCR diversity in healthy chickens and to demonstrate the feasibility of the approach, we characterized the splenic α/β/γ/δ TCR repertoire. Analysis of the repertoires revealed preferential usage of specific V and J combinations in all chains, while the overall features were characteristic of unbiased repertoires. We observed moderate levels of shared complementarity-determining region 3 (CDR3) clonotypes among individual birds within the α and γ chain repertoires, including the most frequently occurring clonotypes. However, the β and δ repertoires were predominantly unique to each bird. Taken together, our TCR repertoire analysis allowed us to decipher the composition, diversity, and functionality of T cells in chickens. This work not only represents a significant step towards understanding avian T cell biology, but will also shed light on host-pathogen interactions, vaccine development, and the evolutionary history of avian immunology

A Cell Culture System to Investigate Marek’s Disease Virus Integration into Host Chromosomes

Marek’s disease virus (MDV) is a highly oncogenic alphaherpesvirus that causes a devastating neoplastic disease in chickens. MDV has been shown to integrate its genome into the telomeres of latently infected and tumor cells, which is crucial for efficient tumor formation. Telomeric repeat arrays present at the ends of the MDV genome facilitate this integration into host telomeres; however, the integration mechanism remains poorly understood. Until now, MDV integration could only be investigated qualitatively upon infection of chickens. To shed further light on the integration mechanism, we established a quantitative integration assay using chicken T cell lines, the target cells for MDV latency and transformation. We optimized the infection conditions and assessed the establishment of latency in these T cells. The MDV genome was efficiently maintained over time, and integration was confirmed in these cells by fluorescence in situ hybridization (FISH). To assess the role of the two distinct viral telomeric repeat arrays in the integration process, we tested various knockout mutants in our in vitro integration assay. Efficient genome maintenance and integration was thereby dependent on the presence of the telomeric repeat arrays in the virus genome. Taken together, we developed and validated a novel in vitro integration assay that will shed light on the integration mechanism of this highly oncogenic virus into host telomeres

Chicken γδ T cells proliferate upon IL-2 and IL-12 treatment and show a restricted receptor repertoire in cell culture

In chickens, γδ T cells represent a large fraction of peripheral T cells; however, their function remains largely unknown. Here, we describe the selective in vitro expansion of γδ T cells from total splenocytes by stimulation with the cytokines IL-2 and IL-12. Under these conditions, γδ T cells proliferated preferentially and reached frequencies of &gt;95% within three weeks. Although IL-2 alone also triggered proliferation, an increased proliferation rate was observed in combination with IL-12. Most of the expanded cells were γδ TCR and CD8 double-positive. Splenocytes sorted into TCR1+CD8+, TCR1highCD8−, and TCR1lowCD8− subsets proliferated well upon dual stimulation with IL-2/IL-12, indicating that none of the three γδ T cell subsets require bystander activation for proliferation. TCR1+CD8+ cells maintained CD8 surface expression during stimulation, whereas CD8− subpopulations showed varied levels of CD8 upregulation, with the highest upregulation observed in the TCR1high subset. Changes in the γδ T-cell receptor repertoire during cell culture from day 0 to day 21 were analyzed by next-generation sequencing of the γδ variable regions. Overall, long-term culture led to a restricted γ and δ chain repertoire, characterized by a reduced number of unique variable region clonotypes, and specific V genes were enriched at day 21. On day 0, the δ chain repertoire was highly diverse, and the predominant clonotypes differed between animals, while the most frequent γ-chain clonotypes were shared between animals. However, on day 21, the most frequent clonotypes in both the γ and δ chain repertoires were different between animals, indicating that selective expansion of dominant clonotypes during stimulation seems to be an individual outcome. In conclusion, IL-2 and IL-12 were sufficient to stimulate the in vitro outgrowth of γδ T cells. Analyses of the TCR repertoire indicate that the culture leads to an expansion of individual T cell clones, which may reflect previous in vivo activation. This system will be instrumental in studying γδ T cell function

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 γ-chain, a subunit that transmits signals upon ligation of many different immunoreceptors. Here we describe the 1.8-Å-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 FcγRs and FcαRI. 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