Unraveling the role of γδ T cells in the pathogenesis of an oncogenic avian herpesvirus.

Marek's disease virus (MDV) is an oncogenic alphaherpesvirus that causes deadly lymphomas in chickens. In chickens, up to 50% of all peripheral T cells are gamma delta (γδ) T cells. Until now, their role in MDV pathogenesis and tumor formation remains poorly understood. To investigate the role of γδ T cells in MDV pathogenesis, we infected recently generated γδ T cell knockout chickens with very virulent MDV. Strikingly, disease and tumor incidence were highly increased in the absence of γδ T cells, indicating that γδ T cells play an important role in the immune response against MDV. In the absence of γδ T cells, virus replication was drastically increased in the thymus and spleen, which are potential sites of T cell transformation. Taken together, our data provide the first evidence that γδ T cells play an important role in the pathogenesis and tumor formation of this highly oncogenic herpesvirus.IMPORTANCEGamma delta (γδ) T cells are the most abundant T cells in chickens, but their role in fighting pathogens remains poorly understood. Marek's disease virus (MDV) is an important veterinary pathogen, that causes one of the most frequent cancers in animals and is used as a model for virus-induced tumor formation. Our study revealed that γδ T cells play a crucial role in combating MDV, as disease and tumor incidence drastically increased in the absence of these cells. γδ T cells restricted virus replication in the key lymphoid organs, thereby decreasing the likelihood of causing tumors and disease. This study provides novel insights into the role of γδ T cells in the pathogenesis of this highly oncogenic virus.

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 ß 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/ß/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 ß 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.

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.

The Discovery of Chicken Foxp3 Demands Redefinition of Avian Regulatory T Cells.

Since the publication of the first chicken genome sequence, we have encountered genes playing key roles in mammalian immunology, but being seemingly absent in birds. One of those was, until recently, Foxp3, the master transcription factor of regulatory T cells in mammals. Therefore, avian regulatory T cell research is still poorly standardized. In this study we identify a chicken ortholog of Foxp3 We prove sequence homology with known mammalian and sauropsid sequences, but also reveal differences in major domains. Expression profiling shows an association of Foxp3 and CD25 expression levels in CD4+CD25+ peripheral T cells and identifies a CD4-CD25+Foxp3high subset of thymic lymphocytes that likely represents yet undescribed avian regulatory T precursor cells. We conclude that Foxp3 is existent in chickens and that it shares certain functional characteristics with its mammalian ortholog. Nevertheless, pathways for regulatory T cell development and Foxp3 function are likely to differ between mammals and birds. The identification and characterization of chicken Foxp3 will help to define avian regulatory T cells and to analyze their functional properties and thereby advance the field 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.

Alpaca (Vicugna pacos), the first nonprimate species with a phosphoantigen-reactive Vγ9Vδ2 T cell subset.

Vγ9Vδ2 T cells are a major γδ T cell population in the human blood expressing a characteristic Vγ9JP rearrangement paired with Vδ2. This cell subset is activated in a TCR-dependent and MHC-unrestricted fashion by so-called phosphoantigens (PAgs). PAgs can be microbial [(E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate, HMBPP] or endogenous (isopentenyl pyrophosphate, IPP) and PAg sensing depends on the expression of B7-like butyrophilin (BTN3A, CD277) molecules. IPP increases in some transformed or aminobisphosphonate-treated cells, rendering those cells a target for Vγ9Vδ2 T cells in immunotherapy. Yet, functional Vγ9Vδ2 T cells have only been described in humans and higher primates. Using a genome-based study, we showed in silico translatable genes encoding Vγ9, Vδ2, and BTN3 in a few nonprimate mammalian species. Here, with the help of new monoclonal antibodies, we directly identified a T cell population in the alpaca (Vicugna pacos), which responds to PAgs in a BTN3-dependent fashion and shows typical TRGV9- and TRDV2-like rearrangements. T cell receptor (TCR) transductants and BTN3-deficient human 293T cells reconstituted with alpaca or human BTN3 or alpaca/human BTN3 chimeras showed that alpaca Vγ9Vδ2 TCRs recognize PAg in the context of human and alpaca BTN3. Furthermore, alpaca BTN3 mediates PAg recognition much better than human BTN3A1 alone and this improved functionality mapped to the transmembrane/cytoplasmic part of alpaca BTN3. In summary, we found remarkable similarities but also instructive differences of PAg-recognition by human and alpaca, which help in better understanding the molecular mechanisms controlling the activation of this prominent population of γδ T cells.

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.

Characterisation of chicken OX40 and OX40L.

The Tumour Necrosis Factor superfamilies of receptors and ligands play a crucial role in the regulation of effective immune responses against pathogens and malignant cells. In chickens, only few members have been identified. Here, we characterise the chicken homologues for mammalian costimulatory molecules OX40 and OX40L, which are involved in sustaining T cell responses. Both genes were identified by virtue of their genomic localisation close to highly conserved genes and their structural relationship to their mammalian homologues. Following cloning and expression of soluble and cell-associated chicken OX40 and OX40L, we confirmed their mutual interaction via ELISA and flow cytometric analyses. In addition, we showed the application of soluble OX40-Fc in staining of chicken cells. Whereas non-activated cells did not express OX40L, activation by IL-2 and IL-12 resulted in upregulation of OX40L on αß and γδ T cell populations. Our results demonstrate the existence of the costimulatory OX40-OX40L system in the chicken and provide the basis for further investigations of chicken T cell responses.

Chicken IL-17A is expressed in αß and γδ T cell subsets and binds to a receptor present on macrophages, and T cells.

IL-17A as important cytokine in host defense has been analysed intensively and various homologous have been identified. To further gain insight into the functional properties of chicken (gg) IL-17A its expression profile was analysed by intracellular cytokine staining. In splenocytes and peripheral blood mononuclear cells gg IL-17A was detected in subsets of CD4+ T cells and γδ T cells. In contrast the gg IL-17A producing populations in intestinal intraepithelial lymphocytes were characterized as either CD3+CD25+ cells or γδ T cells. Furthermore, using FLAG tagged gg IL-17A, binding to its receptor was demonstrated on the macrophage cell line HD11. In peripheral blood IL-17A binding activity was found on αß and γδ T cell subsets, monocytes and a distinct population of CD25high cells. Treatment of HD11 cells with gg IL-17A induced IL-6 mRNA expression and nitric oxide production. These results demonstrate the presence of a αß T helper17 cell subset and IL-17 producing γδ T cells in the chicken.

Identification of Chicken GITR and GITR Ligand, Proof of Their Mutual Interaction, and Analysis of Chicken GITR Tissue Distribution by a Novel Antibody That Reveals Expression on Activated T Cells and Erythrocytes.

Glucocorticoid-induced TNFR (GITR) and its ligand, GITRL, belong to the costimulatory members of the TNF superfamily and are crucially involved in the formation and modulation of an effective immune response, comprising innate as well as adaptive mechanisms. In this study, we identify and describe chicken GITR and GITRL, and provide an initial characterization of the newly developed chGITR-specific mAb 9C5. Structural analyses of the putative chicken molecules GITR and GITRL confirmed the conservation of classic topological features compared with their mammalian homologs and suggested the ability of mutual interaction, which was verified via flow cytometry. Whereas only minute populations of native lymphocytes isolated from spleen, bursa, and thymus expressed GITR, it was strongly upregulated upon activation on αß and γδ T cells, comprising CD4+ as well as CD8+ subsets. In blood, a fraction of CD4+CD25+ T cells constitutively expressed GITR. In addition, virtually all chicken erythrocytes displayed high levels of GITR. Our results verify the existence of both GITR and its ligand, GITRL, in chickens; they provide the basis and novel tools to further characterize their impact within the immune response and reveal the so-far unrecognized expression of GITR on erythrocytes.

Splenic γδ T cell subsets can be separated by a novel mab specific for two CD45 isoforms.

CD45 isoforms have been identified in a variety of different species and mab against various isoforms have been instrumental to define cellular subsets. In the process of generating novel mab against chicken γδ T cells two mab with specificity for CD45 were identified and characterized. The analysis of the chicken CD45 genomic structure suggested three exons being involved in alternative splicing. We cloned and expressed the full length CD45 isoform and three shorter isoforms. While the 7D12 mab reacted with all of these isoforms, the 8B1 mab selectively reacted with two short isoforms lacking either exons 3 and 5 or exons 3, 5 and 6. As expected, the reactivity of 7D12 included all leukocyte subsets, also including thrombocytes. In contrast, the 8B1 mab only reacted with lymphocytes and monocytes. 8B1 expression was found on almost all blood αß T cells, while a γδ T cell subset and virtually all B cells lacked 8B1 reactivity. The fraction of 8B1- αß and γδ cells was larger in splenocytes as compared to PBL and there was also a population of 8B1+ splenic B cells. CD3 stimulation of splenic T cells resulted in upregulation of the 8B1 antigen on all T cells. Three-color immunofluorescence revealed differences in CD28 expression between the 8B1⁺ and 8B1¯ γδ T cell subsets with a higher CD28 expression level on 8B1¯ cells. The CD28 antigen was upregulated upon stimulation of the cells with IL-2 and IL-12. This novel mab will be a useful tool to further analyze chicken γδ T cells in more detail.

γδ T cells represent a major spontaneously cytotoxic cell population in the chicken.

Natural killer cells in the chicken are mainly confined to the intestine, while only small frequencies are detectable in spleen, lung and blood. Here, we compared the spontaneous cytotoxicity of lymphocytes isolated from blood, spleen and intestine using a flow cytometric based cytotoxicity assay. There was no spontaneous cytotoxicity detected in chicken blood preparations. In contrast, freshly prepared splenocytes exhibited a spontaneous cytotoxicity of up to 50% and intestinal epithelial lymphocytes of up to 85%. This cytotoxicity was observed against the RP9 but not against the chicken CU24 target cell line. The observed cytotoxicity was MHC unrestricted since B2B2 derived effector cells killed RP9 target cells (B2B15) equally well compared to MHC mismatched 2D8 targets (B19B19). The cytotoxicity of splenocytes was enhanced by preincubation with IL-2 or strongly increased with IL-2 plus IL-12. By cell sorting, we identified the CD8+γδ T cell subset as the major effectors, whereas both CD8-γδ T cells and CD8+αß T cells had only low cytolytic potential. Within intestinal lymphocyte CD45+cells displayed cytotoxicity as well as sorted γδ T cells and NK cell. In conclusion, the chicken γδ T cells represent a major cytotoxic lymphocyte subset that can lyse target cells in a MHC unrestricted manner.

Generation of glycosylphosphatidylinositol linked chicken IL-17 to generate specific monoclonal antibodies applicable for intracellular cytokine staining.

Interleukin 17 (IL-17) cytokines play a crucial role in host defense and inflammatory diseases. Of the six mammalian IL-17 members five are described in the chicken (gg) genome. A novel method that attached cytokines to the surface of cells via a GPI linker was established to generate two chicken IL-17A and one chicken IL-17F specific mab. Recombinant gg IL-17A and gg IL-17F that showed dimerization in Western blot were used to verify the antibodies specificity. The mab could detect gg IL-17 by intracellular cytokine staining as demonstrated on cells expressing recombinant IL-17. Furthermore IL-17A and lower amounts of IL-17F were detectable in CD4 positive T cells of stimulated splenocytes. In conclusion, we have generated novel tools to analyze chicken IL-17 in more detail and demonstrated that the surface expression of cytokines is a reliable method to generate specific mab applicable for intracellular cytokine staining.

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.

Vγ9 and Vδ2 T cell antigen receptor genes and butyrophilin 3 (BTN3) emerged with placental mammals and are concomitantly preserved in selected species like alpaca (Vicugna pacos).

Human Vγ9Vδ2 T cells recognize phosphorylated products of isoprenoid metabolism (phosphoantigens) PAg with TCR comprising Vγ9JP γ-chains and Vδ2 δ-chains dependent on butyrophilin 3 (BTN3) expressed by antigen-presenting cells. They are massively activated in many infections and show anti-tumor activity and so far, they have been considered to exist only in higher primates. We performed a comprehensive analysis of databases and identified the three genes in species of both placental magnorders, but not in rodents. The common occurrence or loss of in silico translatable Vγ9, Vδ2, and BTN3 genes suggested their co-evolution based on a functional relationship. In the peripheral lymphocytes of alpaca (Vicugna pacos), characteristic Vγ9JP rearrangements and in-frame Vδ2 rearrangements were found and could be co-expressed in a TCR-negative mouse T cell hybridoma where they rescued CD3 expression and function. Finally, database sequence analysis of the extracellular domain of alpaca BTN3 revealed complete conservation of proposed PAg binding residues of human BTN3A1. In summary, we show emergence and preservation of Vγ9 and Vδ2 TCR genes with the gene of the putative antigen-presenting molecule BTN3 in placental mammals and lay the ground for analysis of alpaca as candidate for a first non-primate species to possess Vγ9Vδ2 T cells.

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 CRTAM binds nectin-like 2 ligand and is upregulated on CD8+ αß and γδ T lymphocytes with different kinetics.

During a search for immunomodulatory receptors in the chicken genome, we identified a previously cloned chicken sequence as CRTAM homologue by its overall identity and several conserved sequence features. For further characterization, we generated a CRTAM specific mab. No staining was detectable in freshly isolated cell preparations from thymus, bursa, caecal tonsils, spleen, blood and intestine. Activation of splenocytes with recombinant IL-2 increased rapid CRTAM expression within a 2 h period on about 30% of the cells. These CRTAM(+) cells were identified as CD8(+) γδ T lymphocytes. In contrast, CRTAM expression could not be stimulated on PBL with IL-2, even within a 48 h stimulation period. As a second means of activation, T cell receptor (TCR) crosslinking using an anti-αß-TCR induced CRTAM on both PBL and splenocytes. While CRTAM expression was again rapidly upregulated on splenocytes within 2 h, it took 48 h to reach maximum levels of CRTAM expression in PBL. Strikingly, albeit the stimulation of splenocytes was performed with anti-αß-TCR, CRTAM expression after 2 h was mainly restricted to CD8(+) γδ T lymphocytes, however, the longer anti-TCR stimulation of peripheral blood lymphocytes (PBL) resulted in CRTAM expression on αß T lymphocytes. In order to characterize the potential ligand we cloned and expressed chicken Necl-2, a member of the nectin and nectin-like family which is highly homologous to its mammalian counterpart. Three independent assays including a reporter assay, staining with a CRTAM-Ig fusion protein and a cell conjugate assay confirmed the interaction of CRTAM with Necl-2 which could also be blocked by a soluble CRTAM-Ig fusion protein or a CRTAM specific mab. These results suggest that chicken CRTAM represents an early activation antigen on CD8(+) T cells which binds to Necl-2 and is upregulated with distinct kinetics on αß versus γδ T lymphocytes.