Elsevier

Molecular Immunology

Volume 45, Issue 5, March 2008, Pages 1398-1404
Molecular Immunology

Chicken C-type lectin-like receptor B-NK, expressed on NK and T cell subsets, binds to a ligand on activated splenocytes

https://doi.org/10.1016/j.molimm.2007.08.024Get rights and content

Abstract

B-NK is a C-type lectin with an immunorecptor tyrosine-based inhibition motif, that is located in the vicinity of the chicken MHC and that has been described as a potential chicken NK cell receptor. We have generated an epitope tagged B-NK version for immunization and for biochemical studies. B-NK was expressed as a heavily glycosylated, homodimeric, type II transmembrane protein. With the help of a newly developed B-NK specific mab, the tissue distribution of B-NK has been analyzed. In the blood, B-NK was mainly present on a small population of γδ T cells, whereas in spleen it was expressed by αβ T cells. Moreover, B-NK was also found on CD3CD8+ sorted splenocytes that were in vitro expanded by IL-2 and on embryonic splenocytes, both of which resemble chicken NK cells. In order to characterize cells expressing B-NK ligands, a BWZ.36 based reporter system was employed, that induced β-galactosidase activity upon ligand binding. While potential B-NK ligands such as MHC class I or the C-type lectin B-lec did not induce any signal, a trypsin sensitive B-NK ligand was expressed on phorbol myristate or concanavalin A activated splenocytes, but not unstimulated splenocytes. In summary, B-NK is expressed by NK cells and T cell subsets, and it binds to a ligand on activated cells.

Introduction

NK cells are lymphocytes of the innate immune system that have been defined by their ability to kill certain tumor cells and virally infected cells in an MHC unrestricted manner (Trinchieri, 1995). In recent years, a large number of different cell surface proteins have been defined on NK cells that modulate their activity (Lanier, 2005). Biochemically, these receptors can be divided into types I and II transmembrane proteins. The type I receptors display extracellular Ig domains, whereas the type II receptors are characterized by C-type lectin domains. Many of the NK cell receptors can be further grouped into distinct families based on homologies and their location on chromosomal areas. The leukocyte receptor complex located on human chromosome 19 and mouse chromosome 7 represents such an area and encodes several important NK cell receptor families such as the killer cell Ig-like receptor family (KIR) and the leukocyte Ig-like receptor family (LILR) (Moretta and Moretta, 2004). Most of the KIR and some of the LILR receptors recognize MHC class I molecules. Due to deletions, most KIR genes are only found in primates and absent in rodents. Another group of NK cell receptors is located in the so-called natural killer gene complex (NKC) found on human chromosome 12 and mouse chromosome 6 (Yokoyama and Plougastel, 2003). The mouse Ly49 family that is deleted in humans is found in this area as well as many other C-type lectins that are present on NK cells, such as the NKRP genes, NKG genes and CD94 (Kelley et al., 2005). In the case of NKRP1d, its ligand Clrb is genetically linked in the same locus (Iizuka et al., 2003).

Functionally, most NK cell receptors, regardless of their biochemical nature or chromosomal region, are grouped into activating or inhibitory receptors (Borrego et al., 2002). Activating receptors usually have a short cytoplasmic region lacking intracellular signaling motifs and a basic transmembrane residue that mediates the association to adaptor molecules such as DAP-12, FceRIγ or CD3ζ. In contrast, inhibitory receptors are characterized by their long cytoplasmic tails with immunoreceptor tyrosine-based inhibition motifs (ITIM) that are phosphorylated upon ligand binding, successively leading to the recruitment of intracellular phosphatases and ablation of intracellular signaling. The balance of activating and inhibitory signals finally dictates the cellular response.

In the chicken, NK cells have been defined primarily by their surface phenotype as CD3, Ig lymphocytes that express CD8 and cytoplasmic CD3 proteins (Göbel et al., 1994). In contrast to mammals it appears that the frequency of NK cells in the blood or spleen is quite low. Only very view chicken NK cell receptors have been defined including B-NK. With the help of the chicken genome project, the homologues of mammalian leukocyte receptor complex (LRC) and NKC have been found in the chicken. The chicken LRC contains a large number of Ig-like receptors designated chicken Ig-like receptors (CHIR) (Dennis et al., 2000). Their number exceeds by far the number of genes found in the mammalian LRC, it is highly polymorphic with a lot of allelic variability (Laun et al., 2006, Viertlboeck et al., 2005). One of the genes CHIR-AB1 has recently been found to resemble a high affinity Fc receptor binding to chicken IgY (Viertlboeck et al., 2007). The expression of the CHIR genes seems to be very variable and some of them could well serve as NK cell receptors. In contrast to this vastly extended locus, the chicken NKC located on chromosome 1 contains only two C-type lectins that are most homologous to CD69 and CD94/NKG2 (Chiang et al., 2007). The chicken NKC therefore appears to be very small in comparison with the mammalian counterparts. There are at least several C-type lectins, however, located close to the chicken MHC. Initially, two of them were isolated and by virtue of their location in the MHC or B-locus and their potential expression they were designated B-NK and B-Lec, respectively (Kaufman et al., 1999). By extending the chicken MHC sequence a third C-type lectin designated B-Lec3 was found (Shiina et al., 2007). In addition, at least 13 C-type lectins were found intermingled with nonclassical MHC class I genes in the RFP-Y region (Miller et al., 2004, Rogers et al., 2003).

The B-NK gene has been most intensively studied, including expression analyses by RT-PCR and quantitative PCR, phylogenetic studies and signaling capabilities of its immunoreceptor tyrosine-based inhibition motif (Rogers et al., 2005). The present study clarifies the B-NK surface expression with the help of a newly developed specific mab, it analyzes its biochemical nature and most importantly characterizes a potential B-NK ligand on activated lymphocytes.

Section snippets

Animals

Chicken lines CB (B12/B12) and H.B19 (B19/B19) were hatched at the institute and used for experiments at the age of 6–10 weeks. BALB/c mice were raised at the institute.

Cell preparations

Single cell suspensions of splenocytes were generated by passing the spleen through a stainless steel mesh. Mononuclear cells were prepared from the cell suspensions by density centrifugation on Ficoll-Paque (Amersham Pharmacia Biotech, Freiburg, Germany). CD3CD8+ cells were FACS sorted from splenocytes and in vitro expanded

B-NK is expressed as a highly glycosylated protein dimer

Until now, only the B-NK gene and its signaling capabilities have been characterized, but there was no data regarding biochemical properties or protein expression on lymphocytes. Therefore, we generated a B-NK expression construct that harbours a C-terminal V5 epitope tag. Since B-NK is predicted to resemble a type II transmembrane protein, the V5 epitope tag should be useful as a surface tag of transfected cells.

The construct was initially used for transient transfections of COS7 cells. Cell

Discussion

The B-NK gene was first identified by sequencing the chicken B locus, which is encoding 19 genes including the MHC class I and II genes (Kaufman et al., 1999). It was quite unexpected to find C-type lectin genes in the vicinity of the so-called “minimal essential MHC” where basically all non-MHC genes have been deleted. Moreover, next to B-NK in the opposite transcriptional orientation a second C-type lectin, B-Lec, was found. Whereas B-NK resembles a typical inhibitory receptor with a

Acknowledgements

We thank W. Yokoyama for providing BWZ.36 cells and the NKRP1d-Ly49a-CD3ζ plasmid. These studies were supported by Deutsche Forschungsgemeinschaft Grant GO489/3-5 (to T.W.G.) and Academy of Sciences of the Czech Republic Project AVOZ50520514 (to J.P.).

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Current address: Division of Gene Therapy, University of Ulm, Helmholtzstr. 8/1, 89081 Ulm, Germany.

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