Elsevier

Molecular Immunology

Volume 45, Issue 8, April 2008, Pages 2150-2157
Molecular Immunology

Functional adaptive diversity of the Atlantic salmon T-cell receptor gamma locus

https://doi.org/10.1016/j.molimm.2007.12.007Get rights and content

Abstract

The Atlantic salmon (Salmo salar) T-cell receptor γ (TCRγ) gene has extensive diversity in its capacity for antigen recognition due to the V–J–C gene segments recombinational possibilities, and N-region diversity. This is the first report completely characterizing and annotating a TCRγ gene locus in teleosts. We identified two different TCRγ loci in Atlantic salmon. The first locus, TCRγ 1, spans 260 kbp and contains four tandemly repeated clusters each of which consists of 1–4 variable (V) segments, 1–2 sets of a joining (J) segment and a constant (C) region. In total, 10 V segments, 5 J segments and 5 C regions were found in locus 1. In the second locus, TCRγ 2, a single non-expressed V–J–C cluster was found. Surprisingly, the Atlantic salmon TCRγ loci have a larger number of C regions relative to other teleosts, mouse and human. In addition, each Atlantic salmon TCRγ C region has a different connecting peptide region that may result in distinct cellular responses. Expression data confirm the diverse repertoire found at the genomic level. At least 6 out of 7 functional V segments, all 5 J segments and all 5 C regions found in TCRγ 1 were identified in TCRγ transcription analysis. The identification and characterization of the functional TCRγ 1 along with the associated TCR α/δ locus suggest that Atlantic salmon have a functional γδ T-cell immune component.

Introduction

In vertebrates, the T-cell has a membrane bound T-cell receptor (TCR) that is responsible for antigen recognition in T-cell-mediated immune responses. There are two types of T-cell populations based upon their heteromeric receptors (αβ and γδ). The receptor polypeptide chains are derived from variable (V), joining (J), diversity (D), and constant (C) gene segments. One of each of these segments is randomly selected from the germ line gene pool by a recombination mechanism to generate a wide diversity of TCRs for antigen recognition (Davis, 1990). The γδ TCR shares many structural features common to all TCRs; however, γδ T-cells also have several unique features. For example, in mammals, γδ T-cells localize to epithelial and mucosal sites such as skin and small intestine (Bucy et al., 1989, Itohara et al., 1990, Hayday, 2000). In addition, γδ TCRs bind antigens within the context of MHC molecules but also, like immunoglobulin (Ig), can bind antigens directly (Hayday, 2000). Furthermore, it was recently reported that γδ T-cells can act as antigen presenting cells (Brandes et al., 2005), and it is suggested that γδ T-cells work as a bridge between the innate and the adaptive immune system based on the interpretation that T-cells use their TCR as a pattern recognition receptor (Konigshofer and Chien, 2006).

The γδ T-cells compose less than 5% of peripheral blood T-cell population in human and mouse (Janeway et al., 1988); however, in birds (Sowder et al., 1988, Kubota et al., 1999) and ruminants (Mackay and Hein, 1989, Hein and Dudler, 1993), γδ T-cells compose up to 40% of circulating lymphocytes. The genomic organization of the TCRγ gene locus is also considerably different across species. In humans, the TCRγ locus contains 14 TCRγ V segments, 5 TCRγ J segments and 2 TCRγ C genes (Lefranc and Rabbitts, 1990). The 14 TCRγ V genes are located in a single cluster upstream of the 2 C genes, with 3 J segments upstream of a C region and 2 J segments found in between 2 C regions. In contrast in mice, the TCRγ locus contains three functional V–J–C clusters that have multi V segments, a single J segment and a C gene in each cluster (Vernooij et al., 1993). In ruminants (cow and sheep), the two TCRγ loci are located on two different segments of chromosome 4, and both consist of three tandemly repeated V–J–C clusters (Miccoli et al., 2003, Vaccarelli et al., 2005, Conrad et al., 2007). Thus, TCRγ genes vary in their expression pattern and genomic organization between species.

Jawed fish and mammals are similar in their defense mechanisms against pathogenic infection as they rely on both innate and adaptive immunity. It is believed that the adaptive immune system arose in the gnathostome ancestor about 500 million years ago (Partula et al., 1996, Agrawal et al., 1998, Eason et al., 2004). A fundamental component of the adaptive immune system is the T-cell, which has been considered the ancestral lymphocyte (Richards and Nelson, 2000, Davis and Bjorkman, 1988). Although partial TCRγ sequence data have been reported in lower vertebrates, including nurse shark (Rast et al., 1997), Japanese flounder (Nam et al., 2003) and Xenopus (Haire et al., 2002), the complete characterization of a TCRγ locus in a lower vertebrate has not been fully documented.

The objectives of this study were to completely characterize the Atlantic salmon TCRγ locus and to identify the extent of its possible recombinational diversity and its expression. This will provide important key information for understanding the fish adaptive immune system and the evolution of the TCR in vertebrates.

Section snippets

Screening and sequencing of Atlantic salmon TCRγ locus

An Atlantic salmon CHORI-214 bacterial artificial chromosome (BAC) library, constructed from a Norwegian aquaculture male strain, was obtained from BACPAC Resources, Children's Hospital Oakland Research Institute (CHORI) (Thorsen et al., 2005). Six BAC library filters were hybridized with two 70mer oligo probes based on EST sequence data: TCRG-1 and TCRG-2 (Integrated DNA Technologies) and 5′-end-labeled with γ32P-ATP using T4 polynucleotide kinase (Invitrogen). Labeled probes were added to BAC

Sequence and annotation of the TCRγ locus

Two different BAC contigs containing TCRγ loci were found in the Atlantic salmon BAC library. We refer to them as “TCRγ 1” and “TCRγ 2” in this study. Five Atlantic salmon genomic BAC clones containing the two TCRγ loci were identified and sequenced: 257A09 (215,529 bp), 115J17 (219,415 bp) and 242N16 (229,725 bp containing 2 gaps) for TCRγ 1 (Fig. 1A). A representation of TCRγ 2 is shown in Fig. 1B and is composed of BAC 261P24 (198,050 bp containing 2 gaps) and 272P16 (209,657 bp). The Atlantic

Discussion

This is the first study to completely characterize and annotate a teleost TCRγ locus. We found that the Atlantic salmon has two TCRγ loci, which we named TCRγ 1 and TCRγ 2. Surprisingly, the Atlantic salmon is similar to bovids as they have a larger number of C regions in their TCRγ loci, relative to other teleosts, mouse and human. The Atlantic salmon has four functional V–J–C clusters with an additional C region in TCRγ 1 (Fig. 1A).

Partial genomic data exist for zebrafish and puffer fish TCRγ

Acknowledgments

This study was in part supported by Genome Canada, Genome BC and NSERC. For their assistance in a wide variety of tasks, we would like to thank Jong Leong, Sally Goldes, Johan de Boer, Tricia Lundrigan, Gord Brown and Kris von Schalburg at the University of Victoria.

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