Molecular characterisaton of T cell co-receptors CD3, CD8 and CD4 in Atlantic salmon (Salmo salar)

Abstract

The commercial importance of Atlantic salmon (Salmo salar) in aquaculture has fuelled much research into fish health since outbreaks of infectious disease cause major financial losses. All aspects of immunology are therefore currently of great interest. The aim of this study was to extend the characterisation of T cell markers to enable antibody production and design of expression assays, to study the immune system and immune responses of salmon. Therefore, the genes and cDNAs for the T cell coreceptors CD3, CD8 and CD4 were cloned using a combination of synteny analysis and homology cloning. CD3 in Atlantic salmon consists of three different molecules; a γδ chain, the forerunner of separate γ and δ chains in mammals, an ε chain and a ζ chain (mammals also have an η chain, which is a splice variant of the ζ gene). The translated sequences have low identities to mammalian CD3 sequences (12-34%), but they exhibit similar characteristics with single immunoglobulin-like domains in the γδ and ε genes and immunoreceptor tyrosine-based activation motifs in all cytoplasmic domains. Two copies of the CD3ζ gene were cloned, but these are considered to be alleles. The CD3ε gene has a second copy, but it is a pseudogene containing frame-shifts and stop codons and was poorly expressed compared to the intact CD3ε gene. The CD3γδ gene also appeared to be duplicated and the variants are named CD3γδ-A and CD3γδ -B. Further evidence for the homology of CD3γδ and CD3ε genes was found in their genomic orientation where the pseudo-CD3ε gene is located tail to tail with a CD3γδ gene, similar to the gene organisation in higher vertebrates. There are two CD8 genes in Atlantic salmon, CD8α and CD8β. The synteny of these genes in humans is conserved with the publically available fugu genome sequence, allowing the subsequent identification of the CD8β gene in Atlantic salmon. There are two main transcripts and two alternative transcripts which could result in a putative truncated cytoplasmic domain in CD8α and a severely truncated version of CD8β. The main CD8α and CD8β transcripts were also cloned and sequenced in brown trout (and CD8β in rainbow trout, since only CD8α had been cloned previously). Despite low sequence identity (16-17%) to mammals, these molecules are considered homologs encoding a single immunoglobulin-like extracellular domain anchored by a stalk with many putative glycosylation motifs. The single pass transmembrane domains are followed by short cytoplasmic domains. Unlike mammalian CD8α, the salmon molecule does not contain a CXC, Lck binding, motif in its cytoplasmic domain. This is also true for other published teleost sequences. However, both CD8α and CD8β have motifs in their cytoplasmic domains that are highly conserved between teleosts, implying functional significance. The cloning of CD4 was more complex since two similar molecules were found, type 1 and type 2, containing four and two extracellular immunoglobulin-like domains respectively. These genes were first identified next to each other on a fugu scaffold sequence, showing synteny to CD4 on the human genome. CD4-2 is duplicated with CD4-2a and CD4-2b sub-types, which together with the duplicated CD3 genes illustrates a theme throughout this study: the tetraploid nature of the salmon genome. All the CD4 molecules identified have a Lck binding motif in their cytoplasmic domain and although sequence identities to mammals were low (13-15%) they are homologous to mammalian CD4. At the genomic level all these CD4 genes have the code for the first immunoglobulin-like domain split between two exons, which is a hallmark of CD4 molecules from higher vertebrates. Although two slightly different genomic fragments were isolated for salmon CD4-1, only one cDNA was cloned. In rainbow trout two genes were identified for the CD4-1 gene on a Southern blot, but a second distinct cDNA sequence was not detected. The expression of all these T cell markers is highest in the thymus, when measured using TaqMan assays and RT-qPCR, with the exception of CD4-2a. There was also significant expression in other immunological tissues such as head-kidney, spleen. Characterization of these molecules has led to the production of a sensitive and specific antibody against a CD3ε cytoplasmic peptide, which has been used to show the distribution of T cells in Atlantic salmon tissues using immunohistochemistry. Large numbers of CD3ε expressing cells could be seen in thymus and a lymphoid tissue in gills. A significant amount of CD3ε expression was also seen in head kidney and spleen. CD3ε positive cells were also observed scattered between enterocytes in the hind gut. RT-qPCR of 11 different T cell genes performed on laser capture microdissected material from thymus, gill and hind-gut reflected the CD3ε expression seen using immunohistochemistry. Until recently it was not possible to isolate T cells other than indirectly as immunoglobulin negative cells, due to a lack of reagents. This study paves the way for the development of more antibodies and for the positive isolation of sub-populations of leucocytes, allowing further elucidation of immune mechanisms and cellular studies in Atlantic salmon.