Rainbow trout (Oncorhynchus mykiss) possess multiple novel immunoglobulin- like transcripts containing either an ITAM or ITIMs Anders E. Østergaard, Samuel A.M. Martin, Tiehui Wang, Rene ´ J.M. Stet 1 , Christopher J. Secombes * Scottish Fish Immunology Research Centre, University of Aberdeen, Zoology Building, Tillydrone Avenue, Aberdeen AB24 2TZ, Scotland, UK 1. Introduction The innate immune system is a rapid and efficient barrier against infections, providing a first line of defence able to discriminate between host cells and pathogens. The major effector cells in the innate immune system include professional phagocytes (neutrophils and macrophages) and natural killer (NK) cells. The recognition of pathogens by these cells is carried out by a broad array of germ line-encoded and non-rearranging cell-surface receptors, many of which contain immunoglobulin-like (Ig) domains [1]. These innate immune receptors are structurally diverse and the cellular responses are regulated by either activating or inhibitory signalling pathways. A number of gene families encoding activating or inhibitory receptors and containing Ig domains have been found in clusters, such as the leukocyte receptor complex (LRC) [2] and the triggering receptor expressed on myeloid cells (TREM) cluster [3]. The LRC is a very gene dense region, spanning 1 Mb, which includes the killer cell Ig-like receptors (KIRs), Ig-like transcripts (ILTs), leukocyte Ig-like receptors (LILRs) and the natural cytotoxicity receptor (NCR) NKp46 [4]. The Ig containing proteins have been grouped into multi-gene families based on gene organisation, phylogeny and structure. They recognise conserved pathogen associated molecular patterns (PAMPs) released from or found on the surface of pathogens, which can initiate a signalling cascade [5]. However, ligands for many of the innate immune receptors still remain to be identified. The TREM cluster harbours genes such as TREM1 and 2, as well as the NCR named NKp44 [3,6]. TREM receptors are involved in the amplification and attenuation of the inflammatory response [7,8], while the NKp44 receptor activates NK cells [9]. All these receptors are type I transmembrane proteins characterised by the possession of a variable number of extra-cellular Ig domains of either the C2- type or the novel V-type [9,10]. The Ig domains are followed by a transmembrane region and a cytoplasmic region. Some inhibitory innate immune receptors have a long cytoplasmic region contain- ing one or more immunoreceptor tyrosine-based inhibitory motifs (ITIMs) [11], which blocks NK cell-mediated cytotoxicity [12,13]. The cytoplasmic regions of activating receptors are short and associate with adaptor molecules such as DAP12, CD3z or FceRIg Developmental and Comparative Immunology 33 (2009) 525–532 ARTICLE INFO Article history: Received 4 August 2008 Received in revised form 8 October 2008 Accepted 13 October 2008 Available online 12 November 2008 Keywords: Rainbow trout Innate immune receptors ITAM ITIM Comparative immunology Tissue distribution ABSTRACT The recognition of pathogens by the innate immune system relies on a wide range of inhibitory and activating receptors. Some of these non-rearranging receptors belong to the immunoglobulin superfamily (IgSF) and in teleost fish the novel immune-type receptor (NITR) and the novel immunoglobulin-like transcript (NILT) have been reported. Here we describe the identification and characterisation of three new NILTs from rainbow trout (Oncorhynchus mykiss), with one NILT alternatively spliced into a long isoform containing two Ig domains and a short isoform containing one Ig domain. The cytoplasmic regions contain either immunoreceptor tyrosine-based inhibitory motifs (ITIMs) or an immunoreceptor tyrosine-based activating motif (ITAM) for downstream signalling. Alignment of the various NILT Ig domains revealed a high similarity, especially between Ig domains from NILTs found in this study. Furthermore, a phylogenetic tree showed that NILTs are more closely related to the triggering receptor expressed on myeloid (TREM) cells and NKp44 than to NITRs. The expression of NILTs was studied in six different tissues and two different cell lines, with expression apparent in immunologically important tissues. Expression of NILTs was also shown to be an early event in development, with both eyed eggs and embryos expressing all four genes. The results obtained in this study and future experiments will contribute to our knowledge of the immune system in fish and provide useful information for the control of inflammatory processes in rainbow trout. ß 2008 Elsevier Ltd. All rights reserved. * Corresponding author. Tel.: +44 1224 272872; fax: +44 1224 272396. E-mail address: c.secombes@abdn.ac.uk (C.J. Secombes). 1 In memory of Prof. Rene ´ J.M. Stet (deceased 12 September 2007), who initiated this work. Contents lists available at ScienceDirect Developmental and Comparative Immunology journal homepage: www.elsevier.com/locate/dci 0145-305X/$ – see front matter ß 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.dci.2008.10.002