The novel immune-type receptors (NITRs), which have been described in numerous bony fish species, are encoded by multigene families of inhibitory and activating receptors and are predicted to be functional orthologs to the mammalian natural killer cell receptors (NKRs). Within the zebrafish NITR family, nitr9 is the only gene predicted to encode an activating receptor. However, alternative RNA splicing generates three distinct nitr9 transcripts, each of which encodes a different isoform. Although nitr9 transcripts have been detected in zebrafish lymphocytes, the specific hematopoietic lineage(s) that expresses Nitr9 remains to be determined. In an effort to better understand the role of NITRs in zebrafish immunity, anti-Nitr9 monoclonal antibodies were generated and evaluated for the ability to recognize the three Nitr9 isoforms. The application of these antibodies to flow cytometry should prove to be useful for identifying the specific lymphocyte lineages that express Nitr9 and may permit the isolation of Nitr9-expressing cells that can be directly assessed for cytotoxic (e.g., NK) function. 1. Introduction Mammalian natural killer (NK) cells are large, granular lymphocytes of the innate immune system that express several cell surface receptors to regulate cytotoxic function through a complex network of signaling pathways. NK cell receptors include both activating and inhibitory forms that are proficient in distinguishing neoplastic or virally infected cells from normal host cells [1, 2]. The regulation of NK cell cytotoxicity is dependent on the integration of signals from activating and inhibitory receptors [3]. Although it is postulated that NK cell receptors arose early in vertebrate phylogeny, functional data are based primarily on studies of mammalian NK cell receptors [4]. In order to appreciate the origins and evolution of NK cell receptors and their function, it is critical to define equivalent receptor forms in nonmammalian species. The bony fish represent one of the earliest vertebrate lineages with a functional innate and adaptive immune response that closely parallels that of humans and other mammals [5]. A large multigene family of recently and rapidly evolving inhibitory and activating novel immune-type receptors (NITRs) that share structural and functional characteristics with mammalian NK cell receptors has been identified in multiple fish species [6, 7]. Complete analyses of the NITR gene clusters at the sequence level only have been performed with the zebrafish and medaka genomes [8–11]. Although transcripts of various catfish NITRs
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