Evaluation of zebrafish novel immune-type receptor 9 (NITR9)

Natural killer (NK) cells are lymphocytes of the innate immune system that express several cell surface receptors including both activating and inhibitory forms. The natural killer cell receptors (NKRs) distinguish neoplastic or virally infected cells from normal host cells and regulate cytotoxic function. NKRs of different mammalian species differ dramatically and are the products of different multigene families. Due to the recent and rapid evolution of these receptors it has been difficult to identify orthologs of NK cell receptors (NKRs) in non-mammalian vertebrate species. In this regard, a multigene family of novel immune type receptors (NITRs) has been identified in bony fish species that are structurally similar to mammalian immunoglobulin (Ig)-type NKRs. In zebrafish, 14 NITR gene families have been identified, of which Nitr9 is the only activating receptor. In an effort to better understand the role of Nitr9 in zebrafish immunity, it was necessary to develop tools and methods to enable identification, purification, characterization and function of Nitr9 expressing cells. Chapter II discusses the generation of two anti-Nitr9 monoclonal antibodies (mAbs) that were utilized to determine the Nitr9 protein expression profile in-vivo. Immunofluorescence and flow cytometric analysis of cells transiently transfected with nitr9 demonstrated that the two mAbs can detect Nitr9 expressing cells. However; due to low levels of expression it was difficult to identify and purify Nitr9 expressing cells in-vivo. Thus, the goal of Chapter III was to determine a method to boost Nitr9 expression in-vivo and facilitate the identification and purification of Nitr9 expressing cells with the two mAbs generated in Chapter II. Preliminary results suggest that nitr9 gene expression is higher in the intestine of rag1 deficient zebrafish, which lack a functional adaptive immune response. This may provide a potential model to identify Nitr9 expressing cells.