Cells expressing NKp46 were stained with anti-perforin monoclonal antibody (solid line) or an isotype control (broken line). but not all, express CD16 – characteristics consistent with NK cell populations in other species. We demonstrate that in addition to populations in peripheral blood and secondary lymphoid organs, ovine NKp46+populations are also situated at the mucosal surfaces of the lung, gastro-intestinal tract and non-gravid uterus. Furthermore, we show that purified ovine NKp46+populations cultured in IL-2 and IL-15 have cytotoxic activity that could be enhanced by ligation of NKp46 in re-directed lysis assays. Therefore we conclude that ovine NKp46+cells represent a population that by phenotype, tissue distribution and function correspond to NK cells and that NKp46 is an activating receptor in sheep as in other species. == Introduction == Natural killer (NK) cells are lymphocytes of the innate immune system which through production of cytokines and cytotoxic activity are capable of offering an immediate response to pathogen-infected and transformed host cells [1]. NK cells recognise potential targets through a diverse repertoire of germ-line encoded activating and inhibitory receptors including members of the killer cell Ig-like receptor (KIR), Ly49, and CD94:NKG2 families and the natural cytotoxicity receptors (NCRs) NKp46, NKp30 and Nepafenac NKp44. Induction of NK cell function is dependent on the relative balance of signals received from activating and inhibitory receptors engaged upon interaction with target cells. Through interactions with other cells of the immune system, NK cells have also been found Nepafenac to regulate the development of both innate and adaptive immune responses in a variety of ways. This includes the activation/maturation of antigen-presenting cells [2], providing IFN for the priming of TH1 CD4+T-cells [3], modulating the function of Tregcells [4] and exerting an immunoregulatory effect via the production of IL-10 [5]. Studies in a range of mammalian species have confirmed that NKp46 expression is restricted to NK cells and that it serves as the most reliable NK Rabbit Polyclonal to TF2A1 cell marker available [6-9]. NKp46 is a type I transmembrane glycoprotein with 2 extracellular C2-type Ig-like domains that associate via an arginine residue in the transmembrane region with the ITAM bearing molecules CD3 and FcRI [6,9,10]. In humans, NKp46 has been shown to be a principal activating receptor against a variety of NK cell targets [10,11]. However, with the exceptions of the haemagglutinin of influenza virus and the haemagglutinin-neuraminidase of parainfluenza virus and Newcastle disease virus the ligands for NKp46 are currently unknown [12,13]. The Nepafenac generation of a bovine NKp46-specific antibody has facilitated the study of NK cells in cattle and shown that they contribute to the response against a variety of pathogens includingMycobacterium bovis, Babesia bovisandNeospora caninum[14-16]. At present there is no equivalent antibody in sheep and most previous work has been restricted to the description of NK-like cytotoxicity in ovine PBMC and endometrial cell populations [17-20], although a recent publication has demonstrated that circulating CD16+/CD14-cells in ovine PBMC have the morphological and functional characteristics of NK cells [21]. In this paper we describe the generation of a monoclonal antibody specific for ovine NKp46 and show that cells expressing NKp46 have a phenotype, tissue distribution and cytotoxic function characteristic of NK cells. == Materials and methods == == Animals and tissue preparations == Samples were taken from sheep of various breeds aged between 3 months and 1 year. PBMC were isolated from blood collected in EDTA by density gradient centrifugation (900 g, 30 min) over Ficoll-Paque Plus (Amersham Biosciences, Little Chalfont, UK) and washed three times in PBS/2 mM EDTA. Single-cell suspensions from spleens and lymph nodes were obtained by passing the products of dilacerated tissues through a mesh with a 50 M pore size (BD, San Jose, CA, USA). Tissue samples for immunohistochemical analysis were snap frozen in isopentane/dry ice and mounted in optimal cutting temperature (OCT) compound (Tissue-Tek, Sakura-Finitek, Zoeterwoude, The Netherlands). For samples from the lung, isolated lobes were inflated Nepafenac with a mixture composed of 30% sucrose in water (w/v) and OCT at a ratio of 2:1 prior to snap freezing. Tissue sections cut to 7 m thickness were mounted on poly-L-lysine coated slides and stored at.