NKp46 defines ovine cells that have characteristics corresponding to NK cells

Abstract

Natural killer (NK) cells are well recognized as playing a key role in innate immune defence through cytokine production and cytotoxic activity; additionally recent studies have identified several novel NK cell functions. The ability to study NK cells in the sheep has been restricted due to a lack of specific reagents. We report the generation of a monoclonal antibody specific for ovine NKp46, a receptor which in a number of mammals is expressed exclusively in NK cells. Ovine NKp46+ cells represent a population that is distinct from CD4+ and γδ+ T-cells, B-cells and cells of the monocytic lineage. The NKp46+ cells are heterogenous with respect to expression of CD2 and CD8 and most, 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.

The early intestinal immune response in experimental neonatal ovine cryptosporidiosis is characterized by an increased frequency of perforin expressing NCR1+ NK cells and by NCR1− CD8+ cell recruitment

“…As previously found in the GALTs of other healthy 1–2 month-old lambs [33,34,49], NCR1 labelling of IPP sections (Figure 3A) revealed the presence of NCR1+ cells in the interfollicular T cell areas and domes, and to a lesser degree in the lamina propria and intraepithelial compartment, while few or none were observed in the follicles. There was neither a difference in the density (number of labelled cells per area) nor a change in localization of NCR1+ cells in the infected lambs compared to the controls at any time during the infection (Figure 3A).…”

Characterization of the Th Profile of the Bovine Endometrium during the Oestrous Cycle and Early Pregnancy

“…The sections were fixed in ice-cold acetone for 10 min and air dried for 1 h. Following a rehydration step with 0.05 mM Tris-buffered saline (TBS; pH 7.7), sections were incubated with blocking buffer (TBS supplemented with 10% (v/v) goat serum) for 1 h. All primary antibodies except for mouse anti CD335/NKp46, which was a kind gift from T.Connelly [28], were purchased from VRMD (Pullman, WA 99163, U.S.A.) Single colour immunofluorescence was performed by incubation at 4°C with mouse anti-bovine B-B7 (GB25A, ascites, 1 mg/ml) for B lymphocytes, mouse anti-bovine TcR1-N24 (clone GB21A, ascites, 1 mg⁄mL) for γδT lymphocytes and mouse anti-ovine CD335/NKp46 (clone Gr13.1; ascites, 1 mg⁄ml) for Natural Killer cells [28]. Two-colour immunofluorescence labelling was carried out for CD4 + and Cd8 + T lymphocytes by concomitant incubation of sections with primary antibodies: mouse anti-bovine CD4 (clone IL-A11, ascites, 1 mg ⁄ml) and mouse anti-bovine CD8 (clone BAQ11A, ascites, 1 mg/mL) for T lymphocytes.…”

CD8 T‐cell responses against the immunodominant Theileria parva peptide Tp249–59 are composed of two distinct populations specific for overlapping 11‐mer and 10‐mer epitopes

“…Tetramer‐positive populations within PBMC were analysed using multi‐colour flow cytometry. In brief, PBMC (1 × 10 7 /ml) were stained with phycoerythrin‐labelled tetramers at a final concentration of 20 n m for 30 min at room temperature, washed twice in PBS containing 0·5% FBS before staining with a mixture of monoclonal antibodies against CD4 (IL‐A12), TCR‐ γδ (GB21A), CD21 (CC21, IgG1), NKp46 (EC1.1 IgG1) and CD172a (IL‐A24, IgG1) for 30 min at 4°. After three washes, cells were incubated for 30 min at 4° with Alexa Fluor 647conjugated goat anti‐mouse IgG (Life Technologies, Paisley, UK), washed three times, and then SYTOX Red Dead Cell Stain (Life Technologies) was added.…”