Activating NK cell receptors frequently transmit activating signals via immunoreceptor tyrosine-based activation motifs (ITAMs) present in accessory proteins non-covalently associated with the intracellular region of the activating receptor . Activating NK cell receptors employing this strategy typically express a short cytoplasmic tail lacking ITIMs or other tyrosine signalling motif and possess a basic residue within their transmembrane sequence for association with transmembrane accessory proteins [10, 18, 19]. LLT1 possesses these properties associated with an activating receptor. In the present study, we have examined the signalling pathways
associated with LLT1-stimulated OTX015 IFN-γ production. We determined that the human NK cell line NK92 expresses LLT1 on its surface, and upon ligation with CD161 expressing K562 target cells stimulates IFN-γ production. Using this LLT1:CD161 ligation system, we analysed IFN-γ production in the presence or absence of specific pharmacological inhibitors to determine what signalling pathways are required for LLT1-induced IFN-γ production. These results indicate that LLT1 downstream signalling is likely dependent upon Src-protein tyrosine kinase [Src-PTK], p38 and ERK signalling pathways, but not dependent upon PKC, PI3K or calcineurin. These results were followed up with phosphorylation analysis, which confirmed that the ERK signalling pathway
is associated with Apoptosis Compound Library chemical structure LLT1-mediated IFN-γ production. Finally, we analysed IFN-γ mRNA transcription associated with LLT1 ligation. We found that LLT1 ligation is not associated with any change selleck screening library in detectable IFN-γ mRNA levels, suggesting that LLT1 stimulates IFN-γ production by modulating post-transcriptional or translational events. Tissue culture. NK92 cells were maintained using alpha-MEM
(Hyclone, Logan, UT, USA) with 25% defined Foetal Bovine Serum (Hyclone, Logan, UT, USA) and where appropriate 30 U/ml recombinant human IL-2 (Calbiochem, La Jolla, CA, USA). All other cells were maintained using 4+RPMI 1640 (GibcoBRL, Grand Island, NY, USA; with 10 mm MEM non-essential amino acids, 10 mm HEPES, 100 mm Sodium Pyruvate, 2 mm glutamine and penicillin/streptomycin) with 10% FetalPlex Animal Serum Complex (Gemini Bio-Products, Sacramento, CA, USA) at 37 °C, 5% CO2 in a water-jacketed tissue culture CO2 incubator. Flow cytometry. To evaluate the surface expression of LLT1 on NK92, cells were stained with 5 μg of anti-human OCIL/LLT1 monoclonal antibody (R & D Systems, Minneapolis, MN, USA) and 10 μg of 4C7 mouse anti-human LLT1 monoclonal antibody (Abnova, Taipei, Taiwan) and a PE-conjugated goat anti-mouse IgG polyclonal secondary antibody. In order to confirm the lack of CD161 expression on NK92 cells, cells were stained with mouse anti-human CD161 (Clone DX12; BD Biosciences, San Diego, CA, USA) and an FITC-conjugated goat anti-mouse IgG polyclonal secondary antibody.