Use of transfected Drosophila S2 cells to study NK cell activation.
ABSTRACT Determining the contribution of individual receptors to natural killer (NK) cell function is complicated by the multiplicity of activating and inhibitory NK cell receptors. Mammalian target cells typically express a variety of ligands for NK cell receptors. Engagement of NK cell receptors by antibodies may not mimic activation by natural ligands. To define requirements for activation and dissect the contribution of receptors to NK cell function, we have generated Drosophila Schneider line 2 (S2) cell transfectants expressing ligands for NK cell receptors. The evolutionary distance between Drosophila and mammals greatly reduces the potential of recognition of insect cell molecules by mammalian NK cells. Here, we present methods for maintenance and transfection of S2 cells, as well as protocols for their use in NK cell assays.
- SourceAvailable from: Eric O. Long[show abstract] [hide abstract]
ABSTRACT: Cytotoxic lymphocytes kill target cells through polarized release of the content of lytic granules at the immunological synapse. In human NK cells, signals for granule polarization and for degranulation can be uncoupled: Binding of β(2) integrin LFA-1 to ICAM is sufficient to induce polarization but not degranulation, whereas CD16 binding to IgG triggers unpolarized degranulation. In this study, we investigated the basis for this difference. IL-2-expanded human NK cells were stimulated by incubation with plate-bound ligands of LFA-1 (ICAM-1) and CD16 (human IgG). Surprisingly, LFA-1 elicited signals similar to those induced by CD16, including tyrosine phosphorylation of the TCR ζ-chain, tyrosine kinase Syk, and phospholipase C-γ. Whereas CD16 activated Ca(2+) mobilization and LAT phosphorylation, LFA-1 did not, but induced strong Pyk2 and paxillin phosphorylation. LFA-1-dependent granule polarization was blocked by inhibition of Syk, phospholipase C-γ, and protein kinase C, as well as by paxillin knockdown. Therefore, common signals triggered by CD16 and LFA-1 bifurcate to provide independent control of Ca(2+)-dependent degranulation and paxillin-dependent granule polarization.The Journal of Immunology 03/2011; 186(5):2998-3005. · 5.52 Impact Factor