Optimal lymphocyte activation requires the simultaneous engagement of stimulatory and costimulatory receptors. Stimulatory immunoreceptors are usually composed of a ligand-binding transmembrane protein and noncovalently associated signal-transducing subunits. Here, we report that alternative splicing leads to two distinct NKG2D polypeptides that associate differentially with the DAP10 and KARAP (also known as DAP12) signaling subunits. We found that differential expression of these isoforms and of signaling proteins determined whether NKG2D functioned as a costimulatory receptor in the adaptive immune system (CD8+ T cells) or as both a primary recognition structure and a costimulatory receptor in the innate immune system (natural killer cells and macrophages). This strategy suggests a rationale for the multisubunit structure of stimulatory immunoreceptors.
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Supported by NIH grants (to D. H. R.) and by a Howard Hughes Medical Institute Physician Postdoctoral grant (to A. D.), by institutional grants from INSERM, CNRS and the Ministère de l'Enseignement Supérieur et de la Recherche (to E. V.), and specific grants from Ligue Nationale contre le Cancer (to M. L.) and 'Equipe labellisée La Ligue' (to E. V.).
The authors declare no competing financial interests.
Cell surface expression of NKG2D on cell populations from KARAP-mutant and KARAP-Tg mice. The indicated cell populations were analyzed by flow cytometry for cell surface expression of NKG2D by staining with a mAb. The histograms show electronic gating on the respective cell populations. The MFI of the gated positive cells is indicated above each histogram. (a) Analysis of NKG2D cell surface expression in homozygous KARAP-mutant mice (Δ/Δ), heterozygotes (+/Δ) or wild-type littermates (+/+). (b) Analysis of CD8+ T cells from KARAP-Tg mice and nontransgenic littermates. ND, not done; NS, not stimulated. (JPG 177 kb)
NKG2D-dependent NK cell activation in the absence of KARAP. (a) Freshly isolated NK cells from poly(I·C)-treated KARAP-mutant mice (Δ/Δ, open bars) or wild-type littermates (+/+, solid bars) were stimulated with RMA lymphoma cells transduced or not with the NKG2D ligands Rae-1β or H-60 (left panel) or with the indicated plate-bound antibodies (right panel). Accumulation of IFN-γ was evaluated by intracellular cytokine staining. A representative experiment is shown (n = 3). (b) The cytotoxicity of freshly isolated NK cells from poly(I·C)-treated KARAP-mutant mice (Δ/Δ, open squares) or wild-type littermates (+/+, closed squares) against RMA lymphoma cells transfected or not with Rae-1β or H-60 as indicated. The effector cells were incubated with a control antibody (upper panels) or a mAb to NKG2D (lower panels). A representative experiment is shown (n = 4). (JPG 128 kb)
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Diefenbach, A., Tomasello, E., Lucas, M. et al. Selective associations with signaling proteins determine stimulatory versus costimulatory activity of NKG2D. Nat Immunol 3, 1142–1149 (2002). https://doi.org/10.1038/ni858
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