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.
This is a preview of subscription content, access via your institution
Open Access articles citing this article.
Molecular Cancer Open Access 27 February 2019
Journal for ImmunoTherapy of Cancer Open Access 18 February 2019
Stepwise phosphorylation of p65 promotes NF-κB activation and NK cell responses during target cell recognition
Nature Communications Open Access 25 May 2016
Subscribe to this journal
Receive 12 print issues and online access
$209.00 per year
only $17.42 per issue
Rent or buy this article
Prices vary by article type
Prices may be subject to local taxes which are calculated during checkout
Olcese, L. et al. Human killer cell activatory receptors for MHC class I molecules are included in a multimeric complex expressed by natural killer cells. J. Immunol. 158, 5083–5086 (1997).
Lanier, L.L., Corliss, B.C., Wu, J., Leong, C. & Phillips, J.H. Immunoreceptor DAP12 bearing a tyrosine-based activation motif is involved in activating NK cells. Nature 391, 703–707 (1998).
Tomasello, E. et al. Combined natural killer cell and dendritic cell functional deficiency in KARAP/DAP12 loss-of–function mutant mice. Immunity 13, 355–364 (2000).
Bakker, A.B.H. et al. DAP12-deficient mice fail to develop autoimmunity due to impaired antigen priming. Immunity 13, 345–353 (2000).
Wu, J. et al. An activating immunoreceptor complex formed by NKG2D and DAP10. Science 285, 730–732 (1999).
Chang, C. et al. KAP10, a novel transmembrane adapter protein genetically linked to DAP12 but with unique signaling properties. J. Immunol. 163, 4652–4654 (1999).
Wu, J., Cherwinski, H., Spies, T., Phillips, J.H. & Lanier, L.L. DAP10 and DAP12 form distinct, but functionally cooperative, receptor complexes in natural killer cells. J. Exp. Med. 192, 1059–1067 (2000).
Diefenbach, A., Jamieson, A.M., Liu, S.D., Shastri, N. & Raulet, D.H. Ligands for the murine NKG2D receptor: expression by tumor cells and activation of NK cells and macrophages. Nature Immunol. 1, 119–126 (2000).
Jamieson, A.M. et al. The role of the NKG2D immunoreceptor in immune cell activation and natural killing. Immunity 17, 19–29 (2002).
Cerwenka, A. et al. Retinoic acid early inducible genes define a ligand family for the activating NKG2D receptor in mice. Immunity 12, 721–727 (2000).
Bauer, S. et al. Activation of NK cells and T cells by NKG2D, a receptor for stress-inducible MICA. Science 285, 727–729 (1999).
Cosman, D. et al. ULBPs, novel MHC class I-related molecules, bind to CMV glycoprotein UL16 and stimulate NK cytotoxicity through the NKG2D receptor. Immunity 14, 123–133 (2001).
Groh, V. et al. Cell stress-regulated human major histocompatibility complex class I gene expressed in gastrointestinal epithelium. Proc. Natl. Acad. Sci. USA 93, 12445–12450 (1996).
Diefenbach, A. & Raulet, D.H. Strategies for target cell recognition by natural killer cells. Immunol. Rev. 181, 170–184 (2001).
Diefenbach, A., Jensen, E.R., Jamieson, A.M. & Raulet, D.H. Rae1 and H60 ligands of the NKG2D receptor stimulate tumour immunity. Nature 413, 165–171 (2001).
Pende, D. et al. Role of NKG2D in tumor cell lysis mediated by human NK cells: cooperation with natural cytotoxicity receptors and capability of recognizing tumors of nonepithelial origin. Eur. J. Immunol. 31, 1076–1086 (2001).
Groh, V. et al. Costimulation of CD8αβ T cells by NKG2D via engagement by MIC induced on virus-infected cells. Nature Immunol. 2, 255–260 (2001).
Vance, R.E., Tanamachi, D.M., Hanke, T. & Raulet, D.H. Cloning of a mouse homolog of CD94 extends the family of C-type lectins on murine natural killer cells. Eur. J. Immunol. 27, 3236–3241 (1997).
Ho, E.L. et al. Murine Nkg2d and Cd94 are clustered within the natural killer complex and are expressed independently in natural killer cells. Proc. Natl. Acad. Sci. USA 95, 6320–6325 (1998).
Lucas, M. et al. Massive inflammatory syndrome and lymphocytic immunodeficiency in KARAP/DAP12 transgenic mice. Eur. J. Immunol. 32, 2653–2663 (2002).
Medzhitov, R. & Janeway, C.A. Jr. Decoding the patterns of self and nonself by the innate immune system. Science 296, 298–300 (2002).
Gilfillan, S., Ho, E.L., Cella, M., Yokoyama, W.M. & Colonna1, M. NKG2D recruits two distinct adapters to trigger NK cell activation and costimulation. Nat. Immunol.; published online 11 November 2002 (doi:10.1038/ni857).
Brooks, C.G., Urdal, D.L. & Henney, C.S. Lymphokine-driven “differentiation” of cytotoxic T-cell clones into cells with NK-like specificity: correlations with display of membrane macromolecules. Immunol. Rev. 72, 43–72 (1983).
Mingari, M.C., Moretta, A. & Moretta, L. Regulation of KIR expression in human T cells: a safety mechanism that may impair protective T-cell responses. Immunol. Today 19, 153–157 (1998).
Uhrberg, M. et al. The repertoire of killer cell Ig-like receptor and CD94:NKG2A receptors in T cells: clones sharing identical αβ TCR rearrangement express highly diverse killer cell Ig-like receptor patterns. J. Immunol. 166, 3923–3932 (2001).
Glas, R. et al. Recruitment and activation of natural killer (NK) cells in vivo determined by the target cell phenotype: An adaptive component of NK cell-mediated responses. J. Exp. Med. 191, 129–138 (2000).
Cosson, P., Lankford, S.P., Bonifacino, J.S. & Klausner, R.D. Membrane protein association by potential intramembrane charge pairs. Nature 351, 414–416 (1991).
Klausner, R.D., Lippincott-Schwartz, J. & Bonifacino, J.S. The T cell antigen receptor: insights into organelle biology. Annu. Rev. Cell Biol. 6, 403–431 (1990).
Houchins, J.P., Yabe, T., McSherry, C. & Bach, F.H. DNA sequence analysis of NKG2, a family of related cDNA clones encoding type II integral membrane proteins on human natural killer cells. J. Exp. Med. 173, 1017–1020 (1991).
Wilson, M.J., Haude, A. & Trowsdale, J. The mouse Dap10 gene. Immunogenetics 53, 347–350 (2001).
Howard, F.D., Rodewald, H.R., Kinet, J.P. & Reinherz, E.L. CD3ζ subunit can substitute for the gamma subunit of Fcε receptor type I in assembly and functional expression of the high- affinity IgE receptor: evidence for interreceptor complementation. Proc. Natl. Acad. Sci. USA 87, 7015–7019 (1990).
Orloff, D.G., Ra, C.S., Frank, S.J., Klausner, R.D. & Kinet, J.P. Family of disulphide-linked dimers containing the ζ and ε chains of the T-cell receptor and the γ chain of Fc receptors. Nature 347, 189–191 (1990).
Vivier, E. et al. Tyrosine phosphorylation of the FcγRIII(CD16): ζ complex in human natural killer cells. Induction by antibody-dependent cytotoxicity but not by natural killing. J. Immunol. 146, 206–210 (1991).
Hayes, S.M. & Love, P.E. Distint structure and signaling potential of the γδ TCR complex. Immunity 16, 827–838 (2002).
Tatusova, T.A. & Madden, T.L. BLAST 2 sequences, a new tool for comparing protein and nucleotide sequences. FEMS Microbiol. Lett. 174, 247–250 (1999).
Portnoy, D.A., Jacks, P.S. & Hinrichs, D.J. Role of hemolysin for the intracellular growth of Listeria monocytogenes. J. Exp. Med. 167, 1459–1471 (1988).
Diefenbach, A. et al. Type 1 interferon (IFNα/β) and type 2 nitric oxide synthase regulate the innate immune response to a protozoan parasite. Immunity 8, 77–87 (1998).
Hanke, T. et al. Direct assessment of MHC class I binding by seven Ly49 inhibitory NK cell receptors. Immunity 11, 67–77 (1999).
Diefenbach, A., Schindler, H., Röllinghoff, M., Yokoyama, W.M. & Bogdan, C. Requirement for type 2 NO synthase for IL-12 signaling in innate immunity. Science 284, 951–955 (1999).
Liao, N., Bix, M., Zijlstra, M., Jaenisch, R. & Raulet, D. MHC class I deficiency: susceptibility to natural killer (NK) cells and impaired NK activity. Science 253, 199–202 (1991).
Koo, G.C. & Peppard, J.R. Establishment of monoclonal anti-NK-1.1 antibody. Hybridoma 3, 301–303 (1984).
Coles, M.C., McMahon, C.W., Takizawa, H. & Raulet, D.H. Memory CD8 T lymphocytes express inhibitory MHC-specific Ly49 receptors. Eur. J. Immunol. 30, 236–244 (2000).
Murali-Krishna, K. et al. Counting antigen-specific CD8 T cells: a reevaluation of bystander activation during viral infection. Immunity 8, 177–187 (1998).
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)
About this article
Cite this article
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
This article is cited by
Cellular and Molecular Life Sciences (2021)
Nature Immunology (2020)
Molecular Cancer (2019)
Journal for ImmunoTherapy of Cancer (2019)