Skip to main content

Thank you for visiting You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Selective associations with signaling proteins determine stimulatory versus costimulatory activity of NKG2D

A Corrigendum to this article was published on 01 June 2004


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

Relevant articles

Open Access articles citing this article.

Access options

Rent or buy this article

Prices vary by article type



Prices may be subject to local taxes which are calculated during checkout

Figure 1: Expression of NKG2D splice variants in NK cells, CD8+ T cells and macrophages.
Figure 2: Association of NKG2D-S with DAP10 and KARAP.
Figure 3: Association of NKG2D with DAP10 and KARAP in NK cells and macrophages.
Figure 4: NKG2D-dependent activation of NK cells and macrophages in the absence of KARAP.
Figure 5: NKG2D-DAP10–dependent costimulation of CD8+ T cells.
Figure 6: Function of NKG2D in CD8+ T cells ectopically expressing KARAP.

Accession codes




  1. 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).

    CAS  Google Scholar 

  2. 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).

    Article  CAS  Google Scholar 

  3. 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).

    Article  CAS  Google Scholar 

  4. Bakker, A.B.H. et al. DAP12-deficient mice fail to develop autoimmunity due to impaired antigen priming. Immunity 13, 345–353 (2000).

    Article  CAS  Google Scholar 

  5. Wu, J. et al. An activating immunoreceptor complex formed by NKG2D and DAP10. Science 285, 730–732 (1999).

    Article  CAS  Google Scholar 

  6. 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).

    Google Scholar 

  7. 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).

    Article  CAS  Google Scholar 

  8. 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).

    Article  CAS  Google Scholar 

  9. Jamieson, A.M. et al. The role of the NKG2D immunoreceptor in immune cell activation and natural killing. Immunity 17, 19–29 (2002).

    Article  CAS  Google Scholar 

  10. 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).

    Article  CAS  Google Scholar 

  11. Bauer, S. et al. Activation of NK cells and T cells by NKG2D, a receptor for stress-inducible MICA. Science 285, 727–729 (1999).

    Article  CAS  Google Scholar 

  12. 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).

    Article  CAS  Google Scholar 

  13. 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).

    Article  CAS  Google Scholar 

  14. Diefenbach, A. & Raulet, D.H. Strategies for target cell recognition by natural killer cells. Immunol. Rev. 181, 170–184 (2001).

    Article  CAS  Google Scholar 

  15. 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).

    Article  CAS  Google Scholar 

  16. 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).

    Article  CAS  Google Scholar 

  17. 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).

    Article  CAS  Google Scholar 

  18. 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).

    Article  CAS  Google Scholar 

  19. 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).

    Article  CAS  Google Scholar 

  20. Lucas, M. et al. Massive inflammatory syndrome and lymphocytic immunodeficiency in KARAP/DAP12 transgenic mice. Eur. J. Immunol. 32, 2653–2663 (2002).

    Article  CAS  Google Scholar 

  21. Medzhitov, R. & Janeway, C.A. Jr. Decoding the patterns of self and nonself by the innate immune system. Science 296, 298–300 (2002).

    Article  CAS  Google Scholar 

  22. 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).

  23. 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).

    Article  CAS  Google Scholar 

  24. 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).

    Article  CAS  Google Scholar 

  25. 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).

    Article  CAS  Google Scholar 

  26. 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).

    Article  CAS  Google Scholar 

  27. Cosson, P., Lankford, S.P., Bonifacino, J.S. & Klausner, R.D. Membrane protein association by potential intramembrane charge pairs. Nature 351, 414–416 (1991).

    Article  CAS  Google Scholar 

  28. 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).

    Article  CAS  Google Scholar 

  29. 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).

    Article  CAS  Google Scholar 

  30. Wilson, M.J., Haude, A. & Trowsdale, J. The mouse Dap10 gene. Immunogenetics 53, 347–350 (2001).

    Article  CAS  Google Scholar 

  31. 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).

    Article  CAS  Google Scholar 

  32. 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).

    Article  CAS  Google Scholar 

  33. 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).

    CAS  PubMed  Google Scholar 

  34. Hayes, S.M. & Love, P.E. Distint structure and signaling potential of the γδ TCR complex. Immunity 16, 827–838 (2002).

    Article  CAS  Google Scholar 

  35. 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).

    Article  CAS  Google Scholar 

  36. 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).

    Article  CAS  Google Scholar 

  37. 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).

    Article  CAS  Google Scholar 

  38. Hanke, T. et al. Direct assessment of MHC class I binding by seven Ly49 inhibitory NK cell receptors. Immunity 11, 67–77 (1999).

    Article  CAS  Google Scholar 

  39. 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).

    Article  CAS  Google Scholar 

  40. 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).

    Article  CAS  Google Scholar 

  41. Koo, G.C. & Peppard, J.R. Establishment of monoclonal anti-NK-1.1 antibody. Hybridoma 3, 301–303 (1984).

    Article  CAS  Google Scholar 

  42. 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).

    Article  CAS  Google Scholar 

  43. Murali-Krishna, K. et al. Counting antigen-specific CD8 T cells: a reevaluation of bystander activation during viral infection. Immunity 8, 177–187 (1998).

    Article  CAS  Google Scholar 

Download references


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.).

Author information

Authors and Affiliations


Corresponding authors

Correspondence to Eric Vivier or David H. Raulet.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Web Fig. 1.

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)

Web Fig. 2.

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)

Rights and permissions

Reprints and Permissions

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).

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI:

This article is cited by


Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing