Cancer Immunology, Immunotherapy

, Volume 62, Issue 7, pp 1235–1247 | Cite as

Antibody-dependent cell lysis by NK cells is preserved after sarcoma-induced inhibition of NK cell cytotoxicity

  • Jens H. W. Pahl
  • S. Eriaty N. Ruslan
  • Kitty M. C. Kwappenberg
  • Monique M. van Ostaijen-ten Dam
  • Maarten J. D. van Tol
  • Arjan C. Lankester
  • Marco W. Schilham
Original Article


Osteosarcoma and Ewing’s sarcoma tumor cells are susceptible to IL15-induced or antibody-mediated cytolytic activity of NK cells in short-term cytotoxicity assays. When encountering the tumor environment in vivo, NK cells may be in contact with tumor cells for a prolonged time period. We explored whether a prolonged interaction with sarcoma cells can modulate the activation and cytotoxic activity of NK cells. The 40 h coculture of NK cells with sarcoma cells reversibly interfered with the IL15-induced expression of NKG2D, DNAM-1 and NKp30 and inhibited the cytolytic activity of NK cells. The inhibitory effects on receptor expression required physical contact between NK cells and sarcoma cells and were independent of TGF-β. Five days pre-incubation of NK cells with IL15 prevented the down-regulation of NKG2D and cytolytic activity in subsequent cocultures with sarcoma cells. NK cell FcγRIIIa/CD16 receptor expression and antibody-mediated cytotoxicity were not affected after the coculture. Inhibition of NK cell cytotoxicity was directly linked to the down-regulation of the respective NK cell-activating receptors. Our data demonstrate that the inhibitory effects of sarcoma cells on the cytolytic activity of NK cells do not affect the antibody-dependent cytotoxicity and can be prevented by pre-activation of NK cells with IL15. Thus, the combination of cytokine-activated NK cells and monoclonal antibody therapy may be required to improve tumor targeting and NK cell functionality in the tumor environment.


NK cell NKG2D ADCC IL15 Sarcoma 



The authors thank F. Schaper, M. Verheul and L. Oudejans for technical contributions. We thank T. van Hall, G. de Groot-Swings, J. Suurmond and S. de Jong for kindly providing antibodies against HLA-E, HLA-G, PD-1 and PD-1L, respectively. This work was financially supported by a grant from the foundation ‘Quality of Life Gala 2007,’ the European Commission (EuroBoNeT, grant No 018814) and the Dutch Foundation Children Cancer Free (grant 2009-052).

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

262_2013_1406_MOESM1_ESM.pdf (911 kb)
Supplementary material 1 (PDF 911 kb)


  1. 1.
    Bernstein M, Kovar H, Paulussen M, Randall RL, Schuck A, Teot LA, Juergens H (2006) Ewing’s sarcoma family of tumors: current management. Oncologist 11:503–519PubMedCrossRefGoogle Scholar
  2. 2.
    Hattinger CM, Pasello M, Ferrari S, Picci P, Serra M (2010) Emerging drugs for high-grade osteosarcoma. Expert Opin Emerg Drugs 15:615–634PubMedCrossRefGoogle Scholar
  3. 3.
    van den Berg H, Kroon HM, Slaar A, Hogendoorn PCW (2008) Incidence of biopsy-proven bone tumors in children: a report based on the Dutch pathology registration “PALGA”. J Pediatr Orthop 28:29–35PubMedCrossRefGoogle Scholar
  4. 4.
    Berghuis D, Schilham M.W., Voss HI, Santos SJ, Kloess S, Buddingh EP, Egeler RM, Hogendoorn PCW, Lankester AC (2012) Histone Deacetylase Inhibitors Enhance Expression of NKG2D Ligands in Ewing Sarcoma and Sensitize for Natural Killer Cell-Mediated Cytolysis. Clin Sarcoma Res 2:8Google Scholar
  5. 5.
    Buddingh EP, Schilham MW, Ruslan SE, Berghuis D, Szuhai K, Suurmond J, Taminiau AH, Gelderblom H, Egeler RM, Serra M, Hogendoorn PCW, Lankester AC (2011) Chemotherapy-resistant osteosarcoma is highly susceptible to IL-15-activated allogeneic and autologous NK cells. Cancer Immunol Immunother 60:575–586PubMedCrossRefGoogle Scholar
  6. 6.
    Pahl JH, Ruslan SE, Buddingh EP, Santos SJ, Szuhai K, Serra M, Gelderblom H, Hogendoorn PC, Egeler RM, Schilham MW, Lankester AC (2012) Anti-EGFR antibody cetuximab enhances the cytolytic activity of natural killer cells toward osteosarcoma. Clin Cancer Res 18:432–441PubMedCrossRefGoogle Scholar
  7. 7.
    Verhoeven DHJ, de Hooge AS, Mooiman EC, Santos SJ, ten Dam MM, Gelderblom H, Melief CJ, Hogendoorn PCW, Egeler RM, van Tol MJD, Schilham MW, Lankester AC (2008) NK cells recognize and lyse Ewing sarcoma cells through NKG2D and DNAM-1 receptor dependent pathways. Mol Immunol 45:3917–3925PubMedCrossRefGoogle Scholar
  8. 8.
    Bryceson YT, Long EO (2008) Line of attack: NK cell specificity and integration of signals. Curr Opin Immunol 20:344–352PubMedCrossRefGoogle Scholar
  9. 9.
    Moretta L, Bottino C, Pende D, Castriconi R, Mingari MC, Moretta A (2006) Surface NK receptors and their ligands on tumor cells. Semin Immunol 18:151–158PubMedCrossRefGoogle Scholar
  10. 10.
    Vivier E, Tomasello E, Baratin M, Walzer T, Ugolini S (2008) Functions of natural killer cells. Nat Immunol 9:503–510PubMedCrossRefGoogle Scholar
  11. 11.
    Thielens A, Vivier E, Romagne F (2012) NK cell MHC class i specific receptors (KIR): from biology to clinical intervention. Curr Opin Immunol 24:239–245PubMedCrossRefGoogle Scholar
  12. 12.
    Borrego F, Ulbrecht M, Weiss EH, Coligan JE, Brooks AG (1998) Recognition of human histocompatibility leukocyte antigen (HLA)-E complexed with HLA class I signal sequence-derived peptides by CD94/NKG2 confers protection from natural killer cell-mediated lysis. J Exp Med 187:813–818PubMedCrossRefGoogle Scholar
  13. 13.
    Bacon L, Eagle RA, Meyer M, Easom N, Young NT, Trowsdale J (2004) Two human ULBP/RAET1 molecules with transmembrane regions are ligands for NKG2D. J Immunol 173:1078–1084PubMedGoogle Scholar
  14. 14.
    Bauer S, Groh V, Wu J, Steinle A, Phillips JH, Lanier LL, Spies T (1999) Activation of NK cells and T cells by NKG2D, a receptor for stress-inducible MICA. Science 285:727–729PubMedCrossRefGoogle Scholar
  15. 15.
    Chalupny NJ, Sutherland CL, Lawrence WA, Rein-Weston A, Cosman D (2003) ULBP4 is a novel ligand for human NKG2D. Biochem Biophys Res Commun 305:129–135PubMedCrossRefGoogle Scholar
  16. 16.
    Cosman D, Mullberg J, Sutherland CL, Chin W, Armitage R, Fanslow W, Kubin M, Chalupny NJ (2001) ULBPs, novel MHC class I-related molecules, bind to CMV glycoprotein UL16 and stimulate NK cytotoxicity through the NKG2D receptor. Immunity 14:123–133PubMedCrossRefGoogle Scholar
  17. 17.
    Eagle RA, Traherne JA, Hair JR, Jafferji I, Trowsdale J (2009) ULBP6/RAET1L is an additional human NKG2D ligand. Eur J Immunol 39:3207–3216PubMedCrossRefGoogle Scholar
  18. 18.
    Bottino C, Castriconi R, Pende D, Rivera P, Nanni M, Carnemolla B, Cantoni C, Grassi J, Marcenaro S, Reymond N, Vitale M, Moretta L, Lopez M, Moretta A (2003) Identification of PVR (CD155) and Nectin-2 (CD112) as cell surface ligands for the human DNAM-1 (CD226) activating molecule. J Exp Med 198:557–567PubMedCrossRefGoogle Scholar
  19. 19.
    Brandt CS, Baratin M, Yi EC, Kennedy J, Gao Z, Fox B, Haldeman B, Ostrander CD, Kaifu T, Chabannon C, Moretta A, West R, Xu W, Vivier E, Levin SD (2009) The B7 family member B7–H6 is a tumor cell ligand for the activating natural killer cell receptor NKp30 in humans. J Exp Med 206:1495–1503PubMedCrossRefGoogle Scholar
  20. 20.
    Bryceson YT, March ME, Ljunggren HG, Long EO (2006) Synergy among receptors on resting NK cells for the activation of natural cytotoxicity and cytokine secretion. Blood 107:159–166PubMedCrossRefGoogle Scholar
  21. 21.
    Horng T, Bezbradica JS, Medzhitov R (2007) NKG2D signaling is coupled to the interleukin 15 receptor signaling pathway. Nat Immunol 8:1345–1352PubMedCrossRefGoogle Scholar
  22. 22.
    Lucas M, Schachterle W, Oberle K, Aichele P, Diefenbach A (2007) Dendritic cells prime natural killer cells by trans-presenting interleukin 15. Immunity 26:503–517PubMedCrossRefGoogle Scholar
  23. 23.
    Cerwenka A, Baron JL, Lanier LL (2001) Ectopic expression of retinoic acid early inducible-1 gene (RAE-1) permits natural killer cell-mediated rejection of a MHC class I-bearing tumor in vivo. Proc Natl Acad Sci U S A 98:11521–11526PubMedCrossRefGoogle Scholar
  24. 24.
    Diefenbach A, Jensen ER, Jamieson AM, Raulet DH (2001) Rae1 and H60 ligands of the NKG2D receptor stimulate tumour immunity. Nature 413:165–171PubMedCrossRefGoogle Scholar
  25. 25.
    Kelly JM, Darcy PK, Markby JL, Godfrey DI, Takeda K, Yagita H, Smyth MJ (2002) Induction of tumor-specific T cell memory by NK cell-mediated tumor rejection. Nat Immunol 3:83–90PubMedCrossRefGoogle Scholar
  26. 26.
    Smyth MJ, Swann J, Cretney E, Zerafa N, Yokoyama WM, Hayakawa Y (2005) NKG2D function protects the host from tumor initiation. J Exp Med 202:583–588PubMedCrossRefGoogle Scholar
  27. 27.
    Ruggeri L, Capanni M, Urbani E, Perruccio K, Shlomchik WD, Tosti A, Posati S, Rogaia D, Frassoni F, Aversa F, Martelli MF, Velardi A (2002) Effectiveness of donor natural killer cell alloreactivity in mismatched hematopoietic transplants. Science 295:2097–2100PubMedCrossRefGoogle Scholar
  28. 28.
    Doubrovina ES, Doubrovin MM, Vider E, Sisson RB, O’Reilly RJ, Dupont B, Vyas YM (2003) Evasion from NK cell immunity by MHC class I chain-related molecules expressing colon adenocarcinoma. J Immunol 171:6891–6899PubMedGoogle Scholar
  29. 29.
    Hilpert J, Grosse-Hovest L, Grunebach F, Buechele C, Nuebling T, Raum T, Steinle A, Salih HR (2012) Comprehensive analysis of nkg2d ligand expression and release in leukemia: implications for NKG2D-mediated NK cell responses. J Immunol 189:1360–1371PubMedCrossRefGoogle Scholar
  30. 30.
    Carlsten M, Norell H, Bryceson YT, Poschke I, Schedvins K, Ljunggren HG, Kiessling R, Malmberg KJ (2009) Primary human tumor cells expressing CD155 impair tumor targeting by down-regulating DNAM-1 on NK cells. J Immunol 183:4921–4930PubMedCrossRefGoogle Scholar
  31. 31.
    Groh V, Wu J, Yee C, Spies T (2002) Tumour-derived soluble MIC ligands impair expression of NKG2D and T-cell activation. Nature 419:734–738PubMedCrossRefGoogle Scholar
  32. 32.
    Ogasawara K, Hamerman JA, Hsin H, Chikuma S, Bour-Jordan H, Chen T, Pertel T, Carnaud C, Bluestone JA, Lanier LL (2003) Impairment of NK cell function by NKG2D modulation in NOD mice. Immunity 18:41–51PubMedCrossRefGoogle Scholar
  33. 33.
    Oppenheim DE, Roberts SJ, Clarke SL, Filler R, Lewis JM, Tigelaar RE, Girardi M, Hayday AC (2005) Sustained localized expression of ligand for the activating NKG2D receptor impairs natural cytotoxicity in vivo and reduces tumor immunosurveillance. Nat Immunol 6:928–937PubMedCrossRefGoogle Scholar
  34. 34.
    Castriconi R, Cantoni C, Della CM, Vitale M, Marcenaro E, Conte R, Biassoni R, Bottino C, Moretta L, Moretta A (2003) Transforming growth factor beta 1 inhibits expression of NKp30 and NKG2D receptors: consequences for the NK-mediated killing of dendritic cells. Proc Natl Acad Sci U S A 100:4120–4125PubMedCrossRefGoogle Scholar
  35. 35.
    Rook AH, Kehrl JH, Wakefield LM, Roberts AB, Sporn MB, Burlington DB, Lane HC, Fauci AS (1986) Effects of transforming growth factor beta on the functions of natural killer cells: depressed cytolytic activity and blunting of interferon responsiveness. J Immunol 136:3916–3920PubMedGoogle Scholar
  36. 36.
    Clayton A, Mitchell JP, Court J, Mason MD, Tabi Z (2007) Human tumor-derived exosomes selectively impair lymphocyte responses to interleukin-2. Cancer Res 67:7458–7466PubMedCrossRefGoogle Scholar
  37. 37.
    Clayton A, Mitchell JP, Court J, Linnane S, Mason MD, Tabi Z (2008) Human tumor-derived exosomes down-modulate NKG2D expression. J Immunol 180:7249–7258PubMedGoogle Scholar
  38. 38.
    Wilson EB, El-Jawhari JJ, Neilson AL, Hall GD, Melcher AA, Meade JL, Cook GP (2011) Human tumour immune evasion via TGF-beta blocks nk cell activation but not survival allowing therapeutic restoration of anti-tumour activity. PLoS One 6:e22842PubMedCrossRefGoogle Scholar
  39. 39.
    Waldhauer I, Steinle A (2008) NK cells and cancer immunosurveillance. Oncogene 27:5932–5943PubMedCrossRefGoogle Scholar
  40. 40.
    Coudert JD, Zimmer J, Tomasello E, Cebecauer M, Colonna M, Vivier E, Held W (2005) Altered NKG2D function in NK cells induced by chronic exposure to NKG2D ligand-expressing tumor cells. Blood 106:1711–1717PubMedCrossRefGoogle Scholar
  41. 41.
    Coudert JD, Scarpellino L, Gros F, Vivier E, Held W (2008) Sustained NKG2D engagement induces cross-tolerance of multiple distinct NK cell activation pathways. Blood 111:3571–3578PubMedCrossRefGoogle Scholar
  42. 42.
    Hanaoka N, Jabri B, Dai Z, Ciszewski C, Stevens AM, Yee C, Nakakuma H, Spies T, Groh V (2010) NKG2D initiates caspase-mediated CD3{zeta} degradation and lymphocyte receptor impairments associated with human cancer and autoimmune disease. J Immunol 185:5732–5742PubMedCrossRefGoogle Scholar
  43. 43.
    Roda-Navarro P, Reyburn HT (2009) The traffic of the NKG2D/Dap10 receptor complex during natural killer (NK) cell activation. J Biol Chem 284:16463–16472PubMedCrossRefGoogle Scholar
  44. 44.
    Pietra G, Manzini C, Rivara S, Vitale M, Cantoni C, Petretto A, Balsamo M, Conte R, Benelli R, Minghelli S, Solari N, Gualco M, Queirolo P, Moretta L, Mingari MC (2012) Melanoma cells inhibit natural killer cell function by modulating the expression of activating receptors and cytolytic activity. Cancer Res 72:1407–1415PubMedCrossRefGoogle Scholar
  45. 45.
    Balsamo M, Scordamaglia F, Pietra G, Manzini C, Cantoni C, Boitano M, Queirolo P, Vermi W, Facchetti F, Moretta A, Moretta L, Mingari MC, Vitale M (2009) Melanoma-associated fibroblasts modulate NK cell phenotype and antitumor cytotoxicity. Proc Natl Acad Sci U S A 106:20847–20852PubMedCrossRefGoogle Scholar
  46. 46.
    von Lilienfeld-Toal M, Frank S, Leyendecker C, Feyler S, Jarmin S, Morgan R, Glasmacher A, Marten A, Schmidt-Wolf IG, Brossart P, Cook G (2009) Reduced immune effector cell NKG2D expression and increased levels of soluble NKG2D ligands in multiple myeloma may not be causally linked. Cancer Immunol Immunother 59:829–839CrossRefGoogle Scholar
  47. 47.
    Costello RT, Sivori S, Marcenaro E, Lafage-Pochitaloff M, Mozziconacci MJ, Reviron D, Gastaut JA, Pende D, Olive D, Moretta A (2002) Defective expression and function of natural killer cell-triggering receptors in patients with acute myeloid leukemia. Blood 99:3661–3667PubMedCrossRefGoogle Scholar
  48. 48.
    Fauriat C, Just-Landi S, Mallet F, Arnoulet C, Sainty D, Olive D, Costello RT (2007) Deficient expression of NCR in NK cells from acute myeloid leukemia: evolution during leukemia treatment and impact of leukemia cells in NCRdull phenotype induction. Blood 109:323–330PubMedCrossRefGoogle Scholar
  49. 49.
    Le Maux CB, Moretta A, Vergnon I, Opolon P, Lecluse Y, Grunenwald D, Kubin M, Soria JC, Chouaib S, Mami-Chouaib F (2005) NK cells infiltrating a MHC class I-deficient lung adenocarcinoma display impaired cytotoxic activity toward autologous tumor cells associated with altered NK cell-triggering receptors. J Immunol 175:5790–5798Google Scholar
  50. 50.
    Lee JC, Lee KM, Kim DW, Heo DS (2004) Elevated TGF-beta1 secretion and down-modulation of NKG2D underlies impaired NK cytotoxicity in cancer patients. J Immunol 172:7335–7340PubMedGoogle Scholar
  51. 51.
    Parkhurst MR, Riley JP, Dudley ME, Rosenberg SA (2011) Adoptive transfer of autologous natural killer cells leads to high levels of circulating natural killer cells but does not mediate tumor regression. Clin Cancer Res 17:6287–6297PubMedCrossRefGoogle Scholar
  52. 52.
    Ni J, Miller M, Stojanovic A, Garbi N, Cerwenka A (2012) Sustained effector function of IL-12/15/18-preactivated NK cells against established tumors. J Exp Med 209:2351–2365PubMedCrossRefGoogle Scholar
  53. 53.
    Kono K, Takahashi A, Ichihara F, Sugai H, Fujii H, Matsumoto Y (2002) Impaired antibody-dependent cellular cytotoxicity mediated by herceptin in patients with gastric cancer. Cancer Res 62:5813–5817PubMedGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2013

Authors and Affiliations

  • Jens H. W. Pahl
    • 1
  • S. Eriaty N. Ruslan
    • 1
  • Kitty M. C. Kwappenberg
    • 1
  • Monique M. van Ostaijen-ten Dam
    • 1
  • Maarten J. D. van Tol
    • 1
  • Arjan C. Lankester
    • 1
  • Marco W. Schilham
    • 2
  1. 1.Department of PediatricsLeiden University Medical CentreLeidenThe Netherlands
  2. 2.Department of Pediatrics, Laboratory for Immunology, P3-PLeiden University Medical CentreLeidenThe Netherlands

Personalised recommendations