title: NK cells recognize and lyse Ewing sarcoma cells through NKG2D and DNAM-1 receptor dependent pathways.
abstract: BACKGROUND
  Ewing sarcoma (EWS) is a malignant bone-associated sarcoma, with poor prognosis in case of metastasis or relapse. To explore the feasibility of natural killer (NK) cell mediated immunotherapy and to identify molecular mechanisms involved, the susceptibility of EWS to NK cells was investigated.
  
  
  RESULTS
  All EWS cell lines tested (n=7) were lysed by purified allogeneic NK cells from healthy donors, and the efficacy of lysis was increased by activating NK cells with interleukin-15 (IL-15). FACS analysis and immunohistochemistry revealed that EWS cell lines as well as primary tumor cells expressed ligands for the activating NK cell receptors NKG2D and DNAM-1. NK cell cytotoxicity to EWS cells critically depended on the combination of NKG2D and DNAM-1 signaling, since blocking either of these receptors abrogated lysis by resting NK cells. Cytokine-activated NK cells more efficiently recognized EWS cells, since only combined, but not single blockade of NKG2D and DNAM-1 by antibodies inhibited lysis of EWS cells. Induction or blockade of HLA class I on EWS cells did not significantly influence lysis. This suggests that predominantly activating, rather than inhibitory signals on EWS cells determined susceptibility to NK cell cytotoxicity. NK cell cytotoxicity to EWS cells and K562 was reduced in EWS patients at diagnosis (n=11) compared to age matched controls, despite normal NK cell numbers and increased expression of NKG2D. The impaired function of these NK cells was restored after activation with IL-15 in vitro.
  
  
  CONCLUSIONS
  These results demonstrate that EWS cells are potentially susceptible to NK cell cytotoxicity due to the expression of activating NK cell receptor ligands. The use of cytokine-activated NK cells rather than resting NK cells in immunotherapy may be instrumental to optimize NK cell reactivity to EWS.
authors.affiliation: Division of Immunology, Hematology, Oncology, Bone Marrow Transplantation and Autoimmune Diseases, Department of Pediatrics, Leiden University Medical Center, PO Box 9600, 2300 RC Leiden, The Netherlands.
authors.forename: Dirk H J
authors.initials: DH
authors.lastname: Verhoeven
authors.forename: Alfons S K
authors.initials: AS
authors.lastname: de Hooge
authors.forename: Esther C K
authors.initials: EC
authors.lastname: Mooiman
authors.forename: Susy Justo
authors.initials: SJ
authors.lastname: Santos
authors.forename: Monique M
authors.initials: MM
authors.lastname: ten Dam
authors.forename: Hans
authors.initials: H
authors.lastname: Gelderblom
authors.forename: Cornelis J M
authors.initials: CJ
authors.lastname: Melief
authors.forename: Pancras C W
authors.initials: PC
authors.lastname: Hogendoorn
authors.forename: R Maarten
authors.initials: RM
authors.lastname: Egeler
authors.forename: Maarten J D
authors.initials: MJ
authors.lastname: van Tol
authors.forename: Marco W
authors.initials: MW
authors.lastname: Schilham
authors.forename: Arjan C
authors.initials: AC
authors.lastname: Lankester
mesh_terms: D000293:Adolescent; D000945:Antigens, Differentiation, T-Lymphocyte / Q000276:immunology*; D001859:Bone Neoplasms / Q000276:immunology*; D045744:Cell Line, Tumor; D002648:Child; D002675:Child, Preschool; D016207:Cytokines / Q000276:immunology / Q000494:pharmacology; D003602:Cytotoxicity, Immunologic; D006801:Humans; D007694:Killer Cells, Natural / Q000276:immunology*; D008213:Lymphocyte Activation; D055655:NK Cell Lectin-Like Receptor Subfamily K / Q000276:immunology*; D012512:Sarcoma, Ewing / Q000276:immunology*; D055815:Young Adult; D000098862:T Lineage-Specific Activation Antigen 1
chemical_list: D000945:Antigens, Differentiation, T-Lymphocyte; D000098862:T Lineage-Specific Activation Antigen 1; D016207:Cytokines; C527085:KLRK1 protein, human; D055655:NK Cell Lectin-Like Receptor Subfamily K