Chemo-Resistant Ewing Sarcoma Is Susceptible to Natural Killer Cell-Mediated Cytotoxicity: Implications for Immunotherapy
D Berghuis, DHJ Verhoeven, SJ Santos, EP Buddingh, KL Schaefer, K Scotlandi, RM Egeler, MW Schilham, PCW Hogendoorn, AC Lankester. Leiden University Medical Center, Leiden, Netherlands; Heinrich-Heine University, Dsseldorf, Germany; Rizzoli Orthopaedic Institute, Bologna, Italy
Background: Despite multimodal therapy, patients with refractory or relapsed Ewing sarcoma (EWS) have poor prognoses. To explore the feasibility of natural killer (NK) cell-mediated immunotherapy for patients with advanced-stage EWS, we investigated whether susceptibility of EWS to NK cell-mediated cytolysis is affected by chemo-sensitivity and identified pivotal molecular mechanisms involved in NK cell-mediated cytolysis.
Design: Expression of ligands for inhibitory and activating NK cell receptors was evaluated in chemo-resistant and -sensitive EWS cell lines (n=10) by flow cytometry. Cytotoxicity was determined in chromium release assays, using freshly isolated (resting) and interleukin (IL)-15 activated NK cells obtained from healthy donors. Blocking antibodies against specific ligands/receptors were used to study contribution of these molecules.
Results: All EWS cell lines were lysed by resting NK cells, regardless of chemo-sensitivity, except for one chemo-resistant cell line (CADO-ES). Ligands for the activating NK cell receptors DNAM-1 and NKG2D were expressed by all cell lines, though in heterogeneous patterns. Cytotoxicity depended on these receptors, since blocking either of these receptors abrogated cytolysis by resting NK cells. IL-15 activation of NK cells increased efficacy of lysis in all cell lines, including CADO-ES, and resulted in more efficient recognition of EWS cells, since only combined DNAM-1/NKG2D-blockade inhibited lysis. CADO-ES, resistant to lysis by resting NK cells, is characterized by high levels of HLA class I expression compared to other EWS cell lines; in addition, the HLA class I alleles expressed by CADO-ES are ligands for all inhibitory NK cell receptors (KIR). In this cell line, cytolysis by resting NK cells depended on loss of inhibition, since blocking antibodies against HLA class I reversed resistance. Induction or blockade of HLA class I did not significantly affect lysis in all other cell lines.
Conclusions: The observed susceptibility of chemo-resistant Ewing sarcoma to cytolysis by cytokine-activated NK cells may provide patients with advanced-stage Ewing sarcoma with an additional treatment modality.
Category: Bone & Soft Tissue
Monday, March 9, 2009 1:00 PM
Poster Session II # 11, Monday Afternoon