Elsevier

Molecular Immunology

Volume 45, Issue 15, September 2008, Pages 3917-3925
Molecular Immunology

NK cells recognize and lyse Ewing sarcoma cells through NKG2D and DNAM-1 receptor dependent pathways

https://doi.org/10.1016/j.molimm.2008.06.016Get rights and content

Abstract

Introduction

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.

Methods and 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.

Conclusion

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.

Introduction

Ewing sarcoma (EWS) is the second most common malignant bone tumor occurring in children and young adults. Most patients suffering from EWS have a medullary-centered osseous localization, but a minority of patients presents with a primary soft tissue localization. EWS tumor cells typically bear the CD99 marker on their cell surface, and, in 95% of the cases, the tumor is characterized by EWS/FLI-1 or EWS/ERG gene fusion (Burchill, 2003, Delattre et al., 1994). Treatment of EWS consists of chemotherapy and surgical resection, sometimes in combination with radiotherapy. This treatment strategy results in an overall survival rate of 60–65% in localized disease. However, approximately 25% of children with EWS have detectable metastases at the time of diagnosis, and these patients have a poor prognosis with a 5-year relapse free survival of less than 30%. In case of relapse, this prognosis may even be worse (Cotterill et al., 2000, Bernstein et al., 2006). Since the prognosis of patients presenting with metastasized or relapsed Ewing sarcoma is poor despite current chemotherapeutic and surgical treatment, immunotherapy may provide an additional or adjuvant treatment modality. Besides dendritic cells (DC) and T cells, NK cells may serve as effector or mediator cells of an anti-tumor immune response.

Natural killer (NK) cells are bone marrow derived lymphocytes, which do not bear clonally rearranged antigen-specific receptors. They are cytotoxic against tumor cells and virus infected cells. Activity of NK cells is controlled by a balance between inhibitory and activating signals (Carayannopoulos and Yokoyama, 2004, Moretta et al., 2006). Inhibitory signals are provided by classical as well as non-classical human leukocyte antigen (HLA) class I molecules, which either bind to killer immunoglobulin-like receptors (KIR) or NKG2A/CD94 on NK cells (Parham, 2005, Moretta et al., 2006). These inhibitory signals are counteracted by activating signals, provided by several receptor–ligand combinations. The best characterized activating NK cell receptor is NKG2D, a homodimer with a C-type lectin-like extracellular domain. The activating signal is transduced through recruitment of the adaptor molecule DAP10 and the subsequent activation of the phosphatidylinositol 3-kinase (PI3-K) pathway. NKG2D binds the MIC (MICA/B) and ULBP (ULBP1-4) family of proteins (Raulet, 2003, Hayakawa and Smyth, 2006). These ligands are expressed on tumor cells and virus infected cells and can amongst others be induced by DNA damage (Gasser and Raulet, 2006).

DNAX accessory molecule-1 (DNAM-1, CD226) is an adhesion receptor with two Ig-like domains in the extracellular part, which is expressed on the majority of NK cells, T cells and monocytes (Shibuya et al., 1996). It is associated with LFA-1 and transfers an activating signal through Fyn mediated tyrosine phosphorylation and protein kinase C (PKC) mediated serine phosphorylation of the cytoplasmic domain of DNAM-1 (Shibuya et al., 1998, Shibuya et al., 1999). This activating receptor recognizes the poliovirus receptor (PVR, CD155) and Nectin-2 (CD112) (Bottino et al., 2003). These two members of the Nectin family are not only expressed on healthy neuronal, epithelial, endothelial cells and fibroblasts, but they are also highly expressed on several tumor cells (Campadelli-Fiume et al., 2000). Although the precise role of DNAM-1 in NK and T cell biology is still under investigation, NK cell mediated lysis of neuroblastoma cells and ovarian carcinoma cells was shown to be dependent on the interaction of DNAM-1 with its ligands (Castriconi et al., 2004, Carlsten et al., 2007).

Other activating receptors include the natural cytotoxicity receptors (NCRs): NKp30 and NKp46, which are expressed on all NK cells; NKp44, which can be detected on cytokine-activated NK cells. The ligands for these receptors are largely unknown, although HLA-B-associated transcript 3 (BAT3) was reported to be an activating ligand for NKp30 (Pogge von Strandmann et al., 2007) and influenza hemagglutinin has been demonstrated to be a ligand for NKp46 (Moretta et al., 2006).

We report here that EWS tumor cells are recognized and lysed by resting and with higher efficacy by activated NK cells through NKG2D and DNAM-1 dependent pathways. Furthermore, we report that NK cell cytotoxicity is impaired in patients with EWS by a yet unknown mechanism.

Section snippets

Patients

During 2004–2006, 11 children and adolescents with newly diagnosed Ewing sarcoma with an osseous localization (age average 15.0 years, range 4.9–20.5 years) were enrolled in this study at the Leiden University Medical Center (Departments of Pediatrics and Clinical Oncology) after informed written consent. The study was approved by the Institutional Review Board on Medical Ethics. Peripheral blood mononuclear cells (PBMC) were isolated from blood collected prior to start of chemotherapy by

Ewing sarcoma cell lines are susceptible to lysis by resting and activated NK cells

Resting NK cells have cytotoxic activity to a limited number of human tumor cell lines. To evaluate the sensitivity of Ewing sarcoma to NK cell mediated lysis, chromium release assays were performed using EWS cell lines as targets. As illustrated in Fig. 1A, EWS cells were lysed by freshly isolated resting NK cells obtained from healthy donors. Activation of NK cells with recombinant human IL-15 increased the efficacy of lysis of Ewing sarcoma tumor cells by NK cells (Fig. 1B). Consistent

Discussion and conclusions

In this preclinical study, we report that Ewing sarcoma (EWS) cells were recognized and lysed by resting and, with higher efficacy, by IL-15-activated NK cells from healthy donors. The lysis of EWS by NK cells was dependent on the activating NK cell receptors NKG2D and DNAM-1, whose ligands are commonly expressed by EWS cell lines and primary EWS tumor cells. At diagnosis and prior to chemotherapy, patients suffering from Ewing sarcoma display reduced NK cell cytotoxicity despite normal numbers

Acknowledgements

The authors wish to thank Salvatore Romeo and Ekatherina Jordanova (Department of Pathology, LUMC, Leiden) for assistance with immunohistochemistry.

This research is funded by grants from the Dutch Pediatric Cancer Foundation KiKa (to RME, MJDvT), The Netherlands Organization for Health Research and Development (ZonMw, grant 920-03-267; to DHJV), the Leiden University Medical Center (to DHJV) and the Dutch Cancer Society NKB/KWF (grant no. RUL 2003-2800; to ACL). The Departments of Pediatrics

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