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Histone deacetylase inhibitors enhance expression of NKG2D ligands in Ewing sarcoma and sensitize for natural killer cell-mediated cytolysis
- Background: Ewing sarcoma patients have a poor prognosis despite multimodal therapy. Integration of combination immunotherapeutic strategies into first-/second-line regimens represents promising treatment options, particularly for patients with intrinsic or acquired resistance to conventional therapies. We evaluated the susceptibility of Ewing sarcoma to natural killer cell-based combination immunotherapy, by assessing the capacity of histone deacetylase inhibitors to improve immune recognition and sensitize for natural killer cell cytotoxicity. Methods: Using flow cytometry, ELISA and immunohistochemistry, expression of natural killer cell receptor ligands was assessed in chemotherapy-sensitive/-resistant Ewing sarcoma cell lines, plasma and tumours. Natural killer cell cytotoxicity was evaluated in Chromium release assays. Using ATM/ATR inhibitor caffeine, the contribution of the DNA damage response pathway to histone deacetylase inhibitor-induced ligand expression was assessed. Results: Despite comparable expression of natural killer cell receptor ligands, chemotherapy-resistant Ewing sarcoma exhibited reduced susceptibility to resting natural killer cells. Interleukin-15-activation of natural killer cells overcame this reduced sensitivity. Histone deacetylase inhibitor-pretreatment induced NKG2D-ligand expression in an ATM/ATR-dependent manner and sensitized for NKG2D-dependent cytotoxicity (2/4 cell lines). NKG2D-ligands were expressed in vivo, regardless of chemotherapy-response and disease stage. Soluble NKG2D-ligand plasma concentrations did not differ between patients and controls. Conclusion: Our data provide a rationale for combination immunotherapy involving immune effector and target cell manipulation in first-/second-line treatment regimens for Ewing sarcoma.
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Antibodies used for flow cytometry, NK cell receptor (ligand) blocking, immunohistochemistry and ELISA.
A. Constitutive surface expression of inhibitory (HLA class I) or activating (MICA/B, ULBP1-3, CD112, CD155) natural killer cell receptor ligands in chemotherapy-sensitive (grey) and -resistant (black) Ewing sarcoma cell lines, as assessed by flow cytometry. Results are expressed as the mean ± SD MFI-ratio, obtained in at least two independent experiments. Statistical analysis (t-test) was performed on mean MFI-ratio's (for each ligand) of chemotherapy-sensitive versus -resistant cell lines, revealing no significant differences in expression levels of these ligands (p > 0.05). B. Cytotoxic activity of resting natural killer cells was evaluated in 51Cr release assays using chemotherapy-sensitive (TC71 (blue), SK-ES-1 (purple), SK-N-MC (pink)) and -resistant (STA-ET-2.1 (black), CADO-ES (dark grey), IOR/BER (light grey)) Ewing sarcoma cell lines as target cells. Ewing sarcoma cells were either left untreated (solid bars) or pre-incubated with HLA class I blocking antibody DX17 (checked bars). Results are expressed as the mean ± SD percentage of specific lysis obtained in at least two independent experiments using different healthy donors.
Cytotoxic activity of IL-15-activated natural killer cells was evaluated in 51Cr release assays using chemotherapy-sensitive (TC71 (blue), SK-ES-1 (purple)) and -resistant (STA-ET-2.1 (black), CADO-ES (grey) Ewing sarcoma cell lines as target cells. Ewing sarcoma cells were either left untreated (solid bars) or pre-incubated with NKG2D and DNAM-1 blocking antibodies (checked bars). Results are expressed as the mean ± SD percentage of specific lysis obtained in at least two independent experiments using different healthy donors.
A. Cytotoxicity of resting natural killer cells was evaluated in 51Cr release assays using MS-275-pretreated TC71 and STA-ET2.1 cells. Despite induction of activating NKG2D ligands, no sensitization for natural killer cell cytotoxicity was detectable (at doses up to 1/5 of IC50 value). Similar results were observed for both cell lines upon pre-treatment with NaB and SAHA (not shown). Results are expressed as the mean ± SEM percentages of specific lysis obtained in at least two independent experiments using different healthy donors. B. Upon HDI-pretreatment, persistent dependency of resting natural killer cell-mediated cytotoxicity on signaling via activating receptor NKG2D was demonstrated when 51Cr release assays were performed in the presence of a blocking antibody against NKG2D. Blocking reduced resting natural killer cell-mediated lysis of both untreated and HDI pre-treated cells to similar levels, as demonstrated for CADO-ES upon pre-treatment with NaB. Similar results were obtained for CADO-ES with MS-275 and SAHA, as well as for SK-ES-1 with SAHA (not shown). K562 and EBV B-LCL cell line 107 were used as positive and negative control respectively (not shown). Results are expressed as the mean ± SEM percentages of specific lysis obtained in at least two independent experiments using different healthy donors.
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| Author: | Dagmar Berghuis, Marco Willem Schilham, Hanneke I. Vos, Susy J. Santos, Stephan KlößGND, Emilie P. Buddingh', R. Maarten Egeler, Pancras Cornelis Wilhelmus Hogendoorn, Arjan C. Lankester |
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| URN: | urn:nbn:de:hebis:30:3-240683 |
| DOI: | https://doi.org/10.1186/2045-3329-2-8 |
| ISSN: | 2045-3329 |
| Parent Title (English): | Clinical Sarcoma Research |
| Publisher: | BioMed Central |
| Place of publication: | London |
| Document Type: | Article |
| Language: | English |
| Date of Publication (online): | 2012/03/12 |
| Date of first Publication: | 2012/02/08 |
| Publishing Institution: | Universitätsbibliothek Johann Christian Senckenberg |
| Release Date: | 2012/03/12 |
| Tag: | Ewing sarcoma; chemotherapy-resistance; combination immunotherapy; histone deacetylase inhibitor; natural killer cells; tumour immunology |
| Volume: | 2 |
| Issue: | 8 |
| Page Number: | 13 |
| HeBIS-PPN: | 310993156 |
| Institutes: | Medizin / Medizin |
| Dewey Decimal Classification: | 6 Technik, Medizin, angewandte Wissenschaften / 61 Medizin und Gesundheit / 610 Medizin und Gesundheit |
| Sammlungen: | Universitätspublikationen |
| Licence (German): |  Creative Commons - Namensnennung 3.0 |
