Limits...
Histone deacetylase inhibitors enhance expression of NKG2D ligands in Ewing sarcoma and sensitize for natural killer cell-mediated cytolysis.

Berghuis D, Schilham MW, Vos HI, Santos SJ, Kloess S, Buddingh' EP, Egeler RM, Hogendoorn PC, Lankester AC - Clin Sarcoma Res (2012)

Bottom Line: Interleukin-15-activation of natural killer cells overcame this reduced sensitivity.Soluble NKG2D-ligand plasma concentrations did not differ between patients and controls.Our data provide a rationale for combination immunotherapy involving immune effector and target cell manipulation in first-/second-line treatment regimens for Ewing sarcoma.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Pediatrics, Leiden University Medical Center, Leiden, The Netherlands. a.lankester@lumc.nl.

ABSTRACT

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.

No MeSH data available.


Related in: MedlinePlus

Histone deacetylase inhibitor-induced, ATM/ATR-dependent NKG2D ligand expression sensitizes Ewing sarcoma for natural killer cell cytotoxicity. A-C. Heterogeneous induction of activating NKG2D ligands and/or inhibitory HLA class I molecules in chemotherapy-sensitive (grey) and -resistant (black) cell lines upon pre-treatment with HDI NaB (A), MS-275 (B) and SAHA (C), as assessed by flow cytometry. Results are expressed as mean ± SEM fold increase in MFI-ratio over medium control, obtained in at least three independent experiments. [HDI] = 1/5 IC50 value, except for CADO-ES (NaB 1/20 IC50 value; MS-275 and SAHA 1/10 IC50 value). D. Representative examples of flow cytometry plots for MICB for SK-ES-1 and CADO-ES upon pre-treatment with HDI MS-275 (IC50 values); untreated cells in grey. E. Pre-treatment with caffeine (5 mM) for 2 hours prior to incubation with HDI MS-275 (1/5 of IC50 value) largely abolished HDI-mediated MICB expression (similar results were observed for MICA and ULBP2-3), as assessed by flow cytometry. No such effects were observed for HLA class I expression. Results are expressed as the mean ± SEM fold increase in MFI-ratio over medium control, and are representative of at least two independent experiments. Similar results were obtained for NaB and SAHA (not shown). F. Cytotoxicity of resting natural killer cells was evaluated in 51Cr release assays. Pre-treatment of CADO-ES cells with NaB (1/20 and 1/10 IC50 value) sensitized, in a dose-dependent manner, for natural killer cell cytotoxicity. Statistical analysis (paired t-test) revealed significantly increased sensitivity of NaB pre-treated cells at effector-to-target ratio's > 5:1 (p < 0.05). Similar results were observed for CADO-ES with MS-275 and SAHA, and for SK-ES-1 with SAHA (not shown).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3351702&req=5

Figure 4: Histone deacetylase inhibitor-induced, ATM/ATR-dependent NKG2D ligand expression sensitizes Ewing sarcoma for natural killer cell cytotoxicity. A-C. Heterogeneous induction of activating NKG2D ligands and/or inhibitory HLA class I molecules in chemotherapy-sensitive (grey) and -resistant (black) cell lines upon pre-treatment with HDI NaB (A), MS-275 (B) and SAHA (C), as assessed by flow cytometry. Results are expressed as mean ± SEM fold increase in MFI-ratio over medium control, obtained in at least three independent experiments. [HDI] = 1/5 IC50 value, except for CADO-ES (NaB 1/20 IC50 value; MS-275 and SAHA 1/10 IC50 value). D. Representative examples of flow cytometry plots for MICB for SK-ES-1 and CADO-ES upon pre-treatment with HDI MS-275 (IC50 values); untreated cells in grey. E. Pre-treatment with caffeine (5 mM) for 2 hours prior to incubation with HDI MS-275 (1/5 of IC50 value) largely abolished HDI-mediated MICB expression (similar results were observed for MICA and ULBP2-3), as assessed by flow cytometry. No such effects were observed for HLA class I expression. Results are expressed as the mean ± SEM fold increase in MFI-ratio over medium control, and are representative of at least two independent experiments. Similar results were obtained for NaB and SAHA (not shown). F. Cytotoxicity of resting natural killer cells was evaluated in 51Cr release assays. Pre-treatment of CADO-ES cells with NaB (1/20 and 1/10 IC50 value) sensitized, in a dose-dependent manner, for natural killer cell cytotoxicity. Statistical analysis (paired t-test) revealed significantly increased sensitivity of NaB pre-treated cells at effector-to-target ratio's > 5:1 (p < 0.05). Similar results were observed for CADO-ES with MS-275 and SAHA, and for SK-ES-1 with SAHA (not shown).

Mentions: Flow cytometric evaluation of natural killer cell receptor ligand expression upon 24-hour pre-treatment of the cell lines with defined (IC50-related) concentrations of HDI revealed heterogeneous but consistent induction of several activating NKG2D ligands, in particular MICB, in all cell lines evaluated. The most pronounced effects were observed upon pre-treatment with NaB and MS-275, resulting in up to five-fold induction of MICB (Figure 4A-D). Expression of activating DNAM1 ligands (CD112 and CD155) remained largely unchanged. Induction of HLA class I expression was detectable in cell lines STA-ET2.1 and TC71, whereas in CADO-ES, with relatively high levels of constitutive HLA class I expression (Figure 2A), no induction was observed. Induction of HLA class I expression was demonstrated in the SK-ES-1 cell line as well. However, since constitutive HLA class I expression was hardly detectable in this cell line (Figure 2A), the observed less than two-fold induction by histone deacetylase inhibitors still resulted in marginal HLA class I expression (Figure 4A-C).


Histone deacetylase inhibitors enhance expression of NKG2D ligands in Ewing sarcoma and sensitize for natural killer cell-mediated cytolysis.

Berghuis D, Schilham MW, Vos HI, Santos SJ, Kloess S, Buddingh' EP, Egeler RM, Hogendoorn PC, Lankester AC - Clin Sarcoma Res (2012)

Histone deacetylase inhibitor-induced, ATM/ATR-dependent NKG2D ligand expression sensitizes Ewing sarcoma for natural killer cell cytotoxicity. A-C. Heterogeneous induction of activating NKG2D ligands and/or inhibitory HLA class I molecules in chemotherapy-sensitive (grey) and -resistant (black) cell lines upon pre-treatment with HDI NaB (A), MS-275 (B) and SAHA (C), as assessed by flow cytometry. Results are expressed as mean ± SEM fold increase in MFI-ratio over medium control, obtained in at least three independent experiments. [HDI] = 1/5 IC50 value, except for CADO-ES (NaB 1/20 IC50 value; MS-275 and SAHA 1/10 IC50 value). D. Representative examples of flow cytometry plots for MICB for SK-ES-1 and CADO-ES upon pre-treatment with HDI MS-275 (IC50 values); untreated cells in grey. E. Pre-treatment with caffeine (5 mM) for 2 hours prior to incubation with HDI MS-275 (1/5 of IC50 value) largely abolished HDI-mediated MICB expression (similar results were observed for MICA and ULBP2-3), as assessed by flow cytometry. No such effects were observed for HLA class I expression. Results are expressed as the mean ± SEM fold increase in MFI-ratio over medium control, and are representative of at least two independent experiments. Similar results were obtained for NaB and SAHA (not shown). F. Cytotoxicity of resting natural killer cells was evaluated in 51Cr release assays. Pre-treatment of CADO-ES cells with NaB (1/20 and 1/10 IC50 value) sensitized, in a dose-dependent manner, for natural killer cell cytotoxicity. Statistical analysis (paired t-test) revealed significantly increased sensitivity of NaB pre-treated cells at effector-to-target ratio's > 5:1 (p < 0.05). Similar results were observed for CADO-ES with MS-275 and SAHA, and for SK-ES-1 with SAHA (not shown).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3351702&req=5

Figure 4: Histone deacetylase inhibitor-induced, ATM/ATR-dependent NKG2D ligand expression sensitizes Ewing sarcoma for natural killer cell cytotoxicity. A-C. Heterogeneous induction of activating NKG2D ligands and/or inhibitory HLA class I molecules in chemotherapy-sensitive (grey) and -resistant (black) cell lines upon pre-treatment with HDI NaB (A), MS-275 (B) and SAHA (C), as assessed by flow cytometry. Results are expressed as mean ± SEM fold increase in MFI-ratio over medium control, obtained in at least three independent experiments. [HDI] = 1/5 IC50 value, except for CADO-ES (NaB 1/20 IC50 value; MS-275 and SAHA 1/10 IC50 value). D. Representative examples of flow cytometry plots for MICB for SK-ES-1 and CADO-ES upon pre-treatment with HDI MS-275 (IC50 values); untreated cells in grey. E. Pre-treatment with caffeine (5 mM) for 2 hours prior to incubation with HDI MS-275 (1/5 of IC50 value) largely abolished HDI-mediated MICB expression (similar results were observed for MICA and ULBP2-3), as assessed by flow cytometry. No such effects were observed for HLA class I expression. Results are expressed as the mean ± SEM fold increase in MFI-ratio over medium control, and are representative of at least two independent experiments. Similar results were obtained for NaB and SAHA (not shown). F. Cytotoxicity of resting natural killer cells was evaluated in 51Cr release assays. Pre-treatment of CADO-ES cells with NaB (1/20 and 1/10 IC50 value) sensitized, in a dose-dependent manner, for natural killer cell cytotoxicity. Statistical analysis (paired t-test) revealed significantly increased sensitivity of NaB pre-treated cells at effector-to-target ratio's > 5:1 (p < 0.05). Similar results were observed for CADO-ES with MS-275 and SAHA, and for SK-ES-1 with SAHA (not shown).
Mentions: Flow cytometric evaluation of natural killer cell receptor ligand expression upon 24-hour pre-treatment of the cell lines with defined (IC50-related) concentrations of HDI revealed heterogeneous but consistent induction of several activating NKG2D ligands, in particular MICB, in all cell lines evaluated. The most pronounced effects were observed upon pre-treatment with NaB and MS-275, resulting in up to five-fold induction of MICB (Figure 4A-D). Expression of activating DNAM1 ligands (CD112 and CD155) remained largely unchanged. Induction of HLA class I expression was detectable in cell lines STA-ET2.1 and TC71, whereas in CADO-ES, with relatively high levels of constitutive HLA class I expression (Figure 2A), no induction was observed. Induction of HLA class I expression was demonstrated in the SK-ES-1 cell line as well. However, since constitutive HLA class I expression was hardly detectable in this cell line (Figure 2A), the observed less than two-fold induction by histone deacetylase inhibitors still resulted in marginal HLA class I expression (Figure 4A-C).

Bottom Line: Interleukin-15-activation of natural killer cells overcame this reduced sensitivity.Soluble NKG2D-ligand plasma concentrations did not differ between patients and controls.Our data provide a rationale for combination immunotherapy involving immune effector and target cell manipulation in first-/second-line treatment regimens for Ewing sarcoma.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Pediatrics, Leiden University Medical Center, Leiden, The Netherlands. a.lankester@lumc.nl.

ABSTRACT

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.

No MeSH data available.


Related in: MedlinePlus