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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

Activation of natural killer cells with interleukin-15 restores specific cytolysis of chemotherapy-resistant Ewing sarcoma cells to levels similar to those observed for chemotherapy-sensitive cells. Cytotoxic activity of IL-15-activated natural killer cells was evaluated in 51Cr release assays using chemotherapy-sensitive (grey) and -resistant (black) Ewing sarcoma cell lines TC71 (grey dots), SK-ES-1 (grey squares) and SK-N-MC (grey triangles) respectively STA-ET2.1 (black squares), CADO-ES (black dots) and IOR/BER (black triangles) as target cells. K562 and EBV B-LCL cell line 107 were used as positive and negative control respectively. Results are expressed as the mean ± SEM percentages of specific lysis obtained in at least four independent experiments using different healthy donors.
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Figure 3: Activation of natural killer cells with interleukin-15 restores specific cytolysis of chemotherapy-resistant Ewing sarcoma cells to levels similar to those observed for chemotherapy-sensitive cells. Cytotoxic activity of IL-15-activated natural killer cells was evaluated in 51Cr release assays using chemotherapy-sensitive (grey) and -resistant (black) Ewing sarcoma cell lines TC71 (grey dots), SK-ES-1 (grey squares) and SK-N-MC (grey triangles) respectively STA-ET2.1 (black squares), CADO-ES (black dots) and IOR/BER (black triangles) as target cells. K562 and EBV B-LCL cell line 107 were used as positive and negative control respectively. Results are expressed as the mean ± SEM percentages of specific lysis obtained in at least four independent experiments using different healthy donors.

Mentions: Pre-activation of natural killer cells by either recombinant human IL-15 or co-culture with genetically modified IL-15/4-1BBL expressing K562 feeder cells results in more efficient recognition and lysis of Ewing sarcoma cells [20,23]. Therefore, we evaluated whether the reduced sensitivity of chemotherapy-resistant Ewing sarcoma cell lines to resting natural killer cells could be restored by using IL-15-activated natural killer cells (including cells obtained from donors providing resting natural killer cells). As illustrated in Figure 3, activation of natural killer cells with IL-15 increased specific cytolysis of chemotherapy-resistant Ewing sarcoma to levels similar to those observed for chemotherapy-sensitive cells. Blocking studies using antibodies against NKG2D and DNAM1 revealed comparable contributions of signals provided by these activating natural killer cell receptors to lysis of both chemotherapy-sensitive and -resistant cells (as exemplified for TC71, SK-ES-1, STA-ET2.1 and CADO-ES in additional file 3, figure S2). Together, these data indicate that pre-activation of natural killer cells with IL-15 can overcome resistance of chemotherapy-resistant Ewing sarcoma to natural killer cell-mediated cytolysis.


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)

Activation of natural killer cells with interleukin-15 restores specific cytolysis of chemotherapy-resistant Ewing sarcoma cells to levels similar to those observed for chemotherapy-sensitive cells. Cytotoxic activity of IL-15-activated natural killer cells was evaluated in 51Cr release assays using chemotherapy-sensitive (grey) and -resistant (black) Ewing sarcoma cell lines TC71 (grey dots), SK-ES-1 (grey squares) and SK-N-MC (grey triangles) respectively STA-ET2.1 (black squares), CADO-ES (black dots) and IOR/BER (black triangles) as target cells. K562 and EBV B-LCL cell line 107 were used as positive and negative control respectively. Results are expressed as the mean ± SEM percentages of specific lysis obtained in at least four independent experiments using different healthy donors.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 3: Activation of natural killer cells with interleukin-15 restores specific cytolysis of chemotherapy-resistant Ewing sarcoma cells to levels similar to those observed for chemotherapy-sensitive cells. Cytotoxic activity of IL-15-activated natural killer cells was evaluated in 51Cr release assays using chemotherapy-sensitive (grey) and -resistant (black) Ewing sarcoma cell lines TC71 (grey dots), SK-ES-1 (grey squares) and SK-N-MC (grey triangles) respectively STA-ET2.1 (black squares), CADO-ES (black dots) and IOR/BER (black triangles) as target cells. K562 and EBV B-LCL cell line 107 were used as positive and negative control respectively. Results are expressed as the mean ± SEM percentages of specific lysis obtained in at least four independent experiments using different healthy donors.
Mentions: Pre-activation of natural killer cells by either recombinant human IL-15 or co-culture with genetically modified IL-15/4-1BBL expressing K562 feeder cells results in more efficient recognition and lysis of Ewing sarcoma cells [20,23]. Therefore, we evaluated whether the reduced sensitivity of chemotherapy-resistant Ewing sarcoma cell lines to resting natural killer cells could be restored by using IL-15-activated natural killer cells (including cells obtained from donors providing resting natural killer cells). As illustrated in Figure 3, activation of natural killer cells with IL-15 increased specific cytolysis of chemotherapy-resistant Ewing sarcoma to levels similar to those observed for chemotherapy-sensitive cells. Blocking studies using antibodies against NKG2D and DNAM1 revealed comparable contributions of signals provided by these activating natural killer cell receptors to lysis of both chemotherapy-sensitive and -resistant cells (as exemplified for TC71, SK-ES-1, STA-ET2.1 and CADO-ES in additional file 3, figure S2). Together, these data indicate that pre-activation of natural killer cells with IL-15 can overcome resistance of chemotherapy-resistant Ewing sarcoma to natural killer cell-mediated cytolysis.

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