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Effective immuno-targeting of the IDH1 mutation R132H in a murine model of intracranial glioma.

Pellegatta S, Valletta L, Corbetta C, Patanè M, Zucca I, Riccardi Sirtori F, Bruzzone MG, Fogliatto G, Isacchi A, Pollo B, Finocchiaro G - Acta Neuropathol Commun (2015)

Bottom Line: Presence of the mutation was confirmed by immunoblotting and production of the oncometabolite 2-hydroxyglutarate (2HG), demonstrated by mass spectrometry (LC-MS/MS) performed on cell supernatant.In vitro mIDH1-GL261 had different morphology but similar growth rate than parental GL261 (p-GL261).Immunizations were performed nine days after intracranial implantation of mIDH1- or p-GL261 cells by three subcutaneous injections of five different peptides encompassing the IDH1 mutation site, all emulsified with Montanide ISA-51, in association with GM-CSF.

View Article: PubMed Central - PubMed

ABSTRACT
The R132H mutation of cytosolic isocitrate dehydrogenase (IDH1) is present in the majority of low grade gliomas.Immunotherapy in these tumors has an interesting, still unexploited, therapeutic potential, as they are less immunosuppressive than glioblastomas. Using site-directed mutagenesis we introduced the R132H mutation into the murine glioma cell line GL261,creating mIDH1-GL261. Presence of the mutation was confirmed by immunoblotting and production of the oncometabolite 2-hydroxyglutarate (2HG), demonstrated by mass spectrometry (LC-MS/MS) performed on cell supernatant. In vitro mIDH1-GL261 had different morphology but similar growth rate than parental GL261 (p-GL261). After intracranial injection, MRI suggested that the initial growth rate was slower in mIDH1-GL261 than p-GL261 gliomas but overall survival was similar. mIDH1-GL261 gliomas showed evidence of R132H expression and of intratumoral 2HG production (evaluated by MRS and LC-MS/MS). Immunizations were performed nine days after intracranial implantation of mIDH1- or p-GL261 cells by three subcutaneous injections of five different peptides encompassing the IDH1 mutation site, all emulsified with Montanide ISA-51, in association with GM-CSF. Control mice were injected with four ovalbumin peptides or vehicle. Mice with mIDH1-GL261 but not p-GL261 gliomas treated with mIDH1 peptides survived longer than controls; 25% of them were cured. Immunized mice showed higher amounts of peripheral CD8+ T cells, higher production of IFN-γ, and evidence of anti-mIDH1 antibodies.Immunizations led to intratumoral up-regulation of IFN-γ, granzyme-b and perforin-1 and down-regulation of TGF-β2 and IL-10. These results support the translational potential of immunotherapeutic targeting of gliomas carrying IDH1 mutations.

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Immunization with mutated IDH1 peptides prolongs survival and promotes specific anti-tumor responses. a Experimental schema of in vivo treatments. Two groups or mice injected with mIDH1-GL261 or pGL261 were immunized with three subcutaneous vaccinations on days 9, 16 and 23 after tumor implantation with four peptides (two 9-mer and two 10-mer), or with a single peptide (16-mer) emulsified in Montanide (15 μg/single peptide). A total of 3 μg of GM-CSF/mouse were administered during each immunization. Two group of control mice were treated with GM-CSF and Montanide alone or with ova peptides. On day 19 mice were sacrificed for immune monitoring. b, c Kaplan-Meier curves show that immunized mIDH1-glioma mice (four peptides n = 12; 16-mer peptide n = 12) survived longer than control mice (vehicle, n = 12; ova n = 12). Immunizations of p-GL261 glioma-bearing mice did not modify the survival. d Splenocytes from immunized mice proliferated significantly more than splenocytes from control (vehicle and ova treated) mice in the presence of IDH1-R132H peptides, particularly of peptide 4 (*p < 0.01; **p < 0.005; ***p < 0.001, ****p < 0.0001), not in presence of ova peptides. e Flow cytometry performed on splenocytes shows that CD8+ but not CD4+ T cell percentage increased significantly in immunized mice compared to controls (n = 6 mice/group). No difference between vehicle and ova peptide immunizations. Data are reported in dot plots as the mean% ± SD. f Flow cytometry histograms show that CD3 + CD8+ T cells produced more IFN-γ and expressed less CD62L and more CD49d than controls (vehicle and ova histograms were overlapped). g In vitro MTT cytotoxicity assay reveals that splenocytes from immunized but not from control mice lyse mIDH1-GL261 cells. h Scatter plot shows mIDH1 specific IgG detected in serum from mice immunized with four peptides. Single plots represent the mean of three serum samples/group.
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Fig3: Immunization with mutated IDH1 peptides prolongs survival and promotes specific anti-tumor responses. a Experimental schema of in vivo treatments. Two groups or mice injected with mIDH1-GL261 or pGL261 were immunized with three subcutaneous vaccinations on days 9, 16 and 23 after tumor implantation with four peptides (two 9-mer and two 10-mer), or with a single peptide (16-mer) emulsified in Montanide (15 μg/single peptide). A total of 3 μg of GM-CSF/mouse were administered during each immunization. Two group of control mice were treated with GM-CSF and Montanide alone or with ova peptides. On day 19 mice were sacrificed for immune monitoring. b, c Kaplan-Meier curves show that immunized mIDH1-glioma mice (four peptides n = 12; 16-mer peptide n = 12) survived longer than control mice (vehicle, n = 12; ova n = 12). Immunizations of p-GL261 glioma-bearing mice did not modify the survival. d Splenocytes from immunized mice proliferated significantly more than splenocytes from control (vehicle and ova treated) mice in the presence of IDH1-R132H peptides, particularly of peptide 4 (*p < 0.01; **p < 0.005; ***p < 0.001, ****p < 0.0001), not in presence of ova peptides. e Flow cytometry performed on splenocytes shows that CD8+ but not CD4+ T cell percentage increased significantly in immunized mice compared to controls (n = 6 mice/group). No difference between vehicle and ova peptide immunizations. Data are reported in dot plots as the mean% ± SD. f Flow cytometry histograms show that CD3 + CD8+ T cells produced more IFN-γ and expressed less CD62L and more CD49d than controls (vehicle and ova histograms were overlapped). g In vitro MTT cytotoxicity assay reveals that splenocytes from immunized but not from control mice lyse mIDH1-GL261 cells. h Scatter plot shows mIDH1 specific IgG detected in serum from mice immunized with four peptides. Single plots represent the mean of three serum samples/group.

Mentions: To determine whether IDH1-R132H could be a target for glioma immunotherapy we treated mIDH1-GL261 glioma-bearing mice with four short peptides (two 9-mers and two 10-mers) or one 16-mer peptide, all encompassing the IDH1 region encoding the mutation. Vaccinations with peptides were performed on days 9, 16, and 23 after tumor implantation (Figure 3a). Peptide immunizations significantly increased survival as compared with vehicle-treated and ovalbumin (ova) peptide-treated controls (Figure 3b). Immunization of pGL261-glioma-bearing mice using the same peptides was ineffective (Figure 3c), demonstrating that the immune response induced by peptide vaccines specifically targets mIDH1-expressing cells.Figure 3


Effective immuno-targeting of the IDH1 mutation R132H in a murine model of intracranial glioma.

Pellegatta S, Valletta L, Corbetta C, Patanè M, Zucca I, Riccardi Sirtori F, Bruzzone MG, Fogliatto G, Isacchi A, Pollo B, Finocchiaro G - Acta Neuropathol Commun (2015)

Immunization with mutated IDH1 peptides prolongs survival and promotes specific anti-tumor responses. a Experimental schema of in vivo treatments. Two groups or mice injected with mIDH1-GL261 or pGL261 were immunized with three subcutaneous vaccinations on days 9, 16 and 23 after tumor implantation with four peptides (two 9-mer and two 10-mer), or with a single peptide (16-mer) emulsified in Montanide (15 μg/single peptide). A total of 3 μg of GM-CSF/mouse were administered during each immunization. Two group of control mice were treated with GM-CSF and Montanide alone or with ova peptides. On day 19 mice were sacrificed for immune monitoring. b, c Kaplan-Meier curves show that immunized mIDH1-glioma mice (four peptides n = 12; 16-mer peptide n = 12) survived longer than control mice (vehicle, n = 12; ova n = 12). Immunizations of p-GL261 glioma-bearing mice did not modify the survival. d Splenocytes from immunized mice proliferated significantly more than splenocytes from control (vehicle and ova treated) mice in the presence of IDH1-R132H peptides, particularly of peptide 4 (*p < 0.01; **p < 0.005; ***p < 0.001, ****p < 0.0001), not in presence of ova peptides. e Flow cytometry performed on splenocytes shows that CD8+ but not CD4+ T cell percentage increased significantly in immunized mice compared to controls (n = 6 mice/group). No difference between vehicle and ova peptide immunizations. Data are reported in dot plots as the mean% ± SD. f Flow cytometry histograms show that CD3 + CD8+ T cells produced more IFN-γ and expressed less CD62L and more CD49d than controls (vehicle and ova histograms were overlapped). g In vitro MTT cytotoxicity assay reveals that splenocytes from immunized but not from control mice lyse mIDH1-GL261 cells. h Scatter plot shows mIDH1 specific IgG detected in serum from mice immunized with four peptides. Single plots represent the mean of three serum samples/group.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4359524&req=5

Fig3: Immunization with mutated IDH1 peptides prolongs survival and promotes specific anti-tumor responses. a Experimental schema of in vivo treatments. Two groups or mice injected with mIDH1-GL261 or pGL261 were immunized with three subcutaneous vaccinations on days 9, 16 and 23 after tumor implantation with four peptides (two 9-mer and two 10-mer), or with a single peptide (16-mer) emulsified in Montanide (15 μg/single peptide). A total of 3 μg of GM-CSF/mouse were administered during each immunization. Two group of control mice were treated with GM-CSF and Montanide alone or with ova peptides. On day 19 mice were sacrificed for immune monitoring. b, c Kaplan-Meier curves show that immunized mIDH1-glioma mice (four peptides n = 12; 16-mer peptide n = 12) survived longer than control mice (vehicle, n = 12; ova n = 12). Immunizations of p-GL261 glioma-bearing mice did not modify the survival. d Splenocytes from immunized mice proliferated significantly more than splenocytes from control (vehicle and ova treated) mice in the presence of IDH1-R132H peptides, particularly of peptide 4 (*p < 0.01; **p < 0.005; ***p < 0.001, ****p < 0.0001), not in presence of ova peptides. e Flow cytometry performed on splenocytes shows that CD8+ but not CD4+ T cell percentage increased significantly in immunized mice compared to controls (n = 6 mice/group). No difference between vehicle and ova peptide immunizations. Data are reported in dot plots as the mean% ± SD. f Flow cytometry histograms show that CD3 + CD8+ T cells produced more IFN-γ and expressed less CD62L and more CD49d than controls (vehicle and ova histograms were overlapped). g In vitro MTT cytotoxicity assay reveals that splenocytes from immunized but not from control mice lyse mIDH1-GL261 cells. h Scatter plot shows mIDH1 specific IgG detected in serum from mice immunized with four peptides. Single plots represent the mean of three serum samples/group.
Mentions: To determine whether IDH1-R132H could be a target for glioma immunotherapy we treated mIDH1-GL261 glioma-bearing mice with four short peptides (two 9-mers and two 10-mers) or one 16-mer peptide, all encompassing the IDH1 region encoding the mutation. Vaccinations with peptides were performed on days 9, 16, and 23 after tumor implantation (Figure 3a). Peptide immunizations significantly increased survival as compared with vehicle-treated and ovalbumin (ova) peptide-treated controls (Figure 3b). Immunization of pGL261-glioma-bearing mice using the same peptides was ineffective (Figure 3c), demonstrating that the immune response induced by peptide vaccines specifically targets mIDH1-expressing cells.Figure 3

Bottom Line: Presence of the mutation was confirmed by immunoblotting and production of the oncometabolite 2-hydroxyglutarate (2HG), demonstrated by mass spectrometry (LC-MS/MS) performed on cell supernatant.In vitro mIDH1-GL261 had different morphology but similar growth rate than parental GL261 (p-GL261).Immunizations were performed nine days after intracranial implantation of mIDH1- or p-GL261 cells by three subcutaneous injections of five different peptides encompassing the IDH1 mutation site, all emulsified with Montanide ISA-51, in association with GM-CSF.

View Article: PubMed Central - PubMed

ABSTRACT
The R132H mutation of cytosolic isocitrate dehydrogenase (IDH1) is present in the majority of low grade gliomas.Immunotherapy in these tumors has an interesting, still unexploited, therapeutic potential, as they are less immunosuppressive than glioblastomas. Using site-directed mutagenesis we introduced the R132H mutation into the murine glioma cell line GL261,creating mIDH1-GL261. Presence of the mutation was confirmed by immunoblotting and production of the oncometabolite 2-hydroxyglutarate (2HG), demonstrated by mass spectrometry (LC-MS/MS) performed on cell supernatant. In vitro mIDH1-GL261 had different morphology but similar growth rate than parental GL261 (p-GL261). After intracranial injection, MRI suggested that the initial growth rate was slower in mIDH1-GL261 than p-GL261 gliomas but overall survival was similar. mIDH1-GL261 gliomas showed evidence of R132H expression and of intratumoral 2HG production (evaluated by MRS and LC-MS/MS). Immunizations were performed nine days after intracranial implantation of mIDH1- or p-GL261 cells by three subcutaneous injections of five different peptides encompassing the IDH1 mutation site, all emulsified with Montanide ISA-51, in association with GM-CSF. Control mice were injected with four ovalbumin peptides or vehicle. Mice with mIDH1-GL261 but not p-GL261 gliomas treated with mIDH1 peptides survived longer than controls; 25% of them were cured. Immunized mice showed higher amounts of peripheral CD8+ T cells, higher production of IFN-γ, and evidence of anti-mIDH1 antibodies.Immunizations led to intratumoral up-regulation of IFN-γ, granzyme-b and perforin-1 and down-regulation of TGF-β2 and IL-10. These results support the translational potential of immunotherapeutic targeting of gliomas carrying IDH1 mutations.

Show MeSH
Related in: MedlinePlus