Figure 3

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.