Figure 1.

Generation and validation of the new dTG glioma model. (A) Schematic description of the genetic background of the dTG mouse strain and the workflow to generate the dTG glioma model. The transgenic mouse strain used in the current study lacks expression of mouse MHC class I (${\displaystyle \beta }$2 m) and class II (H-2 IA${\displaystyle \beta }$^b) molecules. However, all nucleated cells (both immune cells and tumor cells) can express the human HLA-A*0201 and the human HLA-DR1*0101. (B) Immunofluorescent staining of DTG- IDH1^R132H cells. Nuclei were visualized with DAPI (blue); intracellular IDH1^R132H protein in yellow (bottom image); isotype control for primary antibody (top image; scale bar, 50 µm). (C) Cell surface expression of HLA-A2 (left panel) and HLA-DR1 (right) on IFNɣ treatment. DTG-R132H cells were cultured in the presence of 50 ng/mL IFNɣ (or solvent only media) for 72 hours and analyzed by FC. (D) Extracellular 2-HG produced by dTG-IDH1^R132H cells in vitro. Within a 6-well plate, 1.5×10⁵ normal human astrocytes (WT astrocytes) and 1.5×10⁵ dTG-IDH1^R132H cells were plated in 2 mL of media and cultured for 72 hours. Levels of 2-HG in the supernatant of each cell line were measured by LC-MS/MS. Data represent technical triplicates of each condition. (E) High levels of 2-HG produced by orthotopically inoculated dTG-IDH1^R132H cells. Tumors were collected from mice injected with dTG-wt (n=5) or dTG-IDH1^R132H (n=4) when tumor burden reached protocol endpoint. Single-cell suspensions were analyzed for the presence of 2-HG. dTG, double transgenic; FC, flow cytometry; HG, hydroxyglutarate; HLA, human leukocyte antigens; MHC, major histocompatibility complex.