Figure 3.

Inhibition of 2-HG production by AG-881 does not alter the tumor-infiltrating myeloid cell population but results in increased recruitment of CD4⁺ T-cells. (A) Mice bearing dTG-IDH1^R132H tumors were treated daily with 10 mg/kg AG-881 or vehicle. Levels of 2-HG in the tumor-bearing hemisphere after 14 days of treatment were measured by LC-MS/MS. (B–D) A separate cohort of mice were treated as described in (A), and single-cell suspensions of the tumors were either analyzed by FC to determine the frequency of CD11b⁺ (B) cells among tumor-infiltrating leukocytes or used to isolate CD11b⁺ cells using a column separation protocol (C, D). 2-HG levels were significantly reduced in CD11b⁺ cells from AG-811-treated mice (C); however, their ability to suppress CD8⁺ ((D), left panel) and CD4⁺ ((D), right panel) T-cell proliferation ex vivo was unchanged. T-cell proliferation as a result of polyclonal CD3/CD28 activation was measured by dilution of CFSE 72 hours post-coculture initiation. (E) Single-cell suspensions generated in (B) were used to examine the frequency for CD8⁺ and CD4⁺ T-cells by FC. In 2-HG quantification and T-cell suppression studies n=3 mice per group. For FC analyses, n=4 in control group and n=5 in AG-881 groups. dTG, double transgenic; FC, flow cytometry; HG, hydroxyglutarate.