Fig. 5
Lack of intracranial tumor eradication and Treg depletion with the anti-GITR IgG 2a antibody/SRS combination. C57BL/6 mice were intracranially inoculated with GL261-luc tumor, randomized to groups of ≥8, and dosed with 200 μl of anti-GITR (2a) (10 mg/kg) on day 10, 13, 16 and/or SRS (10 Gy) on day 10. Mice were followed for survival, a and tumor growth was assessed by bioluminescent imaging b. Mice were sacrificed on day 21, tumor infiltrating Tregs were isolated and analyzed by flow cytometry c. CD11b + CD45+ tumor resident microglia and tumor infiltrating mononuclear cells were isolated on day 21, analyzed by flow cytometry (gated on CD3+ cells), and mean fluorescence intensity (MFI) of FcγRIII and IV expression was calculated d. Flank tumors were established in C57BL/6 mice by subcutaneous inoculation of 2 × 106 GL261-luc cells in a volume of 100 μl, and intracranial tumors were established as in Fig. 1. Mice were dosed i.p with 200 μl of anti-GITR (2a) (10 mg/kg) on days 10, 13, and 16, sacrificed on day 17, and tumor infiltrating lymphocytes were harvested and analyzed by flow cytometry for FoxP3 expression (gated on CD3+ cells) e-f. Symbol and horizontal bar denote single mouse and average value, respectively. *P < .05, ***P < .001. NS, non-significant