Figure 4. Global IDO-deficiency affects Treg levels in brain tumors and draining lymph nodes without effects in the spleen.

Indoleamine 2,3 dioxygenase 1-deficient (IDO^−/−) mice were intracranially-injected (ic.) 4×10⁵ GL261 cells transduced with -scrambled shRNA (vector control, V_c; white bars) or -shRNA specific to IDO (IDO knockdown, IDO_kd; black bars). The (A) frequency and absolute numbers of CD4⁺FoxP3⁺ regulatory T cells isolated from brain, ipsilateral deep and superficial cervical draining lymph nodes (dLN) and spleen of tumor-bearing mice were analyzed at 3 weeks post-intracranial injection (wp-ic). Representative flow cytometric plots demonstrate the gating strategy utilized for the identification of total CD3⁺CD4⁺FoxP3⁺ Tregs in the GL261-cell based orthotopic glioma mouse model. (B) The Kaplan-Meier curve represents mouse survival times over a time course of 150 days (n = 9 – 18 mice/group) and represent the combined data from 3 independent experiments. (C) RasB8 mice that were IDO -competent (white bars) or -deficient were analyzed for the frequency and (E) absolute numbers of Tregs. Representative flow cytometric plots demonstrate the gating strategy utilized for the identification of total CD3⁺CD4⁺FoxP3⁺ Tregs in the RasB8 transgenic glioma mouse model. For all experiments, Tregs were initially first identified for the co-expression of CD3 and CD4, prior to FoxP3. Bar graphs in figures A and C are shown as mean ± SEM and are representative of two independent experiments (n = 3 – 5 mice/group). *p < 0.05; **p < 0.01; ***p < 0.001.