Patients with GBM possess an increased frequency of T_Regs after TMZ treatment that can be reduced by a single administration of an anti–IL-2Rα mAb. (A-B) Percentages and absolute numbers of T_Regs (CD4⁺CD25⁺Foxp3⁺) from leukapheresis (pre-TMZ and post-TMZ and daclizumab) and peripheral blood (post-TMZ) samples were determined by CBC counts and FACS analysis. T_Reg levels from preoperative samples (pre-TMZ, ∼ day − 100), before initial vaccination and daclizumab (post-TMZ, day 0) and ∼ 7 weeks after vaccination and daclizumab (post-TMZ and daclizumab, day ∼ 50) were assessed. (A) Frequency of T_Regs by paired t test, *P = .0236 and **P = .0061. (B) Total CD4⁺, CD4⁺Foxp3⁺, and CD4⁺CD25⁺Foxp3⁺ T-cell frequencies from leukapheresis and peripheral blood samples from a representative patient were determined before and after daclizumab administration (day 0). Gating strategies for flow cytometric analyses were as follows and as shown in supplemental Figure 12: (1) For CD4⁺ T cells, all cells were displayed using forward and side scatter, and the lymphocyte population was selected. Lymphocytes were then displayed by side scatter and CD4 on a dot plot. The CD4⁺ population was then selected out of this lymphocyte population. (2) For Foxp3⁺ of CD4⁺, an identical gating strategy as described in 1 was used to select CD4⁺ T cells. The CD4⁺ T cells were then displayed by dot plot against Foxp3, and all Foxp3⁺ cells were selected. (3) For CD25⁺Foxp3⁺ of CD4⁺, an identical gating strategy as described in (1) was used to select the CD4⁺ population. The selected CD4⁺ population was then displayed by dot plot as CD25 versus Foxp3. CD25 and Foxp3 double-positive cells were then selected out of the CD4⁺ population.