Frequency of functional T_Regs increases after lymphodepletive TMZ. (A) C57BL/6 mice were treated with 5 Gy of TBI (n = 3), single-dose TMZ (200 mg/kg; n = 5), or multiday TMZ (60 mg/kg/5 day; n = 5), and then CBCs were compared with untreated controls (n = 5). Total lymphocyte counts in peripheral blood were monitored over the course of 28 days; a representative experiment shown (n = 3). Postlymphodepletion values were evaluated for statistical significance (day 2): untreated vs 200 mg/kg TMZ, 60 mg/kg/5 day TMZ, and TBI, P < .0001 for all comparisons; and TBI vs 200 mg/kg TMZ and 60 mg/kg/5 day TMZ, P = .2042 and P = .0168, respectively. (B-C) The frequency and absolute number of CD4⁺ and CD8⁺ T cells and CD4⁺CD25⁺Foxp3⁺ T_Regs in the peripheral blood of untreated (n = 5), lymphodepletive TMZ-treated (n = 5), and TMZ-lymphodepleted mice who also received OT-I transfer (n = 5) were monitored by FACS analysis 1 week after TMZ administration (frequency CD4⁺, *P = .0396 and **P = .0216; frequency T_Regs, *P = .0006 and **P = .0019). For FACS analysis of all murine T-cell populations (supplemental Figure 11, available on the Blood Web site; see the Supplemental Materials link at the top of the online article), total lymphocytes were first gated by side scatter and forward scatter. For CD4⁺ and CD8⁺ T-cell selection, total CD3⁺ T cells were selected from the lymphocyte gate by displaying side scatter by CD3⁺. CD4⁺ or CD8⁺ T cells were selected from the CD3⁺ T-cell population by displaying CD4⁺ by CD8⁺ and gating the desired population. For T_Regs, lymphocytes were first gated by forward and side scatter, and from this gate CD4⁺ T cells were selected from side scatter. CD4⁺ T cells were displayed by CD25 versus Foxp3. For CD4⁺CD25⁺Foxp3⁺ T_Regs, double-positive cells were selected and for CD4⁺Foxp3⁺ T_Regs all Foxp3⁺ T cells were selected. Representative experiments are shown; all experiments performed in at least triplicate. (D) C57BL/6 mice were treated with 200 mg/kg single dose TMZ or 60 mg/kg/5day. Three days after the completion of TMZ treatment, CD4⁺CD25⁻ responder T cells and CD4⁺CD25⁺ T_Regs were isolated and cultured for 3 days in the presence of α-CD3e beads (all wells in triplicate). Proliferation was assessed after a 16-hour incubation with [³H]thymidine. (E) To determine the ratio of effector cells to T_Regs, the absolute number of peripheral blood CD8⁺ cells was divided by the absolute number of peripheral blood T_Regs (n = 5/cohort), ratios from representative experiments (B-C). *P = .0354.