Fig. 3.

Comparative study of the characterization of Treg splenocytes by CD25 surface staining or by Foxp3 intracellular staining. Four groups of three rats were either inoculated with RG2 cells on day-12 or left tumor-free (Naive). Treatment, which consisted of vehicle alone (Untreated), TMZ 30 mg/kg per day for 5 days (Std TMZ) or TMZ 0.5 mg/kg per day for 3 weeks (Metronomic TMZ) was administered to tumor-bearing rats. Animals were sacrificed on day 21. Isolated splenocytes were stained with antiCD3, antiCD4, and antiCD25 or with antiCD3, antiCD4, and antiFoxp3 mAbs and then submitted to flow cytometry analysis. a Results expressed as mean ± SD of the Treg/CD4+ levels (%) in the four groups of rats, Treg being either characterized as CD4+CD25+ cells (open bars) or as CD4+Foxp3+ cells (filled bars). b FCM analysis of spleen cells isolated from an untreated (left) and a metronomic TMZ-treated rat (right), both bearing RG2 tumors. Cells were surface stained with antiCD3-FITC and antiCD4-APC, then submitted to intracellular staining with antiFoxp3-PE. These plots (gated on the CD3+ population) are representative of the results obtained with three rats in each group (percentage of cells in the CD4+ quadrants are indicated)