Evaluation of Treg frequency in PB and LN of NHL patients before and after vaccination. (A) Schematic comparison of CD4⁺CD25⁺FOXP3⁺ T-cell frequencies in healthy donor PB, patient PB and LNs before vaccination, and responding and nonresponding patient PB after vaccination. Statistically significant differences, by 2-sided Student t test, are reported (*P ≤ .05; **P ≤ .01; ***P ≤ .001). (B) Representative examples of Treg frequencies in prevaccination versus postvaccination PB of the complete responding patient no. 14 (i), the nonresponding patient no. 7 (ii), and LN specimens collected before vaccination, 6 months after vaccination at the time of partial remission, and 12 months after vaccination at disease progression from the partial responding patient no. 5 (iii). All plots were gated on CD3⁺CD4⁺ cells. Data were acquired using BD CellQuest software version 3.3 on the 4-color analysis flow cytometer BD FACSCalibur (Becton Dickinson, Lincoln Park, NJ). Analyses were performed using FlowJo 8.7.1 software version for Macintosh (TreeStar, Eugene, OR).