Changes in levels of Treg cells following immunization and/or administration of denileukin diftitox. PBMCs before and after vaccination for each cohort were analyzed by flow cytometry for expression of CD4, CD25, and Foxp3. (A) Cohort 0: PBMCs were collected before and after TRICOM-CEA vaccination. (B) Cohort 1: PBMCs were collected before TRICOM-CEA vaccination and denileukin diftitox administration (LPA), before vaccination and after denileukin diftitox (week 0), and weeks 1, 3, 6, 9, and following the final vaccine dose. (C) Cohort 2: before TRICOM-CEA vaccination and denileukin diftitox (LPA), before vaccination and after 1 dose of denileukin diftitox (week 0) and weeks 1, 2, 3, 5, 6, 8, 9, and following the final vaccination. Arrows indicate time of denileukin diftitox administration. The data are presented as the mean change in percentage of CD4⁺CD25⁺ cells that are 90% and greater Foxp3⁺ plus or minus SE for patients completing 4 vaccinations for each cohort (5 patients analyzed for each cohort). The data for each cohort are presented as the mean percentage change in the percentage of CD4⁺CD25^highFoxp3⁺ cells from baseline, defined as the time of the leukapheresis (LPA) before any denileukin diftitox. Arrows indicate time of administration of denileukin diftitox.