Tumor-specific regulatory cells maintain suppressor activity in the absence of antigen. CFSE-labeled Thy1.1⁺ TgCD4⁺ T cells were transferred into A20HA-bearing mice that were immunized with vacHA 2 weeks later. Thy1.1⁺CFSE_low TMTregs were isolated after 5 days and transferred into NT BALB/c mice. Forty days after transfer, the recipient mice were challenged with vacHA and spleen cells were harvested 5 days later. (A) Surface staining of long-term TMTregs. Plots shown are gated on Thy1.1⁺ donor cells. Cells from NT mice that received naive TgCD4⁺ T cells without or with vaccination were included as naive or effector controls, respectively. The number in each plot indicates the percentage of positive cells in the gated population. (B) Thy1.1⁺ cells recovered from those described in panel A were sorted and stimulated with peptide-pulsed BALB/c splenocytes. Purified CD4⁺ T cells from Rag^–/– 6.5 Tg mice were used as responders in the suppression assay. Results are shown as mean ± SE of triplicate cultures.