Successive DC encounters lead to enhanced expansion in a TCR affinity–dependent manner. (A–E) Prf1-deficient mice received s.c. injections of N4-, Q4-, and T4-pulsed DCs, followed by transfer of 10⁴ GFP⁺ OT-I T cells 1 d later. After 60 h, mice received a second wave of unpulsed or N4-, Q4-, or T4-pulsed DCs. Flow cytometry was performed on day 6. (A) Percentage of FSC^high cells on day 6 after transfer. (B and C) Percentage of CD25⁺ OT-I T cells (B) and CD25 MFI levels (normalized to unpulsed DC-exposed OT-I T cell values; C). (A–C) Data are pooled from three independent experiments with total of nine mice per condition. (D and E) Percentage (D) and total cell numbers (E) of OT-I T cells on day 6 after a second transfer of unpulsed or peptide-pulsed DCs 60 h after T cell transfer. Data are pooled from two independent experiments with total of six mice per condition. Bars show the mean. Statistical significance was analyzed by Mann-Whitney (A–C) or Kruskal-Wallis tests with Sidak’s posttest (D and E). **, P < 0.01; ***, P < 0.001.