A12-responsive T cells are not Tregs. Inguinal lymph node cells from DR1 Tg mice immunized 10 days earlier with A12/CFA (left panel), A2/CFA (middle panel), or Ova/CFA (right panel) were cultured directly ex vivo with anti CD25, CD4+, VB8+, VB14+, and intracellular FoxP3. Selective gating was performed on the CD4+, VB8+VB14+ cells. Before intracellular staining, a sample of the A12-specific cells was stained with DR1-A12 tetramer to confirm that the VB8+, VB14+ population was enriched for tetramer+ cells (15% tetramer+). The experiments were repeated after 4 days of culture with the A12 peptide in vitro, and the results were similar to those shown. These data indicate that the numbers of Foxp3+, CD25+ Treg cells remain at background levels after immunization with A12/CFA.