CD4^highCD25⁺ alloantigen-specific Treg can be continuously expanded by CD40-activated B cells in large-scale without loss of function, and exogenous IL-2 does not enhance this cell expansion. Freshly purified naive CD4⁺CD25⁻ T cells were cocultured with CD40-activated allogeneic B cells for the indicated time. (A) The percentages of CD4^highCD25⁺ and CD4^mediumCD25⁻ cells in the cultures (n = 10). (B) Expansion of CD4^highCD25⁺ alloantigen-specific Treg from 10 different persons. The expansion was normalized for the CD4^highCD25⁺ cells, and the fold increase of the CD4^highCD25⁺ was shown. (C) Naive CD4⁺CD25⁻ were cocultured with CD40-activated allogeneic B cells with or without IL-2. The expansion was normalized for the CD4^highCD25⁺ cells, and the fold increase of the CD4^highCD25⁺ is shown (n = 4). (D) Absolute numbers of CD4^highCD25⁺ alloantigen-specific Treg generated from 10⁶ naive CD4⁺CD25⁻ T cells (n = 10). (E) CD4^highCD25⁺ alloantigen-specific Treg induced and expanded by CD40-activated B cells for 21 days remain functional. Freshly purified naive CD4⁺CD25⁻ T cells (responder) were cocultured with CD40-activated allogeneic B cells (target antigen) to induce and expand CD4^highCD25⁺ Treg for 21 days with replacement of B cells every 7 days. The sorted CD4^highCD25⁺ and CD4^mediumCD25⁻ cells were added into the MLR culture system as described in “Mixed lymphocyte reaction assays.” Data shown here are representative of 3 independent experiments.