Polyclonal expansion of sorted CD4⁺CD25^high T cells with bead-coupled anti-CD3/anti-CD28 Abs and high-dose IL-2. Sorted CD4⁺CD25^high T cells can be expanded polyclonally with bead-coupled anti-CD3/anti-CD28 Abs and high-dose IL-2 without down-regulation of LN homing receptors or loss of suppressive function. (A) Sorted CD4⁺CD25^high T cells (n = 7 individual cultures) were stimulated with anti-CD3/anti-CD28–coated beads plus IL-2. Symbols indicate different donors (n = 5; same as in Figure 2). (B) TCR Vβ-repertoire of CD4⁺CD25^high and CD4⁺CD25^- T cells before and after expansion for 24 days with Ab-coated beads and IL-2. One of 5 different cultures with cells from 5 different donors. (C) Surface expression of CD25, CD62L, CCR7, and CD27, and intracellular expression of CTLA-4 of CD4⁺CD25^high T cells (gray histograms in i and ii; and iv and vi) and CD4⁺CD25^- T cells (solid-line histograms in i and ii; and iii and v) after expansion with Ab-coated beads and IL-2 for 3 weeks. Dotted line indicates isotype control. Numbers indicate the percentage of cells within each quadrant. (D) CD4⁺ T_resp cells were stimulated with allogeneic PBMCs for 5 days. CD4⁺CD25^high T cells, freshly isolated or expanded with Ab-coated beads for 13 or 22 days and then rested for 2 days, were added at variable numbers to obtain the indicated ratios. One of 3 independent experiments using cells from different donors. Error bars represent standard deviations from triplicate wells.