Generation of anergic and regulatory T cells after coculture with CTLA4-KDEL–transfected DCs. (A) Purified T cells (10⁶) were cocultured with DCs (10:1 ratio), which were either untransfected or transfected with CTLA4-KDEL or mock-KDEL. After 5 days, T cells were rested in culture for another 5 days. The cells were then put into fresh culture with mDCs from either the same donor (i-ii) or third-party (iii-iv) mDCs at 5:1 ratio in the presence (ii,iv) or absence (i,iii) of 10 U/mL exogenous rIL-2. The proliferation of T cells was determined by ³H-thymidine incorporation on days 3, 5, and 7. The results are expressed as the mean ± SD of triplicate wells. (B) T cells exposed to untransfected DCs, mock-KDEL–transfected DCs, or CTLA4-KDEL–transfected DCs were analyzed for expression of p27^kip1 and β-actin expression by Western blotting. The density of the p27^kip1 bands (normalized to β-actin) are shown below, as the mean ± SD of 3 experiments. (C) The T cells generated following incubation with untransfected or with CTLA4-KDEL or mock-transfected DCs were added at varying numbers to an MLR between fresh T cells (from the same donor) and DCs from either the original stimulator or third-party DCs. ³H-thymidine incorporation was measured on day 5. All data are representative of 3 experiments and show the mean ± SD of triplicate cultures.