The N-terminus of FoxP3 induces T-cell anergy. (A) Suppression of FoxP3 expression breaks Treg anergy. Purified mouse CD4⁺ CD25⁺ T cells were infected with lentivirus that carry GFP only or GFP with siRNA against FoxP3. GFP⁺ cells were sorted and restimulated with anti-CD3 plus anti-CD28 for 24 hours. Cell proliferation was examined by [³H]-thymidine incorporation. Error bars represent data from 3 independent experiments. (B) Suppression of FoxP3 restores AP-1 DNA-binding in Tregs. GFP⁺ cells from panel A were restimulated with anti-CD3 plus anti-CD28. Nuclear extracts were isolated, and AP-1 DNA-binding was determined. (C) Expression of FoxP3 N-terminus inhibits T-cell proliferation. Mouse CD4⁺ CD25⁻ T cells were infected with retroviruses that carry GFP only, GFP with FoxP3 or with FoxP3 N-terminus. GFP⁺ T cells were sorted and restimulated with anti-CD3 plus anti-CD28 for 24 hours. Cell proliferation was examined by [³H]-thymidine. Error bars represent data from 3 independent experiments. (D) Lack of suppressive functions of T cells that carry FoxP3 N-terminus. CD4⁺ CD25⁻ T cells were isolated and plated at 10⁵ cells/well in 96-well plate with anti-CD3 plus anti-CD28. GFP⁺ cells in panel C were added to the wells at the indicated ratios. Cells were cultured for 24 hours followed by adding 1 μCi/well H-thymidine and cultured for another 16 hours. Error bars represent data from 3 independent experiments.