The generation of T cells with regulatory capacity requires immediate vicinity of cells but is independent of Foxp3. (A) To in vitro cocultures of naive, CFSE-labeled CD4⁺ T cells and mature DC loaded with antigenic peptide (pOVA 323-339), TofDC or TofB were added directly and via a semipermeable transwell membrane. At 72 hours, proliferation of the naive T cells was determined by FACS. Close cell contact is needed for exertion of the regulatory effect of TofB, as separation from naive T cells plus DC via a transwell membrane abrogates the effect. (B-D) The level of the transcription factor Foxp3 was determined via intracellular staining and FACS after 72 hours of cocultures of naive splenic CD4⁺ T cells with peptide-loaded mature DC or B cells, respectively. (B) Preferential induction of Foxp3⁺ T cells could not be observed after B-cell contact; equally low numbers were found in Tof B and TofDC. (C) The Foxp3⁺ T cells are also CD25⁺. Depicted is the result for the CD4⁺ TofB population only. (D) The positivity of Foxp3 is independent of the CD62L level. Depicted is the result for the CD4⁺ TofB population only. The data shown are representative of at least 3 independent experiments.