CD25 and cell contact dependent uptake of dendritic cell IL-2 by Treg cells.

<p><b>A–C:</b> CFSE-labeled DO11.10 Tregs were stimulated with OVA peptide and different DC transfectants as in materials and methods. Panels show representative images of mCherry transfected dendritic cell (A) and IL-2mCherry transfected dendritic cell (B) in conjugate formation with Treg cell. Panels C: Three-dimensional rendering of a Treg cell localizes the IL-2mCherry to vesicles within Treg. <b>D&E:</b> DC-Treg contact facilitates IL-2mCherry transfer from DCs to Tregs that can be blocked by anti-CD25. DO11.10 CD4⁺CD25⁺ Treg or CD4⁺CD25⁻ T effector (Teff) cells were cultured in triplicate in upper chambers of transwells separated from (grey bars) or in lower chambers of transwells in contact with (black bars) BMDCs from IL-2^−/− B6 mice that were transfected with IL-2mCherry (DC-IL2mCh) or control mCherry (DCmCh) and analyzed by flow cytometry as in materials and methods. Indicated cultures contained anti-CD25 antibody (antiCD25). <b>D:</b> Bars are the mCherry MFI means ± SE of gated Thy1.2high cells from triplicate lower chambers (black) and upper chambers (gray). <b>E:</b> Bars are the CD25 (left panel) and mCherry (right panel) MFI means ± SE of gated Thy1.2high cells from triplicate lower chambers (black). Statistical analysis was performed by paired <i>t</i>-test for within well samples and by student t-test for between well samples. Experiment was performed three times yielding similar results, representative experiments are shown.