Dendritic Cell IL-2 Required for Treg Function Figure 6. CD25 and cell contact dependent uptake of dendritic cell IL-2 by Treg cells. A–C: 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. D&E: 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+CD252 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-22/2 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). D: Bars are the mCherry MFI means 6 SE of gated Thy1.2high cells from triplicate lower chambers (black) and upper chambers (gray). E: Bars are the CD25 (left panel) and mCherry (right panel) MFI means 6 SE of gated Thy1.2high cells from triplicate lower chambers (black). Statistical analysis was performed by paired t-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. doi:10.1371/journal.pone.0043609.g006 Figure 7. Dendritic cell IL-2 increases Treg phenotype. DO11.10 CD4+CD25+ Treg cells were cultured without or with BMDCs from either WT or IL-22/2 KO (on B6 background) mice in transwells and the level of CD25 (A) and Foxp3 (B) on Treg cells was measured by flow cytometry as in materials and methods. A: Bars are the mean 6 SE of mean fluorescent intensity (MFI) of CD25 levels on Thy1.2-gated Tregs of triplicate samples from one of four representative experiments. Statistical analysis was by Tukey’s test for all pairwise comparisons. B: Bars are the mean 6 SE of mean fluorescent intensity (MFI) of Foxp3 levels on Thy1.2-gated Tregs of triplicate samples from a single experiment. Statistical analysis was by Tukey’s test for all pairwise comparisons. doi:10.1371/journal.pone.0043609.g007 production by CD4+ Teff cells does not depend on exogenous IL- 2, we were able to test the effects of both anti-CD25 and exogenous IL-2 on Tregs in our system. DO11.10 Tregs were added to mixed lymphocyte cultures containing DO11.10 CD4+ Teff cells (responder cells) and allogeneic BMDCs or splenic DCs (stimulator cells) from B6 mice, and the ability of Tregs to suppress the late phase of IL-2 secretion by DO11.10 CD4+ Teff cells (beyond 24 h) was measured by ELISPOT. CD4+ Teff cells showed similar late-phase IL-2 response when stimulated with allogeneic DCs from either wild- type or IL-22/2 B6 mice (Figure 9A&B, compare black bars). Thus, CD4+ Teff function is independent of DC IL-2. Tregs suppressed IL-2 secretion by alloreactive DO11.10 CD4+ Teff responder cells by more than 95% when added to mixed lymphocyte cultures that contained allogeneic wild-type C57BL/ 6 DCs. In contrast, Tregs showed no suppression of alloreactive DO11.10 CD4+ Teff cells when added to cultures that contained allogeneic IL-22/2 B6 DCs (Figure 9A&B, compare gray bars). These data indicate that in vitro Treg suppressor function depends entirely on IL-2 supplied by DCs, but not on IL-2 produced by Teff CD4+ cells. PLOS ONE | www.plosone.org 6 September 2012 | Volume 7 | Issue 9 | e43609