Figure 3. Tregs preserve dendritic cell transcription of IL-2. A: DO11.10 Tregs and C57BL/6 splenic DCs were cultured together or in separate tubes for 5 and 24 h, IL-2 message was measured in cultured cells by RT-qPCR as in materials and methods. Bars are the mean 6 SE of IL-2 message in the cultures. Statistical analysis was by one-way repeated measures ANOVA and Tukey’s test for all pairwise multiple comparison procedure. B&C: BMDCs were transfected with the IL-2 reporter plasmid (pIL2P8.4-EGFP), a negative control plasmid (pcDNA3.1/V5-His B), and a positive control plasmid to assess transfection efficiency (pAcGFP-N1). Transfectants were analyzed for IL-2-promoter activity in cells as in materials and methods. B: Panels show histograms of the level of promoter-driven GFP in gated CD11c+ cells. The experiment was repeated with similar results. C: pIL-2P8.4- EGFP-transfected BMDCs were cultured unstimulated or with CpG-B, with and without Treg cells for 40 h. Bars are the mean 6 SE of IL-2- promter-EGFP+ BMDCs from two experiments. doi:10.1371/journal.pone.0043609.g003 DCs completely restored the detection of IL-2-secreting DCs in IL-2 ELISPOT, whereas the addition of isotype control antibody had no effect (Figure 5). Thus, in the absence of Tregs, ELISPOT plates capture the IL-2 that DCs secrete. In the presence of Tregs, via their CD25 surface receptor, Tregs capture the IL-2 that DCs secrete. Tregs require CD25 and cell-cell contact with dendritic cells to take up IL-2 Utilizing two-photon laser-scanning microscopy to characterize Treg behavior in vivo during development of autoimmune priming in diabetes-prone mice, Tang et. al, reported that Treg cells in pancreatic draining lymph node form stable conjugates with dendritic cells but not with CD4+ Teff cells [51]. Therefore, we tested whether DCs formed conjugates with Tregs to allow IL-2 transfer. We transfected BMDCs with plasmids encoding IL-2- mCherry fusion protein (or control mCherry), and we performed multi-color live cell imaging with epifluorescence microscopy to track IL-2 delivery from DCs. We first examined cultures of DO11.10 Tregs that were stimulated with OVA peptide and BALB/c transfected BMDCs. DCs that were transfected with mCherry displayed the mCherry throughout their cytoplasm, whereas DCs that were transfected with IL-2-mCherry fusion protein displayed the IL-2-mCherry in intracellular vesicles (compare mCherry distribution in Figures 6A and 6B). Figure 6B shows that IL-2-mCherry was transferred to a Treg cell Dendritic Cell IL-2 Required for Treg Function conjugated with an IL-2-mCherry-expressing DC. Figure 6C illustrates that the IL-2-mCherry was located within the Treg cell, indicating that IL-2 is endocytosed by the Treg and not merely on the cell surface. However, we also detected some IL-2-mCherry Treg cells that were not conjugated to DCs (not shown). Because the images represent a snap shot of the co-culture after 24 hours, we were not able to determine whether the later mCherry Treg cells were ever conjugated to IL-2-producing DCs. To explore the question of whether DCs transferred IL-2 to Tregs via direct cell contact we made use of the transwell system. We transfected IL-22/2 BMDCs with plasmids encoding IL-2- mCherry fusion protein or mCherry, and we cultured the transfected cells in the lower chamber of transwell plates. Alloreactive DO11.10 Tregs were labeled with carboxylfluorescein succinimide ester (CFSE) and added to both lower and upper chambers. Flow cytometry was performed on the Treg from upper and lower chambers. The Tregs that were co-cultured in contact with IL-2-mCherry-producing DCs in the same lower transwell chamber (Figure 6D, black bars) took up significantly more IL-2- mCherry than Tregs that were cultured in the upper chamber separately from the DCs (Figure 6D, gray bars; compare top pair of gray and black bars, p,.001). The addition of anti-CD25 to the transwell plate blocked uptake of IL-2-mCherry by Tregs co- cultured in the lower chamber in contact with IL-2-mCherry- producing DCs to the background level (Figure D, p,.001). These results indicate that cell-cell contact and CD25 are required for efficient transfer of IL-2 from the DC to the Treg cell. Compared to Tregs, there was no transfer of IL-2-mCherry to CD4+ Teff cells that were co-cultured in lower chambers in contact with IL-2-mCherry-producing DCs (Figure 6E right panel, top vs. bottom bars, p,.001). This was not from lack of CD25 expression on CD4+ Teff cells. The level of CD25 on the stimulated CD4+ Teff cells in contact with IL-2-mCherry- producing DCs was the same as the level of CD25 on the Treg cells in contact with control, mCherry-expressing DCs (Figure 6E left panel, middle vs. bottom bars, not significant). These data suggest that IL-2-producing DCs preferentially form conjugates with Tregs to facilitate juxtacrine delivery of IL-2. Dendritic cell IL-2 increases Treg expression of CD25 and Foxp3 IL-2 increases expression of CD25 on CD4+ cells [52] and Foxp3 in Tregs [29], [42], [53]. We noted that Tregs in contact with IL-2-mCherry-producing DCs that took up IL-2mCherry expressed higher levels of CD25 than Treg cells in contact with control, mCherry-expressing DCs (Figure 6E left panel, top vs. middle bars, p,.001). To further examine whether DC-secreted IL-2 affects Treg phenotype we compared the baseline level of CD25 and Foxp3 protein in DO11.10 Tregs that had been cultured alone with the levels of CD25 and Foxp3 protein in Tregs cultured with either wild-type or IL-22/2 KO BMDCs. Figure 7A shows that Tregs co-cultured with wild-type DCs expressed significantly higher levels of CD25 compared with Tregs cultured alone (p = ,.001, Tukey’s Test) or with DCs from IL-22/2 mice (p = .008, Tukey’s Test). Figure 7B shows that Tregs co-cultured with wild-type DCs expressed significantly higher levels of Foxp3 compared with Tregs cultured alone (p = .002, Tukey’s Test). In contrast, there was no difference in the level of expression of Foxp3 in Tregs co-cultured with DCs from IL-22/2 mice compared with Tregs cultured alone (p = 0.8, Tukey’s Test). These data indicate a biological response to IL-2 by Tregs in culture with IL-2-sufficient DCs and provide further evidence that DCs can supply IL-2 to Tregs. PLOS ONE | www.plosone.org 4 September 2012 | Volume 7 | Issue 9 | e43609