Figure 1. CD25 localizes to Treg:dendritic cell contact site. Foxp3-GFP Treg cells from C.CgFoxp3tm2Tch/J mice were co-cultured with BALB/cJ splenic DCs and anti-CD3 with or without unlabeled anti- CD25 antibody. Live cell confocal imaging was performed on Treg:DC conjugates 24 h later as in materials and methods. Anti-CD25-APC was added to the cultures prior to collecting imaging data. Left: DIC/GFP/ APC projection images of Treg:DC conjugate from culture without blocking, unlabeled anti-CD25 (upper) and culture with blocking, unlabeled anti-CD25 (lower). Right: The corresponding LCS-Lite analysis of the relative intensity of anti-CD25-APC (y-axis) on Treg membrane not contacting (purple) or in contact with (green) the DC across multiple z-stacks (x-axis). doi:10.1371/journal.pone.0043609.g001 Figure 3B, left panel shows approximately 1% of CD11c+ BMDCs with endogenous IL-2 promoter activity, within the range of BMDCs that secreted IL-2 protein in ELISPOT assays in Figure 2C. Figure 3C shows a higher percentage of DCs with endogenous IL-2 promoter activity when DCs were co-cultured with Tregs compared to DCs cultured alone. Thus, by four separate readouts—IL-2 promoter activity, message, intracellular protein, and secreted protein—we conclude that a small subpop- ulation of DCs constitutively makes IL-2 protein. Moreover, interaction with Treg may be necessary to sustain IL-2 producing DCs. IL-2-producing dendritic cells form conjugates with Treg cells We performed flow cytometric analysis of BMDCs and splenic DCs from C57Bl/6-Tg-IL2p8-4 transgenic mice (gift from Ellen Rothenberg, California Institute of Technology) to define markers for the DC subset with IL-2 promoter activity. These studies showed that the subset was CD11c+/CD4 2 /CD8 2 /CD252/ CD1032/CD32/Siglec-H2/CD2052, indicating that IL-2-tran- scribing DCs belong to a non-plasmacytoid, non-CD8a DC population. In the absence of unique subset markers we relied on DCs that were transfected with the IL-2-reporter plasmid pIL2p8.4EGFP to track the behavior of DCs with IL-2 promoter activity. We then examined IL-2 promoter activity of C57BL/6 DCs that were non- conjugated or conjugated with alloreactive DO11.10 CD4+CD25+ Tregs or CD4+ Teff cells. Figure 4 shows an enrichment of DCs with IL-2 promoter activity in the Treg-DC conjugates. One interpretation of these data is that IL-2-producing DCs interact preferentially with Tregs in culture. Tregs intercept dendritic cell IL-2 Data from Figure 4 also suggests that IL-2 promoter activity is preserved in DCs in conjugate formation with Tregs. We then examined the effect of Tregs on IL-2 protein secretion by the DCs. We cultured C57BL/6 DCs with and without alloreactive Dendritic Cell IL-2 Required for Treg Function Figure 2. A small subpopulation of dendritic cells secrete IL-2 protein. A: CD11c+ dendritic cells contain IL-2 protein. DCs were stained for intracytoplasmic IL-2 as in materials and methods. Panels show FACS contour plots of BMDCs stained with anti-IL-2 (right) or isotype control antibody (left). The experiments were repeated with similar results. B: Progressive purification of CD11c+ dendritic cells enriches for IL-2 producing cells. The frequency of spontaneous IL-2 secreting BMDCs was measured by IL-2 ELISPOT assay as in materials and methods. Bars show mean 6 SE of % IL-2-producing cells in triplicate samples of unpurified, 1X and 2X MACS-purified CD11c+ BMDCs. Statistical analysis was by one-way repeated measures ANOVA and Tukey’s test for all pairwise multiple comparison procedure. C: Dendritic cells were doubly-purified and the percentage of IL-2- producing bone marrow-derived DCs ranged from 0.7 to 2.4% (N = 7), and the percentage of IL-2-producing splenic-derived DCs ranged from 1.2 to 2.0 (N = 3). doi:10.1371/journal.pone.0043609.g002 DO11.10 Tregs in IL-2 ELISPOT plates and measured the number of IL-2-producing DCs. In addition, since it has been reported that DCs produce IL-2 in response to toll-like receptor (TLR) signals [48], [49], [50] we examined the effect of different TLR agonists on the DCs in our system. About 2% of freshly isolated splenic DCs (Figure 5, left panel) and BMDCs (Figure 5, right panel) secreted IL-2 constitutively. Treatment with the TLR9 agonist CpG-B increased the number of IL-2-secreting DCs (Figure 5), whereas treatment with the TLR4 agonist LPS (lipopolysaccharide) did not alter the number of IL-2-secreting DCs (not shown). Tregs profoundly abrogated detection of IL-2-secreted protein by DC. We did not detect secreted IL-2 in more than a dozen experiments where Tregs were co-cultured with DCs in IL-2 ELISPOT, regardless of whether we measured constitutive, CpG- B-induced, or LPS-induced IL-2 responses (Figure 5 and data not shown). Because IL-2 promoter activity was preserved in DCs that were conjugated to Tregs (Figure 4), and because CD25 localized to the interface between Treg-DC conjugates (Figure 1), we reasoned that DCs secrete IL-2 directly to adjacent Tregs (Figure 5). In further support of this interpretation, we demon- strated that the addition of anti-CD25 to co-cultures of Tregs and PLOS ONE | www.plosone.org 3 September 2012 | Volume 7 | Issue 9 | e43609