Dendritic Cell IL-2 Required for Treg Function Figure 8. Dendritic cell IL-2 regulates Treg suppression of CD4+ proliferation. A: The Panels show proliferation of anti-CD3 stimulated CFSE-labeled C57BL/6 CD4+CD252 Teff cells co-cultured with WT or IL-22/2 KO (on B6 background) BMDCs, with and without C57BL/6 Treg cells at a Treg:Teff ratio of 2.5:10. Numbers indicate percentage of Teffs that have divided. B: CD4+CD252 Teff cell proliferation was measured by flow cytometry, and the % of Treg suppression of Teff cell proliferation was calculated as in materials and methods. Bars show percent Treg inhibition of proliferation of CD4+CD252 Teff cells that were co-cultured with IL-22/2 KO (gray) or WT (black) B6 BMDCs and Tregs at Treg:Teff ratio of 2.5:10 (A) or 1:10 (B–D), and stimulated as specified in columns A–D and materials and methods. doi:10.1371/journal.pone.0043609.g008 The addition of anti-CD25 to cultures containing alloreactive DO11.10 CD4+ Teff cells and wild-type allogeneic DCs did not alter the IL-2 response by CD4+ Teff cells (Figure 9D, compare open and closed triangles). In contrast, the addition of anti-CD25 blocked the Treg-mediated suppression of the late-phase IL-2 response by CD4+ Teff cells (Figure 9C&D, compare open and closed circles). The addition of exogenous IL-2 to the mixed lymphocyte cultures containing alloreactive DO11.10 CD4+ Teff cells and DCs from IL-22/2 (B6 background) mice did not change their late-phase IL-2 response (Figure 9E&F, compare triangles). In contrast, the addition of exogenous IL-2 at doses that exceeded 500 U/ml restored Treg-mediated suppression of CD4+ Teff cells’ late-phase IL-2 response (Figure 9E&F, compare circles). These data confirm that Tregs require IL-2 from DCs to attain suppressor function. Moreover, in the absence of IL-2-producing DCs, the recovery of Treg suppressor function requires exogenous IL-2 at concentrations higher than 500 U/ml, suggesting that DCs utilize spatial control to target delivery of relatively higher concentrations of IL-2 to Tregs. Discussion Our data demonstrate a mechanism whereby dendritic cells play a vital role in controlling Treg cell suppressor function in vitro by supplying essential IL-2 to Tregs. First, we show that a subset of dendritic cells constitutively secrete IL-2. Second, we show Tregs consume DC IL-2, as evident by the increased expression of CD25 and Foxp3 by Tregs cultured with IL-2-sufficient dendritic cells. Third, we show that dendritic cells provide the essential source of IL-2 to Tregs via cell-cell contact and CD25-dependent juxtacrine delivery. Finally, we show that IL-2 from dendritic cells regulates Treg suppression in vitro. A subset of dendritic cells constitutively secretes IL-2 Freshly isolated mouse splenic and bone marrow-derived dendritic cells constitutively secreted IL-2 protein in IL-2 ELI- SPOT plates (Figures 2B&C and 5). The IL-2 ELISPOT specifically detected IL-2 protein, since dendritic cells from IL-2 knockout mice produced no IL-2 spots when cultured on IL-2 ELISPOT plates, nor did wild-type dendritic cells produce spots when cultured on plates that were coated with an irrelevant isotype control antibody (not shown). Stimulation of either splenic or bone marrow-derived dendritic cells with CpG-B consistently enhanced IL-2 production (Figure 5). We confirmed that dendritic cells were the source of the IL-2 by intracellular detection of IL-2 protein (Figure 2A) and IL-2 promoter activity (Figure 3B) in CD11c+ cells. We observed IL-2 promoter activity in bone marrow-derived CD11c+ dendritic cells that were transfected with either the pIL2P8.4EGFP (Figure 3B) or the pIL2P7.4REYFP (not shown) reporter constructs—both constructs with sense IL-2 promoter fragments driving expression of fluorescent protein in the cell—but not in dendritic cells that were transfected with an anti-sense IL-2 promoter construct (not shown). To our knowledge ours is the first report of constitutive secretion of IL-2 by splenic dendritic cells and bone marrow-derived DCs. Granucci et al. first reported that dendritic cells produce IL-2. Their gene expression microarray studies of bone marrow-derived dendritic cells revealed upregulated IL-2 message in dendritic cells that were stimulated with gram-negative bacteria [56]. Subse- quently Granucci et al. showed that stimulating bone marrow- derived or splenic dendritic cells through CD40 or with various microbial products that signal through TLR 2, 4, or 9, increases IL-2 transcription and secretion [57]. Other investigators have independently confirmed that LPS-matured bone marrow-derived dendritic cells produce IL-2 [46], [58]. Marguti et al. further reported that co-culture with allogeneic apoptotic cells induces IL- 2 production by immature dendritic cells and enhances IL-2 production by mature dendritic cells [59]. However, these studies did not report any IL-2 production by unstimulated dendritic cells. An important implication of our finding is that IL-2-producing dendritic cells may control Treg fitness in vivo even in the absence of infection or other danger signals. PLOS ONE | www.plosone.org 7 September 2012 | Volume 7 | Issue 9 | e43609