of IL-2, finding no difference in Treg proliferation in response to wild-type or IL-2 knockout dendritic cells. They concluded that a contaminating T-cell population was the source of the small amounts of IL-2 measured in the cultures that contained IL-2 knockout dendritic cells [64]. Marguti et al. similarly found that dendritic cells produce IL-2 and expand Tregs in culture. These investigators did not determine the identity of the IL-2 source in their cultures but speculated it to be activated CD4+ cells [59]. Brinster and Shevach also reported that dendritic cells induce Treg proliferation. However, unable to detect IL-2-producing dendritic cells, they concluded that DC-driven proliferation of Tregs was primarily independent of IL-2 [62]. Data from Figure 5 are consistent with a CD25-mediated consumption of dendritic cell IL-2 by Tregs. These data provide compelling in vitro evidence in favor of the dendritic cell as the important source of IL-2. Dendritic cell IL-2 regulates Treg suppressive function in vitro Darrasse-Je`ze et al. showed that ablation of dendritic cells in vivo caused progressive loss of Tregs and of Foxp3 expression in Tregs as well as the development of autoimmunity [66]. Though the authors speculated that the mechanism by which dendritic cells sustain Treg homeostasis involves DC expression of MHC class II, the finding that in vivo the DC levels are proportional to the level of Foxp3 expression in Tregs [66] is consistent with the model that Treg functional fitness is maintained by IL-2-secreting dendritic cells. Under some experimental conditions, other DC cytokines (e.g., IL-15) can support Treg proliferation [67]. Unlike Treg proliferation, Treg function depends more exclusively on IL-2. Wuest et al. recently compared the in vitro suppressor function of Tregs that were pre-activated with different common, gamma- chain family cytokines: IL-2, IL-7, IL-15, and IL-21. Although IL- 2, IL-7, and IL-15 each triggered STAT5 phosphorylation and upregulated Foxp3 in Tregs, only Tregs that were incubated with IL-2 showed potent inhibitory function [42]. If Tregs require IL-2 for their suppressor function, then the finding that wild-type Tregs effectively suppress IL-2-deficient T-cells [45] lends credence to a non-T-cell source of IL-2, such as dendritic cells. Natural Tregs are known to express high levels of IL-2 receptor alpha chain (CD25) [68]. Figure 7 clearly demonstrates that Tregs require DC IL-2 to upregulate CD25, and Foxp3 levels are maintained at a higher level when Tregs are cultured in the presence of IL-2-sufficient dendritic cells. Tregs failed to upregulate CD25 upon stimulation with IL-22/2 dendritic cells, ruling out autocrine IL-2 signaling from the Treg as the source driving CD25 expression. Figures 8 and 9 clearly demonstrate that Tregs require dendritic cell IL-2 to exhibit suppressor function. Tregs failed to suppress wild-type CD4+ Teff cells when IL-22/2 dendritic cells are substituted for wild-type dendritic cells in Figure 9. Moreover, data presented in Figure 9 rule out contaminating CD4+ Teff cells as the paracrine source of IL-2 in our in vitro system. Of note, although CD4+ Teff cells secreted IL-2 in cultures with IL-22/2 DCs, this IL-2 did not appear to be sufficient for Treg function. Instead, exogenous IL-2 was needed to restore the suppressor function of Treg (Figure 9E&F). Our data herein identify the dendritic cell as the necessary supplier of IL-2 to manifest Treg suppressor function. The Tregs in all of our experiments were stimulated through their TCR, either with anti-CD3, OVA peptide, or alloantigen. Therefore we have not formally tested whether IL-2 from DCs can impart Treg suppressive function independent of TCR signaling. Although IL-2 stimulation of CD25 promoter activity has been shown without TCR signaling [52], it seems implausible that Dendritic Cell IL-2 Required for Treg Function Tregs would gain suppressor function in the absence of TCR activation [69]. The ability of IL-2-producing DCs to equip Tregs for suppressive function in vivo remains to be determined. Our results do not rule out the possibility that IL-2-producing BMDCs may be an artifact of the in vitro culture conditions for generating BMDCs. However, that we find IL-2-producing DCs in freshly isolated spleen (Figures 2C and 3A) lends credibility to the existence of such cells in vivo. Furthermore, reports by Sgouroudis et al. that LPS-matured BMDCs secrete IL-2 enabling them to expand Treg cells in vivo [46] add promise that our in vitro findings will have in vivo significance. Delivery of essential IL-2 to Tregs facilitated by DC-Treg cell-cell contact and CD25 IL-2-producing dendritic cells showed a preference for conju- gate formation with Treg cells compared with CD4+ Teff cells (Figure 4). Onishi et al. previously noted that compared with CD4+ Teff cells, Tregs are much more efficient in forming conjugates with DCs in vitro and outcompete Teff cells in this process in mixed co-cultures [70]. Tang et al. reported that Treg cells form lasting and stable conjugates with CD11c+ DCs in pancreatic draining lymph node of NOD mice following priming with autoantigen [51]. Importantly, that Tregs rarely encounter Teff but readily engage antigen bearing dendritic cells prior to the inhibition of Teff activation by dendritic cells, places the dendritic cells at the center of Treg cell function in vivo [51]. It is intriguing that the most IL-2-producing DCs are contained within the Treg- conjugated DCs (Figure 4). Perhaps Treg and IL-2-producing cells express unique sets of chemokines and chemokine receptors that promote special chemoattraction between these two relatively sparse populations, such that IL-2-producing dendritic cells and Tregs seek out each other. Indeed, unique chemokine receptor and ligands utilized between DCs and Tregs have been reported [71]. Because IL-2 transcription is preserved in DCs in conjugate formation with Tregs, yet DC secretion of IL-2 onto ELISPOT plates is not seen, we speculate that IL-2 secretion is redirected towards Tregs in contact with the DCs. We explored this possibility using genetically-modified dendritic cells that constitu- tively secrete an IL-2-mCherry fusion protein. The imaging and flow cytometry studies in Figure 6 confirmed that DCs preferen- tially deliver IL-2 to Treg cells and not Teff cells. Epifluorescence microscopy of the DC-Treg conjugates showed IL-2mCherry inside both DCs and Tregs in a vesicular-like distribution pattern (Figure 6B&C). The IL-2mCherry distribution pattern in the Tregs is consistent with receptor-mediated IL-2 cytokine endocy- tosis and post-endocytosis receptor-ligand complex processing [72], [73]. Moreover, contact between DCs and Tregs facilitates IL-2 uptake by Tregs (Figure 6D). This process depends on expression of CD25 on the Treg, because antibody against CD25 ablated the transfer of IL-2mCherry from DCs to Tregs. Our data suggest that Tregs are uniquely capable of uptake of IL-2 from dendritic cells and utilize a cell-cell contact to facilitate this process. Altogether these data expand on the current knowledge of IL-2 requirement for Treg fitness and function and document a crucial role for cell-cell contact with dendritic cells and CD25 on Tregs for IL-2 delivery to Tregs. It is known that Tregs require lower concentrations of IL-2 than Teff cells for in vivo expansion [38]. This may be accomplished in part by a subset of dendritic cells with the ability to spatially control the concentration and target of IL-2 secretion. PLOS ONE | www.plosone.org 9 September 2012 | Volume 7 | Issue 9 | e43609