Treg-extrinsic source and (ii) an intact IL-2 receptor on Treg cells, suggesting that the formation of a functional IL-2/IL-2Rabc quaternary complex is necessary for optimizing Treg fitness. IL- 22/2, IL-2Ra2/2, or IL-2Rb2/2 KO mice have decreased numbers of natural CD4+CD25+ Tregs [26], [27], [28], [29] and suffer from autoimmunity [30], [31], [32] or fatal lymphoprolif- erative disease [29]. Wild-type Tregs, after adoptive transfer to IL- 2Rb2/2 KO mice, engraft and undergo normal homeostatic proliferation in peripheral lymph nodes [33] and rescue mice from autoimmunity [34]. In contrast, wild-type Tregs, after adoptive transfer to IL-22/2 KO mice, fail to expand in the periphery and fail to prevent autoimmunity [27]. In spontaneous experimental autoimmune encephalomyelitis (EAE) secondary to Treg dysfunc- tion, the adoptive transfer of CD4+ T cells from either wild-type or IL-22/2 KO mice conferred protection from EAE, whereas adoptive transfer of CD4+ T cells from IL-2Ra2/2 KO mice did not [35]. The enforced expression in the IL-2Rb2/2 KO mice of a transgenic chimeric receptor—composed of the extracellular domain of wild-type IL-2Rb fused to the cytoplasmic domain of the IL-7Ra—rescued the IL-2Rb2/2 KO mice from autoimmu- nity. In contrast, the transgenic expression of either the wild-type IL-7R or the chimeric receptor composed of extracytoplasmic domain of IL-7Ra fused to the cytoplasmic domain of IL-2Rb did not [36]. This failure of Tregs to thrive in the absence of a Treg- extrinsic source of IL-2 or access to the components of the IL-2 receptor that confer high affinity binding of IL-2 indicates that Tregs require an ongoing supply of IL-2 for survival. Similarly, the in vivo treatment of mice with either an antibody to neutralize IL-2 or anti-CD25 triggers autoimmune disease [30], [31], [32]. The short-term neutralization of circulating IL-2 by anti-IL-2 monoclonal antibody reduces the number of Tregs in the periphery and elicits autoimmune gastritis in BALB/c mice and diabetes and other autoimmune manifestations in non-obese diabetic (NOD) mice [37]. Furthermore, administration of a relatively ‘‘lower’’ dose of IL-2 (complexed with anti-IL-2) promotes survival of Tregs within islets and retards the develop- ment of diabetes in NOD mice [38] and prevents autoimmunity in IL-22/2/Bim2/2 double KO mice [39]. Likewise, Treg suppressor function in vitro requires that Tregs have intact IL-2 receptors and a Treg-extrinsic supply of IL-2. Tregs suppress proliferation of CD4+CD252 cells as well as their transcription and secretion of IL-2; this Treg suppressor function is blocked by antibody against CD25 [40] or a combination of antibodies against CD25 and CD122 [40], [41]. Tregs that are pre-activated and treated with supplemental IL-2 show 20-fold greater capacity for in vitro suppression compared with Tregs that are pre-activated in the absence of IL-2 [40], [41]. Moreover, though treatment of Tregs with IL-2, IL-7, or IL-15 triggered STAT5 phosphorylation and increased forkhead box protein 3 (Foxp3) in Tregs, only treatment with IL-2 conferred potent suppressor function to Tregs [42]. Taken together these data favor a model whereby Treg suppressor function depends on IL-2 driving the formation of high affinity IL-2R on the Treg cell. Tregs constitutively express all three chains of the IL-2R [27], [43]. However, because Tregs do not produce IL-2 [28], [40] their suppressor function is controlled through IL-2 supplied by a nearby cell [22], [36]. Until now the identity of that IL-2 source has not been conclusively verified. The activated non-Treg CD4+ cell has been touted as a logical candidate IL-2 [37]. This possibility is supported by the fact that in vivo development of Tregs occurs when the IL-2 source is restricted to other non-Treg CD4+ cells [44]. However, other studies have put forth the possibility that the Dendritic Cell IL-2 Required for Treg Function IL-2 source may be a non-T cell [45] such as the dendritic cell [46], [47]. The aim of our in vitro experiments was to determine whether dendritic cells supply IL-2 to Tregs and, if so, the mechanism of that delivery. We demonstrate here that DCs cells are a bona fide source of IL-2 for Tregs, even in the absence of an inflammatory microenvironment. Tregs lose the ability to suppress wild-type CD4+CD252 cells (Teff) when IL-2 from DCs is restricted, either by blocking with anti-CD25 or by substituting DCs from IL-22/2 KO mice. The dendritic cells favor close interaction with Treg and preferentially transfer their IL-2 to Treg via a cell-cell, contact- dependent juxtacrine process. Thus, we identify for the first time the existence of a unique subset of IL-2-producing dendritic cells that provide Tregs with a supply of critical IL-2 needed to gain suppressor function. Furthermore, we show for the first time that dendritic cell IL-2 regulates Treg Foxp3 concentration and suppression function in vitro. These results provide insights that should help inform the design of interventions aimed at enhancing or inhibiting Treg function. Results Small subpopulation of dendritic cells constitutively produces IL-2 The observation from live-cell imaging studies that CD25 localized at the interface between some Treg-DC conjugates (Figure 1) prompted our investigation of DCs as a source of IL-2 to Tregs. We examined bone marrow-derived dendritic cells (BMDCs) and splenic DCs for their capacity to produce IL-2 constitutively. We first confirmed that splenic DCs and bone marrow-derived BMDCs transcribed IL-2 mRNA (not shown), using reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR). Figure 2A demonstrates by flow cytometry that 1.9% of gated CD11c+ BMDCs contained intracytoplasmic IL-2 protein. Likewise, we detected intracytoplasmic IL-2 protein in a similarly small subpopulation of splenic DCs (not shown). Using IL-2 ELISPOT assay and progressively purified DCs, we confirmed that a subset of DCs secreted IL-2 (Figure 2B). Progressive enrichment of CD11c+ cells (61% to 98% after two rounds of magnetic bead selection), increased the percentage of IL- 2-secreting DCs significantly (0.04 to 1% of the DC population). In a series of experiments performed over several months on doubly- selected BMDCs or splenic DCs and shown in Figure 2C, the percent of IL-2-producing BMDCs ranged from 0.7 to 2.4% (mean was 1.3%60.7, N = 7), and the percent of IL-2 producing, splenic DCs ranged from 1.2 to 2.0% (mean was 1.5%60.4, N = 3). There was no significant difference between the percent- ages of IL-2 producing cells from the two sources of dendritic cells in Figure 2C (p = 0.62, t-test). Tregs preserve dendritic cell transcription of IL-2 We then cultured C57BL/6 DCs alone or with alloreactive Tregs from the DO11.10 mouse and measured IL-2 message by RT-qPCR. Figure 3A shows that IL-2 message waned as DCs were cultured alone over time but was significantly higher in cells that were co-cultured with Tregs (p = 0.038, Tukey Test). To corroborate these observations and further confirm that IL-2 was coming from the DC, we transfected BMDCs with an IL-2 promoter-driven reporter plasmid, pIL2p8.4EGFP (Figure 3B, left panel), which uses the 8.4 kb IL-2 promoter fragment to drive enhanced green fluorescent protein (EGFP) transcription. This method allowed us to use flow cytometry to detect individual DCs with endogenous IL-2 promoter activity. After adjusting for the 23% transfection efficiency of the cells (see Figure 3B, right panel), PLOS ONE | www.plosone.org 2 September 2012 | Volume 7 | Issue 9 | e43609