Dendritic Cell IL-2 Required for Treg Function Figure 9. Treg suppression of the MLR-IL-2 response requires dendritic cell IL-2. Panels A–F show Treg suppression of IL-2-secreting cells in primary MLR cultures. MLR cultures of DO11.10 CD4+CD252 cells and allogeneic DCs from WT or IL-22/2 KO littermates were incubated overnight with or without DO11.10 Tregs, washed, and transferred to IL-2-ELISPOT plates to measure IL-2-secreting cells as per materials and methods. A&B: Tregs fail to suppress MLR-IL-2 response in cultures containing IL-22/2 KO DCs. Bars are mean 6 SE IL-2 producing cells/culture from N = 5 independent experiments with splenic DCs (A) and N = 3–4 independent experiments with BMDCs (B). * indicates p,.001 (A) and p,.005 (B) compared with all other conditions, Holm-Sidak method of multiple pairwise comparison. C&D: Anti-CD25 blocks Treg suppression of MLR-IL-2 response. MLR cultures of DO11.10 CD4+CD252 cells and allogeneic WT splenic DCs (C) or BMDCs (D), with or without Treg, were treated with anti- CD25 or isotype control antibody, washed, then transferred to IL-2-ELISPOT plates as in material and methods. C: Symbols are mean 6 SE IL-2 producing cells/culture from N = 6 independent experiments. * indicates p,.001, anti-CD25 versus isotype control, Holm-Sidak method of multiple pairwise comparison. D: Symbols are mean 6 SE IL-2-producing cells of triplicate cultures from one of four representative experiments. * indicates p,.001, Treg treatment groups anti-CD25 versus isotype control, Tukey’s test for all pairwise multiple comparison. E&F: Exogenous IL-2 restores Treg suppression of MLR cultures containing IL-2KO DCs. MLR cultures of DO11.10 CD4+CD252 cells and allogeneic IL-22/2 KO splenic DCs (E) or BMDCs (F), with or without Treg, were treated with exogenous IL-2, washed, then transferred to IL-2 ELISPOT plates as in material and methods. Symbols are mean 6 SE IL-2 producing cells of triplicate cultures from one of three representative experiments. * indicates p,.001, IL-2 treatment DC/CD4eff + Treg versus DC/CD4eff control, Tukey’s test for all pairwise multiple comparison. doi:10.1371/journal.pone.0043609.g009 Our use of the ELISPOT assay, a more sensitive assay than ELISA [60], [61], likely enhanced our ability to detect IL-2- producing dendritic cells in the absence of any stimulation. We did not detect IL-2 in any culture supernatant using the ELISA assay (not shown), whereas we consistently detected IL-2-producing DCs by ELISPOT. We found it necessary to modify the ELISPOT assay to detect IL-2-producing DCs. Culturing the DCs directly in the ELISPOT plates optimized detection of IL-2 spots. No spots were detected if we pre-cultured the DC preparations in tubes overnight and washed the cells before transfer to ELISPOT plates (not shown)—the method used to detect IL-2-producing T cells. Although IL-2-producing DCs were detected, as yet other unique markers for these cells have not been identified. Conversely, two groups were unable to measure IL-2 message or protein in bone marrow-derived dendritic cells, whether or not DCs were matured with LPS [62], [63]. The inconsistent reports of IL-2 production by dendritic cells may be explained by differences in DC culture conditions. Sauma et al. demonstrated that adding IL-4 during the differentiation of bone marrow- derived dendritic cells inhibits IL-2 secretion by the dendritic cells [58]. Of note, investigators who matured bone marrow-derived dendritic cells with IL-4 did not detect IL-2 [62], [63], whereas investigators who omitted IL-4 during differentiation or matura- tion detected IL-2 [56], [57], [58], [59], [64]. Similarly, we omitted IL-4 from our bone marrow-derived culture conditions and detected IL-2-producing dendritic cells. Tregs consume dendritic cell IL-2 The expansion of Tregs depends on a paracrine source of IL-2 [21]. Reports that dendritic cells trigger Treg cell proliferation in vitro in the absence of exogenous IL-2 raise the possibility that dendritic cell IL-2 may control Treg proliferation in vitro and Treg expansion and homeostasis in vivo. Guiducci et al. showed that Treg proliferation in vitro and Treg survival in vivo depends on IL-2 secretion from dendritic cells, which is prompted by CD40- CD40L interaction between dendritic cells and Tregs [47]. Suffner et al. reported that adoptively transferred Tregs divided less in DC-depleted recipient mice [65]. Sgouroudis et al. reported higher IL-2 transcription in LPS-matured BMDCs from the diabetes- protective NOD.B6 Idd3 congenic mouse compared to BMDCs from the diabetes-prone NOD mouse [46]. In vitro, Tregs from either disease-prone or disease-resistant mice proliferate more to NOD.B6 Idd3 BMDCs compared to NOD BMDCs, and anti-IL-2 blocks the proliferation. Moreover, in vivo priming with NOD.B6 Idd3 BMDCs expands Foxp3+ Treg cells in the draining pancreatic lymph node of recipient NOD congenic mice [46]. Yamazaki et al. showed that dendritic cells produce IL-2 and expand Treg cells in culture, but they dismissed dendritic cells as the paracrine source PLOS ONE | www.plosone.org 8 September 2012 | Volume 7 | Issue 9 | e43609