Dendritic Cell IL-2 Required for Treg Function Figure 4. IL-2-producing dendritic cells form conjugates with Treg cells. BMDCs were transfected with IL-2 reporter plasmid (pIL2P8.4-EGFP) or control plasmids and analyzed for IL-2-promoter activity in cells as in materials and methods. pIL-2P8.4-EGFP-transfected C57BL/6 BMDCs were cultured with DO11.10 Treg cells or DO11.10 CD4+CD252 Teff cells for 40 h. Panels show histograms of the level of IL-2-promoter-EGFP+ activity in gated populations: DCs conjugated with Treg or Teff cells, and non-conjugated cells. doi:10.1371/journal.pone.0043609.g004 Dendritic cell IL-2 is essential for in vitro suppressor function of CD4+CD25+ Tregs The suppressor function of mouse Tregs has been linked to their level of Foxp3. The finding that DC-secreted IL-2 increases Treg expression of Foxp3 (Figure 7B) raised the possibility that DC- secreted IL-2 imparts Treg suppressor function. We directly Figure 5. Tregs interfere with dendritic cell paracrine secretion of IL-2. Treg interference of dendritic cell paracrine secretion of IL-2 depends on CD25. C57BL/6 splenic DCs (left) and BMDCs (right) were cultured in IL-2 ELISPOT plates unstimulated or with CpG-B, with and without DO11.10 Treg cells, anti-CD25, or isotype control, and the number of IL-2 secreting DCs was measured as in materials and methods. Bars are the mean 6 SE of IL-2 secreting DCs in triplicate samples of one of six representative experiments with splenic DCs (left) and one of three representative experiments with BMDCs (right). Statistical analysis by one-way repeated measures ANOVA and Tukey’s test for all pairwise multiple comparison procedure. doi:10.1371/journal.pone.0043609.g005 examined the effect of DC-supplied IL-2 on Treg function by conducting Treg suppressor assays with either DCs from wild-type or IL-22/2 mice. We utilized two kinds of Treg suppressor assays. The first type was the standard Treg suppressor assay, which measures Treg inhibition of proliferation of polyclonally-activated CFSE-labeled conventional CD4+ Teff cells. CD4+ Teff cells proliferate to a similar degree when stimulated with anti-CD3 in the presence of syngeneic DCs from either wild-type or IL-22/2 mice (84% versus 81% of cells proliferated, respectively, Figure 8A and Figure 8B, column A). The addition of syngeneic Tregs to cultures that contain wild-type DCs decreased proliferation of CD4+ Teff cells from 84% to 13% (85% suppression). In contrast, Tregs were minimally suppressive (3% suppression) when added to cultures that contained DCs from IL-22/2 mice. These data indicate that IL-2 from DCs is required for Treg suppressor function, but not for CD3-induced proliferation of CD4+ Teff cells. We performed the Treg suppressor assays in systems using DO11.10 Treg cells stimulated with wild-type or IL-22/2 DCs plus OVA peptide (Figure 8B, column B) and DO11.10 Treg stimulated with wild-type or IL-22/2 DCs (Figure 8B, columns C and D). Across different stimulation protocols, IL-2 KO DCs rendered Tregs less suppressive of CD4+ Teff cell proliferation (42% versus 88%, p = .029, Mann-Whitney Rank Sum Test). We were unable to test the effect of anti-CD25 or exogenous IL-2 on Treg suppressor function in this system because anti-CD25 and exogenous IL-2 alter baseline proliferation of CD3-stimulated CD4+ Teff cells. Utilizing the second type of Treg suppressor assay, we tested Treg-mediated suppression of the late-phase of IL-2 production by CD4+ Teff cells. Though Tregs do not inhibit the early phase of IL-2 production by activated CD4+ Teff cells, Tregs have been shown to truncate IL-2 transcription and suppress the late-phase of IL-2 production by activated CD4+ Teff cells [54], [55]. One advantage of the second suppressor assay is that, because IL-2 PLOS ONE | www.plosone.org 5 September 2012 | Volume 7 | Issue 9 | e43609