at 1:1 for 5 and 24 h. The Tregs and DCs from separate tubes were combined and cells were immediately harvested from cultures for detection of IL-2 message by RT-qPRC. To detect IL-2 promoter activity in dendritic cells, C57BL/6 or WT BMDCs were transfected with the IL-2 promoter-driven EGFP reporter plasmid (pIL2P8.4EGFP, kindly provided by Dr. Ellen Rothenberg, California institute of Technology, Pasadena, CA, [78]), negative control plasmid pcDNA3.1/V5-His B (Invitrogen, Grand Island, NY, USA), and positive control plasmid pAcGFP-N1 (ClonTech Laboratories), using Lonza/ AMAXA-Immature DC Nucleofection Kit (Allendale, NJ, USA) according to manufacturer’s specifications, and 24–40 h later cells were stained with anti-CD11c-PE-Cy7 (eBioscience) and analyzed for GFP+/CD11c+ or EGFP+/CD11c+ double positive cells using a BD LSR flow cytometer and FlowJo software (Tree Star, Inc., Ashland, OR). Transfection efficiency was determined by the percentage of GFP+ DCs in BMDCs that were transfected with pAcGFP-N1 and ranged from 15–35% in all experiments. In some experiments C57BL/6 transfectants were stimulated with CpG-B (1 mg/ml) and/or co-cultured with DO11.10 Treg or DO11.10 CD4+CD252 cells at a 10:1 ratio of Treg:DC or Teff:DC, and cells were stained with anti-CD11c-APC, anti- Thy1.2-PE-Cy7, and anti-CD25-PE (all from eBioscience). Multiparameter gating was used to measure IL-2 promoter- driven EGFP activity in different conjugated and nonconjugated populations. Treg phenotype and function DO11.10 Treg cells were cultured alone in the upper chamber of transwell plates and with WT or IL-22/2 (B6 background) DCs in the lower chamber of transwell plates. Cells were harvested from upper and lower chambers and treated with Live/Dead Fixable Dead Cell Stain (Invitrogen, Life Technologies), blocked with anti-CD16/32 (eBioscience) and stained with anti-Thy1.2- APC/anti-CD25-PE or isotype control Ab Rat IgG2a-APC and Rat IgG1-PE (BD Biosciences), fixed and permeabilized using Foxp3 Staining Buffer Set (eBioscience), and stained with anti- Foxp3-FITC or isotype control Rat IgG2a-FITC (eBioscience). CD25 and Foxp3 were measured on gated live Thy1.2+ cells by flow cytometry. To measure Treg suppression of CD4 T-cell proliferation, na¨ıve conventional CD4+ T cells (CD4+CD252CD62L+) from C57BL/ 6 mice were labeled with CFSE (1 mM, Sigma-Aldrich) and cultured for 4 d at 16105 cells/ml in 12675 mm polystyrene tubes in RPMI complete media with anti-CD3e (0.3 mg/ml, 145- 2C11, eBioscience) along with purified DCs from WT or IL-22/2 (B6 background) mice (2.66104 cells/ml), with or without purified C57BL/6 CD4+CD25+ Treg cells (2.66104 cells/ml). Cells were harvested, blocked with anti-CD16/CD32 then stained with anti- CD11c-APC and anti-CD4-APC-Alexa750 (all from eBioscience). Hoechst 33258 (Sigma-Aldrich) was added for live/dead discrim- ination. Proliferation of CFSE-labeled conventional CD4+ cells (Teff) was measured on BD-LSR II flow cytometer and analyzed using FlowJo software (Tree Star, Inc.). In other experiments DO11.10 Treg (104 cells/ml) and CFSE-labeled Teff cells (105 cells/ml) were cultured with 104 allogeneic DCs from WT and IL- 22/2 (B6 background) mice with or without OVA peptide 323– 339 (100 mM, Sigma-Aldrich) for 4 d in 12675 mm polystyrene tubes in RPMI complete media and proliferation of Teff was measured as above. Proliferation was measured by flow cytometry, and the % of Treg suppression of Teff cell proliferation was calculated by: Dendritic Cell IL-2 Required for Treg Function ½1{(Teff prolif eration with Treg= Teff prolif eration without Treg) X100: To measure Treg suppression of CD4 cell IL-2 production, 105 DO11.10 CD4+CD252 Teff were stimulated with 104 allogeneic DCs from WT and IL-22/2 (B6 background) mice overnight in 96-well plates with or without co-cultured 104 DO11.10 CD4+CD25+ DO11.10 Tregs. In some experiments anti-CD25 (PC61) or isotype control Rat IgG1 (both BD Biosciences) was added (3 to 30 mg/ml). In other experiments recombinant murine IL-2 (eBioscience) was added (100 to 1000 U/ml). Cells were harvested, washed, then replated on IL-2 ELISPOT plates for 24 h. IL-2 spots were developed and quantified by ImmunoSpot (Cellular Technology Limited). Confocal microscopy Foxp3-GFP Treg cells from C.CgFoxp3tm2Tch/J mice were stimulated with anti-CD3 (1 mg/ml) and unlabeled BALB/cJ splenic DCs for 6 h in presence or absence of unlabeled anti- CD25 antibody (PC61, 30 mg/ml). Live cell confocal imaging was performed using Leica TCS SP2 inverted confocal microscope (Leica Microsystems, Wetzlar, Germany). After identifying DC:Treg conjugates, anti-CD25-APC (7D4, Southern Biotec, Birmingham, AL, USA) was added to cultures and conjugates were imaged. Normarski differential interference contract (DIC), GFP and APC channels were collected sequentially at 0.3–0.5 micron z steps. LCS Lite software (Leica Microsystems) was used to quantitate CD25-APC intensity on Treg membrane not contacting and in contact with DC. Tracking and transfer of IL-2 between DCs and Tregs To visualize the transfer of IL-2 between DCs and Tregs by microscopy, BMDCs were transfected with IL-2mCherry or mCherry-expressing plasmids using Lonza/AMAXA-Immature DC Nucleofection Kit according to manufacturer’s specifications. In detail, engineered primers were used to delete the IL-2 gene stop codon, and the amplified product was sub-cloned into P- mCherry-N3 and related plasmids. These plasmids encoding IL-2 fluorescent fusion proteins were transfected into CD11c+ enriched, BMDCs by Amaxa nucleofection. We measured cytokine-induced proliferation of the HT-2 cell line (ATCC# CRL-1841) and CD25 up-regulation on Tregs to confirm that IL-2-mCherry and IL-2 had similar biological activity (not shown). Transfected DCs from BALB/c mice were washed and co-cultured for 24 h with purified and CFSE-labeled DO11.10 CD4+CD25+ Tregs or conventional CD4+ cells (CD4+CD252, Teff) and OVA peptide 323–339 (100 mM, Sigma-Aldrich). Live-cell epifluorescence imaging was performed to track IL-2mCherry uptake by Tregs and Teff utilizing Olympus X-81 microscope with temperature and CO2 environmental controls and SlideBook software (Intel- ligent Imaging Innovations, Denver, CO, USA). To quantify the transfer of IL-2 from DCs to Tregs, BMDCs from IL-22/2 B6 mice were transfected with IL-2mCherry or control mCherry plasmid, washed and co-cultured with purified DO11.10 CD4+CD25+ Tregs or DO11.10 conventional CD4+ cells (CD4+CD252, Teff) in transwell culture plates with or without 30 mg/ml anti-CD25 (PC61, BD Biosciences) for 24 h at 37uC and 5% CO2 in triplicates. DCs were cultured in the lower chamber of the transwells. Tregs or Teff were cultured in both the lower and upper chambers. Anti-CD3 antibody (10 mg/ml) was added to the wells with Teff to further upregulate CD25 expression so that PLOS ONE | www.plosone.org 11 September 2012 | Volume 7 | Issue 9 | e43609