Next steps towards immunotherapeutic applications To build on this understanding, future experiments are needed to determine the phenotype(s) of IL-2-producing dendritic cells and why these cells prefer conjugate formation with the Tregs. A next step to regulate the supply of IL-2 to Tregs will be to elucidate the mechanism that sustains DC transcription of IL-2. Further- more, potential clinical applications of these findings will depend on whether IL-2-producing DCs regulate Treg function in vivo as well as whether similar DCs exist in humans to control Treg function. In vivo studies by Tang et al. support this line of investigation; they found Treg-DC contact preceded the inhibition of Teff cell activation by dendritic cells [51]. Additional studies utilizing DC-lineage-specific IL-2 knock-out and knock-in mice should address the in vivo relevance of our findings. Even so, our in vitro findings warrant the immediate consider- ation of a few practical applications. To our knowledge immunotherapy protocols utilizing dendritic cells that have been manipulated ex vivo to become immunogenic or tolerogenic have not addressed the potential of IL-2-producing DCs to activate immunosuppression by Tregs [74]. We recommend that dendritic cells used in immunotherapy for cancer (or infectious diseases) be routinely screened for IL-2-producing DCs because inadvertent delivery of IL-2-producing DCs may undermine the effectiveness of DC-based therapy by promoting Treg suppressor function. Secondly, because we were able to increase Foxp3 message by co- culturing Tregs with IL-2 transfected DCs, we suggest that this methodology may offer an alternative approach to preferentially expand Tregs over Teff cells ex vivo. Summary We show that a subset of dendritic cells constitutively secretes IL-2. We show this DC-produced IL-2 is delivered to and consumed by natural Tregs in vitro. We also show a juxtacrine mechanism enables the preferential delivery of IL-2 to Tregs, but not to Teff cells. Furthermore, we show that dendritic cell IL-2 is essential for acquisition of Treg suppressor function in vitro. These discoveries expand our knowledge of IL-2 requirement for Treg fitness and function and may help inform future experiments aimed at designing interventions to enhance or inhibit Treg function. Materials and Methods Mice and cell lines C57BL/6, C.CgFoxp3tm2Tch/J, BALB/cJ mice, DO11.10 TCR transgenic breeding pairs and B6.129P2-Il2tm1Hor/J hetero- zygous breeding pairs were purchased from The Jackson Laboratory (Bar Harbor, ME) and bred at The University of Iowa Animal Care Facilities. Wild-type IL-2 sufficient mice (WT) and homozygous IL-22/2 mice were identified from litters by genotyping tail DNA. The transgenic DO11.10 TCR recognizes OVA peptide 323–339 with H-2d [75] and the alloantigen H-2b in the absence of OVA peptide [76], [77]. C57Bl/6-Tg-IL2p8-4 transgenic mice were kindly provided by Drs. M. Yui and E. Rothenberg, California Institute of Technology [78]. C57BL/6- Tg-IL2p8-4 mice have 3 copies of the IL2p8-EGFP transgene and have been useful for tracking IL-2 positive cells through the expression of IL-2 promoter-driven EGFP [79]. All studies were conducted using protocols reviewed and approved #0591101, 0890902 by the Veterans Affairs and The University of Iowa Institutional Animal Care and Use Committee and in accordance with the Guide to the Care and Use of Laboratory Animals and the Animal Welfare Regulations (CFR, Title 9, Chapter 1, Subchapter A). Dendritic Cell IL-2 Required for Treg Function Dendritic cell and CD4+ cell subset purification Dendritic cells were purified from single-cell suspensions of splenocytes or BMDC cultures from indicated mice by one or two passes through AutoMACS using the Pan DC isolation kit (Miltenyi Biotec, Auburn, CA, USA) according to manufacturer’s specifications. Enriched splenic DCs and BMDCs were blocked with anti-CD16/CD32 then stained with CD11c-APC (both eBioscience, San Diego, CA, USA) and analyzed for CD11c+ by flow cytometer. After 2 passes DCs purity (CD11c+) was 97–99%. For BMDCs, bone marrow was harvested from mouse tibias and femurs and grown in complete DMEM (Dulbecco’s modified Eagle’s medium) for 6 d with intermittent feeding every 48 h with 20 ng/mL GM-CSF (Miltenyi Biotec). CD4+CD25+ (Treg) and CD4+CD252 (Teff) populations were obtained from spleen cell suspensions from indicated mice using AutoMACS and the mouse Treg isolation kit (Miltenyi Biotec) according to manufacturer’s specifications. FACS analysis of the subset populations showed CD4+CD25+ purity .97–98%. Dendritic production of IL-2 To detect intracytoplasmic IL-2 protein in dendritic cells, purified C57BL/6 or WT BMDCs were cultured 5 h with golgistop (eBioscience) followed with straining with Live/Dead Fixable Violet dead cell stain kit for 405 nm excitation (Invitrogen, Life Technologies). Cells were surface stained with anti-CD11c- APC, CD8a-FITC, CD205-PE-Cy7 then intracellular stained with anti-IL-2-PE or isotype control Rat IgG2b-PE (all antibodies from eBioscience) and analyzed by flow cytometry. To detect secreted IL-2 protein, purified spleen DCs or BMDCs (104/well) from C57BL/6 were cultured directly in IL-2-coated ELISPOT plates. Whatman ELISPOT plates were pre-coated with IL-2 capture antibody JES6-IA12, 2 mg/ml (eBiosciences) for O/N in 4uC. Plates were blocked with 150 ml/well 1xPBS-BSA 5% for 45 min at room temperature (RT), conditioned with 150 ml/well complete DMEM for 45 min at RT and then washed 4 times with 1XPBS before use. Dendritic cells were then cultured in ELISPOT plates for 24 h at 37uC in complete DMEM media, or in media supplemented with LPS (E Coli strain 0127:B8, 1 mg/ ml, Sigma-Aldrich, St. Louis, MO, USA), CpG-A or CpG-B (each 1 mg/ml and kindly provided by Dr. Petar Lenert, The University of Iowa). In some experiments DO11.10 Tregs were added 1:1 with C57BL/6 splenic DCs and BMDCs with or without anti- CD25 (PC61) or isotype control antibody Rat IgG1 (both BD Biosciences, San Diego, CA, USA), final concentration 3–30 mg/ ml. IL-2 ELISPOT plates were thoroughly washed with 1xPBS 24 h later and IL-2 spots were detected using JES6-5H, 100 ml/ well (eBioscience) for O/N at 4u C, then goat anti-rat Biotin-HRP (Sigma-Alrich) for O/N at 4uC, then AEC (3-amino-9-ethylcar- bazole, Sigma-Aldrich) substrate as described [77]. Spots were counted and analyzed with the ImmunoSpot analyzer (Cellular Technology Limited, Shaker Heights, OH, USA). To detect IL-2 message, total RNA was isolated (RNeasy, Qiagen, Valencia, CA, USA) from C57BL/6 splenic DCs or BMDCs and cDNA was synthesized from 100 ng RNA/sample using Invitrogen SuperScript First strand synthesis system. IL-2 transcription was measured by RT-qPCR on triplicate samples using FAM tagged mouse IL-2 primers (Mm00434256_m1, Applied Biosystems) and ABI PRISM 7700 Sequence Detection System according to manufacturer’s protocols. Samples were standardized for GAPDH VIC-TAMARA and quantification of the gene of interest is given by 2 – delta Ct, where delta Ct is obtained by calculating the difference between Ct of the gene of interest and GAPDH. In one experiment C57BLl/6 splenic DCs and DO11.10 Treg cells were cultured in separate tube or together PLOS ONE | www.plosone.org 10 September 2012 | Volume 7 | Issue 9 | e43609