CD25 expression on Tregs and Teff were comparable. The DC:T cells ratio was between 1–1.6:1. Cells were collected from top and bottom wells, blocked with anti-CD16/CD32 (eBioscience) and stained with anti-CD25-PE and Thy1.2-FITC or isotype control antibodies: rat IgG2b, k-FITC, and rat IgG1, l-PE (all eBioscience). Hoechst 33258 (Sigma-Aldrich) was added to discriminate live and dead cells. Cells were analyzed by BD LSR flow cytometer and FlowJo software (Tree Star, Inc.). Mean fluorescent intensity of mCherry and CD25 of Thy1.2high gated cells was expressed as mean 6 SEM. Statistical analysis Data on the frequency of spontaneous IL-2 secreting BMDCs and Treg interference of DC secretion of IL-2 were analyzed using one-way, repeated measures ANOVA and Tukey’s test for single- step, pairwise multiple comparison procedure. Mean fluorescent intensities of Foxp3 levels on Thy1.2-gated Tregs were compared using Student’s t-test, or as specified in results, using SigmaStat References 1. Sakaguchi S, Ono M, Setoguchi R, Yagi H, Hori S, et al. (2006) Foxp3+ CD25+ CD4+ natural regulatory T cells in dominant self-tolerance and autoimmune disease. Immunol Rev 212: 8–27. 2. Wood KJ, Sakaguchi S (2003) Regulatory T cells in transplantation tolerance. Nat Rev Immunol 3: 199–210. 3. Schulze Zur Wiesch J, Thomssen A, Hartjen P, To´th I, Lehmann C, et al. (2011) Comprehensive analysis of frequency and phenotype of T regulatory cells in HIV infection: CD39 expression of FoxP3+ T regulatory cells correlates with progressive disease. J Virol 85: 1287–1297. 4. Curiel TJ (2008) Regulatory T cells and treatment of cancer. Curr Opin Immunol 20: 241–246. 5. Welters MJ, Kenter GG, de Vos van Steenwijk PJ, Lo¨wik MJ, Berends-van der Meer DM, et al. (2010) Success or failure of vaccination for HPV16-positive vulvar lesions correlates with kinetics and phenotype of induced T-cell responses. Proc Natl Acad Sci U S A 107: 11895–11899. 6. Riley JL, June CH, Blazar BR (2009) Human T regulatory cells as therapeutic agents: Take a billion or so of these and call me in the morning. Immunity 30: 656–665. 7. Shuntharalingam G, Perry MR, Ward S, Brett SJ, Castello-Cortes A, et al. (2006) Cytokine storm in a phase 1 trial of the anti-CD28 monoclonal antibody TGN1412. N Engl J Med 355: 1018–1028. 8. Brusko T, Bluestone J (2008) Clinical application of regulatory T cells for treatment of type 1 diabetes and transplantation. Eur J Immunol 38: 931–934. 9. Putnam AL, Brusko TM, Lee MR, Liu W, Szot GL, et al. (2009) Expansion of human regulatory T-cells from patients with type 1 diabetes. Diabetes 58: 652– 662. 10. Battaglia M, Roncarolo MG (2011) Immune intervention with T regulatory cells: past lessons and future perspectives for type 1 diabetes. Semin Immunol 23: 182–194. 11. Feng G, Wood KJ, Bushell A (2008) Interferon-gamma conditioning ex vivo generates CD25+CD62L+Foxp3+ regulatory T cells that prevent allograft rejection: potential avenues for cellular therapy. Transplantation 86: 578–589. 12. Tang Q, Bluestone JA, Kang SM (2012) CD4(+)Foxp3(+) regulatory T cell therapy in transplantation. J Mol Cell Biol 4: 11–21. 13. Edinger M, Hoffmann P (2011) Regulatory T cells in stem cell transplantation: strategies and first clinical experiences. Curr Opin Immunol 23: 679–684. 14. Bruno L, Merkenschlager M (2008) Directing T cell differentiation and function with small molecule inhibitors. Cell Cycle 7: 2296–2298. 15. Brusko TM, Putnam AL, Bluestone JA (2008) Human regulatory T cells: role in autoimmune disease and therapeutic opportunities. Immunol Rev 223: 371–390. 16. Ruter J, Barnett BG, Kryczek I, Brumlik MJ, Daniel BJ, et al. (2009) Altering regulatory T cell function in cancer immunotherapy: a novel means to boost the efficacy of cancer vaccines. Front Biosci 14: 1761–1770. 17. Welters MJ, Piersma SJ, van der Burg SH (2008) T-regulatory cells in tumour- specific vaccination strategies. Expert Opin Biol Ther 8: 1365–1379. 18. Wei S, Kryczek I, Edwards RP, Zou L, Szeliga W, et al. (2007) Interleukin-2 administration alters the CD4+FOXP3+ T-cell pool and tumor trafficking in patients with ovarian carcinoma. Cancer Res 67: 7487–7494. 19. Berntsen A, Brimnes MK, thor Straten P, Svane IM (2010) Increase of circulating CD4+CD25highFoxp3+ regulatory T cells in patients with metastatic renal cell carcinoma during treatment with dendritic cell vaccination and low- dose interleukin-2. J Immunother 33: 425–434. 20. Beyer M, Schumak B, Weihrauch MR, Andres B, Giese T, et al. (2012) In vivo expansion of naive CD4+ CD25(high) FOXP3+ regulatory T cells in patients with colorectal carcinoma after IL-2 administration. PLoS One 7: e30422. 21. Cheng G, Yu A, Malek TR (2011) T-cell tolerance and the multi-functional role of IL-2R signaling in T-regulatory cells. Immunol Rev 241: 63–76. Dendritic Cell IL-2 Required for Treg Function software (San Jose, CA). Data for Treg suppression of MLR-IL-2 response were analyzed using the Holm-Sidak method of multiple pairwise comparisons or Tukey’s test for pairwise comparison. Acknowledgments We acknowledge the following core facilities of The University of Iowa: Flow Cytometry Facility, Central Microscopy, Carver Center for Imaging and DNA Facility. We thank Dr. Nicholas Zavazava for critical discussions, Qizhi Tang for manuscript review, Lauren Jensen for technical assistance, Teresa Ruggle for graphics assistance, and Judith Hower for manuscript editing. The contents do not represent the views of the Department of Veterans Affairs or the United States Government. Author Contributions Conceived and designed the experiments: KK TR MN EF. Performed the experiments: KK TR MN. Analyzed the data: KK TR MN EF. Contributed reagents/materials/analysis tools: EF. Wrote the paper: EF KK TR MN. 22. Barron L, Dooms H, Hoyer KK, Kuswanto W, Hofmann J, et al. (2010) Cutting edge: mechanisms of IL-2-dependent maintenance of functional regulatory T cells. J Immunol 185: 6426–6430. 23. Smith KA (2006) The structure of IL2 bound to the three chains of the IL2 receptor and how signaling occurs. Med Immunol 5: 3. 24. Wang X, Rickert M, Garcia KC (2005) Structure of the quaternary complex of interleukin-2 with its alpha, beta, and gammac receptors. Science 310: 1159– 1163. 25. Stauber DJ, Debler EW, Horton PA, Smith KA, Wilson IA (2006) Crystal structure of the IL-2 signaling complex: paradigm for a heterotrimeric cytokine receptor. Proc Natl Acad Sci U S A 103: 2788–2793. 26. Almeida AR, Legrand N, Papiernik M, Freitas AA (2002) Homeostasis of peripheral CD4+ T cells: IL-2R alpha and IL-2 shape a population of regulatory cells that controls CD4+ T cell numbers. J Immunol 169: 4850–4860. 27. Malek TR, Yu A, Vincek V, Scibelli P, Kong L (2002) CD4 regulatory T cells prevent lethal autoimmunity in IL-2Rbeta-deficient mice. Implications for the nonredundant function of IL-2. Immunity 17: 167–178. 28. Papiernik M, de Moraes ML, Pontoux C, Vasseur F, Pe´nit C (1998) Regulatory CD4 T cells: expression of IL-2R alpha chain, resistance to clonal deletion and IL-2 dependency. Int Immunol 10: 371–378. 29. Soper DM, Kasprowicz DJ, Ziegler SF (2007) IL-2Rbeta links IL-2R signaling with Foxp3 expression. Eur J Immunol 37: 1817–1826. 30. Sadlack B, Lo¨hler J, Schorle H, Klebb G, Haber H, et al. (1995) Generalized autoimmune disease in interleukin-2-deficient mice is triggered by an uncontrolled activation and proliferation of CD4+ T cells. Eur J Immunol 25: 3053–3059. 31. Suzuki H, Ku¨ ndig TM, Furlonger C, Wakeham A, Timms E, et al. (1995) Deregulated T cell activation and autoimmunity in mice lacking interleukin-2 receptor beta. Science 268: 1472–1476. 32. Willerford DM, Chen J, Ferry JA, Davidson L, Ma A, et al. (1995) Interleukin-2 receptor alpha chain regulates the size and content of the peripheral lymphoid compartment. Immunity 3: 521–530. 33. Bayer AL, Yu A, Adeegbe D, Malek TR (2005) Essential role for interleukin-2 for CD4(+)CD25(+) T regulatory cell development during the neonatal period. J Exp Med 201: 769–777. 34. Malek TR (2003) The main function of IL-2 is to promote the development of T regulatory cells. J Leukoc Biol 74: 961–965. 35. Furtado GC, Curotto de Lafaille MA, Kutchukhidze N, Lafaille JJ (2002) Interleukin 2 signaling is required for CD4(+) regulatory T cell function. J Exp Med 196: 851–857. 36. Yu A, Malek TR (2006) Selective availability of IL-2 is a major determinant controlling the production of CD4+CD25+Foxp3+ T regulatory cells. J Immunol 177: 5115–5121. 37. Setoguchi R, Hori S, Takahashi T, Sakaguchi S (2005) Homeostatic maintenance of natural Foxp3(+) CD25(+) CD4(+) regulatory T cells by interleukin (IL)-2 and induction of autoimmune disease by IL-2 neutralization. J Exp Med 201: 723–735. 38. Tang Q, Adams JY, Penaranda C, Melli K, Piaggio E, et al. (2008) Central role of defective interleukin-2 production in the triggering of islet autoimmune destruction. Immunity 28: 687–697. 39. Barron L, Dooms H, Hoyer KK, Kuswanto W, Hofmann J, et al. (2010) Cutting edge: mechanisms of IL-2-dependent maintenance of functional regulatory T cells. J Immunol 185: 6426–6430. 40. Thornton AM, Donovan EE, Piccirillo CA, Shevach EM (2004) Cutting edge: IL-2 is critically required for the in vitro activation of CD4+CD25+ T cell suppressor function. J Immunol 172: 6519–6523. PLOS ONE | www.plosone.org 12 September 2012 | Volume 7 | Issue 9 | e43609