Cell and Tissue Research

, Volume 339, Issue 3, pp 585–595 | Cite as

Inducible regulatory T cells (iTregs) from recent-onset type 1 diabetes subjects show increased in vitro suppression and higher ITCH levels compared with controls

  • Sanja Glisic
  • Sarah Ehlenbach
  • Parthav Jailwala
  • Jill Waukau
  • Srikanta Jana
  • Soumitra Ghosh
Regular Article


CD4+CD25+high regulatory T cells (Tregs) play a pivotal role in the control of the immune response. A growing body of evidence suggests the reduced function of these cells in autoimmune diseases, including type 1 diabetes (T1D). Restoration of their function can potentially delay further disease development. In the present study, we have converted conventional effector T cells into induced Tregs (iTregs) in recent-onset (RO) T1D (n=9) and compared them with the same cells generated in controls (n=12) and in long-standing (LS) T1D subjects (n=9). The functional potential of in-vitro-generated Tregs was measured by using an in vitro proliferation assay. We noted that the suppressive potential of iTregs exceeded that of natural regulatory T cells (nTregs) only in the RO T1D subjects. We showed that iTregs from RO T1D subjects had increased expression of Foxp3, E3 ubiquitin ligase (ITCH) and TGF-β-inducible early gene 1 (TIEG1) compared with control and LS T1D subjects. We also expanded natural, thymically derived Tregs (nTregs) and compared the functional ability of these cells between subject groups. Expanded cells from all three subject groups were suppressive. RO T1D subjects were the only group in which both iTregs and expanded Tregs were functional, suggesting that the inflammatory milieu impacts in vitro Treg generation. Future longitudinal studies should delineate the actual contribution of the stage of disease to the quality of in-vitro-generated Tregs.


Type 1 diabetes Tregs Induced Tregs Expansion E3 ubiquitin ligase Apoptosis Human 



The authors thank Hope Alberts, Jeffrey Woodliff, and Corbett Reynbold for FACS services, Marilyn Koppen and Joanna Kramer for patient recruitment, and Karen Zeqiri for administrative support. They also express their sincere gratitude to all study participants for their generosity and enthusiastic support of this work.


  1. ABI (1997) Relative quantitation of gene expression; ABI prism 7700 sequence detection system. User bulletin no. 2. PE Applied Biosystems, Foster City, USAGoogle Scholar
  2. Allan SE, Crome SQ, Crellin NK, Passerini L, Steiner TS, Bacchetta R, Roncarolo MG, Levings MK (2007) Activation-induced FOXP3 in human T effector cells does not suppress proliferation or cytokine production. Int Immunol 19:345–354CrossRefPubMedGoogle Scholar
  3. Baecher-Allan C, Brown JA, Freeman GJ, Hafler DA (2003) CD4+CD25+ regulatory cells from human peripheral blood express very high levels of CD25 ex vivo. Novartis Found Symp 252:67–88 (discussion 88–91, 106–114)CrossRefPubMedGoogle Scholar
  4. Bai Y, Yang C, Hu K, Elly C, Liu YC (2004) Itch E3 ligase-mediated regulation of TGF-beta signaling by modulating smad2 phosphorylation. Mol Cell 15:825–831CrossRefPubMedGoogle Scholar
  5. Banerjee DK, Dhodapkar MV, Matayeva E, Steinman RM, Dhodapkar KM (2006) Expansion of FOXP3high regulatory T cells by human dendritic cells (DCs) in vitro and after injection of cytokine-matured DCs in myeloma patients. Blood 108:2655–2661CrossRefPubMedGoogle Scholar
  6. Battaglia M, Stabilini A, Roncarolo MG (2005) Rapamycin selectively expands CD4+CD25+FoxP3+ regulatory T cells. Blood 105:4743–4748CrossRefPubMedGoogle Scholar
  7. Battaglia M, Stabilini A, Migliavacca B, Horejs-Hoeck J, Kaupper T, Roncarolo MG (2006) Rapamycin promotes expansion of functional CD4+CD25+FOXP3+ regulatory T cells of both healthy subjects and type 1 diabetic patients. J Immunol 177:8338–8347PubMedGoogle Scholar
  8. Brusko TM, Wasserfall CH, Clare-Salzler MJ, Schatz DA, Atkinson MA (2005) Functional defects and the influence of age on the frequency of CD4+ CD25+ T-cells in type 1 diabetes. Diabetes 54:1407–1414CrossRefPubMedGoogle Scholar
  9. Brusko TZS, Putnam A, Lee M, McClymont S, Liu W, Nishimura M, Koya R, Tree T, Riley J, Bluestone J (2009) Development of engineered antigen-specific human regulatory T cells for the treatment of type 1 diabetes by TCR gene transfer. Clin Immunol 131:S42–S43CrossRefGoogle Scholar
  10. Caruso C, Buffa S, Candore G, Colonna-Romano G, Dunn-Walters D, Kipling D, Pawelec G (2009) Mechanisms of immunosenescence. Immun Ageing 6:10CrossRefPubMedGoogle Scholar
  11. Chen W, Jin W, Hardegen N, Lei KJ, Li L, Marinos N, McGrady G, Wahl SM (2003) Conversion of peripheral CD4+CD25- naive T cells to CD4+CD25+ regulatory T cells by TGF-beta induction of transcription factor Foxp3. J Exp Med 198:1875–1886CrossRefPubMedGoogle Scholar
  12. Danke NA, Koelle DM, Yee C, Beheray S, Kwok WW (2004) Autoreactive T cells in healthy individuals. J Immunol 172:5967–5972PubMedGoogle Scholar
  13. Earle KE, Tang Q, Zhou X, Liu W, Zhu S, Bonyhadi ML, Bluestone JA (2005) In vitro expanded human CD4+CD25+ regulatory T cells suppress effector T cell proliferation. Clin Immunol 115:3–9CrossRefPubMedGoogle Scholar
  14. Elpek KG, Yolcu ES, Franke DD, Lacelle C, Schabowsky RH, Shirwan H (2007) Ex vivo expansion of CD4+CD25+FoxP3+ T regulatory cells based on synergy between IL-2 and 4–1BB signaling. J Immunol 179:7295–7304PubMedGoogle Scholar
  15. Fantini MC, Becker C, Monteleone G, Pallone F, Galle PR, Neurath MF (2004) Cutting edge: TGF-beta induces a regulatory phenotype in CD4+CD25- T cells through Foxp3 induction and down-regulation of Smad7. J Immunol 172:5149–5153PubMedGoogle Scholar
  16. Fantini MC, Becker C, Tubbe I, Nikolaev A, Lehr HA, Galle P, Neurath MF (2006) Transforming growth factor beta induced FoxP3+ regulatory T cells suppress Th1 mediated experimental colitis. Gut 55:671–680CrossRefPubMedGoogle Scholar
  17. Gavin MA, Torgerson TR, Houston E, DeRoos P, Ho WY, Stray-Pedersen A, Ocheltree EL, Greenberg PD, Ochs HD, Rudensky AY (2006) Single-cell analysis of normal and FOXP3-mutant human T cells: FOXP3 expression without regulatory T cell development. Proc Natl Acad Sci USA 103:6659–6664CrossRefPubMedGoogle Scholar
  18. Glisic-Milosavljevic S, Waukau J, Jana S, Jailwala P, Rovensky J, Ghosh S (2005) Comparison of apoptosis and mortality measurements in peripheral blood mononuclear cells (PBMCs) using multiple methods. Cell Prolif 38:301–311CrossRefPubMedGoogle Scholar
  19. Glisic-Milosavljevic S, Waukau J, Jailwala P, Jana S, Khoo HJ, Albertz H, Woodliff J, Koppen M, Alemzadeh R, Hagopian W, Ghosh S (2007a) At-risk and recent-onset type 1 diabetic subjects have increased apoptosis in the CD4+CD25+high T-cell fraction. PLoS ONE 2:e146CrossRefPubMedGoogle Scholar
  20. Glisic-Milosavljevic S, Wang T, Koppen M, Kramer J, Ehlenbach S, Waukau J, Jailwala P, Jana S, Alemzadeh R, Ghosh S (2007b) Dynamic changes in CD4+ CD25+(high) T cell apoptosis after the diagnosis of type 1 diabetes. Clin Exp Immunol 150:75–82PubMedCrossRefGoogle Scholar
  21. Godfrey WR, Ge YG, Spoden DJ, Levine BL, June CH, Blazar BR, Porter SB (2004) In vitro-expanded human CD4(+)CD25(+) T-regulatory cells can markedly inhibit allogeneic dendritic cell-stimulated MLR cultures. Blood 104:453–461CrossRefPubMedGoogle Scholar
  22. Hamdi H, Godot V, Maillot MC, Prejean MV, Cohen N, Krzysiek R, Lemoine FM, Zou W, Emilie D (2007) Induction of antigen-specific regulatory T lymphocytes by human dendritic cells expressing the glucocorticoid-induced leucine zipper. Blood 110:211–219CrossRefPubMedGoogle Scholar
  23. Herold KC, Hagopian W, Auger JA, Poumian-Ruiz E, Taylor L, Donaldson D, Gitelman SE, Harlan DM, Xu D, Zivin RA, Bluestone JA (2002) Anti-CD3 monoclonal antibody in new-onset type 1 diabetes mellitus. N Engl J Med 346:1692–1698CrossRefPubMedGoogle Scholar
  24. Hoffmann P, Eder R, Kunz-Schughart LA, Andreesen R, Edinger M (2004) Large-scale in vitro expansion of polyclonal human CD4(+)CD25high regulatory T cells. Blood 104:895–903CrossRefPubMedGoogle Scholar
  25. Hori S, Sakaguchi S (2004) Foxp3: a critical regulator of the development and function of regulatory T cells. Microbes Infect 6:745–751CrossRefPubMedGoogle Scholar
  26. Huter EN, Stummvoll GH, DiPaolo RJ, Glass DD, Shevach EM (2008) Cutting edge: antigen-specific TGF beta-induced regulatory T cells suppress Th17-mediated autoimmune disease. J Immunol 181:8209–8213PubMedGoogle Scholar
  27. Jaeckel E, Boehmer H von, Manns MP (2005) Antigen-specific FoxP3-transduced T-cells can control established type 1 diabetes. Diabetes 54:306–310CrossRefPubMedGoogle Scholar
  28. Lindley S, Dayan CM, Bishop A, Roep BO, Peakman M, Tree TI (2005) Defective suppressor function in CD4(+)CD25(+) T-cells from patients with type 1 diabetes. Diabetes 54:92–99CrossRefPubMedGoogle Scholar
  29. Levings MK, Sangregorio R, Roncarolo MG (2001) Human cd25(+)cd4(+) T regulatory cells suppress naive and memory T cell proliferation and can be expanded in vitro without loss of function. J Exp Med 193:1295–1302CrossRefPubMedGoogle Scholar
  30. Long SA, Buckner JH (2008) Combination of rapamycin and IL-2 increases de novo induction of human CD4(+)CD25(+)FOXP3(+) T cells. J Autoimmun 30:293–302CrossRefPubMedGoogle Scholar
  31. Ludewig B, Junt T, Hengartner H, Zinkernagel RM (2001) Dendritic cells in autoimmune diseases. Curr Opin Immunol 13:657–662CrossRefPubMedGoogle Scholar
  32. Marie JC, Letterio JJ, Gavin M, Rudensky AY (2005) TGF-beta1 maintains suppressor function and Foxp3 expression in CD4+CD25+ regulatory T cells. J Exp Med 201:1061–1067CrossRefPubMedGoogle Scholar
  33. Melino G, Gallagher E, Aqeilan RI, Knight R, Peschiaroli A, Rossi M, Scialpi F, Malatesta M, Zocchi L, Browne G, Ciechanover A, Bernassola F (2008) Itch: a HECT-type E3 ligase regulating immunity, skin and cancer. Cell Death Differ 15:1103–1112CrossRefPubMedGoogle Scholar
  34. Morgan RA, Dudley ME, Wunderlich JR, Hughes MS, Yang JC, Sherry RM, Royal RE, Topalian SL, Kammula US, Restifo NP, Zheng Z, Nahvi A, Vries CR de, Rogers-Freezer LJ, Mavroukakis SA, Rosenberg SA (2006) Cancer regression in patients after transfer of genetically engineered lymphocytes. Science 314:126–129CrossRefPubMedGoogle Scholar
  35. Park HB, Paik DJ, Jang E, Hong S, Youn J (2004) Acquisition of anergic and suppressive activities in transforming growth factor-beta-costimulated CD4+CD25- T cells. Int immunol 16:1203–1213CrossRefPubMedGoogle Scholar
  36. Peters JH, Hilbrands LB, Koenen HJ, Joosten I (2008) Ex vivo generation of human alloantigen-specific regulatory T cells from CD4(pos)CD25(high) T cells for immunotherapy. PLoS ONE 3:e2233CrossRefPubMedGoogle Scholar
  37. Putnam AL, Brusko TM, Lee MR, Liu W, Szot GL, Ghosh T, Atkinson MA, Bluestone JA (2008) Expansion of human regulatory T cells from patients with type 1 diabetes. Diabetes 58:652–662CrossRefPubMedGoogle Scholar
  38. Riley JL, June CH, Blazar BR (2009) Human T regulatory cell therapy: take a billion or so and call me in the morning. Immunity 30:656–665CrossRefPubMedGoogle Scholar
  39. Roederer M (2001) Spectral compensation for flow cytometry: visualization artifacts, limitations, and caveats. Cytometry 45:194–205CrossRefPubMedGoogle Scholar
  40. Schubert LA, Jeffery E, Zhang Y, Ramsdell F, Ziegler SF (2001) Scurfin (FOXP3) acts as a repressor of transcription and regulates T cell activation. J Biol Chem 276:37672–37679CrossRefPubMedGoogle Scholar
  41. Skapenko A, Kalden JR, Lipsky PE, Schulze-Koops H (2005) The IL-4 receptor alpha-chain-binding cytokines, IL-4 and IL-13, induce forkhead box P3-expressing CD25+CD4+ regulatory T cells from CD25-CD4+ precursors. J Immunol 175:6107–6116PubMedGoogle Scholar
  42. Tang Q, Henriksen KJ, Bi M, Finger EB, Szot G, Ye J, Masteller EL, McDevitt H, Bonyhadi M, Bluestone JA (2004) In vitro-expanded antigen-specific regulatory T cells suppress autoimmune diabetes. J Exp Med 199:1455–1465CrossRefPubMedGoogle Scholar
  43. Tran DQ, Ramsey H, Shevach EM (2007) Induction of FOXP3 expression in naive human CD4+FOXP3 T cells by T-cell receptor stimulation is transforming growth factor-beta dependent but does not confer a regulatory phenotype. Blood 110:2983–2990CrossRefPubMedGoogle Scholar
  44. Tu W, Lau YL, Zheng J, Liu Y, Chan PL, Mao H, Dionis K, Schneider P, Lewis DB (2008) Efficient generation of human alloantigen-specific CD4+ regulatory T cells from naive precursors by CD40-activated B cells. Blood 112:2554–2562CrossRefPubMedGoogle Scholar
  45. Venuprasad K, Huang H, Harada Y, Elly C, Subramaniam M, Spelsberg T, Su J, Liu YC (2008) The E3 ubiquitin ligase Itch regulates expression of transcription factor Foxp3 and airway inflammation by enhancing the function of transcription factor TIEG1. Nat Immunol 9:245–253CrossRefPubMedGoogle Scholar
  46. von Herrath M, Sanda S, Herold K (2007) Type 1 diabetes as a relapsing-remitting disease? Nat Rev Immunol 7:988–994CrossRefGoogle Scholar
  47. Waldron-Lynch F, Herold KC (2009) Advances in Type 1 diabetes therapeutics: immunomodulation and beta-cell salvage. Endocrinol Metab Clin North Am 38:303–317CrossRefPubMedGoogle Scholar
  48. Walker MR, Kasprowicz DJ, Gersuk VH, Benard A, Van Landeghen M, Buckner JH, Ziegler SF (2003) Induction of FoxP3 and acquisition of T regulatory activity by stimulated human CD4+CD25- T cells. J Clin Invest 112:1437–1443PubMedGoogle Scholar
  49. Walker MR, Carson BD, Nepom GT, Ziegler SF, Buckner JH (2005) De novo generation of antigen-specific CD4+CD25+ regulatory T cells from human CD4+CD25- cells. Proc Natl Acad Sci USA 102:4103–4108CrossRefPubMedGoogle Scholar
  50. Wang C, Wen T, Routy JP, Bernard NF, Sekaly RP, Watts TH (2007a) 4–1BBL induces TNF receptor-associated factor 1-dependent Bim modulation in human T cells and is a critical component in the costimulation-dependent rescue of functionally impaired HIV-specific CD8 T cells. J Immunol 179:8252–8263PubMedGoogle Scholar
  51. Wang J, Ioan-Facsinay A, Voort EI van der, Huizinga TW, Toes RE (2007b) Transient expression of FOXP3 in human activated nonregulatory CD4+ T cells. Eur J Immunol 37:129–138CrossRefPubMedGoogle Scholar
  52. Yagi H, Nomura T, Nakamura K, Yamazaki S, Kitawaki T, Hori S, Maeda M, Onodera M, Uchiyama T, Fujii S, Sakaguchi S (2004) Crucial role of FOXP3 in the development and function of human CD25+CD4+ regulatory T cells. Int Immunol 16:1643–1656CrossRefPubMedGoogle Scholar
  53. Zheng SG, Gray JD, Ohtsuka K, Yamagiwa S, Horwitz DA (2002) Generation ex vivo of TGF-beta-producing regulatory T cells from CD4+CD25- precursors. J Immunol 169:4183–4189PubMedGoogle Scholar
  54. Zheng SG, Wang JH, Gray JD, Soucier H, Horwitz DA (2004) Natural and induced CD4+CD25+ cells educate CD4+CD25- cells to develop suppressive activity: the role of IL-2, TGF-beta, and IL-10. J Immunol 172:5213–5221PubMedGoogle Scholar

Copyright information

© Springer-Verlag 2010

Authors and Affiliations

  • Sanja Glisic
    • 1
  • Sarah Ehlenbach
    • 1
  • Parthav Jailwala
    • 1
  • Jill Waukau
    • 1
  • Srikanta Jana
    • 1
  • Soumitra Ghosh
    • 1
  1. 1.Max McGee National Center for Juvenile Diabetes and Human Molecular Genetic Center, Department of PediatricsMedical College of Wisconsin, Children’s Hospital of WisconsinMilwaukeeUSA

Personalised recommendations