Inflammation Research

, Volume 62, Issue 2, pp 201–212 | Cite as

The effect of ionizing radiation on the homeostasis and functional integrity of murine splenic regulatory T cells

  • Andrea Balogh
  • Eszter Persa
  • Enikő Noémi Bogdándi
  • Anett Benedek
  • Hargita Hegyesi
  • Géza Sáfrány
  • Katalin Lumniczky
Original Research Paper



Radiotherapy affects antitumor immune responses; therefore, it is important to study radiation effects on various compartments of the immune system. Here we report radiation effects on the homeostasis and function of regulatory T (Treg) cells, which are important in down-regulating antitumor immune responses.


C57Bl/6 mice were irradiated with 2 Gy and alterations in splenic lymphocyte fractions analyzed at different intervals.


Total CD4+ numbers showed stronger decrease after irradiation than CD4+Foxp3+ Tregs. Tregs were less prone to radiation-induced apoptosis than CD4+Foxp3− T cells. The ratio of CD4+Foxp3− and CD4+Foxp3+ fractions within the proliferating CD4+ pool progressively changed from 74:26 in control animals to 59:41 eleven days after irradiation, demonstrating a more dynamic increase in the proliferation and regeneration of the Treg pool. The CD4+Foxp3+ fraction expressing cell-surface CTLA4, an antigen associated with Treg cell activation increased from 5.3 % in unirradiated mice to 10.5 % three days after irradiation. The expression of IL-10 mRNA was moderately upregulated, while TGF-β expression was not affected. On the other hand, irradiation reduced Treg capacity to suppress effector T cell proliferation by 2.5-fold.


Tregs are more radioresistant, less prone to radiation-induced apoptosis, and have faster repopulation kinetics than CD4+Foxp3− cells, but irradiated Tregs are functionally compromised, having a reduced suppressive capacity.


Regulatory T cells Irradiation Apoptosis Ki67 CTLA4 



The authors wish to thank Dr. Serge Candeias for the careful reading of the manuscript and for helpful suggestions in manuscript writing. The authors thank the expert technical assistance of Ms. Erzsébet Fekete. This work was supported by the following grants: European Union FP6-036465/2006 (NOTE), European Union FP7- CEREBRAD-295552/2011 and Hungarian OTKA (Hungarian Scientific Research Fund) K77766 and ETT (Medical Research Council) 827-1/2009.


  1. 1.
    Formenti SC, Demaria S. Systemic effects of local radiotherapy. Lancet Oncol. 2009;10:718–26.PubMedCrossRefGoogle Scholar
  2. 2.
    Reits EA, Hodge JW, Herberts CA, Groothuis TTA, Chakraborty M, et al. Radiation modulates the peptide repertoire, enhances MHC class I expression, and induces successful antitumor immunotherapy. J Exp Med. 2006;203:1259–71.PubMedCrossRefGoogle Scholar
  3. 3.
    Biswas S, Guix M, Rinehart C, Dugger TC, Chytil A, et al. Inhibition of TGF-beta with neutralizing antibodies prevents radiation-induced acceleration of metastatic cancer progression. J Clin Invest. 2007;117:1305–13.PubMedCrossRefGoogle Scholar
  4. 4.
    Demaria S, Ng B, Devitt ML, Babb JS, Kawashima N, et al. Ionizing radiation inhibition of distant untreated tumors (abscopal effect) is immune mediated. Int J Radiat Oncol Biol Phys. 2004;58:862–70.PubMedCrossRefGoogle Scholar
  5. 5.
    Shiku H. Importance of CD4+ Helper T-cells in antitumor immunity. Int J Hematol. 2003;77:435–8.PubMedCrossRefGoogle Scholar
  6. 6.
    Corthay A, Skovseth DK, Lundin KU, Rosjo E, Omholt H, et al. Primary antitumor immune response mediated by CD4+ T cells. Immunity. 2005;22:371–83.PubMedCrossRefGoogle Scholar
  7. 7.
    Hung K, Hayashi R, Lafond-Walker A, Lowenstein C, et al. The central role of CD4+ T cells in the antitumor immune response. J Exp Med. 1998;188:2357–68.PubMedCrossRefGoogle Scholar
  8. 8.
    North RJ. Radiation-induced, immunologically mediated regression of an established tumor as an example of successful therapeutic immunomanipulation. Preferential elimination of suppressor T cells allows sustained production of effector T cells. J Exp Med. 1986;164:1652–66.PubMedCrossRefGoogle Scholar
  9. 9.
    Sakaguchi S, Fukuma K, Kuribayashi K, Masuda T. Organ-specific autoimmune diseases induced in mice by elimination of T cell subset. J Exp Med. 1985;161:72–87.PubMedCrossRefGoogle Scholar
  10. 10.
    Itoh M, Takahashi T, Sakaguchi N, Kuniyasu Y, Shimizu J, et al. Thymus and autoimmunity: production of CD4+CD25+ naturally anergic and suppressive T cells as a key function of the thymus in maintaining immunologic self tolerance. J Immunol. 1999;162:5317–26.PubMedGoogle Scholar
  11. 11.
    Cvetanovich GL, Hafler DA. Human regulatory T cells in autoimmune diseases. Curr Opin Immunol. 2010;22:753–60.PubMedCrossRefGoogle Scholar
  12. 12.
    Costantino CM, Baecher-Allan CM, Hafler DA. Human regulatory T cells and autoimmunity. Eur J Immunol. 2008;38:921–4.PubMedCrossRefGoogle Scholar
  13. 13.
    Wood KJ, Sakaguchi S. Regulatory T cells in transplantation tolerance. Nat Rev Immunol. 2003;3:199–210.PubMedCrossRefGoogle Scholar
  14. 14.
    Wang HY, Wang RF. Regulatory T cells and cancer. Curr Opin Immunol. 2007;19:217–23.PubMedCrossRefGoogle Scholar
  15. 15.
    Nishikawa H, Sakaguchi S. Regulatory T cells in tumor immunity. Int J Cancer. 2010;15:759–67.Google Scholar
  16. 16.
    Awwad M, North RJ. Sublethal, whole-body ionizing irradiation can be tumor promotive or tumor destructive depending on the stage of development of underlying antitumor immunity. Cancer Immunol Immunother. 1988;26:55–60.PubMedCrossRefGoogle Scholar
  17. 17.
    Dunn PL, North RJ. Selective radiation resistance of immunologically induced T cells as the basis for irradiation-induced T-cell-mediated regression of immunogenic tumor. J Leukoc Biol. 1991;49:388–96.PubMedGoogle Scholar
  18. 18.
    Nisco SJ, Hissink RJ, Vriens PW, Hoyt EG, Reitz BA, et al. In vivo studies of the maintenance of peripheral transplant tolerance after cyclosporine. Radiosensitive antigen-specific suppressor cells mediate lasting graft protection against primed effector cells. Transplantation. 1995;59:1444–52.PubMedCrossRefGoogle Scholar
  19. 19.
    Cao M, Cabrera R, Xu Y, Liu C, Nelson D. Different radiosensitivity of CD4+CD25+ regulatory T cells and effector T cells to low dose gamma irradiation in vitro. Int J Radiat Biol. 2011;87:71–80.PubMedCrossRefGoogle Scholar
  20. 20.
    Komatsu N, Hori S. Full restoration of peripheral Foxp3 regulatory T cell pool by radioresistant host cells in scurfy bone marrow chimeras. PNAS. 2007;104:8959–64.PubMedCrossRefGoogle Scholar
  21. 21.
    Qu Y, Jin S, Zhang A, Zhang B, Shi X, et al. Gamma-ray resistance of regulatory CD4+CD25+Foxp3+ T cells in mice. Radiat Res. 2010;173:148–57.PubMedCrossRefGoogle Scholar
  22. 22.
    Qu Y, Zhang B, Liu S, Zhang A, Wu T, et al. 2-Gy whole-body irradiation significantly alters the balance of CD4+CD25- T effector cells and CD4+CD25+Foxp3+ T regulatory cells in mice. Cell Mol Immunol. 2010;7:419–27.PubMedCrossRefGoogle Scholar
  23. 23.
    Bogdándi EN, Balogh A, Felgyinszki N, Szatmári T, Persa E, et al. Effects of low-dose radiation on the immune system of mice after total-body irradiation. Radiat Res. 2010;174:480–9.PubMedCrossRefGoogle Scholar
  24. 24.
    Takahashi T, Tagami T, Yamazaki S, Uede T, Shimizu J, et al. Immunologic self-tolerance maintained by CD25(+)CD4(+) regulatory T cells constitutively expressing cytotoxic T lymphocyte-associated antigen 4. J Exp Med. 2000;192:303–10.PubMedCrossRefGoogle Scholar
  25. 25.
    Tran DQ, Andersson J, Wang R, Ramsey H, Unutmaz D, et al. GARP (LRRC32) is essential for the surface expression of latent TGF-b on platelets and activated FOXP3+ regulatory T cells. PNAS. 2009;106:13445–50.PubMedCrossRefGoogle Scholar
  26. 26.
    Wang R, Kozhaya L, Mercer F, Khaitan A, Fujii H, et al. Expression of GARP selectively identifies activated human FOXP3+ regulatory T cells. PNAS. 2009;106:13439–44.PubMedGoogle Scholar
  27. 27.
    Matsumura S, Wang B, Kawashima N, Braunstein S, Badura M, et al. Radiation-induced CXCL16 release by breast cancer cells attracts effector T cells. J Immunol. 2008;181:3099–107.PubMedGoogle Scholar
  28. 28.
    Lugade AA, Moran JP, Gerber SA, Rose RC, Frelinger JG, et al. Local radiation therapy of B16 melanoma tumors increases the generation of tumor antigen-specific effector cells that traffic to the tumor. J Immunol. 2005;174:7516–23.PubMedGoogle Scholar
  29. 29.
    Timke C, Winnenthal HS, Klug F, Roeder FF, Bonertz A, et al. Randomized controlled phase I/II study to investigate immune stimulatory effects by low dose radiotherapy in primarily operable pancreatic cancer. BMC Cancer. 2011;11:134.PubMedCrossRefGoogle Scholar
  30. 30.
    Tatsuta K, Tanaka S, Tajiri T, Shibata S, Komaru A, et al. Complete elimination of established neuroblastoma by synergistic action of gamma-irradiation and DCs treated with rSeV expressing interferon-beta gene. Gene Ther. 2009;16:240–51.PubMedCrossRefGoogle Scholar
  31. 31.
    Wang YS, Tsang YW, Chi CH, Chang CC, Chu RM, et al. Synergistic anti-tumor effect of combination radio- and immunotherapy by electro-gene therapy plus intra-tumor injection of dendritic cells. Cancer Lett. 2008;266:275–85.PubMedCrossRefGoogle Scholar
  32. 32.
    Teitz-Tennenbaum S, Li Q, Rynkiewicz S, Ito F, Davis MA, et al. Radiotherapy potentiates the therapeutic efficacy of intratumoral dendritic cell administration. Cancer Res. 2003;63:8466–75.PubMedGoogle Scholar
  33. 33.
    Safwat A, Aggerholm N, Roitt I, Overgaard J, Hokland M. Low-dose total body irradiation augments the therapeutic effect of interleukin-2 in a mouse model for metastatic malignant melanoma. J Exp Ther Oncol. 2003;3:161–8.PubMedCrossRefGoogle Scholar
  34. 34.
    Lumniczky K, Désaknai S, Mangel L, Szende B, Hamada H, et al. Local tumor irradiation augments the anti-tumor effect of cytokine producing autologous cancer cell vaccines in a murine glioma model. Cancer Gene Ther. 2002;9:44–52.PubMedCrossRefGoogle Scholar
  35. 35.
    Lee WC, Wu TJ, Chou HS, Yu MC, Hsu PY, et al. The impact of CD4+CD25+ T cells in the tumor microenvironment of hepatocellular carcinoma. Surgery. 2012;151:213–22.PubMedCrossRefGoogle Scholar
  36. 36.
    Yamagami W, Susumu N, Tanaka H, Hirasawa A, Banno K, et al. Immunofluorescence-detected infiltration of CD4+FOXP3+ regulatory T cells is relevant to the prognosis of patients with endometrial cancer. Int J Gynecol Cancer. 2011;21:1628–34.PubMedCrossRefGoogle Scholar
  37. 37.
    Winerdal ME, Marits P, Winerdal M, Hasan M, Rosenblatt R, et al. FOXP3 and survival in urinary bladder cancer. BJU Int. 2011;108:1672–8.PubMedCrossRefGoogle Scholar
  38. 38.
    Tao H, Mimura Y, Aoe K, Kobayashi S, Yamamoto H, et al. Prognostic potential of FOXP3 expression in non-small cell lung cancer cells combined with tumor-infiltrating regulatory T cells. Lung Cancer. 2012;75:95–101.PubMedCrossRefGoogle Scholar
  39. 39.
    Knol AC, Nguyen JM, Quéreux G, Brocard A, Khammari A, et al. Prognostic value of tumor-infiltrating Foxp3+ T-cell subpopulations in metastatic melanoma. Exp Dermatol. 2011;20:430–4.PubMedCrossRefGoogle Scholar
  40. 40.
    Agarwalla P, Barnard Z, Fecci P, Dranoff G, Curry WT Jr. Sequential immunotherapy by vaccination With GM-CSF-expressing glioma cells and CTLA-4 blockade effectively treats established murine intracranial tumors. J Immunother. 2012;35:385–9.PubMedCrossRefGoogle Scholar
  41. 41.
    Sundstedt A, Celander M, Eriksson H, Törngren M, Hedlund G. Monotherapeutically nonactive CTLA-4 blockade results in greatly enhanced antitumor Effects when combined with tumor-targeted superantigens in a B16 melanoma model. J Immunother. 2012;35:344–53.PubMedCrossRefGoogle Scholar
  42. 42.
    Curran MA, Kim M, Montalvo W, Al-Shamkhani A, Allison JP. Combination CTLA-4 blockade and 4–1BB activation enhances tumor rejection by increasing T-cell infiltration, proliferation, and cytokine production. PLoS ONE. 2011;6:e19499.PubMedCrossRefGoogle Scholar
  43. 43.
    Calabrò L, Danielli R, Sigalotti L, Maio M. Clinical studies with anti-CTLA-4 antibodies in non-melanoma indications. Semin Oncol. 2010;37:460–7.PubMedCrossRefGoogle Scholar
  44. 44.
    Scheier B, Amaria R, Lewis K, Gonzalez R. Novel therapies in melanoma. Immunotherapy. 2011;3:1461–9.PubMedCrossRefGoogle Scholar
  45. 45.
    Fong L, Kwek SS, O’Brien S, Kavanagh B, McNeel DG, et al. Potentiating endogenous antitumor immunity to prostate cancer through combination immunotherapy with CTLA4 blockade and GM-CSF. Cancer Res. 2009;69:609–15.PubMedCrossRefGoogle Scholar
  46. 46.
    Bayer AL, Jones M, Chirinos J, de Armas L, Schreiber TH, et al. Host CD4+CD25+ T cells can expand and comprise a major component of the Treg compartment after experimental HCT. Blood. 2009;113:733–43.PubMedCrossRefGoogle Scholar
  47. 47.
    Gridley DS, Luo-Owen X, Rizvi A, Makinde AY, Pecaut MJ, et al. Low-dose photon and simulated solar particle event proton effects on Foxp3+ T regulatory cells and other leukocytes. Technol Cancer Res Treat. 2010;9:637–49.PubMedGoogle Scholar
  48. 48.
    Lemon JA, Rollo CD, McFarlane NM, Boreham DR. Radiation-induced apoptosis in mouse lymphocytes is modified by a complex dietary supplement: the effect of genotype and gender. Mutagenesis. 2008;23:465–72.PubMedCrossRefGoogle Scholar
  49. 49.
    Cui YF, Gao YB, Yang H, Xiong CQ, Xia GW, et al. Apoptosis of circulating lymphocytes induced by whole body gamma-irradiation and its mechanism. J Environ Pathol Toxicol Oncol. 1999;18:185–9.PubMedGoogle Scholar
  50. 50.
    Mohamood AS, Trujillo CJ, Zheng D, Jie C, Murillo FM, et al. Gld mutation of Fas ligand increases the frequency and up-regulates cell survival genes in CD251CD41 TR cells. Int Immunol. 2006;18:1265–77.PubMedCrossRefGoogle Scholar
  51. 51.
    Fritzsching B, Oberle N, Eberhardt N, Quick S, Haas J, et al. In contrast to effector T cells, CD4+CD25+FoxP3+ regulatory T cells are highly susceptible to CD95 ligand- but not to TCR-mediated cell death. J. Immunol. 2005;175(32–36):52.Google Scholar
  52. 52.
    Fujimori Y, Saheki K, Itoi H, Okamamoto T, Kakishita E. Increased expression of Fas (APO-1, CD95) on CD4+ and CD8+ T lymphocytes during total body irradiation. Acta Haematol. 2000;104:193–6.PubMedCrossRefGoogle Scholar
  53. 53.
    Curotto de Lafaille MA, Lino AC, Kutchukhidze N, Lafaille JJ. CD25− T cells generate CD25+Foxp3+ regulatory T Cells by peripheral expansion. J Immunol. 2004;173:7259–68.PubMedGoogle Scholar
  54. 54.
    Haribhai M, Lin W, Edwards B, Ziegelbauer J, Salzman NH, et al. A central role for induced regulatory T cells in tolerance induction in experimental colitis. J Immunol. 2009;182:3461–8.PubMedCrossRefGoogle Scholar
  55. 55.
    Suffner J, Hochweller K, Kühnle MC, Li X, Kroczek RA, et al. Dendritic cells support homeostatic expansion of Foxp3+ regulatory T cells in Foxp3.LuciDTR mice. J Immunol. 2010;184:1810–20.PubMedCrossRefGoogle Scholar
  56. 56.
    Amundson SA, Do KT, Shahab S, Bittner M, Meltzer P, et al. Identification of potential mRNA biomarkers in peripheral blood lymphocytes for human exposure to ionizing radiation. Radiat Res. 2000;154:342–6.PubMedCrossRefGoogle Scholar
  57. 57.
    Han SK, Song JY, Yun YS, Yi SY. Gamma irradiation-reduced IFN-gamma expression, STAT1 signals, and cell-mediated immunity. J Biochem Mol Biol. 2002;35:583–9.PubMedCrossRefGoogle Scholar
  58. 58.
    Gridley DS, Dutta-Roy R, Andres ML, Nelson GA, Pecaut MJ. Acute effects of iron-particle radiation on immunity. Part II: Leukocyte activation, cytokines and adhesion. Radiat Res. 2006;165:78–87.PubMedCrossRefGoogle Scholar
  59. 59.
    Dummer CD, Carpio VN, Gonçalves LF, Manfro RC, Veronese FV. FOXP3+ regulatory T cells: from suppression of rejection to induction of renal allograft tolerance. Transpl Immunol. 2012;26:1–10.PubMedCrossRefGoogle Scholar
  60. 60.
    Askenasy N, Kaminitz A, Yarkoni S. Mechanisms of T regulatory cell function. Autoimmun Rev. 2008;7:370–5.PubMedCrossRefGoogle Scholar
  61. 61.
    Billiard F, Buard V, Benderitter M, Linard C. Abdominal γ-radiation induces an accumulation of function-impaired regulatory T cells in the small intestine. Int J Radiat Oncol Biol Phys. 2011;80:869–76.PubMedCrossRefGoogle Scholar
  62. 62.
    Oida T, Weiner HL. TGF-beta induces surface LAP expression on murine CD4 T cells independent of Foxp3 induction. PLoS ONE. 2010;5:15523.CrossRefGoogle Scholar
  63. 63.
    Wing K, Fehervari Z, Sakaguchi S. Emerging possibilities in the development and function of regulatory T cells. Int Immunol. 2006;18:991–1000.PubMedCrossRefGoogle Scholar
  64. 64.
    Wing K, Onishi Y, Prieto-Martin P, Yamaguchi T, Miyara M, et al. CTLA-4 control over Foxp3+ regulatory T cell function. Science. 2008;322:271–5.PubMedCrossRefGoogle Scholar
  65. 65.
    Read S, Malmstrom V, Powrie F. Cytotoxic T lymphocyte-associated antigen 4 plays an essential role in the function of CD25(+)CD4(+) regulatory cells that control intestinal inflammation. J Exp Med. 2000;192:295–302.PubMedCrossRefGoogle Scholar
  66. 66.
    Cao M, Cabrera R, Xu Y, Liu C, Nelson D. Gamma irradiation alters the phenotype and function of CD4+CD25+ regulatory T cells. Cell Biol Int. 2009;33:565–71.PubMedCrossRefGoogle Scholar
  67. 67.
    Schmidt A, Oberle N, Krammer PH. Molecular mechanisms of Treg-mediated T cell suppression. Front Immunol. 2012;3:51.PubMedGoogle Scholar
  68. 68.
    Baecher-Allan C, Brown JA, Freeman GJ, Hafler DA. CD4+CD25high regulatory cells in human peripheral blood. J Immunol. 2001;167:1245–53.PubMedGoogle Scholar
  69. 69.
    Levings MK, Sangregorio R, Roncarolo MG. Human cd25(+)cd4(+) t regulatory cells suppress naïve and memory T cell proliferation and can be expanded in vitro without loss of function. J Exp Med. 2001;193:1295–302.PubMedCrossRefGoogle Scholar
  70. 70.
    Schmidt A, Oberle N, Weiß EM, Vobis D, Frischbutter S, et al. Human regulatory T cells rapidly suppress T cell receptor-induced Ca2+, NF-kB, and NFAT signaling in conventional T cells. Sci Signal. 2011;4:ra90.PubMedCrossRefGoogle Scholar
  71. 71.
    Birebent B, Lorho R, Lechartier H, de Guibert S, Alizadeh M, et al. Suppressive properties of human CD4+CD25+ regulatory T cells are dependent on CTLA4 expression. Eur J Immunol. 2004;34:3485–96.PubMedCrossRefGoogle Scholar

Copyright information

© Springer Basel 2012

Authors and Affiliations

  • Andrea Balogh
    • 1
  • Eszter Persa
    • 1
  • Enikő Noémi Bogdándi
    • 1
  • Anett Benedek
    • 1
  • Hargita Hegyesi
    • 2
  • Géza Sáfrány
    • 2
  • Katalin Lumniczky
    • 1
  1. 1.Division of Cellular and Immune RadiobiologyFrédéric Joliot-Curie National Research Institute for Radiobiology and RadiohygieneBudapestHungary
  2. 2.Division of Molecular and Tumor RadiobiologyFrédéric Joliot-Curie National Research Institute for Radiobiology and RadiohygieneBudapestHungary

Personalised recommendations