, Volume 51, Issue 3, pp 478–489 | Cite as

Levels of regulatory T cells CD69+NKG2D+IL-10+ are increased in patients with autoimmune thyroid disorders

  • Ana Rodríguez-Muñoz
  • Marlen Vitales-Noyola
  • Ana Ramos-Levi
  • Ana Serrano-Somavilla
  • Roberto González-Amaro
  • Mónica Marazuela
Original Article


Regulatory T (Treg) cells play an important role in the pathogenesis of autoimmune thyroid disorders (AITD). New subsets of CD4+CD69+ and CD4+NKG2D+ T lymphocytes that behave as regulatory cells have been recently reported. The role of these immunoregulatory lymphocytes has not been previously explored in AITD. We analyzed by multi-parametric flow cytometry different Treg cell subsets in peripheral blood from 32 patients with AITD and 19 controls, and in thyroid tissue from seven patients. The suppressive activity was measured by an assay of inhibition of lymphocyte activation. We found a significant increased percentage of CD4+CD69+IL-10+, CD4+CD69+NKG2D+, and CD4+CD69+IL-10+NKG2D+ cells, in peripheral blood from GD patients compared to controls. The increase in CD4+CD69+IL-10+ and CD4+CD69+IL-10+NKG2D+ T cells was especially remarkable in patients with active Graves’ ophthalmopathy (GO), and a significant positive correlation between GO activity and CD4+CD69+IL-10+ or CD4+CD69+IL-10+NKG2D+ cells was also found. In addition, these cells were increased in patients with a more severe and/or prolonged disease. Thyroid from AITD patients showed an increased proportion of CD69+ regulatory T cells subpopulations compared to autologous peripheral blood. The presence of CD69+, NKG2D+, and IL-10+ cells was confirmed by immunofluorescence microscopy. In vitro functional assays showed that CD69+ Treg cells exerted an important suppressive effect on the activation of T effector cells in controls, but not in AITD patients. Our findings suggest that the levels of CD69+ regulatory lymphocytes are increased in AITD patients, but they are apparently unable to down-modulate the autoimmune response and tissue damage.


Treg cells CD69 NKG2D IL-10 Hashimoto thyroiditis Graves’ disease 


Conflict of interest

The authors declare no conflict of interest.


This work has received the following Grants: Proyectos de Investigación en Salud (FIS) PI13-01414, PIE-0041 (funded by Instituto de Salud Carlos III) and S2011/BMD-2328 TIRONET (funded by Comunidad de Madrid) (to Mónica Marazuela). Ayuda Predoctoral de Formación en Investigación en Salud (PFIS) FI11/00668 (funded by Instituto de Salud Carlos III) (to Ana Rodríguez-Muñoz).


  1. 1.
    A.P. Weetman, Autoimmune thyroid disease. Autoimmunity 37(4), 337–340 (2004). doi: 10.1080/08916930410001705394 CrossRefPubMedGoogle Scholar
  2. 2.
    G. Stassi, R. De Maria, Autoimmune thyroid disease: new models of cell death in autoimmunity. Nat. Rev. Immunol. 2(3), 195–204 (2002). doi: 10.1038/nri750 CrossRefPubMedGoogle Scholar
  3. 3.
    S.Z. Josefowicz, L.F. Lu, A.Y. Rudensky, Regulatory T cells: mechanisms of differentiation and function. Annu. Rev. Immunol. 30, 531–564 (2012). doi: 10.1146/annurev.immunol.25.022106.141623 CrossRefPubMedGoogle Scholar
  4. 4.
    A. Schmidt, N. Oberle, P.H. Krammer, Molecular mechanisms of treg-mediated T cell suppression. Front. Immunol. 3, 51 (2012). doi: 10.3389/fimmu.2012.00051 PubMedCentralPubMedGoogle Scholar
  5. 5.
    E.M. Shevach, A.M. Thornton, tTregs, pTregs, and iTregs: similarities and differences. Immunol. Rev. 259(1), 88–102 (2014). doi: 10.1111/imr.12160 PubMedCentralCrossRefPubMedGoogle Scholar
  6. 6.
    S. Sakaguchi, K. Wing, Y. Onishi, P. Prieto-Martin, T. Yamaguchi, Regulatory T cells: how do they suppress immune responses? Int. Immunol. 21(10), 1105–1111 (2009). doi: 10.1093/intimm/dxp095 CrossRefPubMedGoogle Scholar
  7. 7.
    S. Sakaguchi, K. Wing, M. Miyara, Regulatory T cells - a brief history and perspective. Eur. J. Immunol. 37(Suppl 1), S116–S123 (2007). doi: 10.1002/eji.200737593 CrossRefPubMedGoogle Scholar
  8. 8.
    L.W. Collison, V. Chaturvedi, A.L. Henderson, P.R. Giacomin, C. Guy, J. Bankoti, D. Finkelstein, K. Forbes, C.J. Workman, S.A. Brown, J.E. Rehg, M.L. Jones, H.T. Ni, D. Artis, M.J. Turk, D.A. Vignali, IL-35-mediated induction of a potent regulatory T cell population. Nat. Immunol. 11(12), 1093–1101 (2010). doi: 10.1038/ni.1952 PubMedCentralCrossRefPubMedGoogle Scholar
  9. 9.
    M. Battaglia, S. Gregori, R. Bacchetta, M.G. Roncarolo, Tr1 cells: from discovery to their clinical application. Semin. Immunol. 18(2), 120–127 (2006). doi: 10.1016/j.smim.2006.01.007 CrossRefPubMedGoogle Scholar
  10. 10.
    R.K. Dinesh, B.J. Skaggs, A. La Cava, B.H. Hahn, R.P. Singh, CD8 + Tregs in lupus, autoimmunity, and beyond. Autoimmun. Rev. 9(8), 560–568 (2010). doi: 10.1016/j.autrev.2010.03.006 PubMedCentralCrossRefPubMedGoogle Scholar
  11. 11.
    R.A. Peterson, Regulatory T-cells: diverse phenotypes integral to immune homeostasis and suppression. Toxicol. Pathol. 40(2), 186–204 (2012). doi: 10.1177/0192623311430693 CrossRefPubMedGoogle Scholar
  12. 12.
    Y. Han, Q. Guo, M. Zhang, Z. Chen, X. Cao, CD69 + CD4 + CD25- T cells, a new subset of regulatory T cells, suppress T cell proliferation through membrane-bound TGF-beta 1. J. Immunol. 182(1), 111–120 (2009). doi: 10.4049/jimmunol.182.1.111 CrossRefPubMedGoogle Scholar
  13. 13.
    J. Zhu, A. Feng, J. Sun, Z. Jiang, G. Zhang, K. Wang, S. Hu, X. Qu, Increased CD4(+) CD69(+) CD25(-) T cells in patients with hepatocellular carcinoma are associated with tumor progression. J. Gastroenterol. Hepatol. 26(10), 1519–1526 (2011). doi: 10.1111/j.1440-1746.2011.06765.x CrossRefPubMedGoogle Scholar
  14. 14.
    S. Bauer, V. Groh, J. Wu, A. Steinle, J.H. Phillips, L.L. Lanier, T. Spies, Activation of NK cells and T cells by NKG2D, a receptor for stress-inducible MICA. Science 285(5428), 727–729 (1999). doi: 10.1126/science.285.5428.727 CrossRefPubMedGoogle Scholar
  15. 15.
    Z. Dai, C.J. Turtle, G.C. Booth, S.R. Riddell, T.A. Gooley, A.M. Stevens, T. Spies, V. Groh, Normally occurring NKG2D + CD4 + T cells are immunosuppressive and inversely correlated with disease activity in juvenile-onset lupus. J. Exp. Med. 206(4), 793–805 (2009). doi: 10.1084/jem.20081648 PubMedCentralCrossRefPubMedGoogle Scholar
  16. 16.
    M. Vitales-Noyola, L. Doníz-Padilla, C. Álvarez-Quiroga, A. Monsiváis-Urenda, H. Portillo-Salazar, R. González-Amaro, Quantitative and functional analysis of CD69 + NKG2D + T regulatory cells in healthy subjects. Hum. Immunol. (2015). doi: 10.1016/j.humimm.2015.06.003
  17. 17.
    S. Bhattacharyya, S. Ghosh, P.L. Jhonson, S.K. Bhattacharya, S. Majumdar, Immunomodulatory role of interleukin-10 in visceral leishmaniasis: defective activation of protein kinase C-mediated signal transduction events. Infect. Immun. 69(3), 1499–1507 (2001). doi: 10.1128/IAI.69.3.1499-1507.2001 PubMedCentralCrossRefPubMedGoogle Scholar
  18. 18.
    K. Asadullah, R. Sabat, M. Friedrich, H.D. Volk, W. Sterry, Interleukin-10: an important immunoregulatory cytokine with major impact on psoriasis. Curr. Drug Targets Inflamm. Allergy 3(2), 185–192 (2004). doi: 10.2174/1568010043343886 CrossRefPubMedGoogle Scholar
  19. 19.
    E. Niesen, J. Schmidt, T. Flecken, R. Thimme, Suppressive effect of interleukin 10 on priming of naive hepatitis C virus-specific CD8 + T cells. J. Infect. Dis. 211(5), 821–826 (2015). doi: 10.1093/infdis/jiu541 CrossRefPubMedGoogle Scholar
  20. 20.
    R. de Waal Malefyt, J. Abrams, B. Bennett, C.G. Figdor, J.E. de Vries, Interleukin 10(IL-10) inhibits cytokine synthesis by human monocytes: an autoregulatory role of IL-10 produced by monocytes. J. Exp. Med. 174(5), 1209-1220 (1991). doi: 10.1084/jem.174.5.1209
  21. 21.
    Yssel, H., De Waal Malefyt, R., Roncarolo, M.G., Abrams, J.S., Lahesmaa, R., Spits, H., de Vries, J.E.: IL-10 is produced by subsets of human CD4 + T cell clones and peripheral blood T cells. J Immunol 149(7), 2378-2384 (1992)Google Scholar
  22. 22.
    G. Del Prete, M. De Carli, F. Almerigogna, M.G. Giudizi, R. Biagiotti, S. Romagnani, Human IL-10 is produced by both type 1 helper (Th1) and type 2 helper (Th2) T cell clones and inhibits their antigen-specific proliferation and cytokine production. J Immunol 150(2), 353–360 (1993)PubMedGoogle Scholar
  23. 23.
    D.F. Fiorentino, M.W. Bond, T.R. Mosmann, Two types of mouse T helper cell. IV. Th2 clones secrete a factor that inhibits cytokine production by Th1 clones. J. Exp. Med. 170(6), 2081–2095 (1989). doi: 10.1084/jem.170.6.2081 CrossRefPubMedGoogle Scholar
  24. 24.
    P.L. Vieira, J.R. Christensen, S. Minaee, E.J. O’Neill, F.J. Barrat, A. Boonstra, T. Barthlott, B. Stockinger, D.C. Wraith, A. O’Garra, IL-10-secreting regulatory T cells do not express Foxp3 but have comparable regulatory function to naturally occurring CD4 + CD25 + regulatory T cells. J. Immunol. 172(10), 5986–5993 (2004). doi: 10.4049/jimmunol.172.10.5986 CrossRefPubMedGoogle Scholar
  25. 25.
    M. Asano, M. Toda, N. Sakaguchi, S. Sakaguchi, Autoimmune disease as a consequence of developmental abnormality of a T cell subpopulation. J. Exp. Med. 184(2), 387–396 (1996). doi: 10.1084/jem.184.2.387 CrossRefPubMedGoogle Scholar
  26. 26.
    A. Kojima, Y. Tanaka-Kojima, T. Sakakura, Y. Nishizuka, Spontaneous development of autoimmune thyroiditis in neonatally thymectomized mice. Lab. Invest. 34(6), 550–557 (1976)PubMedGoogle Scholar
  27. 27.
    H. Xue, X. Yu, L. Ma, S. Song, Y. Li, L. Zhang, T. Yang, H. Liu, The possible role of CD4CD25 Foxp3/CD4 IL-17A cell imbalance in the autoimmunity of patients with Hashimoto thyroiditis. Endocrine (2015). doi: 10.1007/s12020-015-0569-y PubMedGoogle Scholar
  28. 28.
    N. Figueroa-Vega, M. Alfonso-Perez, I. Benedicto, F. Sanchez-Madrid, R. Gonzalez-Amaro, M. Marazuela, Increased circulating pro-inflammatory cytokines and Th17 lymphocytes in Hashimoto’s thyroiditis. J. Clin. Endocrinol. Metab. 95(2), 953–962 (2010). doi: 10.1210/jc.2009-1719 CrossRefPubMedGoogle Scholar
  29. 29.
    M. Marazuela, M.A. Garcia-Lopez, N. Figueroa-Vega, H. de la Fuente, B. Alvarado-Sanchez, A. Monsivais-Urenda, F. Sanchez-Madrid, R. Gonzalez-Amaro, Regulatory T cells in human autoimmune thyroid disease. J. Clin. Endocrinol. Metab. 91(9), 3639–3646 (2006). doi: 10.1210/jc.2005-2337 CrossRefPubMedGoogle Scholar
  30. 30.
    L. Bartalena, L. Baldeschi, A. Dickinson, A. Eckstein, P. Kendall-Taylor, C. Marcocci, M. Mourits, P. Perros, K. Boboridis, A. Boschi, N. Curro, C. Daumerie, G.J. Kahaly, G.E. Krassas, C.M. Lane, J.H. Lazarus, M. Marino, M. Nardi, C. Neoh, J. Orgiazzi, S. Pearce, A. Pinchera, S. Pitz, M. Salvi, P. Sivelli, M. Stahl, G. von Arx, W.M. Wiersinga, Consensus statement of the European Group on Graves’ orbitopathy (EUGOGO) on management of GO. Eur. J. Endocrinol. 158(3), 273–285 (2008). doi: 10.1530/EJE-07-0666 CrossRefPubMedGoogle Scholar
  31. 31.
    N. Figueroa-Vega, P. Sanz-Cameno, R. Moreno-Otero, F. Sanchez-Madrid, R. Gonzalez-Amaro, M. Marazuela, Serum levels of angiogenic molecules in autoimmune thyroid diseases and their correlation with laboratory and clinical features. J. Clin. Endocrinol. Metab. 94(4), 1145–1153 (2009). doi: 10.1210/jc.2008-1571 CrossRefPubMedGoogle Scholar
  32. 32.
    S. Leskela, A. Rodriguez-Munoz, H. de la Fuente, N. Figueroa-Vega, P. Bonay, P. Martin, A. Serrano, F. Sanchez-Madrid, R. Gonzalez-Amaro, M. Marazuela, Plasmacytoid dendritic cells in patients with autoimmune thyroid disease. J. Clin. Endocrinol. Metab. 98(7), 2822–2833 (2013). doi: 10.1210/jc.2013-1273 CrossRefPubMedGoogle Scholar
  33. 33.
    J.B. Canavan, B. Afzali, C. Scotta, H. Fazekasova, F.C. Edozie, T.T. Macdonald, M.P. Hernandez-Fuentes, G. Lombardi, G.M. Lord, A rapid diagnostic test for human regulatory T-cell function to enable regulatory T-cell therapy. Blood 119(8), e57–e66 (2012). doi: 10.1182/blood-2011-09-380048 CrossRefPubMedGoogle Scholar
  34. 34.
    P.K. Chattopadhyay, J. Yu, M. Roederer, A live-cell assay to detect antigen-specific CD4 + T cells with diverse cytokine profiles. Nat. Med. 11(10), 1113–1117 (2005). doi: 10.1038/nm1293 CrossRefPubMedGoogle Scholar
  35. 35.
    A.P. Weetman, A.M. McGregor, Autoimmune thyroid disease: further developments in our understanding. Endocr. Rev. 15(6), 788–830 (1994)PubMedGoogle Scholar
  36. 36.
    S. Sakaguchi, Regulatory T cells: key controllers of immunologic self-tolerance. Cell 101(5), 455–458 (2000). doi: 10.1016/S0092-8674(00)80856-9 CrossRefPubMedGoogle Scholar
  37. 37.
    K. Otsubo, H. Kanegane, I. Kobayashi, T. Miyawaki, IPEX syndrome and human Treg cells. Nihon Rinsho Meneki Gakkai Kaishi 33(4), 196–206 (2010). doi: 10.2177/jsci.33.196 CrossRefPubMedGoogle Scholar
  38. 38.
    P. Castro-Sanchez, J.M. Martin-Villa, Gut immune system and oral tolerance. Br. J. Nutr. 109(Suppl 2), S3–11 (2013). doi: 10.1017/S0007114512005223 CrossRefPubMedGoogle Scholar
  39. 39.
    D. Sancho, M. Gomez, F. Viedma, E. Esplugues, M. Gordon-Alonso, M.A. Garcia-Lopez, H. de la Fuente, A.C. Martinez, P. Lauzurica, F. Sanchez-Madrid, CD69 downregulates autoimmune reactivity through active transforming growth factor-beta production in collagen-induced arthritis. J Clin Invest 112(6), 872–882 (2003). doi: 10.1172/JCI19112112/6/872 PubMedCentralCrossRefPubMedGoogle Scholar
  40. 40.
    R. Gandhi, M.F. Farez, Y. Wang, D. Kozoriz, F.J. Quintana, H.L. Weiner, Cutting edge: human latency-associated peptide + T cells: a novel regulatory T cell subset. J Immunol 184(9), 4620–4624 (2010). doi: 10.4049/jimmunol.0903329 PubMedCentralCrossRefPubMedGoogle Scholar
  41. 41.
    M. Bonelli, A. Savitskaya, K. von Dalwigk, C.W. Steiner, D. Aletaha, J.S. Smolen, C. Scheinecker, Quantitative and qualitative deficiencies of regulatory T cells in patients with systemic lupus erythematosus (SLE). Int. Immunol. 20(7), 861–868 (2008). doi: 10.1093/intimm/dxn044 CrossRefPubMedGoogle Scholar
  42. 42.
    M.H. Garcia-Hernandez, B. Alvarado-Sanchez, M.Z. Calvo-Turrubiartes, M. Salgado-Bustamante, C.Y. Rodriguez-Pinal, L.R. Gamez-Lopez, R. Gonzalez-Amaro, D.P. Portales-Perez, Regulatory T Cells in children with intestinal parasite infection. Parasite Immunol. 31(10), 597–603 (2009). doi: 10.1111/j.1365-3024.2009.01149.x CrossRefPubMedGoogle Scholar
  43. 43.
    A. Saez-Borderias, M. Guma, A. Angulo, B. Bellosillo, D. Pende, M. Lopez-Botet, Expression and function of NKG2D in CD4 + T cells specific for human cytomegalovirus. Eur. J. Immunol. 36(12), 3198–3206 (2006). doi: 10.1002/eji.200636682 CrossRefPubMedGoogle Scholar
  44. 44.
    V. Groh, A. Bruhl, H. El-Gabalawy, J.L. Nelson, T. Spies, Stimulation of T cell autoreactivity by anomalous expression of NKG2D and its MIC ligands in rheumatoid arthritis. Proc Natl Acad Sci USA 100(16), 9452–9457 (2003). doi: 10.1073/pnas.16328071001632807100 PubMedCentralCrossRefPubMedGoogle Scholar
  45. 45.
    M. Allez, V. Tieng, A. Nakazawa, X. Treton, V. Pacault, N. Dulphy, S. Caillat-Zucman, P. Paul, J.M. Gornet, C. Douay, S. Ravet, R. Tamouza, D. Charron, M. Lemann, L. Mayer, A. Toubert, CD4 + NKG2D + T cells in Crohn’s disease mediate inflammatory and cytotoxic responses through MICA interactions. Gastroenterology 132(7), 2346–2358 (2007). doi: 10.1053/j.gastro.2007.03.025 CrossRefPubMedGoogle Scholar
  46. 46.
    R. Gonzalez-Amaro, F. Sanchez-Madrid, Cell adhesion molecules: selectins and integrins. Crit. Rev. Immunol. 19(5–6), 389–429 (1999)PubMedGoogle Scholar
  47. 47.
    D. Cao, V. Malmstrom, C. Baecher-Allan, D. Hafler, L. Klareskog, C. Trollmo, Isolation and functional characterization of regulatory CD25brightCD4 + T cells from the target organ of patients with rheumatoid arthritis. Eur. J. Immunol. 33(1), 215–223 (2003). doi: 10.1002/immu.200390024 CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  • Ana Rodríguez-Muñoz
    • 1
  • Marlen Vitales-Noyola
    • 2
  • Ana Ramos-Levi
    • 1
  • Ana Serrano-Somavilla
    • 1
  • Roberto González-Amaro
    • 2
  • Mónica Marazuela
    • 1
  1. 1.Department of Endocrinology, Hospital Universitario de la Princesa, Instituto de Investigación Sanitaria PrincesaUniversidad Autónoma de MadridMadridSpain
  2. 2.Department of Immunology, School of MedicineUASLPSan Luis PotosíMexico

Personalised recommendations