mimmunМедицинская иммунологияMedical Immunology (Russia)1563-06252313-741XSPb RAACI10.15789/1563-0625-2008-2-3-173-180mimmun-122Research ArticleОРИГИНАЛЬНЫЕ СТАТЬИORIGINAL ARTICLESГЕНЕРАЦИЯ В КУЛЬТУРЕ IN VITRO И ХАРАКТЕРИСТИКА РЕГУЛЯТОРНЫХ Т-КЛЕТОК ЧЕЛОВЕКАIN VITRO GENERATION AND CHARACTERIZATION OF HUMAN REGULATORY T-CELLSКургановаЕ. В.KurganovaE. V.

630099, г. Новосибирск, ул. Ядринцевская 1

ct_lab@mail.ruШевелаЕ. Я.ShevelaE. Ya.ct_lab@mail.ruТихоноваМ. А.TihonovaM. A.ct_lab@mail.ruОстанинА. А.OstaninA. A.ct_lab@mail.ruЧерныхЕ. Р.ChernykhE. R.ct_lab@mail.ruГУ НИИ клинической иммунологии СО РАМН, г. НовосибирскRussian Federation200816072014102-3173180Copyright © Курганова Е.В., Шевела Е.Я., Тихонова М.А., Останин А.А., Черных Е.Р., 20142014Курганова Е.В., Шевела Е.Я., Тихонова М.А., Останин А.А., Черных Е.Р.Kurganova E.V., Shevela E.Y., Tihonova M.A., Ostanin A.A., Chernykh E.R.Данная работа распространяется под лицензией Creative Commons Attribution 4.0.This work is licensed under a Creative Commons Attribution 4.0 License.https://www.mimmun.ru/mimmun/article/view/122

Резюме. Работа посвящена оценке эффективности генерации и характеристике регуляторных Т-клеток (Treg), полученных из мононуклеарных клеток крови здоровых доноров при поликлональной активации анти-CD3-антителами и IL-2 в отсутствии (модель 1) и в присутствии (модель 2) иммуносупрессорных агентов (витамина D3 и дексаметазона). Показано, что Т-клетки, генерированные в первой модели, характеризуются сниженным пролиферативным ответом на анти-CD3 и не проявляют супрессорной активности. Т-клетки, полученные в присутствии витамина D3 и дексаметазона, находятся в состоянии анергии, которое частично нивелируется экзогенным IL-2, а также обладают супрессорной активностью, опосредуемой как через прямые межклеточные взаимодействия, так и через продукцию растворимых факторов, в частности, IL-10. Полученные данные позволяют отнести Т-клетки, генерированные во второй модели, к индуцибельным IL-10-продуцирующим регуляторным Т-клеткам (Tr1). Тем не менее, выявленное среди них увеличение FOXP3-экспрессирующих CD4+Т-клеток свидетельствует также о возможной генерации естественных регуляторных клеток.

Abstract. The aim of present study was to evaluate efficacy of generation and characterization of regulatory T cells (Treg). The Treg were derived from peripheral mononuclear cells of healthy donors, using activation with anti-CD3mAb and IL-2, either in absence (model 1), or in the presence of immunosuppressive agents (vitamin D3 and dexamethasone, model 2). It was shown, that the Treg, obtained in model 1, are characterized by decreased anti-CD3-induced proliferative response and lack of suppressive activity. Treg, obtained in presence of vitamin D3 and dexamethasone, proved to be profoundly anergic. Such a state may be partially abolished by exogenic IL-2. These cells are also characterized by suppressive activity mediated by both cell-to-cell contacts and soluble factors, such as IL-10. Hence, the regulatory T cells generated in model 2, can be referred as inducible Treg (Tr1). Nevertheless, increased ratio of cells expressing CD4+FOXP3+ among Treg obtained by model 2, may also suggest an ability to generate natural regulatory cells. (Med. Immunol., 2008, vol. 10, n 2-3, pp 173-180).

ТregCD4CD25CD4FoxP3IL-10витамин D3дексаметазонТregCD4CD25CD4FoxP3IL-10vitamin D3dexamethasoneReferencesКурганова Е.В., Тихонова М.А., Стрельцова Е.И., Останин А.А., Черных Е.Р., Козлов В.А. Регуляторные Т-клетки с супрессорной активностью при хирургическом сепсисе // Мед. иммунология. – 2006. – Т. 8, № 1. – С. 51-60.Курганова Е.В., Тихонова М.А., Стрельцова Е.И., Останин А.А., Черных Е.Р., Козлов В.А. Регуляторные Т-клетки с супрессорной активностью при хирургическом сепсисе // Мед. иммунология. – 2006. – Т. 8, № 1. – С. 51-60.Ярилин А.А., Донецкова А.Д. Естественные регуляторные Т-клетки и фактор FOXP3 // Иммунология. – 2006. – № 3. – С. 176-188.Ярилин А.А., Донецкова А.Д. Естественные регуляторные Т-клетки и фактор FOXP3 // Иммунология. – 2006. – № 3. – С. 176-188.Allan S.E., Crome S.Q., Crellin N.K., Passerini L., Steiner T.S., Bacchetta R., Roncarolo M.G., Levings M.K. Activation-induced FOXP3 in human T effector cells does not suppress proliferation or cytokine production // International Immunology. – 2007. – Vol. 19, N 4. – P. 345-354.Allan S.E., Crome S.Q., Crellin N.K., Passerini L., Steiner T.S., Bacchetta R., Roncarolo M.G., Levings M.K. Activation-induced FOXP3 in human T effector cells does not suppress proliferation or cytokine production // International Immunology. – 2007. – Vol. 19, N 4. – P. 345-354.Alroy I., Towers T.L., Freedman L.P. Transcriptional repression of the interleukin-2 gene by vitamin D3: direct inhibition of NFATp/AP-1 complex formation by a nuclear hormone receptor // Mol. Cell. Biol. – 1995. – Vol. 15. – P. 5789-5799.Alroy I., Towers T.L., Freedman L.P. Transcriptional repression of the interleukin-2 gene by vitamin D3: direct inhibition of NFATp/AP-1 complex formation by a nuclear hormone receptor // Mol. Cell. Biol. – 1995. – Vol. 15. – P. 5789-5799.Baecher-Allan C., Hafler D.A. Suppressor T cells in human diseases // J. Exp. Med. – 2004. – Vol. 200, N 3. – P. 273-276.Baecher-Allan C., Hafler D.A. Suppressor T cells in human diseases // J. Exp. Med. – 2004. – Vol. 200, N 3. – P. 273-276.Balandina A., Lecart S., Dartevelle P., Saoudi A., Berrih-Aknin S. Functional defect of regulatory CD4+CD25+ T cells in the thymus of patients with autoimmune myasthenia gravis // Blood. – 2005. – Vol. 105. – P. 735-741.Balandina A., Lecart S., Dartevelle P., Saoudi A., Berrih-Aknin S. Functional defect of regulatory CD4+CD25+ T cells in the thymus of patients with autoimmune myasthenia gravis // Blood. – 2005. – Vol. 105. – P. 735-741.Baratelli F., Lin Y., Zhu L., Yang S.C., Heuze-Vourc’h N., Zeng G., Reckamp K., Dohadwala M., Sharma S., Dubinett S.M. Prostaglandin E2 induces FOXP3 gene expression and T regulatory cell function in human CD4+ T cells // J. Immunol. – 2005. – Vol. 175. – P. 1483-1490.Baratelli F., Lin Y., Zhu L., Yang S.C., Heuze-Vourc’h N., Zeng G., Reckamp K., Dohadwala M., Sharma S., Dubinett S.M. Prostaglandin E2 induces FOXP3 gene expression and T regulatory cell function in human CD4+ T cells // J. Immunol. – 2005. – Vol. 175. – P. 1483-1490.Barrat F.J., Cua D.J., Boonstra A., Richards D.F., Crain C., Savelkoul H.F., de Waal-Malefyt R., Coffman R.L., Hawrylowicz C.M., O'Garra A. In vitro generation of interleukin 10-producing regu-latory CD4+ T cells is induced by immunosuppressive drugs and inhibited by T helper type 1 (Th1)- and Th2-inducing cytokines // J. Exp. Med. – 2002. – Vol. 195, N 5. – P. 603-616.Barrat F.J., Cua D.J., Boonstra A., Richards D.F., Crain C., Savelkoul H.F., de Waal-Malefyt R., Coffman R.L., Hawrylowicz C.M., O'Garra A. In vitro generation of interleukin 10-producing regu-latory CD4+ T cells is induced by immunosuppressive drugs and inhibited by T helper type 1 (Th1)- and Th2-inducing cytokines // J. Exp. Med. – 2002. – Vol. 195, N 5. – P. 603-616.Bisikirska B.C., Herold K.C. Use of anti-CD3 monoclonal antibody to induce immune regulation in type 1 diabetes // Ann. N.Y. Acad. Sci. – 2004. – Vol. 1037. – P. 1-9.Bisikirska B.C., Herold K.C. Use of anti-CD3 monoclonal antibody to induce immune regulation in type 1 diabetes // Ann. N.Y. Acad. Sci. – 2004. – Vol. 1037. – P. 1-9.Dieckmann D., Bruett C.H., Ploettner H., Lutz M.B., Schuler G. Human CD4+CD25+ regulatory, contact-dependent T cells induce interleukin 10-producing, contact-independent type 1-like regulatory T cells // J. Exp. Med. – 2002. – Vol. 196, N 2. – P. 247-253.Dieckmann D., Bruett C.H., Ploettner H., Lutz M.B., Schuler G. Human CD4+CD25+ regulatory, contact-dependent T cells induce interleukin 10-producing, contact-independent type 1-like regulatory T cells // J. Exp. Med. – 2002. – Vol. 196, N 2. – P. 247-253.Fantini M.C., Becker C., Monteleone G., Pallone F., Galle P.R., Neurath M.F. TGF-beta induces a regulatory phenotype in CD4+CD25- T cells through Foxp3 induction and down-regulation of Smad7 // J. Immunol. – 2004. – Vol. 172, N 9. – P. 5149-5153.Fantini M.C., Becker C., Monteleone G., Pallone F., Galle P.R., Neurath M.F. TGF-beta induces a regulatory phenotype in CD4+CD25- T cells through Foxp3 induction and down-regulation of Smad7 // J. Immunol. – 2004. – Vol. 172, N 9. – P. 5149-5153.Hoffmann P., Eder R., Kunz-Schughart L.A., Andreesen R., Edinger M. Large-scale in vitro expansion of polyclonal human CD4+CD25high regulatory T cells // Blood. – 2004. – Vol. 104, N 3. – P. 895-903.Hoffmann P., Eder R., Kunz-Schughart L.A., Andreesen R., Edinger M. Large-scale in vitro expansion of polyclonal human CD4+CD25high regulatory T cells // Blood. – 2004. – Vol. 104, N 3. – P. 895-903.Jonuleit H., Schmitt E., Stassen M., Tuettenberg A., Knop J., Enk A.H. Identification and functional characterization of human CD4+CD25+ T cells with regulatory properties isolated from peripheral blood // J. Exp. Med. – 2001. – Vol. 193, N 11. – P. 1285-1294.Jonuleit H., Schmitt E., Stassen M., Tuettenberg A., Knop J., Enk A.H. Identification and functional characterization of human CD4+CD25+ T cells with regulatory properties isolated from peripheral blood // J. Exp. Med. – 2001. – Vol. 193, N 11. – P. 1285-1294.Jonuleit H., Schmitt E., Schuler G., Knop J., Enk A.H. Induction of interleukin 10-producing, non-proliferating CD4+ T cells with regulatory properties by repetitive stimulation with allogeneic immature human dendritic cells // J. Exp. Med. – 2000. – Vol. 192. – P. 1213-1222.Jonuleit H., Schmitt E., Schuler G., Knop J., Enk A.H. Induction of interleukin 10-producing, non-proliferating CD4+ T cells with regulatory properties by repetitive stimulation with allogeneic immature human dendritic cells // J. Exp. Med. – 2000. – Vol. 192. – P. 1213-1222.Kohm A.P., Williams J.S., Bickford A.L., McMahon J.S., Chatenoud L., Bach J.F., Bluestone J.A., Miller S.D. Treatment with nonmitogenic anti-CD3 monoclonal antibody induces CD4+ T cell unresponsiveness and functional reversal of established experimental autoimmune encephalomyelitis // J. Immunol. – 2005. – Vol. 174. – P. 4525-4534.Kohm A.P., Williams J.S., Bickford A.L., McMahon J.S., Chatenoud L., Bach J.F., Bluestone J.A., Miller S.D. Treatment with nonmitogenic anti-CD3 monoclonal antibody induces CD4+ T cell unresponsiveness and functional reversal of established experimental autoimmune encephalomyelitis // J. Immunol. – 2005. – Vol. 174. – P. 4525-4534.Levings M.K., Sangregorio R., Sartirana C., Moschin A.L., Battaglia M., Orban P.C., Roncarolo M.G. Human CD4+CD25+ T suppressor cell clones produce transforming growth factor β, but not IL-10, and are distinct from type 1 T regulatory cells // J. Exp. Med. – 2002 – Vol. 196, N 10. – P. 1335-1346.Levings M.K., Sangregorio R., Sartirana C., Moschin A.L., Battaglia M., Orban P.C., Roncarolo M.G. Human CD4+CD25+ T suppressor cell clones produce transforming growth factor β, but not IL-10, and are distinct from type 1 T regulatory cells // J. Exp. Med. – 2002 – Vol. 196, N 10. – P. 1335-1346.Levings M.K., Sangregorio R., Roncarolo M.G. Human CD25+CD4+ T cells suppress naive and memory T-cell proliferation and can be expanded in vitro without loss of suppressor function // J. Exp. Med. – 2001. – Vol. 193. – P. 1295-1302.Levings M.K., Sangregorio R., Roncarolo M.G. Human CD25+CD4+ T cells suppress naive and memory T-cell proliferation and can be expanded in vitro without loss of suppressor function // J. Exp. Med. – 2001. – Vol. 193. – P. 1295-1302.Levings M.K., Roncarolo M.G. T-regulatory 1 cells: a novel subset of CD4 T cells with immunoregulatory properties // J. Allergy Clin. Immunol. – 2000. – Vol. 106. – P. 109-112.Levings M.K., Roncarolo M.G. T-regulatory 1 cells: a novel subset of CD4 T cells with immunoregulatory properties // J. Allergy Clin. Immunol. – 2000. – Vol. 106. – P. 109-112.Lindley S., Dayan C.M., Bishop A., Roep B.O., Peakman M., Tree T.I. Defective suppressor function in CD4+CD25+ T-cells from patients with type 1 diabetes // Diabetes. – 2005. – Vol. 54. – P. 92-99.Lindley S., Dayan C.M., Bishop A., Roep B.O., Peakman M., Tree T.I. Defective suppressor function in CD4+CD25+ T-cells from patients with type 1 diabetes // Diabetes. – 2005. – Vol. 54. – P. 92-99.Li ng E.M. Relation of CD4+CD25+ regulatory T-cells suppression of allergen-driven T-cell activation to atopic status and expression of allergic disease // Lancet. – 2004. – Vol. 363. – P. 608-615.Li ng E.M. Relation of CD4+CD25+ regulatory T-cells suppression of allergen-driven T-cell activation to atopic status and expression of allergic disease // Lancet. – 2004. – Vol. 363. – P. 608-615.Morgan M.E., Sutmuller R.P., Witteveen H.J., van Duivenvoorde L.M., Zanelli E., Melief C.J., Snijders A., Offringa R., de Vries R.R., Toes R.E. CD25+ cell depletion hastens the onset of severe disease in collagen-induced arthritis // J. Arthritis Rheum. – 2003. – Vol. 48. – P. 1452-1460.Morgan M.E., Sutmuller R.P., Witteveen H.J., van Duivenvoorde L.M., Zanelli E., Melief C.J., Snijders A., Offringa R., de Vries R.R., Toes R.E. CD25+ cell depletion hastens the onset of severe disease in collagen-induced arthritis // J. Arthritis Rheum. – 2003. – Vol. 48. – P. 1452-1460.Ng W.F., Duggan P.J., Ponchel F., Matarese G., Lombardi G., Edwards A.D., Isaacs J.D., Lechler R.I. Human CD4+CD25+ cells: a naturally occurring population of regulatory T cells // Blood. – 2001. – Vol. 98. – P. 2736-2744.Ng W.F., Duggan P.J., Ponchel F., Matarese G., Lombardi G., Edwards A.D., Isaacs J.D., Lechler R.I. Human CD4+CD25+ cells: a naturally occurring population of regulatory T cells // Blood. – 2001. – Vol. 98. – P. 2736-2744.Penna G., Roncari A., Amuchastegui S., Daniel K.C., Berti E., Colonna M., Adorini L. Expression of the inhibitory receptor ILT3 on dendritic cells is dispensable for induction of CD4+Foxp3+ regulatory T cells by 1,25-dihydroxyvitamin D3 // Blood. – 2005. – Vol. 106, N 10. – P. 3490-3497.Penna G., Roncari A., Amuchastegui S., Daniel K.C., Berti E., Colonna M., Adorini L. Expression of the inhibitory receptor ILT3 on dendritic cells is dispensable for induction of CD4+Foxp3+ regulatory T cells by 1,25-dihydroxyvitamin D3 // Blood. – 2005. – Vol. 106, N 10. – P. 3490-3497.Piemonti L., Monti P., Sironi M., Fraticelli P., Leone B.E., Dal Cin E., Allavena P., Di Carlo V. Vitamin D3 affect differentiation, maturation, and function of human monocyte-derived dendritic cells // J. Immunol. – 2000. – Vol. 164. – P. 4443-4451.Piemonti L., Monti P., Sironi M., Fraticelli P., Leone B.E., Dal Cin E., Allavena P., Di Carlo V. Vitamin D3 affect differentiation, maturation, and function of human monocyte-derived dendritic cells // J. Immunol. – 2000. – Vol. 164. – P. 4443-4451.Ramsdell F. Foxp3 and natural regulatory T cells: key to a cell lineage? // Immunity. – 2003. – Vol. 19. – P. 165-168.Ramsdell F. Foxp3 and natural regulatory T cells: key to a cell lineage? // Immunity. – 2003. – Vol. 19. – P. 165-168.Robinson D.S., Larche M., Durham S.R. Tregs and allergic disease // J. Clin. Invest. – 2004. – Vol. 114, N 10. – P. 1389-1397.Robinson D.S., Larche M., Durham S.R. Tregs and allergic disease // J. Clin. Invest. – 2004. – Vol. 114, N 10. – P. 1389-1397.Sakaguchi S. Naturally arising CD4+ regulatory T cells for immunologic self-tolerance and negative control of immune responses // Annu. Rev. Immunol. – 2004. – Vol. 22. – P. 531-562.Sakaguchi S. Naturally arising CD4+ regulatory T cells for immunologic self-tolerance and negative control of immune responses // Annu. Rev. Immunol. – 2004. – Vol. 22. – P. 531-562.Sakaguchi S. Regulatory T cells: key controllers of immunologic self-tolerance // Cell. – 2002. – Vol. 101. – P. 455-458.Sakaguchi S. Regulatory T cells: key controllers of immunologic self-tolerance // Cell. – 2002. – Vol. 101. – P. 455-458.Shevach E.M. Regulatory T cells in autoimmunity // Annu. Rev. Immunol. – 2000. – Vol. 18. – P. 423-449.Shevach E.M. Regulatory T cells in autoimmunity // Annu. Rev. Immunol. – 2000. – Vol. 18. – P. 423-449.Suri-Payer E., Amar A.Z., Thornton A.M., Shevach E.M. CD4+CD25+ T cells inhibit both the induction and effector function of autoreactive T cells and represent a unique lineage of immunoregulatory cells // J. Immunol. – 1998. – Vol. 160. – P. 1212-1218.Suri-Payer E., Amar A.Z., Thornton A.M., Shevach E.M. CD4+CD25+ T cells inhibit both the induction and effector function of autoreactive T cells and represent a unique lineage of immunoregulatory cells // J. Immunol. – 1998. – Vol. 160. – P. 1212-1218.Viglietta V., Baecher-Allan C., Weiner H.L., Hafler D.A. Loss of functional suppression by CD4+CD25+ regulatory T cells in patients with multiple sclerosis // J. Exp. Med. – 2004. – Vol. 199, N 7. – P. 971-979.Viglietta V., Baecher-Allan C., Weiner H.L., Hafler D.A. Loss of functional suppression by CD4+CD25+ regulatory T cells in patients with multiple sclerosis // J. Exp. Med. – 2004. – Vol. 199, N 7. – P. 971-979.Xystrakis E., Kusumakar S., Boswell S., Peek E., Urry Z., Richards D.F., Adikibi T., Pridgeon C., Dallman M., Loke T.K., Robinson D.S., Barrat F.J., O'Garra A., Lavender P., Lee T.H., Corrigan C., Hawrylowicz C.M. Reversing the defective induction of IL-10-secreting regulatory T cells in glucocorticoid-resistant asthma patients // J. Clin. Invest. – 2006. – Vol. 116, N 1. – P. 146-155.Xystrakis E., Kusumakar S., Boswell S., Peek E., Urry Z., Richards D.F., Adikibi T., Pridgeon C., Dallman M., Loke T.K., Robinson D.S., Barrat F.J., O'Garra A., Lavender P., Lee T.H., Corrigan C., Hawrylowicz C.M. Reversing the defective induction of IL-10-secreting regulatory T cells in glucocorticoid-resistant asthma patients // J. Clin. Invest. – 2006. – Vol. 116, N 1. – P. 146-155.Yagi H., Nomura T., Nakamura K., Yamazaki S., Kitawaki T., Hori S., Maeda M., Onodera M., Uchiyama T., Fujii S., Sakaguchi S. Crucial role of FOXP3 in the development and function of human CD25+CD4+ regulatory T cells // Int. Immunol. – 2004. – Vol. 16, N 11. – P. 1643-1656.Yagi H., Nomura T., Nakamura K., Yamazaki S., Kitawaki T., Hori S., Maeda M., Onodera M., Uchiyama T., Fujii S., Sakaguchi S. Crucial role of FOXP3 in the development and function of human CD25+CD4+ regulatory T cells // Int. Immunol. – 2004. – Vol. 16, N 11. – P. 1643-1656.

The authors declare that there are no conflicts of interest present.