AGE-DEPENDENT CHANGES OF T-REGULATORY AND Th17 SUBSET LEVELS IN PERIPHERAL BLOOD FROM HEALTHY HUMANS

Age-dependent development of Th17 and Treg lymphocyte subsets in healthy  humans is studied insufficiently. The  present  study aimed  to investigate  quantitative characteristics of Th17  and  Treg subsets in peripheral blood of healthy  subjects for various age groups.

352 healthy  humans (168 female  and 184 male), one month to 85 years old, were subject to examination, including 79 infants in their first year of life; 34 children at aged 1 year to 2 years 11 months; 24, at 3 to 4 years 11 months; 28, at the age of 5 to 6 years 11 months; 25 children aged 7-8 years 11 month; 36 children aged 9 to 11 years 11 months; 39 adolescents aged 12 to 14 years 11 months; 26 adolescents aged 15 to 18 years; 25 young adults aged 20 to 35 years; 11 adults at 36 to 49 years old; 16 adults aged 59-70 years, and 9 elderly people over 70 years old. The study was performed with capillary blood in children under 2 years, and venous blood taken in elder persons.  The basic and ‘minor’ subsets of peripheral blood lymphocytes were evaluated by flow cytometry using four-color staining of whole blood and following erythrocyte lysis. We used the following surface markers: CD3, CD4, CD8, CD25, CD127, CD161, CD45R0 for lymphocyte subsets detection.

It has been  shown  that  Treg percentage (a ratio  of CD4⁺CD25^hiCD127^low/neg  in the CD3⁺CD4⁺  gate) did not  depend on  age of the  people  under  study, and  can  be approximated by a linear  function. The  absolute number of Tregs in childhood is progressively  decreased and,  after 10 years old, it reaches  plateau  values. This age-dependent relationship may be approximated by a logarithmic function. Evaluation of Th17 subset levels demonstrated a strong  relative  and  absolute  age-dependent growth  of this  cell  subpopulation. Percentage and absolute  numbers of Th17 lymphocyte (share  of CD4⁺CD161⁺CD45R0⁺ in the CD3⁺CD4⁺gate), can be approximated by a square function. The age of 10-12 years seems to be critical to the immune system formation. We suppose  the process of the immune system development to be completed at this age, and maturation of the immune cell populations is then  observed.  A decrease in both relative and absolute  numbers of Treg and Th17 lymphocyte subsets was found in elderly persons (> 70 years old). Our data on peripheral blood Tregs and Th17 subsets, with respect  to their percentage and absolute  numbers in healthy  humans, may be used as age-related reference values.

1. Борисов А.Г., Савченко А.А., Кудрявцев И.В., Мошев А.В., Козлов В.А. Характеристика субпопуляционного состава Ти В-лимфоцитов у больных хроническими обструктивными заболеваниями легких при использовании вакцины для профилактики синегнойной инфекции // Медицинская иммунология, 2015. Т. 17, № 5. С. 431-442. [Borisov A.G., Savchenko A.A., Kudryavtsev I.V., Moshev A.V., Kozlov V.A. Characteristics of Tand B-cell subpopulation profile in the patients with chronic obstructive pulmonary disease treated with vaccine against Pseudomonas aeruginosa. Meditsinskaya Immunologiya = Medical Immunology (Russia), 2015, Vol. 17, no. 5, pp. 431-442. (In Russ.)] doi: 10.15789/1563-0625-2015-5-431-442.

2. Никонова М.Ф., Донецкова А.Д., Сидорович О.И., Лусс Л.В., Ярилин А.А. Особенности экспрессии генов транскрипционных факторов, контролирующих дифференцировку адаптивных субпопуляций CD4+Т-лимфоцитов, при аллергии // Иммунология, 2011. Т. 32, № 4. С. 189-191. [Nikonova M.F., Donetskova A.D., Sidorovich O.I., Luss L.V., Yarilin A.A. Peculiarities of expression of the genes encoding for transcription factors controlling differentiation of adaptive CD4+ T-lymphocyte subpopulations in allergy. Immunologiya = Immunology, 2011, Vol. 32, no. 4, pp. 189-191. (In Russ.)]

3. Пухальский А.Л., Топтыгина А.П. Сравнительное изучение механизмов антипролиферативного действия низких концентраций мафосфамида и циклоспорина А // Бюллетень экспериментальной биологии и медицины, 1993. Т. 115, № 5. С. 514-515. [Pukhalskiy L.A., Toptygina A.P. Comparative study of mechanisms of antiproliferative action of mafosfamide and cyclosporine A in low doses. Byulleten eksperimental’noy biologii i meditsiny = Bulletin of Experimental Biology and Medicine, 1993, Vol. 115, no. 5, pp. 563-565. (In Russ.)].

4. Топтыгина А.П., Семикина Е.Л., Алешкин В.А. Регуляция иммунного ответа у детей, привитых против кори, краснухи и эпидемического паротита // Иммунология, 2012. Т. 33, № 4. С. 175-179. [Toptygina A.P., Semikina E.L., Alioshkin V.A. Immune response regulation in children vaccinated with measles, rubella and mumps. Immunologiya = Immunology, 2012, Vol. 33, no. 4, pp. 175-179. (In Russ.)]

5. Топтыгина А.П., Семикина Е.Л., Копыльцова Е.А., Алешкин В.А. Возрастные особенности формирования гуморального звена иммунного ответа у детей // Медицинская иммунология, 2012. Т. 14, № 4-5. С. 289-294. [Toptygina A.P., Semikina E.L., Kopyltsova E.A., Alioshkin V.A. Age related characteristics of humoral immunity maturation in children. Meditsinskaya immunologiya = Medical Immunology (Russia), 2012, Vol. 14, no. 4-5, pp. 289-294. (In Russ.)] doi: 10/15789/1563-0625-2012-4-5-289-294.

6. Хайдуков С.В., Байдун Л.А., Зурочка А.В., Тотолян Арег А. Стандартизованная технология «Исследование субпопуляционного состава лимфоцитов периферической крови с применением проточных цитофлюориметров-анализаторов» (Проект) // Медицинская иммунология, 2012. Т. 14, № 3. С. 255-268. [Khaydukov S.V., Baydun L.A., Zurochka A.V., Totolian Areg A. Standardized technology “Research of lymphocytes subpopulation composition in peripheral blood using flow cytometry analyzers” (Draft). Meditsinskaya immunologiya = Medical Immunology (Russia), 2012, Vol. 14, no. 3, pp. 255-268. (In Russ.)] doi: 10/15789/1563-0625-2012-3-255-268.

7. Ярилин А.А Иммунология. М.: ГЭОТАР-Медия, 2010. 752 с. [Yarilin A.A. Immunology]. Moscow: GEOTAR-Media, 2010. 752 p.

8. Abbas A.K., Benoist C., Bluestone J.A., Campbell D.J., Ghosh S., Hori S., Jiang S., Kuchroo V.K., Mathis D., Roncarolo M.G., Rudensky A., Sakaguchi S., Shevach E.M., Vignali D.A., Ziegler S.F. Regulatory T cells: recommendations to simplify the nomenclature. Nat. Immunol., 2013, Vol. 14, pp. 307-308.

9. Cevenini E., Monti D., Franceschi C. Inflammageing. Curr. Opin. Clin. Nutr. Metab. Care 2013, Vol. 16, pp. 14-20.

10. Chougnet C.A., Tripathi P., Lages C.S., Raynor J., Sholl A., Fink P., Plas D.R., Hildeman D.A. A major role for Bim in regulatory T cell homeostasis. J. Immunol., 2011, Vol. 186, pp. 156-163.

11. Cosmi L., Palma R., Santarlasci V., Maggi L., Capone M., Frosali F., Rodolico G., Querci V., Abbate G., Angeli R., Berrino L., Fambrini M., Caproni M., Tonelli F., Lazzeri E., Parronchi P., Liotta F., Maggi E., Romagnani S., Annunziato F. Human interleukin 17-producing cells originate from a CD161+CD4+ T cell precursor. J. Exp. Med., 2008, Vol. 205, no. 8, pp. 1903-1916.

12. den Braber I., Mugwagwa T., Vrisekoop N., Westera L., Mogling R., de Boer A.B., Willems N., Schrijver E.H., Spierenburg G., Gaiser K., Mui E., Otto S.A., Ruitter A.F., Ackermans M.T., Miedema F., Borghans J.A., de Boer R.J., Tesselaar K. Maintenance of peripheral naive T cells is sustained by thymus output in mice but not humans. Immunity, 2012, Vol. 36, no. 2, pp. 288-297.

13. Dong H., Qu S., Chen X., Zhu H., Tai X., Pan J. Changes in the cytokine expression of peripheral Treg and Th17 cells in children with rotavirus enteritis. Exp. Ther. Med. 2015, Vol. 10, no. 2, pp. 679-682.

14. Fontenot J.D., Rasmussen J.P., Williams L.M., Dooley J.L., Farr A.G., Rudensky A.Y. Regulatory T cell lineage specification by the forkhead transcription factor Foxp3. Immunity, 2005, Vol. 22, pp. 329-341.

15. Gregg R., Smith C.M., Clark F.J., Dunnion D., Khan N., Chakraverty R., Nayak L., Moss P.A. The number of human peripheral blood CD4+CD25high regulatory T cells increases with age. Clin. Exp. Immunol. 2005, Vol. 140, pp. 540-546.

16. Grindebacke H., Stenstad H., Quiding-Järbrink M., Waldenström J., Adlerberth I., Wold A.E., Rudin A. Dynamic development of homing receptor expression and memory cell differentiation of infant CD4+CD25high regulatory T cells. J. Immunol., 2009, Vol. 183, pp. 4360-4370.

17. Korn T., Bettelli E., Oukka M., Kuchroo V.K. IL-17 and Th17 cells. Annu. Rev. Immunol., 2009, Vol. 27, pp. 485-517.

18. Lages C.S., Suffia I., Velilla P.A., Huang B., Warshaw G., Hildeman D.A., Belkaid Y., Chougnet C. Functional regulatory T cells accumulate in aged hosts and promote chronic infectious disease reactivation. J. Immunol., 2008, Vol. 181, pp. 1835-1848.

19. Li Y.Y., Wei S.G., Zhao X., Jia Y.Z., Zhang Y.F., Sun S.Z. Th17/Treg cell expression in children with primary nephritic syndrome and the effects of ox-LDL on Th17/Treg cells. Genet. Mol. Res., 2016, Vol. 15, no. 2, pp. 1-8.

20. Li Q., Wang Y., Wang Y., Chen K., Zhou Q., Wei W., Wang Y. Treg/Th17 ratio acts as a novel indicator for acute coronary syndrome. Cell Biochem. Biophys., 2014, Vol. 70, no. 2, pp. 1489-1498.

21. Mosmann T.R., Cherwinski H., Bond M.W., Giedlin M.A., Coffman R.L. Two types of murine helper T cell clone. I. Definition according to profiles of lymphokine activities and secreted proteins, J. Immunol., 1986, Vol. 136, pp. 2348-2357.

22. Prelog M., Hilligardt D., Schmidt C.A., Przybylski G.K., Leierer J., Almanzar G., El Hajj N., Lesch K.P., Arolt V., Zwanzger P.,Haaf T., Domschke K. Hypermethylation of FOXP3 Promoter and Premature Aging of the Immune System in Female Patients with Panic Disorder? PLoS One, 2016, Vol.11, no. 6, e0157930. doi: 10.1371/journal.pone.0157930.

23. Pukhalsky A.L., Toptygina A.P., Viktorov V.V. Immunosuppressive action of cyclophosphamide in mice: contribution of some factors to determination of strain differences. Int. J. Immunopharmacol., 1993, Vol. 15, no. 4, pp. 509-514.

24. Unutmaz D. RORC2: the master of human Th17 cell programming. Eur. J. Immunol., 2009, Vol. 39, no. 6, pp. 1452-1455.

25. Sakaguchi S., Sakaguchi N., Asano M., Itoh M., Toda M. Immunologic self-tolerance maintained by activated T cells expressing IL-2 receptor alpha-chains (CD25). Breakdown of a single mechanism of self-tolerance causes various autoimmune diseases. J. Immunol., 1995, Vol. 155, pp. 1151-1164.

26. Seddiki N., Santner-Nanan B., Martinson J., Zaunders J., Sasson S., Landay A., Solomon M., Selby W., Alexander S.I., Nanan R., Kelleher A., Fazekas de St Groth B. Expression of interleukin (IL)-2 and IL-7 receptors discriminates between human regulatory and activated T cells. J. Exp. Med., 2006, Vol. 203, no. 7, pp. 1693-1700.

27. Schmitt V., Rink L., Uciechowski P. The Th17/Treg balance is disturbed during aging. Exp. Gerontol., 2013, Vol. 48, no. 12, pp. 1379-1386.

28. Sharma S., Dominguez A.L., Lustgarten J. High accumulation of T regulatory cells prevents the activation of immune responses in aged animals. J. Immunol., 2006, Vol. 177, pp. 8348-8355.