MULTICOLOR FLOW CYTOMETRIC ANALYSIS OF CYTOTOXIC T CELL SUBSETS

Multiparameteric flow analysis has offered an ability of simultaneous analysis of multiple molecules at the single-cell level. Peripheral blood cells from 110 healthy subjects aged 18-65 years (59 males and 51 females) were stained with antibodies to CD3, CD4, CD8, CD27, CD28, CD45, CD45RA and CD62L, and analyzed using different gating strategies. The first one was based on initial analysis of CD45RA and CD62L expression, and CD3+CD8+ cells were divided into naïve population (CD45RA+CD62L+) comprising approx. 30% of the CD3+CD8+ subset; central memory cells (CD45RA–CD62L+, ~11%), effector memory cells (EM; CD45RA–CD62L–, ~35%) and «terminally differentiated» effector memory cells (TEMRA, CD45RA+CD62L–, ~24% of total CD8+ subset). As based on expression of CD27 and CD28 in EM and TEMRA, some further populations were distinguished, i.e., CD27+CD28+ (termed as EM1, about 19% from CD3+CD8+); CD27+CD28– (EM2, ~5%), CD27–CD28– (EM3, ~9%) and CD27–CD28+ (EM4, ~2%). Appropriate subsets were identified within TEMRA population, as follows: CD27+CD28+ (pE1, ~3%), CD27+CD28– (pE2, ~5%) and CD27–CD28– (E, ~15%). The second approach was based on initial
expression of CD27 and CD28, followed by analysis of CD45RA and CD62L expression on CD27+CD28+subset. Total cytotoxic T cell population was divided into naïve – CD27+CD28+CD45RA+CD62L+ (~30% from CD3+CD8+ subset), central memory (CD27+CD28+CD45RA–CD62L+, ca.~12% of total), transitional memory cells (CD27+CD28+CD45RA–CD62L–, approx.~12%), as well as effector memory cells and effector
cells (CD27+CD28я, ~11% и CD27–CD28–, ~24%, respectively). Expression of CD45RA and CD62L was not analyzed for the latter two populations. Frequencies of all cell populations, identified by means of two different gating strategies, were expressed as percentages of the total CD3+CD8+ and absolute cell counts. Using the gating strategy based on initial analysis of CD45RA and CD62L, some correlations between naïve CD3+CD8+frequencies and donor age were revealed (r = -0.646, р < 0.001, and r = -0.562, р < 0.001, respectively). Relative and absolute counts of ЕМ3 (r = 0.474, р < 0.001 and r = 0.435, р < 0.001, respectively) and Е subsets (r = 0.393, р < 0.001 and r = 0.375, р < 0.001, respectively) CD3+CD8+ subsets showed linear increase with age. Usage of another gating strategy based on CD27 and CD28 expression revealed age-dependent changes in relative and absolute frequencies of naïve CD3+CD8+ (r = -0.638, р < 0.001 and r = -0.530, р < 0.001, respectively). Meanwhile, CD27–CD28– subset accumulated linearly with age (r = 0.495, р < 0.001 and r = 0.442, р < 0.001, respectively). The results suggest that differences in subset distribution are responsible for age-related changes in CD8+ cells.

1. 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. [Baidun L.A., Zurochka A.V., Totolian Areg A. Standard technology «Flow cytometric immunophenotyping of peripheral blood lymphocytes» (draft version). Meditsinskaya immunologia = Medical Immunology (Russia), 2012, Vol. 14, no. 3. pp. 255-268. doi: 10.15789/1563-0625-2012-3-255-268 (In Russ.)]

2. Борисов А.Г., Савченко А.А., Смирнова С.В. К вопросу о классификации нарушений функционального состояния иммунной системы // Сибирский медицинский журнал, 2008. Т. 23, № 3-1. С. 13-18. [Borisov A.G., Savchenko A.A., Smirnova S.V. Оn classification of immune system functional status injuries. Sibirskiy meditsinskiy zhurnal = The Siberian Medical Journal, 2008, Vol. 23, no. 1-3, pp. 13-18. (In Russ.)]

3. Зурочка А.В., Хайдуков С.В., Кудрявцев И.В., Черешнев В.А. Проточная цитометрия в медицине и биологии. Екатеринбург: РИО УрО РАН, 2013. 552 с. [Zurochka A.V., Khaidukov S.V., Kudryavtsev I.V., Chereshnev V.A. Flow cytometry in medicine and biology]. Ekaterinburg: RIO UrO RAN, 2013. 552 p.

4. Иванова И.П., Селедцов В.И., Мамаев С.В., Козлов В.А. Содержание отдельных субпопуляций Т-клеток памяти у больных ревматоидным артритом в процессе лечения Т-клеточной вакциной // Вестник Уральской медицинской академической науки, 2011. № 2-2 (35). С. 25-26. [Ivanova I.P., Seledtsov V.I., Mamaev S.V., Kozlov V.A. Memory T-cells distinct subpopulations levels in rheumatoid arthritis patients during T-cell vaccination. Vestnik Ural`skoy meditsinskoy akademicheskoy nauki = Bulletin of the Ural Medical Academic Science, 2011, no. 2-2 (35), pp. 25-26. (In Russ.)]

5. Иванова И.П., Савкин И.В., Селедцова Г.В., Шишков А.А., Селедцов В.И. Фенотипические характеристики и внутриклеточные цитокины Т-клеток памяти у больных рассеянным склерозом после Т-клеточной вакцинации // Бюллетень Восточно-Сибирского научного центра СО РАМН, 2012. № 3-2. С. 79-82. [Ivanova I.P., Savkin I.V., Seledtsova G.V., Shishkov A.A., Seledtsov V.I. Phenotypic characterization and intracellular cytokines of memory T-cells in multiple sclerosis patients after T-cell vaccination. Byulleten` Vostochno-Sibirskogo nauchnogo tsentra SO RAMN = Bulletin of the East Siberian Scientific Center SB RAMS, 2012, no. 3-2, pp. 79-82. (In Russ.)]

6. Кудрявцев И.В. Т-клетки памяти: основные популяции и стадии дифференцировки // Российский иммунологический журнал, 2014. Т. 8 (17), № 4. С. 947-964. [Kudryavtsev I.V. Memory T cells: major populations and stages of differentiation. Rossiyskiy immunologicheskiy zhurnal = Russian Journal of Immunology, 2014, Vol. 8 (17), no. 4, pp. 947-964. (In Russ.)]

7. Кудрявцев И.В, Борисов А.Г., Волков А.Е., Савченко А.А., Серебрякова М.К., Полевщиков А.В. Анализ уровня экспрессии CD56 и CD57 цитотоксическими Т-лимфоцитами различного уровня дифференцировки // Тихоокеанский медицинский журнал, 2015. № 2. С. 30-35. [Kudryavtsev I.V., Borisov A.G., Volkov A.E., Savchenko A.A., Serebryakova M.K.,. Polevschikov A.V. CD56 and CD57 expression by distinct populations of human cytotoxic CD8+T-lymphocytes. Tikhookeanskiy meditsinskiy zhurnal = Pacific Medical Journal, 2015, no. 2, pp. 30-35. (In Russ.)]

8. Кудрявцев И.В., Елезов Д.С. Анализ основных популяций цитотоксических Т-лимфоцитов периферической крови на основании уровня экспрессии CD27, CD28, CD45R0 и CD62L // Российский иммунологический журнал, 2013. Т. 7 (16), № 2-3 (1). С. 57-61. [Kudryavtsev I.V., Elezov D.S. Analysis of the main populations of cytotoxic T lymphocytes of peripheral blood on the basis level or the expression of CD27, CD28, CD45R0 and CD62L. Rossiyskiy immunologicheskiy zhurnal = Russian Journal of Immunology, 2013, Vol. 7 (16), no. 2-3 (1), pp. 57-61. (In Russ.)]

9. Кудрявцев И.В., Субботовская А.И. Опыт измерения параметров иммунного статуса с использованием шести-цветного цитофлуоримерического анализа // Медицинская иммунология, 2015. Т. 17, № 1. С. 19-26. [Kudryavtsev I.V., Subbotovskaya A.I. Application of six-color flow cytometric analysis for immune profile monitoring. Meditsinskaya Immunologiya = Medical Immunology (Russia), 2015, Vol. 17, no. 1, pp. 19-26. doi: 10.15789/1563-0625-2015-1-19-26 (In Russ.)]

10. Лагерева Ю.Г., Черешнев В.А., Еременко А.Ю., Бейкин Я.Б. Содержание CD4+ и CD8+ Т-клеток памяти, наивных и терминально-дифференцированных Т-эффекторов в цереброспинальной жидкости при менингеальной форме энтеровирусной инфекции у детей // Российский иммунологический журнал, 2014. Т. 8 (17). № 3. С. 823-825. [Lagereva J.G., Chereshnev V.A., Eremenko A.J., Beykin J.B. CD4 + and CD8 + memory Т-cells, naive and terminally differentiated T lymphocytes in cerebrospinal fluid during acute stage of enteroviral meningitis in children. Rossiyskiy immunologicheskiy zhurnal = Russian Journal of Immunology, 2014, Vol. 8 (17), no. 3, pp. 823-825. (In Russ.)]

11. Петухова Г.Д., Лосев И.В., Донина С.А., Найхин А.Н. Формирование CD4+ и CD8+ Т-клеток иммунологической памяти у волонтеров после вакцинации живыми аттенуированными гриппозными вакцинами А (H5N2) и А (H1N1)PDM2009 // Инфекция и иммунитет, 2014. Т. 4, № 1. С. 84-85. [Petukhova G.D., Losev I.V., Donina S.A., Na’khin A.N. The formation of CD4+ and CD8+ memory T cells in volunteers blood after immunization with modified-live vaccine against flu virus А (H5N2) and А (H1N1)PDM2009. Infektsiya i immunitet = Russian Journal of Infection and Immunity, 2014, Vol. 4, no. 1, pp. 84-85. doi: 10.15789/2220-7619-2014-1-49-99 (In Russ.)]

12. Семенов А.В., Арсентьева Н.А., Елезов Д.С., Кудрявцев И.В., Эсауленко Е.В., Тотолян А.А. Особенности популяционного состава CXCR3-положительных лимфоцитов периферической крови больных хроническим вирусным гепатитом C // Журнал микробиологии, эпидемиологии и иммунобиологии, 2013. № 6. С. 69-76. [Semenov A.V., Arsentieva N.A., Elezov D.S., Kudryavtsev I.V., Esaulenko E.V., Totolyan A.A. Features of population composition of peripheral blood CXCR3-positive lymphocytes in chronic viral hepatitis C patients. Zhurnal mikrobiologii, epidemiologii i immunobiologii = Journal of Microbiology, Epidemiology and Immunobiology, 2013, no. 6, pp. 69-76. (In Russ.)]

13. Сохоневич Н.А., Хазиахматова О.Г., Юрова К.А., Шуплетова В.В., Литвинова Л.С. Фенотипическая характеристика и функциональные особенности Т- и В-клеток иммунной памяти // Цитология, 2015. Т. 57, № 5. С. 311-318. [Sokhonevich N.A., Khaziakhmatova O.G., Yurova K.A., Shupletsova V.V., Litvinova L.S. Phenotypic characterization and functional features of memory T- and B-cells. Tsitologiya = Сytology, 2015, Vol. 57, no. 5, pp. 311-318. (In Russ.)]

14. Ярилин А.А. Иммунология. М.: ГЭОТАР-Медиа, 2010. 752 c. [Yarilin A.A. Immunology]. Moscow: GEOTAR-Media, 2010. 752 p.

15. Akbar A.N., Terry L., Timms A., Beverley P.C., Janossy G. Loss of CD45R and gain of UCHL1 reactivity is a feature of primed T cells. J. Immunol., 1988, Vol. 140, no. 7, pp. 2171-2178.

16. Appay V., Dunbar P.R., Callan M., Klenerman P., Gillespie G.M., Papagno L., Ogg G.S., King A., Lechner F., Spina C.A., Little S., Havlir D.V., Richman D.D., Gruener N., Pape G., Waters A., Easterbrook P., Salio M., Cerundolo V., McMichael A.J., Rowland-Jones S.L. Memory CD8+ T cells vary in differentiation phenotype in different persistent virus infections. Nat. Med., 2002, Vol. 8, no. 4, pp. 379-385.

17. Appay V., van Lier R.A., Sallusto F., Roederer M. Phenotype and function of human T lymphocyte subsets: consensus and issues. Cytometry A, 2008, Vol. 73, no. 11, pp. 975-983.

18. Badr G., Bedard N., Abdel-Hakeem M.S., Trautmann L., Willems B., Villeneuve J.P., Haddad E.K., Sekaly R.P., Bruneau J., Shoukry N.H. Early interferon therapy for hepatitis C virus infection rescues polyfunctional, long-lived CD8+ memory T cells. J. Virol., 2008, Vol. 82, no. 20, pp. 10017-10031.

19. Fritsch R.D., Shen X., Sims G.P., Hathcock K.S., Hodes R.J., Lipsky P.E. Stepwise differentiation of CD4 memory T cells defined by expression of CCR7 and CD27. J. Immunol., 2005, Vol. 175, no. 10, pp. 6489-6497.

20. Geginat J., Lanzavecchia A., Sallusto F. Proliferation and differentiation potential of human CD8+ memory T-cell subsets in response to antigen or homeostatic cytokines. Blood, 2003, Vol. 101, no. 11, pp. 4260-4266.

21. Hamann D., Baars P.A., Rep M.H., Hooibrink B., Kerkhof-Garde S.R., Klein M.R., van Lier R.A. Phenotypic and functional separation of memory and effector human CD8+ T cells. J. Exp. Med., 1997, Vol. 186, no. 9, pp. 1407-1418.

22. Kambayashi T., Assarsson E., Lukacher A.E., Ljunggren H.G, Jensen P.E. Memory CD8+ T cells provide an early source of IFN-gamma. J. Immunol., 2003, Vol. 170, no. 5, pp. 2399-2408.

23. Killian M.S., Johnson C., Teque F., Fujimura S., Levy J.A. Natural suppression of human immunodeficiency virus type 1 replication is mediated by transitional memory CD8+ T cells. J. Virol., 2011, Vol. 85, no. 4, pp. 1696-1705.

24. Kohler S., Wagner U., Pierer M., Kimmig S., Oppmann B., Möwes B., Julke K., Romagnani C., Thiel A. Post-thymic in vivo proliferation of naive CD4+ T cells constrains the TCR repertoire in healthy human adults. Eur. J. Immunol., 2005, Vol. 35, no. 6, pp. 1987-1994.

25. Larbi A., Fulop T. From «truly naïve» to «exhausted senescent» T cells: when markers predict functionality. Cytometry A, 2014, Vol. 85, no. 1, pp. 25-35.

26. Mahnke Y.D., Roederer M. Optimizing a multicolor immunophenotyping assay. Clin. Lab. Med. 2007, Vol. 27, pp. 469-485.

27. Moro-Garcia M.A., Alonso-Arias R., Lepez-Larrea C. Molecular mechanisms involved in the aging of the T-cell immune response. Curr. Genomics, 2012, Vol. 13, no. 8, pp. 589-602.

28. Naylor K., Li G., Vallejo A.N., Lee W.W., Koetz K., Bryl E., Witkowski J., Fulbright J., Weyand C.M., Goronzy J.J. The influence of age on T cell generation and TCR diversity. J. Immunol., 2005, Vol. 174, no. 11, pp. 7446-7452.

29. Newell E.W., Sigal N., Bendall S.C., Nolan G.P., Davis M.M. Cytometry by time-of-flight shows combinatorial cytokine expression and virus-specific cell niches within a continuum of CD8+ T cell phenotypes. Immunity, 2012, Vol. 36, no. 1, pp. 142-152.

30. Okada R., Kondo T., Matsuki F., Takata H., Takiguchi M. Phenotypic classification of human CD4+ T cell subsets and their differentiation. Int. Immunol., 2008, Vol. 20, no. 9, pp. 1189-1199.

31. Picker L.J., Treer J.R., Ferguson-Darnell B., Collins P.A., Buck D., Terstappen L.W. Control of lymphocyte recirculation in man. I. Differential regulation of the peripheral lymph node homing receptor L-selectin on T cells during the virgin to memory cell transition. J. Immunol., 1993, Vol. 150, no. 3, pp. 1105-1121.

32. Qi Q., Liu Y., Cheng Y., Glanville J., Zhang D., Lee J.Y., Olshen R.A., Weyand C.M., Boyd S.D., Goronzy J.J. Diversity and clonal selection in the human T-cell repertoire. PNAS, 2014, Vol. 111, no. 36, pp. 13139-13144.

33. Romero P., Zippelius A., Kurth I., Pittet M.J., Touvrey C., Iancu E.M., Corthesy P., Devevre E., Speiser D.E., Rufer N. Four functionally distinct populations of human effector-memory CD8+ T lymphocytes. J. Immunol., 2007, Vol. 178, no. 7, pp. 4112-4119.

34. Rufer N., Zippelius A., Batard P., Pittet M.J., Kurth I., Corthesy P., Cerottini J.C., Leyvraz S., Roosnek E., Nabholz M., Romero P. Ex vivo characterization of human CD8+ T subsets with distinct replicative history and partial effector functions. Blood, 2003, Vol. 102, no. 5, pp. 1779-1787.

35. Sallusto F., Lenig D., Förster R., Lipp M., Lanzavecchia A. Two subsets of memory T lymphocytes with distinct homing potentials and effector functions. Nature, 1999, Vol. 401, no. 6754, pp. 708-712.

36. Saule P., Trauet J., Dutriez V., Lekeux V., Dessaint J.P., Labalette M. Accumulation of memory T cells from childhood to old age: central and effector memory cells in CD4(+) versus effector memory and terminally differentiated memory cells in CD8(+) compartment. Mech. Ageing. Dev., 2006, Vol. 127, no. 3, pp. 274-281.

37. Sharpe A.H. Mechanisms of costimulation. Immunol. Rev., 2009, Vol. 229, no. 1, pp. 5-11.

38. Sperling A.I., Auger J.A., Ehst B.D., Rulifson I.C., Thompson C.B., Bluestone J.A. CD28/B7 interactions deliver a unique signal to naive T cells that regulates cell survival but not early proliferation. J. Immunol., 1996, Vol. 157, no. 9, pp. 3909-3917.

39. van Aalderen M.C., Remmerswaal E.B., ten Berge I.J., van Lier R.A. Blood and beyond: properties of circulating and tissue-resident human virus-specific αβ CD8(+) T cells. Eur. J. Immunol., 2014, Vol. 44, no. 4, pp. 934-944.

40. van Epps P., Banks R., Aung H., Betts M.R., Canaday D.H. Age-related differences in polyfunctional T cell responses. Immun. Ageing., 2014, Vol. 11, p. 14.