Abstract

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 naive 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 naive – 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 naive 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 naive 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.

Highlights

  • Медицинская Иммунология Medical Immunology (Russia)/Meditsinskaya Immunologiya ванного на первоначальной экспрессии CD45RA и CD62L, обнаружена значительная обратная зависимость между возрастом и уровнем «наивных» CD3+CD8+ клеток

  • Еще в 1988 году было показано, что CD45R (в настоящее время – CD45RA) может рассматриваться в качестве маркера «наивных» или «не праймированных» Т-клеток, тогда как антитело UCHL1, распознающее CD45R0, выявляет Т-клетки иммунологической памяти [15]

  • 7. Кудрявцев И.В, Борисов А.Г., Волков А.Е., Савченко А.А., Серебрякова М.К., Полевщиков А.В

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Summary

МНОГОЦВЕТНОЙ ПРОТОЧНОЙ ЦИТОМЕТРИИ

Кудрявцев И.В.1, 2, 3, Борисов А.Г.4, 5, Кробинец И.И.6, Савченко А.А.4, 5, Серебрякова М.К.1. В этом случае выявляют «наивные» цитотоксический Т-клетки фенотипом CD27+CD28+CD45RA+CD62L+ (~30% от CD3+CD8+), клетки центральной памяти – CD27+CD28+CD45RA–CD62L+ (~12%), переходные или «транзиторные» клетки памяти – CD27+CD28+CD45RA–CD62L– (~12%), а также клетки эффекторной памяти и эффекторные клетки (CD27+CD28–, ~11% и CD27–CD28–, ~24% соответственно), оценка CD45RA и CD62L на которых не производится. Медицинская Иммунология Medical Immunology (Russia)/Meditsinskaya Immunologiya ванного на первоначальной экспрессии CD45RA и CD62L, обнаружена значительная обратная зависимость между возрастом и уровнем «наивных» CD3+CD8+ клеток (для относительных значений r = -0,646 при р < 0,001, для абсолютных – r = -0,562 при р < 0,001). В рамках применения подхода, основанного на первоначальной оценке экспрессии CD27 и CD28, обнаружена аналогичная зависимость для «наивных» клеток (r = -0,638 при р < 0,001 и r = -0,530 при р < 0,001 соответственно). Ключевые слова: проточная цитометрия, многоцветный анализ, субпопуляции цитотоксических Т-лимфоцитов, CD45RA и CD62L, CD27 и CD28

MULTICOLOR FLOW CYTOMETRIC ANALYSIS OF CYTOTOXIC
Материалы и методы
Результаты и обсуждение
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