MULTICOLOR FLOW CYTOMETRIC ANALYSIS OF CYTOTOXIC T CELL SUBSETS

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 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 initialexpression 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 effectorcells (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.

Keywords

• flow cytometry
• multicolor analysis
• cytotoxic t cell subsets
• cd45ra/cd62l coexpression
• cd27/cd28 phenotype