Multiparametric Flow Cytometric Analysis of the Kinetics of Surface Molecule Expression after Polyclonal Activation of Human Peripheral Blood T Lymphocytes

Abstract

In this report we have analysed the kinetics of modulation of human peripheral blood T lymphocyte membrane molecules upon activation with optimal amounts of phytohaemagglutinin (PHA) and concanavalin A (ConA). The following activation-related and differentiation/adhesion molecules were selectively and concomitantly investigated on CD4+ and CD8+ subsets by dual colour flow cytometry: CD69, CD25 and CD71; CD2, CD45RA and L-selectin. Cultures were assayed after 24, 48, 72, 120 and 168 h of incubation with PHA and ConA. This approach allowed a comprehensive evaluation of membrane phenomena occurring during activation of normal resting human T lymphocytes. Data show that the kinetics of expression of these molecules follows a precise and consistent time-course with no major differences between CD4 and CD8 subsets. CD69 expression peaked at 24 h, whereas CD25 and CD71 expression peaked at 48/72 h with some differences between PHA and ConA activation. L-selectin expression started an evident decrease in step with culture time whose magnitude was dependent on the lectin used, being higher with PHA than with ConA. Conversely, the expression of CD45RA remained stable for 72 h and then briskly decreased with no major differences between PHA and ConA activation. CD2 molecules increased with time in number and density, although the percentage of positive cells remained essentially constant (greater than 85%). After 48/72 h of stimulation about 10% of cells co-expressed CD4 and CD8 molecules. To ascertain whether the phenomenon was restricted to cells in a particular activation state, the phenotype of cells in the diverse phases of the cell cycle was established. Results obtained show that only actively proliferating cells, that is cells in S and G2-M phases, co-expressed the two molecules, suggesting that such a phenomenon reflects a momentary dysregulation of the normal sequence of gene expression. The present data are also discussed in the light of the dynamic role of T lymphocyte activation and adhesion molecules in regulating cell-cell interactions, tissue localization and eventual immunological function.