Quantitative analysis of surface marker densities after exposure of T-cells to concanavalin A (Con A): A sensitive early index of cellular activation

The kinetics of expression of activation-linked T-cell surface markers were analyzed in T-cells obtained from normal donors. The cells were cocultured in triplicate for 0, 1, 24, 48, and 72 hr in the presence of Con A in RPMI 1640. The density of HLA-DR, interleukin 2, and transferrin receptors (IL-2-R and TR, respectively) on the surface of CD3- and CD8-positive cells was computed by a mathematical model based on fluorescence intensity vector analysis, adjusted for cell size, utilizing two-color flow cytometry. The results were compared with controls obtained at the same time with control cells cultured in RPMI 1640 alone. There was a significant increase in the mean density of HLA-DR on the surface of CD3- and CD8-positive cells as early as 1 hr after exposure to Con A when compared with controls (250 and 300%, respectively; P < 0.0001). The mean density of IL-2-R and TR on CD3⁺ cells increased by 265 and 208%, respectively; P < 0.06 and P = n.s., respectively, when compared with control cells. The mean density of Class II MHC products on CD3⁺ and CD8⁺ cells treated with RPMI alone increased by 202 and 468%, 234 and 540%, and 1375 and 2442%, respectively, at 24, 48, and 72 hr of culture. In contrast, the mean cell surface density of these markers in cells treated with Con A increased by 614 and 1962%, 3304 and 7231%, and 8665 and 22,619%, respectively (P < 0.00001) at the corresponding times following exposure to Con A. The density of IL-2-R and TR on CD3⁺ cells exposed to Con A also increased significantly at 24, 48, and 72 hr (P < 0.0001). At the same times, the relative percentage of cell subsets bearing these particular markers increased by 78, 138, and 175% at 24, 48, and 72 hr, respectively. The data suggest that objective quantitative evidence of lymphocyte activation after exposure to Con A may be obtained as early as 1 hr after antigen stimulation, and before significant changes in cell numbers occur. Measurement of cell surface marker densities may provide a useful index for the detection and quantitation of cell activation in the early phase of antigenic stimulation.