Proliferation pattern and phenotype of day 8 CD4+CD45RO+ Tck cells after cytokine stimulation. Freshly isolated CD4+CD45RO+ cells were labeled with 2.5 μmol/l carboxyfluoroscein succinimidyl ester and stimulated with IL-2, IL-6 and tumour necrosis factor (TNF)-α for 8 days. (a) The number of cell divisions was analyzed on day 8 using FlowJo 7.2.2 software (Tree Star, Inc., Oregon, USA). Cells were counterstained with antibodies against (b) CD25, (c) CD69, (d) CD18 and (e) CD49d, with mean fluorescent intensity (MFI) of these markers assessed in the undivided (M1) peak versus the divided (M2 to M7) fractions. Results are shown in the gates. Figure representative of four independent experiments. Tck cell, cytokine-activated T cell.