Figure 2.

Zeatin riboside inhibits activation marker expression by CD4⁺ and CD8⁺ T cells; T-cell viability is unaffected by zeatin riboside exposure. Purified CD3⁺CD4⁺ or CD3⁺CD8⁺ T cells (200 000 per well) were incubated on immobilized anti-CD3 mAb for 6 (b and e) or 24 (a, c and d) h in the presence of 1 µM CGS21680 (CGS) or varying concentrations of zeatin riboside (or vehicle control) and 10 µM ZM241385 (or vehicle control). Cell surface expression of CD25 (a and d), CD69 (b and e) and CD40L (c) was assessed by FACS. CD3⁺ T cells were gated on for analysis. Data are shown as the mean±s.e.m. from three independent experiments performed in quadruplicate. *P<0.05 vs. double vehicle-treated control (treated with neither zeatin riboside nor ZM241385) as assessed by one-way ANOVA followed by Dunnett's multiple comparison test. ^#P<0.05 vs. corresponding single vehicle samples (treated with equivalent concentrations of zeatin riboside, but no ZM241385) as assessed by unpaired t-test. Viability of cells treated with 0, 31.25, 62.5, 125, 250, 500 or 1000 µM zeatin riboside was assessed via staining with Alexa Fluor 488 annexin V and propidium iodide (f and g); viable cells are defined as annexin V-negative/PI-negative cell populations. Data are shown as the mean±s.e.m. from three independent experiments; one representative dot plot for each cell subset is included to illustrate characteristic fluorescence intensity levels. ANOVA, analysis of variance; mAb, monoclonal antibody; PI, propidium iodide.