Rhodamine 123: a useful probe for monitoring T cell activation

The T cell activation pathway involves an increase in mitochondrial activity. This can be evaluated in individual cells using the fluorescent probe rhodamine 123 (Rh123) and flow cytometry. Peripheral blood mononuclear cells (PBMC) were stimulated with optimal concentrations of phytohaemagglutinin (PHA), superantigens (Sag) SEA and SEC2, and allogeneic cells. Activation kinetics were followed at days 1, 2, 4 and 7. In all activation conditions, Rh123 uptake was augmented with the CD25 expression, cell size, and DNA synthesis. Rh123 uptake reflected an increase in mitochondrial activity and mass, as assessed by experiments in which Rh123 was substituted for by the 10-nonyl acridine orange, which stains mitochondria in an energy-independent manner. The spectral characteristics of Rh123 allowed us to double stain cells with Rh123 and phycoerythrin-conjugated monoclonal antibodies. In PHA-activated cultures, CD4+ and CD8+ cells incorporated essentially the same amount of Rh123 at all time points, suggesting that the two subsets did not differ in their activation kinetics. Accordingly, after 1 week of culture, no significant modification in the CD4/CD8 ratio was observed. Sag-activated CD4+ cells incorporated a higher amount of Rh123 than did CD8+ cells and preferentially expanded after 1 week of culture as indicated by the increase in the CD4/CD8 ratio. The different behavior of the CD4 and CD8 subsets observed by dual color flow cytometry in the PHA and Sag models was confirmed using purified CD4+ and CD8+ cell preparations obtained by immunomagnetic sorting. CD4+ cells were also the preferential target in the allogeneic model, although the magnitude of the phenomenon was lower than in the Sag model. Present data indicate that Rh123 is a reliable marker for monitoring the mitochondrial compartment during T cell activation. The possibility of phenotyping Rh123-stained cells adds to the applicability of the probe.