Flow cytometry used for the analysis of calcium signaling induced by antigen-specific T-cell activation

Background: In this study, the effect of antigen-presentingcells (APC), peptide concentration, and CD28 costimulationon calcium signaling, induced by antigen-specificT-cell activation, was studied by flow cytometry.Methods: We used two experimental approaches, whichdiffered in their time scale and in the duration of the Tcell-APC interaction, to measure the increase of intracellularfree calcium levels ([Ca2]i) in activated T cells: (1)Fluo-3–loaded T cells were activated by cocentrifugationwith peptide-loaded APC and the kinetics of fluorescenceintensity changes was monitored continuously and (2)peptide-loaded APC and T cells were mixed, cocultured,and the fluorescence intensity was measured at varioustime intervals.Results: The calcium signal of T cells was dependent onthe APC as demonstrated by the ratio of cells exhibitinghigh versus low fluorescence intensity and by the magnitudeof the calcium signal in the activated population.Short-term interaction of T cells with less potent APC orwith efficient APC in the presence of low antigen concentrationresulted in decreased calcium signaling. CD28-mediated costimulation enhanced the magnitude and sustainedthe increase of intracellular calcium levels. In linewith the strong and sustained calcium signals, the activationof the calcium-dependent transcription factors NF-AT,AP-1, and NF-B was induced.Conclusions: Flow cytometric methods, feasible for therapid and flexible analysis of calcium signaling upon antigen-specificT-cell activation, were established. Kinetics ofthe increase of mean fluorescence intensity reflected thecalcium response of the total cell population whereasstatistical analysis of fluorescence intensity at selectedtime points provided information on the activation state ofsingle cells. Cytometry 47:207–216, 2002