Flow cytometry used for the analysis of calcium signaling induced by antigen-specific T-cell activation.
BACKGROUND
In this study, the effect of antigen-presenting cells (APC), peptide concentration, and CD28 costimulation on calcium signaling, induced by antigen-specific T-cell activation, was studied by flow cytometry.


METHODS
We used two experimental approaches, which differed in their time scale and in the duration of the T cell-APC interaction, to measure the increase of intracellular free calcium levels ([Ca(2+)](i)) in activated T cells: (1) Fluo-3-loaded T cells were activated by cocentrifugation with peptide-loaded APC and the kinetics of fluorescence intensity changes was monitored continuously and (2) peptide-loaded APC and T cells were mixed, cocultured, and the fluorescence intensity was measured at various time intervals.


RESULTS
The calcium signal of T cells was dependent on the APC as demonstrated by the ratio of cells exhibiting high versus low fluorescence intensity and by the magnitude of the calcium signal in the activated population. Short-term interaction of T cells with less potent APC or with efficient APC in the presence of low antigen concentration resulted in decreased calcium signaling. CD28-mediated costimulation enhanced the magnitude and sustained the increase of intracellular calcium levels. In line with the strong and sustained calcium signals, the activation of the calcium-dependent transcription factors NF-AT, AP-1, and NF-kappaB was induced.


CONCLUSIONS
Flow cytometric methods, feasible for the rapid and flexible analysis of calcium signaling upon antigen-specific T-cell activation, were established. Kinetics of the increase of mean fluorescence intensity reflected the calcium response of the total cell population whereas statistical analysis of fluorescence intensity at selected time points provided information on the activation state of single cells.
Réthi
Bence
B
Department of Immunology, Eötvös L. University, Göd, Hungary. has@ludens.elte.hu
Detre
Cynthia
C
Gogolák
Péter
P
Kolonics
Attila
A
Magócsi
Mária
M
Rajnavölgyi
Eva
E
D000818:Animals; D000938:Antigen-Presenting Cells / Q000166:cytology / Q000276:immunology / Q000378:metabolism*; D000941:Antigens / Q000276:immunology*; D000954:Antigens, Surface / Q000276:immunology; D018122:B7-1 Antigen / Q000276:immunology; D018106:CD28 Antigens / Q000276:immunology; D002118:Calcium / Q000378:metabolism*; D020013:Calcium Signaling / Q000502:physiology*; D002450:Cell Communication / Q000276:immunology; D004268:DNA-Binding Proteins / Q000276:immunology; D005434:Flow Cytometry / Q000379:methods*; D008213:Lymphocyte Activation / Q000276:immunology*; D051379:Mice; D008807:Mice, Inbred BALB C; D016328:NF-kappa B / Q000276:immunology; D050778:NFATC Transcription Factors; D009687:Nuclear Proteins; D013601:T-Lymphocytes / Q000166:cytology / Q000276:immunology / Q000378:metabolism*; D018808:Transcription Factor AP-1 / Q000276:immunology; D014157:Transcription Factors / Q000276:immunology; D014407:Tumor Cells, Cultured
D000941:Antigens; D000954:Antigens, Surface; D018122:B7-1 Antigen; D018106:CD28 Antigens; D004268:DNA-Binding Proteins; D016328:NF-kappa B; D050778:NFATC Transcription Factors; D009687:Nuclear Proteins; D018808:Transcription Factor AP-1; D014157:Transcription Factors; D002118:Calcium