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

Bence Réthi, Cynthia Detre, Péter Gogolák, Attila Kolonics, Mária Magócsi, Éva Rajnavölgyi

Research output: Contribution to journalArticle

21 Citations (Scopus)

Abstract

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 ([Ca2+]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-κB 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.

Original languageEnglish
Pages (from-to)207-216
Number of pages10
JournalCytometry
Volume47
Issue number4
DOIs
Publication statusPublished - Apr 1 2002

Fingerprint

CD27 Antigens
Calcium Signaling
Antigen-Presenting Cells
Flow Cytometry
Calcium
T-Lymphocytes
Fluorescence
Peptides
NFATC Transcription Factors
Viral Tumor Antigens
Transcription Factor AP-1
Cell Communication
Population
Antigens

Keywords

  • Antigen-presenting cell
  • Calcium signal
  • Costimulation
  • T-cell activation

ASJC Scopus subject areas

  • Hematology
  • Cell Biology
  • Pathology and Forensic Medicine
  • Biophysics
  • Endocrinology

Cite this

Flow cytometry used for the analysis of calcium signaling induced by antigen-specific T-cell activation. / Réthi, Bence; Detre, Cynthia; Gogolák, Péter; Kolonics, Attila; Magócsi, Mária; Rajnavölgyi, Éva.

In: Cytometry, Vol. 47, No. 4, 01.04.2002, p. 207-216.

Research output: Contribution to journalArticle

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N2 - 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 ([Ca2+]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-κB 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.

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