title: Phospho-flow cytometry based analysis of differences in T cell receptor signaling between regulatory T cells and CD4+ T cells.
abstract: CD4+ T regulatory cells (Tregs) are activated during auto-immune, injury, and inflammatory responses, however, the molecular events that trigger Treg activation are poorly understood. The purpose of this study was to investigate whether Tregs (FoxP3+ CD4+ T cells) and non-Treg CD4+ T cells might display differences in T cell receptor (TCR) dependent signaling responses following in vitro or in vivo stimulation. This study used phospho-flow cytometry as a tool to profile the kinetics and extent of TCR signaling (ZAP-70 and PKC-θ phosphorylation and expression) in Tregs and non-Tregs. We found that in vitro stimulation with anti-CD3ε induces early and transient activation of ZAP-70 and PKC-θ in both Tregs and non-Tregs. However, the response in Tregs was more rapid and higher in magnitude than responses seen in non-Tregs. In contrast, bacterial superantigen or antigen-specific TCR stimulation did not significantly activate these signaling pathways in Tregs or non-Tregs. Additional experiments tested the kinetics of in vivo TCR signaling in Tregs and non-Tregs in mice challenged with bacterial superantigen. The results of these experiments showed that superantigen rapidly activated ZAP-70 and PKC-θ in lymph node Tregs, but not in non-Tregs. In summary, we demonstrate the versatility of using phospho-flow cytometry to measure cell signaling in CD4+ T cells. The results of these in vitro and in vivo studies demonstrate that Tregs and non-Treg CD4+ T cells show marked differences in their reactivity to TCR-dependent stimulation and contribute new insights into basic mechanisms that lead to Treg activation.
authors.affiliation: Department of Surgery (Immunology), Brigham and Women's Hospital and Harvard Medical School, 75 Francis Street, Boston, MA 02115, United States.
authors.forename: Marc
authors.initials: M
authors.lastname: Hanschen
authors.forename: Goro
authors.initials: G
authors.lastname: Tajima
authors.forename: Fionnuala
authors.initials: F
authors.lastname: O'Leary
authors.forename: Kimberly
authors.initials: K
authors.lastname: Hoang
authors.forename: Kimiko
authors.initials: K
authors.lastname: Ikeda
authors.forename: James A
authors.initials: JA
authors.lastname: Lederer
mesh_terms: D000818:Animals; D015496:CD4-Positive T-Lymphocytes / Q000276:immunology; D004768:Enterotoxins / Q000276:immunology; D005434:Flow Cytometry / Q000379:methods; D051858:Forkhead Transcription Factors / Q000276:immunology*; D007527:Isoenzymes / Q000276:immunology; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D008822:Mice, Transgenic; D011493:Protein Kinase C / Q000276:immunology; D000074121:Protein Kinase C-theta; D011948:Receptors, Antigen, T-Cell / Q000276:immunology*; D015398:Signal Transduction / Q000276:immunology; D018709:Statistics, Nonparametric; D050378:T-Lymphocytes, Regulatory / Q000276:immunology*; D051746:ZAP-70 Protein-Tyrosine Kinase / Q000276:immunology
chemical_list: D004768:Enterotoxins; D051858:Forkhead Transcription Factors; C498833:Foxp3 protein, mouse; D007527:Isoenzymes; D011948:Receptors, Antigen, T-Cell; C031818:enterotoxin B, staphylococcal; D051746:ZAP-70 Protein-Tyrosine Kinase; C497262:Zap70 protein, mouse; C489593:Prkcq protein, mouse; D011493:Protein Kinase C; D000074121:Protein Kinase C-theta