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Updated: May 6, 2026
Real-time Live Imaging of T-cell Signaling Complex Formation
Published on: June 23, 2013
A quantitative assessment of costimulation and phosphatase activity on microclusters in early T cell signaling
J Joris Witsenburg1, Heike Glauner, Jörg P Müller
1Department of Biochemistry, Nijmegen Centre for Molecular Life Sciences, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands.
Abstract:
T cell signaling is triggered through stimulation of the T cell receptor and costimulatory receptors. Receptor activation leads to the formation of membrane-proximal protein microclusters. These clusters undergo tyrosine phosphorylation and organize multiprotein complexes thereby acting as molecular signaling platforms. Little is known about how the quantity and phosphorylation levels of microclusters are affected by costimulatory signals and the activity of specific signaling proteins. We combined micrometer-sized, microcontact printed, striped patterns of different stimuli and simultaneous analysis of different cell strains with image processing protocols to address this problem. First, we validated the stimulation protocol by showing that high expression levels CD28 result in increased cell spreading. Subsequently, we addressed the role of costimulation and a specific phosphotyrosine phosphatase in cluster formation by including a SHP2 knock-down strain in our system. Distinguishing cell strains using carboxyfluorescein succinimidyl ester enabled a comparison within single samples. SHP2 exerted its effect by lowering phosphorylation levels of individual clusters while CD28 costimulation mainly increased the number of signaling clusters and cell spreading. These effects were observed for general tyrosine phosphorylation of clusters and for phosphorylated PLCγ1. Our analysis enables a clear distinction between factors determining the number of microclusters and those that act on these signaling platforms.
Insights
Co-stimulation via CD28 increases the number of T cell signaling microclusters and cell spreading. SHP2 phosphatase reduces microcluster phosphorylation, distinguishing factors that control microcluster quantity versus their signaling activity.
Area of Science:
- Immunology
- Cell Biology
- Biophysics
Background:
- T cell activation relies on T cell receptor (TCR) and costimulatory receptor signaling.
- Receptor stimulation forms membrane-proximal protein microclusters that act as signaling platforms.
- The impact of costimulation and specific signaling proteins on microcluster dynamics is not fully understood.
Purpose of the Study:
- To investigate how CD28 costimulation and SHP2 phosphatase activity influence T cell microcluster formation and phosphorylation.
- To differentiate between factors controlling microcluster quantity and those affecting their signaling state.
Main Methods:
- Utilized microcontact printing of patterned stimuli to control receptor engagement.
- Employed simultaneous analysis of different T cell strains (wild-type vs. SHP2 knock-down) labeled with carboxyfluorescein succinimidyl ester.
- Integrated advanced image processing protocols to quantify microcluster characteristics.
Main Results:
- High CD28 expression led to increased T cell spreading and a greater number of signaling microclusters.
- SHP2 knockdown resulted in higher phosphorylation levels within individual microclusters.
- Both general tyrosine phosphorylation and phosphorylated PLCγ1 levels were affected by CD28 and SHP2.
Conclusions:
- CD28 costimulation primarily increases the number of signaling microclusters and cell spreading.
- SHP2 phosphatase regulates the phosphorylation state of existing microclusters.
- This study distinguishes mechanisms governing microcluster quantity from those modulating their signaling activity.
