A Quantitative Assessment of Costimulation and Phosphatase Activity on Microclusters in Early T Cell Signaling

J Joris Witsenburg,Merel J W Adjobo-Hermans,Heike Glauner,Roland Brock,Jörg P Müller,Günter Roth,Johannes M M Groenewoud,Frank-Dietmar Böhmer

doi:10.1371/journal.pone.0079277

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.

Highlights

The formation of membrane-proximal protein clusters upon engagement of the T cell receptor (TCR) is a hallmark of early T cell signaling [1,2,3]
When addressing the impact of SH2containing protein tyrosine phosphatase 2 (SHP2) on the phosphorylation of signaling microclusters, we show that the deficiency of this protein-tyrosine phosphatase (PTP) leads to a significant increase in overall phosphotyrosine levels and, phosphorylation of PLCc
Cells with high levels of Cluster of differentiation 28 (CD28) expression have increased surface contact areas but lower local tyrosine phosphorylation when stimulated with aCD28 on microstructured surfaces

Summary

Introduction

The formation of membrane-proximal protein clusters upon engagement of the T cell receptor (TCR) is a hallmark of early T cell signaling [1,2,3]. Cluster formation is the result of protein interactions, driven by phosphorylation of immunoreceptor tyrosine-based activation motifs (ITAMs) in the TCR complex itself and of tyrosines in scaffolding proteins such as the linker for activation of T cells (LAT) [4,5,6,7] and reorganization of the cytoskeleton [8] but the exact mechanisms remain to be further elucidated [9] These protein clusters represent the molecular platforms of early T cell signaling and coalesce to form an immunological synapse (IS) [2,10,11,12,13,14,15,16,17]. One of the downstream effectors is phospholipase C-c1 (PLCc1) for which CD28 costimulation leads to increased activation and tyrosine phosphorylation [28,29]

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