A Multimodal Platform for Simultaneous T-Cell Imaging, Defined Activation, and Mechanobiological Characterization.

Martin Fölser,Viktoria Motsch,Johannes B Huppa,René Platzer,Gerhard J Schütz

doi:10.3390/cells10020235

Abstract

T-cell antigen recognition is accompanied by extensive morphological rearrangements of the contact zone between the T-cell and the antigen-presenting cell (APC). This process involves binding of the T-cell receptor (TCR) complex to antigenic peptides presented via MHC on the APC surface, the interaction of costimulatory and adhesion proteins, remodeling of the actin cytoskeleton, and the initiation of downstream signaling processes such as the release of intracellular calcium. However, multiparametric time-resolved analysis of these processes is hampered by the difficulty in recording the different readout modalities at high quality in parallel. In this study, we present a platform for simultaneous quantification of TCR distribution via total internal reflection fluorescence microscopy, of intracellular calcium levels, and of T-cell-exerted forces via atomic force microscopy (AFM). In our method, AFM cantilevers were used to bring single T-cells into contact with the activating surface. We designed the platform specifically to enable the study of T-cell triggering via functionalized fluid-supported lipid bilayers, which represent a widely accepted model system to stimulate T-cells in an antigen-specific manner. In this paper, we showcase the possibilities of this platform using primary transgenic T-cells triggered specifically via their cognate antigen presented by MHCII.

Highlights

T-cells recognize antigen presented at the surface of antigen-presenting cells (APCs) with exquisite sensitivity and specificity
Each plate was built from a silicon frame (Figure S1a) that had been glued onto a glass coverslip to enable simultaneous fluorescence microscopy and atomic force microscopy (AFM) experiments
We investigated the formation of close contacts between the T-cell surface and functionalized Supported lipid bilayers (SLB) using total internal reflection fluorescence (TIRF) microscopy of the T-cell receptor (TCR) labeled using H57-AF647

Summary

Introduction

T-cells recognize antigen presented at the surface of antigen-presenting cells (APCs) with exquisite sensitivity and specificity. While the mechanisms for transducing TCR-pMHC binding into a subcellular signal are still enigmatic [1,2], this process eventually leads to the phosphorylation of immunoreceptor tyrosine-based activation motifs (ITAMs) on the TCR-associated CD3 subunits. A variety of non-exclusive models have been proposed to explain signal transduction, including the kinetic proofreading of single TCR-pMHC binding events [3,4], the serial triggering of multiple TCR complexes by single pMHC molecules [5], or the kinetic segregation of bulky proteins from the narrow cleft of the immunological synapse [6]. Data have indicated mechanical force sensor capabilities in the TCR complex [7]. To this date, the extent to which the different models contribute to the correct picture of

Methods

Results

Conclusion