Characterization of the Elasticity of CD4+ T Cells: An Approach Based on Peak Force Quantitative Nanomechanical Mapping

Abstract

CD4⁺ T cells are essential players in orchestrating the specific immune response against intracellular pathogens, and in inhibiting tumor development in an early stage. The activation of T cells is triggered by engagement of T cell receptors (TCRs). Here, CD3 and CD28 molecules are key factors, (co)stimulating signaling pathways essential for activation and proliferation of CD4⁺ T cells. T cell activation induces the formation of a tight mechanical bond between T cell and target cell, the so-called immunological synapse (IS). Due to this, mechanical cell properties, including stiffness, play a significant role in modulating cell functions. In the past, many approaches were made to investigate mechanical properties of immune cells, including micropipette aspiration, microplate-based rheometry, techniques based on deformation during cytometry, or the use of optical tweezers. However, the stiffness of T lymphocytes at a subcellular level at the IS still remains largely elusive. With this protocol, we introduce a method based on atomic force microscopy (AFM), to investigate the local cellular stiffness of T cells on functionalized glass/Polydimethylsiloxan (PDMS) surfaces, which mimicks focal stimulation of target cells inducing IS formation by T cells. By applying the peak force nanomechanical mapping (QNM) technique, cellular surface structures and the local stiffness are determined simultaneously, with a resolution of approximately 60 nm. This protocol can be easily adapted to investigate the mechanical impact of numerous factors influencing IS formation and T cell activation. Graphical abstract: Overview of the experimental workflow. Individual experimental steps are shown on the left, hands on and incubation times for each step are shown right.

Keywords: CD4+ T cell; AFM; Elasticity mapping; Peak Force QNM; Stiffness.

Video 1.. Sample preparation.

Figure 1.. Experimental setup for CD4⁺ T cell sample and AFM preparation.

A. Incubation of CD4⁺ T cells on functionalized surfaces (glass coverslips: top; PDMS substrate on glass slide: bottom). The samples were kept in a wet chamber during a 15-min incubation step at 37°C, to avoid drying (for more details on sample preparation, refer to Procedure part B, C, D and Video 1). B. Microscopic view of CD4⁺ T cells forming an IS on the functionalized substrate after 15 min of incubation. C. Functionalized glass coverslip mounted onto a glass slide after sample preparation. D. Functionalized piece of PDMS after sample preparation. E. Equipment used to insert a cantilever into the cantilever holder (I: tweezers, II: cantilever holder, III: preparation plate, VI: box of cantilevers). F. Detailed view of cantilever holder mounted onto the preparation plate, with adequately inserted cantilever.

Video 2.. Data analysis and image export.

Figure 2.. AFM-based elasticity mapping (PeakForce QNM) and data analysis.

A. Ideal cantilever orientation during elasticity mapping of a quarter CD4⁺ T cell, after shifting the X- and Y-offsets. The resulting elasticity map is indicated as a blue square, and the height profile as a black frame. B, C. Height profile and according elasticity channel (DMT) of a quarter of a T cell after data capture. Shown are scan size and elasticity data scale. D. 3D overlay of height profile and elasticity map, with scan dimensions and elasticity data scale. E. Examples of data maps showing measurement artifacts; top, left: lamellipodia region appears covered by blurry structures (single filopodia are unintentionally detached by the movement of the cantilever tip); top, right and bottom: blurry areas surrounding cell bodies (cantilever edge colliding with the cell body) (see also Notes 1, 2).