Functionalized Bead Assay to Measure Three-dimensional Traction Forces during T-cell Activation

When T-cells probe their environment for antigens, the bond between the T-cell receptor (TCR) and the peptide-loaded major histocompatibility complex (MHC) is put under tension, thereby influencing the antigen discrimination. Yet, the quantification of such forces in the context of T-cell signaling is technically challenging. Here, we developed a traction force microscopy platform which allows for quantifying the pulls and pushes exerted via T-cell microvilli, in both tangential and normal directions, during T-cell activation. We immobilized specific T-cell activating antibodies on the marker beads used to read out the hydrogel deformation. Microvilli targeted the functionalized beads, as confirmed by superresolution microscopy of the local actin organization. Moreover, we found that cellular components, such as actin, TCR, and CD45 reorganize upon interaction with the beads, such that actin forms a vortex-like ring structure around the beads and TCR is enriched at the bead surface, whereas CD45 is excluded from bead-microvilli contacts.