A, B Spreading of T cells over TCR/CD28-activating bead surface. Isolated human resting T cells were conjugated with anti-CD3/CD28/LAP beads and incubated at 37°C for 45 min and analysed by fluorescence microscopy. Outline of T cells was marked with fluorescent Alexa488 anti-actin mAb (in green in merged image, please note weak binding of Alexa488 mAb to protein G beads), protein G beads with Alexa555 IgG1 mAb (in red). A) Anti-CD3/CD28/LAP beads triggered spread of cells over bead surface leading to their partial or complete engulfment. Actin was depleted from the centre of bead/ T cell contact region and actin-rich lamellipodia formed at the periphery. B) TCR/CD28-mediated bead-engulfment by T cells and actin reorganisation was suppressed in conjugates with TGFβ1-presenting activating beads. Scale bar =10 μm. C) Bar graphs indicate diameter of contact area of 23 (0 ng/ml TGFβ1) or 14 (20 ng/ml TGFβ1) bead/cell conjugates in two independent experiments. Boxes mark the 25% and 75% percentiles and error bars 5% and 95% percentiles of the contact secants. *** mark significant difference of p < 0.0001 in two-tailed Mann–Whitney testing. Please note that 5% and 25% percentiles in data sets of 0 ng/ml TGFβ1 cannot be graphed separately.