7B). In addition, the proliferation of LPL knock-down T cells was attenuated (Fig. 7C). Since the shorter calcium signal of knock-down DAPT nmr T cells is the consequence of a reduced contact time with APC (Fig. 7A and B), it was tempting to speculate that antibody stimulation of T cells neutralizes this effect as this kind of stimulation is independent on T-cell/APC contact time. This was indeed the case (Supporting Information Fig. 8). In marked contrast to stimulation via APC, stimulation via antibodies induced an equal calcium influx in both control and LPL knock-down T cells. Furthermore, there was no inhibition

of proliferation if LPL knock-down T cells were stimulated via crosslinked antibodies (Supporting Information Fig. 8). Thus, the attenuated proliferation of LPL knock-down T cells upon stimulation with APC may at least rely in part on the reduced contact time and the short calcium signals. The activation of antigen-specific T cells is initiated by interaction of T cells with APC bearing the cognate antigen. Thereby, an ordered contact zone, called IS,

is established. The actin cytoskeleton is indispensable for spatial arrangements of a multitude of receptors and proteins that finally define a mature IS at the T-cell/APC contact zone 9, 30. Intracellular proteins regulating the selective spatio-temporal receptor accumulation to the IS were, however, so far not known. Employing RNAi-guided experiments here, we demonstrate that the actin-bundling EGFR inhibitor protein LPL is crucial for the stabilization of LFA-1 in the IS, the duration of T-cell/APC contact formation and sustained calcium signaling. Thus, LPL is an important regulator for the temporal organization of IS formation and T-cell

activation. There are a couple of evidences that the initial relocalization of LPL in the IS is dependent on actin polymerization. Thus, LPL completely colocalized with F-actin in the IS and deletion of the actin-binding domains of LPL interfered with its relocalization. Moreover, it was described that T-plastin binds only to newly formed actin enough filaments, a process which can be considered as actin–plastin copolymerization 14, 15. Therefore, it is likely that the actin/LPL copolymerization in the IS is the major force that brings LPL in the IS. Vice versa, LPL seemed to stabilize F-actin since LPL knock-down T cells had a reduced amount of total F-actin. Given that LPL might protect F-actin from being depolymerized by cofilin 16, F-actin depolymerization may be enhanced in LPL knock-down cells, whereas actin polymerization may be comparable with control cells. In this scenario, F-actin fibers were smaller, which could contribute to the reduced synapse size. Our data show that actin polymerization and accumulation in the T-cell/APC contact zone are required but not sufficient to establish a mature IS.