1B). Next, plexA1 expression was siRNA

ablated in T cells to evaluate its importance for their expansion driven by allogeneic mDC. Though the scrambled RNA also reduced to some extent the efficiency of proliferation, this was much more pronounced upon plexA1 silencing (Fig. 1C, and inset for RNA silencing control). Similarly, ectopic selleck kinase inhibitor expression of dominant negative, but not full length plexA1 (nor that of an unrelated eGFP-expression plasmid), efficiently abrogated allogeneic T-cell expansion though transfection efficiencies were around 25% only as detected by flow cytometry for the VSV-G-tag of the respective constructs (Fig. 1D, and inset for expression control). To relate their functional requirement to subcellular localization, we Belnacasan analyzed redistribution of plexA1/NP-1 in fixed allogeneic DC/T-cell

conjugates (Fig. 2). CD3 and plexA1 inefficiently translocated towards interfaces in the rarely detectable iDC/T-cell conjugates (Fig. 2A, exemplified in the upper row). In about 80% of mDC/T-cell conjugates, however, interface recruitment of plexA1, and there, co-localization with CD3 were observed (Fig. 2A, exemplified in the bottom row, and in Fig. 2B, fourth panel). PlexA1 interface accumulation was similarly efficient in autologous conjugates involving superantigen(SA)-loaded mDC (not shown). As reported earlier 32, a fraction of NP-1 was also detected within allogeneic mDC/T-cell interfaces (an example is shown in Fig. 2B). Collectively, these data indicate that plexA1 and, to a more limited extent, NP-1 are components of the IS. The instability of MV-DC/T-cell conjugates prevented direct analyses of potential alteration of plexA1/NP-1 redistribution 10. Since IS recruitment of plexA1 specifically in T cells was not yet reported, we confirmed redistribution of this molecule and NP-1 towards stimulatory interfaces

by replacement of mDC by αCD3/CD28-coated beads (Fig. 2C). In line with our flow cytometry data, especially plexA1 was mainly detected in intracellular PDK4 compartments from where it was effciently recruited towards the bead interfaces in about 50% of conjugating T cells (Fig. 2C, upper row and right graph), and this also referred to NP-1 (Fig. 2C, bottom row and right graph). Pre-exposure to MV dramatically decreased the percentage of T cells that are able to polarize these molecules towards the interface (Fig. 2C graphs). This was dependent on the interaction of T cells with the MV gp complex since translocation was recovered in the presence of antibodies directed against the MV H protein. Moreover, plexA1/NP-1 efficiently translocated towards the interfaces in T cells exposed to a recombinant MV expressing VSV-G protein instead of the MV gps (MGV) (Fig. 2C).