Fig. 6

SHP-1 knockdown abolished TRAIL-R-mediated inhibition on Lck phosphorylation, lipid raft recruitment and T cell activation. a Immunoblotting of phosphorylation of Lck and other proximal TCR signaling molecules (30 min), (b) immunoblotting of Lck and other proximal TCR signaling molecules in pooled raft fractions (30 min), (c) representative confocal images of co-localization of GM-1 with Lck (30 min), (d) flow cytometry of T cell activation markers CD69 and CD25 (24 h) (Scale bar, 4 µm), and (e) intracellular staining of IL-2 from murine splenic CD4.⁺ T cells transfected with scramble or SHP-1 siRNA, followed by stimulation with anti-CD3 (3 µg/mL) and anti-CD28 (2 µg/mL) Abs in the presence/absence of TRAIL (0–1 µg/mL) for 24 h. Quantification of phosphorylated protein levels are shown at the bottom of the panel. Data d–e represent analyses from three independent experiments and statistics determined using Mann–Whitney U test. NS, not significant; *** p < 0.001. f Model of TRAIL-R/SHP-1 repressing TCR signaling and T cell activation through dephosphorylation of Lck at Y394. Upon TCR ligation, Lck binds to, and phosphorylates TCR and Zap70 to form an immunological synapse in the lipid raft, which transduces proximal TCR signaling downstream, resulting in T cell activation (Left). TRAIL engagement reduces Lck (Y394) phosphorylation along with the increased phosphorylated SHP-1 in the TRAIL-R/SHP-1/Lck complex, leading to the dissociation of the co-receptor-Lck complex from the lipid raft, which in turn limits downstream TCR signaling to restrain T cell activation (Right)