Fig. 1

TRAIL-R inhibits proximal TCR signaling and T cell activation. a Flow cytometry analyses of T cell activation markers—CD69 and CD25 (24 h), (b) carboxyfluorescein diacetate succinimidyl diester dilution assays (96 h), and (c) intracellular staining of IL-2 (24 h) on Trail-r^+/+ or Trail-r^–/– murine splenic CD4⁺ T cells stimulated with anti-CD3 (3 µg/ml) and anti-CD28 (2 µg/mL) Abs in the presence/absence of TRAIL (0–1 µg/mL). Data in a–c represent analyses from three independent experiments; statistical significance determined by Mann–Whitney U test; NS, no significance; * p < 0.05; *** p < 0.001. d Trail-r^+/+ murine splenic CD4⁺ T cells were stimulated with CD3 (3 µg/mL) and CD28 (2 µg/ml) Abs in the presence/absence of TRAIL (10 µg/mL) for 24 h. Total RNA was extracted and sequenced on the Illumina platform. Principal component analysis represented correlations among groups. Network analysis of biological process Gene Ontology term enrichment among significant genes in the indicated comparisons. Node color is proportional to p-adjusted enrichment value; node size proportional to number of genes in each Gene Ontology term. Heatmap showing two differentially expressed genes in TCR signaling pathway. Color bars indicate scores of log₂-fold change for each comparison. e ELISAs of lysates from Trail-r^+/+ or Trail-r^–/– murine splenic CD4⁺ T cells stimulated with CD3 (3 µg/mL) and CD28 (2 µg/ml) Abs in the presence/absence of TRAIL (10 µg/mL) for 24 h. Data represent analyses from three independent experiments; statistical significance determined by Mann–Whitney U test; NS, no significance; * p < 0.05; *** p < 0.001. f Immunoblotting of phosphorylation of TCR tyrosine kinases in Trail-r^+/+ or Trail-r^−/− murine splenic CD4⁺ T cells at indicated time points. Quantification of phosphorylated protein levels are shown at the bottom of the panel. Data represent analyses from at least three independent experiments; statistical significance determined by two-tailed Student’s t test; NS, no significance; *** p < 0.001. g Trail-r^+/+ or Trail-r^−/− CD4⁺ T cells were stimulated with anti-CD3 (3 µg/mL) and anti-CD28 (2 µg/mL) Abs in presence/absence of TRAIL (10 µg/mL) for 30 min, then lysed and fractionated as raft and non-raft layers; Lck, ZAP70, LAT, and PLCγ1 were immunoblotted from pooled raft or non-raft fractions. h Representative confocal images of Trail-r^+/+ murine splenic CD4⁺ T cells following staining with anti-GM-1 (red) and anti-Lck (green), or anti-ZAP70 (green) Abs, as indicated. Colocalization of GM-1 with Lck or ZAP70 is indicated by yellow areas. Scale bar: 4 µm