Figure 2

TCR-induced L-selectin downregulation on T-cells requires ADAM17. (A–D) ADAM17^WT, ADAM17^ΔZn/ΔZn RAG-1^−/− chimeras (A,B), B6 and L∆P mice (C,D) were injected intraperitoneally with 10 µg of SEB. After 4 h, L-selectin expression on Vβ8⁺ and Vβ8⁻ T cells isolated from lymph nodes was determined by flow cytometry. Representative histograms show L-selectin expression on Vβ8⁺ (dashed line) and Vβ8⁻ (solid line) T-cells versus an isotype control (control) (A,C). Scatter plots show mean ± SEM (n = 3–5 mice) (B,D). (E–G) Cell surface levels of L-selectin on flow-sorted 868 TCR⁺ Molt3 cells expressing wildtype (E–G) or ΔM-N (E) L-selectin were determined by flow cytometry after incubation for 1 h with SLY peptide-pulsed antigen-presenting cells at a ratio of 1:3. SLY peptide stimulation was conducted in the absence of inhibitors (E), the presence of selective ADAM10 inhibitor GI or dual ADAM10/ADAM17 inhibitor GW (F), or the presence of blocking ADAM17 antibody D1(A12) or control human IgG (G). (H) TCR downregulation on 868 TCR⁺ Molt3 cells was determined by flow cytometry in the absence or presence of 30 μM GW. Percentages for L-selectin and TCR expression were obtained by subtracting the median fluorescence intensity (MFI) of the isotype-matched control from the MFI of each sample and normalizing to non-incubated cells stored on ice (100% expression). Cells were gated as live, single lymphocytes, and antigen-presenting cells were excluded using CD19 expression. Red dashed lines indicate 100% expression (E–H) and maximal downregulation (F,G). Symbols in panels (B) and (D) show data from individual mice, and horizontal bars indicate means. Results in panels (E–H) are mean ± SEM (n = 3–5). Statistical analysis used unpaired Student’s 2-tailed t test. *P < 0.05; ***P < 0.001.