Attenuation of TCR/CD3 signal transduction following CD26 stimulation in CBTCs. (A) CD26-activated protein tyrosine phosphorylation. PBTCs and CBTCs (5 × 106) were incubated in media alone or stimulated with CD26 cross-linking (1F7; 10 μg/mL) for the indicated time periods at 37°C. Equal amounts of total protein loaded from samples were analyzed by immunoblotting with antiphosphotyrosine Ab (4G10). Molecular weight markers are indicated. Results are representative of 10 different donors. (B) Reduced phosphorylation of Lck, ZAP-70, and LAT stimulated with anti-CD26 compared with anti-CD3. PBTCs and CBTCs (5 × 106) were stimulated with anti-CD3 (OKT-3; 10 μg/mL) or anti-CD26 (1F7; 10 μg/mL) for 2 minutes at 37°C. Equal amounts of total protein loaded from samples were analyzed by immunoblotting with antiphosphotyrosine (4G10) or anti-phosphor-Y319 ZAP-70. The blots were stripped and reprobed for Lck, ZAP-70, and LAT. Differences in the intensity of the phosphorylation proteins were assessed using densitometric scanning and expressed graphically. These data are representative of 10 different donors.
