Fig. 8.
Dasatinib blocks antigen-induced TCR downregulation and tetramer internalization from the cell surface. A. Mel13 CTL were pre-treated with PBS ± 50 nM dasatinib and exposed to C1R-A2 B cells previously pulsed with 10-6M ELAGIGILTV peptide or medium alone for 4 h at 37 °C. Cells were subsequently stained with anti-TCR-FITC (clone BMA 031; Serotec) and anti-CD8-APC (clone RPA-T8; BD Pharmingen) mAbs for 30 min on ice, washed twice and resuspended in PBS. Data were acquired on a FACSCalibur flow cytometer (BD) and analyzed using FlowJo software. B. 105 ILA1 CTL were pre-treated with PBS (i & ii) or PBS + 50 nM dasatinib (iii & iv) for 30 min at 37 °C, then stained with 20μg/ml HLA A2/ILAKFLHWL-Alexa488 tetramer for 15 min at 37 °C. Microscopy was performed as described in the Materials and methods.