Figure 2.

The CLIP peptide binds to both DP4 and DP4^84DEAV87 with greater binding strength than the known DP4-restricted MAGE-A3_243–258 peptide. (a) Parental K562 or K562 cells expressing one of DP4 or DP4^84DEAV87 were pulsed with 100 μM of CLIP, TT_947–967, or an equivalent volume of DMSO for 18 hrs. Surface CLIP expression was then analyzed by flow cytometry on the indicated K562 transfected cells following staining with CLIP-specific mAb. (b) A K562 cell-based competitive binding assay was performed using K562-based transfected cells encoding either DP4 or DP4^84DEAV87, pulsed with graded concentrations of CLIP, MAGE-A3_243–258, or MART1_27–35 peptide in the presence of 2 μM biotin-conjugated reference peptide, HIV ENV_31–45. Cells were then stained by phycoerythrin-conjugated streptavidin and analyzed by flow cytometry. Percent inhibition of binding by CLIP peptide was calculated by Mean Fluorescence Intensity (MFI) using the following formula: %inhibition = [1 − (MFI with CLIP peptide/MFI without CLIP peptide)] × 100.