CTLs recognize 8- to 10-mer peptides on MHC class I molecules. Recent studies have shown that human CTLs kill autologous tumor cells in an HLA-restricted and peptide-specific manner, and that artificial peptides can stimulate tumor-specific CTLs both in vitro and in vivo. Accordingly, several human clinical trials using such peptides are ongoing worldwide. In such methods, the amount of peptide-MHC complexes that remain on the cell surface of APCs after peptide administration is crucial, because CTL activation depends on the number of ligated TCRs and co-stimulation. However, it remains uncertain how many peptide-MHC complexes are reconstituted and remain on live cells after peptide administration. We herein examined the binding affinities of five HLA-A*0201 restricted peptides - four TAAs and one HIV antigen - to HLA-A*0201 molecules and their decay rates on a live B cell line using tandem mass spectrometry. Our experiments showed that nearly 105 peptide-MHC complexes per cell could be reconstituted on a cell surface by pulsing a high dose of peptide even if the binding affinities were intermediate or low. However, the decay rates observed for these peptide-MHC complexes on a B cell line were faster than previously estimated. © American Society for Histocompatibility and Immunogenetics, 2001.