Briefly, the relative binding (RB) affinity of each PSCPL sublibrary was determined as RB (PSCPL)=ED 50 (X 9 )/ ED 50 (PSCPL) (where ED 50 is the concentration needed to half-saturate a low concentration of MHC-I molecules) and normalized so that the sum of the RB values of the 20 naturally occurring amino acids equals 20 (since peptides with a given amino acid in a given position are 20 times more frequent in the corresponding PSCPL sublibrary than in the completely random X 9 library). A RB value above 2 was considered as the corresponding position and amino acid being favored, whereas a RB value below 0.5 was considered as being unfavorable (these thresholds represent the 95% confidence intervals). An anchor position (AP) value was calculated by the equation ∑(RB−1) 2 . A primary anchor position is characterized by one or few amino acids being strongly preferred and many amino acids being unacceptable. We have arbitrarily defined anchor residues as having an AP value above 15 ). The peptide-SLA-I*0401 binding activity of each sublibrary was determined using previously published biochemical binding assay (ELISA) (Sylvester-Hvid et al. 2002 ) (with the modifications described above).