nor MW: HLA A1, A3, B8, and B*3501) and HLA-B*3508 LCLs (donor CA: HLA A30, A32, B42, and B*3508) that had been irradiated (200 rads) and incubated at 37 C overnight to enhance BZLF1 expression (18). These LCLs were selected for these experiments on the basis of relatively high lev- els of BZLF1 expression as measured using flow cytometry and a mouse mAb to BZLF1 (BZ.1). Peptides were recovered from cell lysates after treatment with 0.5% TFA and acid extraction from the cell pellet as de- scribed elsewhere (37). After ultrafiltration (3-kD cutoff) and preconcentra- tion, peptides were collected in 50- l fractions as described previously (38). Peptides were separated by RP-HPLC using a SMART system HPLC (GE Healthcare) with a vydac C18 column (1 mm [inside diameter] 25 cm) and eluted using an optimized linear gradient of acetonitrile in aqueous 0.09% TFA. Peptide fractions were used in chromium release assays to sen- sitize HLA-B*3508 LCL target cells for lysis by a CTL clone from the B*3508 donor SB. In brief, 5 l of each undiluted or diluted fraction was added to 15 l of target cells for 90 min before CTL addition. To test for toxicity, each fraction was also added to chromium-labeled target cells without CTL addition, but no toxicity was detected, even at the lowest di- lution (1:4; unpublished data). HLA-B*3501 fraction 32 was also character- ized by liquid chromatography–mass spectrometry using an Ultra ion trap mass spectrometer coupled to an 1100 nanoLC (both Agilent Technologies) as described previously (38, 39) and found to contain the APQP 10-mer peptide after MS/MS fragmentation of the [M 2H]2 species of target mass m/z 529.2 D (unpublished data). Online supplemental material. Fig. S1 shows an alternate conformation of the polymorphic residue 156 in the HLA-B*3508–APQP complex. 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