HLA-B*3501 versus HLA-B*3508 cells, we acid eluted class I–bound peptides from EBV-infected cells expressing each of these HLA alleles and subjected extracts to HPLC fractionation. As mentioned in the previous paragraph, only a small proportion of cells in LCLs express the BZLF1 anti- gen, which posed a potential problem for these experiments. Because levels vary slightly between different cell lines, we used flow cytometry and an mAb for BZLF1 to test several HLA-B*3501 and HLA-B*3508 LCLs from our stored panel, and those expressing the highest levels were selected for further analysis (HLA-B*3501 LCL, 7.3% BZLF1 ; B*3508 LCL, 3.8% BZLF1 ). Ionizing radiation has been shown to increase the amount of viral reactivation in LCLs (18); thus, to ensure that levels of BZLF1 expression were sufficient for the peptide elution experiments, the LCLs were -irradiated (200 rads) and incubated at 37 C over- night before peptide recovery. This procedure nearly dou- bled the percentage of cells in our cell lines that were posi- tive for BZLF1 expression (HLA-B*3501 LCL, 12.3% BZLF1 ; B*3508 LCL, 6.5% BZLF1 ). Additional control experiments using ELISPOT assays similar to those shown in Fig. 3 C showed that processing and presentation of the APQP peptide were not inhibited by the irradiation step (unpublished data). The HPLC fractions from extracts from the two cell lines that were predicted to include the APQP peptide (based on a parallel HPLC run with synthetic APQP peptide) were tested at varying dilutions for recognition by an APQP-specific CTL clone (Fig. 4, A and B). To allow an estimate of the concentra- tion of APQP in each fraction, synthetic APQP was tested in parallel for CTL recognition at varying concentrations (Fig. 4 C). Half-maximum lysis was observed with 48 pg/ml of syn- thetic peptide (Fig. 4 C), and similar levels of lysis were re- corded when HLA-B*3501 fraction 32 was used at a dilution of 1:32 (Fig. 4 A), suggesting that the concentration of APQP in this fraction was 1,540 pg/ml. Similar extrapolation with data from the other fractions allowed an estimate of the APQP concentration in each (Fig. 4 D). The total amount of APQP peptide eluted off each cell line (from 8 108 cells) was esti- mated at 90 pg for HLA-B*3501 and 40 pg for B*3508. Be- cause there were approximately twice as many BZLF1-express- ing cells in the original HLA-B*3501 population, these data suggest that similar levels of the APQP peptide are presented by virus-infected cells expressing these highly homologous HLA alleles, indicating that other factors must control the im- munogenicity of this EBV T cell epitope. Further analysis by liquid chromatography–mass spectrometry of the peptide rep- ertoire in fraction 32 from the B*3501 LCLs confirmed that the APQP 10-mer peptide was present (not depicted). Structural analyses To determine the effect that the polymorphic residue 156 has on peptide conformation, we determined the structures of HLA-B*3501 and HLA-B*3508 bound to APQP to 2.0 Å (Rfactor, 21.3%; Rfree, 23.3%) and 1.8 Å (Rfactor, 20.9%; Rfree, 23.4%), respectively (Table I). These two binary complexes JEM VOL. 202, November 7, 2005 A R T I C L E Figure 4. EBV-infected cells expressing HLA-B*3501 or HLA-B*3508 present similar levels of the APQP peptide after endogenous pro- cessing. Approximately 8 108 LCLs from the HLA-B3501 donor MW or the HLA-B3508 donor CA were irradiated and incubated overnight to enhance expression of BZLF1 (donor MW, 12.3% of cells BZLF1 ; donor CA, 6.5% of cells BZLF1 ). Cells were then lysed in 0.5% TFA, homogenized, and subjected to ultrafiltration and HPLC fractionation. Fractions 30–33 from (A) the HLA-B*3501 LCL and (B) the HLA-B*3508 LCL that were predicted to include the APQP peptide (based on a parallel HPLC run with synthetic APQP peptide) were tested at varying dilutions for their ability to sensitize HLA-B*3508 target cells to lysis by an APQP-specific CTL clone (see panel D for the graph legend; E/T ratio 2:1). The broken lines at the points of inflection on the dose-response curves mark the fraction dilution that led to 37% lysis. Toxicity controls consisting of target cells incubated with fractions in the absence of CTLs were negative (not depicted). (C) To allow an estimate of the concentration of the APQP peptide in each fraction, synthetic APQP was tested in parallel for CTL recognition at varying con- centrations. Half-maximum lysis (37%) was measured at a synthetic peptide concentration of 48 pg/ml. (D) The concentration of synthetic peptide that led to 37% lysis (48 pg/ml) was divided by the fraction dilution that also led to 37% lysis to give an estimate of the amount of APQP in each fraction and a total amount of the peptide eluted from each cell populations. crystallize in the same space group under identical conditions with isomorphous unit cells and, importantly, the bound epitopes do not participate in crystal contacts. Accordingly, conformational differences that are observed between these structures can be attributed to the polymorphic amino acid at position 156. The electron density for the bound peptides and contacting residues was very clear in the HLA-B*3501- APQP and the HLA-B*3508-APQP structures (Fig. 5). The analysis below links changes in peptide conformation to the local effect of the polymorphism at position 156. Like the previously determined HLA-B35 structure (19), the APQP epitope is bound in a slightly bulged mode, with the N and COOH termini anchored in the A and F pockets, respectively. 10 out of the 12 direct H bonds between the peptide and HLA-B*3508 are located within these pockets (Table II), the majority of which are conserved between all solved HLA B35 structures (16, 19–21). The P1-C /P - C distance in the HLA-B*3508–APQP complex compares 1253