Bzlf1

The following stimulatory agents were used in this study: Overlapping peptide pools of EBV-derived proteins BZLF1 (59 peptides) and EBNA3A (234 peptides) (JPT Peptide Technologies, Berlin, Germany), consisting of 15mers overlapping 11 amino acids in a concentration of 1 µg/ml. The optimal assay concentration of both peptide pools was identified in previous titration experiments. Phytohemagglutinin (PHA-L) (Sigma-Aldrich Chemie, Schnelldorf, Germany) was used as a mitogen for stimulation in a concentration of 2 µg/ml. All experiments were performed in triplicates when cells were stimulated with antigen (BZLF1, EBNA3A) or in six replicates for the PHA-L-stimulated cells. RPMI-10 was added as a negative control in triplicates and anti-CD3 (in a dilution of 1:1000, mAb CD3-2, Mabtech AB, Nacka Strand, Sweden) was used as a positive control in a single well for each donor.

For multiVST generation and functional studies, pepmixes (15mers overlapping by 11aa) spanning AdV (Hexon, Penton), BKV (large T, VP1), CMV (IE1, pp65), EBV (EBNA1, BZLF1, LMP2), and HHV6 (U11, U14, U90) (JPT Peptide Technologies, Berlin, Germany) were synthesized. Lyophilized pepmixes were reconstituted in dimethyl sulfoxide (DMSO) (Sigma-Aldrich) and stored at -80°C.

Two types of synthetic peptides consisting of 24 HLA class I-restricted peptides and 9 overlapping peptide pools were utilized. All HLA class I-restricted peptides were kindly provided by Professor Alan B. Rickinson (The University of Birmingham, Birmingham, United Kingdom) and their sequences and identities are listed in Supplementary Table 2. The HLA class-I restricted peptides were diluted into 2 μg/mL in CTL-Test Medium (Cellular Technology Limited, United States) and 100 μl was added to each well in 96-well plate. Fifteen-mer peptides overlapped by 11 amino acids spanning each of the EBV latent proteins, EBNA1, EBNA3A, EBNA3B, EBNA3C and LMP2 and the lytic proteins, BZLF1, BRLF1, BMLF1 and GP350 were purchased from JPT Peptide Technologies, Berlin, Germany. The lyophilized peptide pools of each EBV protein were reconstituted according to manufacturer’s instruction. Final concentration of overlapping peptides was 10 μg/ml per million cells. The HLA class-I restricted peptides were used in the ELISPOT assay and overlapping peptides were used in the flow cytometric analysis.