Overview of screening strategy.

<p>PBMCs were screened by <i>ex vivo</i> ELISpot analysis for recognition of 187 overlapping 15mer peptides spanning the entire IE1 and IE2. Pools of positively recognized peptides were used to expand the T cells for 12–14 days and subsequently analyzed for CD4⁺ and CD8⁺ T cell recognition using ICS and flow cytometric analysis. <i>CD4</i>⁺<i>T cell epitope deconvolution:</i> The recognized 15mer peptide and the donor's HLA class II molecules were submitted to NetMHCIIpan to predict the HLA class II restriction element and the peptide core sequence interacting with the HLA class II molecule. The interaction was subsequently validated using a biochemical HLA class II binding assay. For a selection of the recognized epitopes, H₆-tagged peptides were produced and used to generate peptide-HLA class II tetramers, which were subsequently used for validation of T cell specificity and HLA class II restriction. <i>CD8⁺ T cell epitope deconvolution:</i> The 15mer peptides recognized by a given donor together with the donors HLA class I molecules were submitted to the HLArestrictor to predict the optimal size epitope and HLA-restriction. Interaction between the predicted epitope and HLA class I molecule was validated by biochemical affinity- and stability assays. The T cell specificity and HLA class I restriction was validated by peptide-HLA class I tetramer staining.