Schematic of the experimental design for proteome-wide screening of the memory CD8+ T cell response to EBV lytic cycle antigens in healthy virus carriers.

EBV-specific CD8+ memory T cells in the blood of healthy carriers constitute a small fraction of the resting lymphocyte pool and require selective re-activation before expansion in vitro. To achieve this, dendritic cells are first prepared from the healthy carrier by GMCSF/IL4 treatment of CD14+ blood monocytes and then exposed to an IE, E and L antigen-containing lysate made from EBV lytically-infected HEK293 cells. Lysate-loaded dendritic cells displaying cross-presented lytic antigens are then co-cultured with PBMCs of the healthy carrier, leading to re-activation of the EBV lytic antigen-specific memory population. After 3 days, CD8+ T cells now expressing the CD137 activation marker are then selected by CD3+,CD8+,CD137+ fluorescent sorting. Thereafter the sorted cells are expanded in vitro for two weeks in mitogen/IL2-containing medium (as used for IM cell expansion) and tested as an effector population. Testing is carried out on transiently transfected MJS cells using the relevant HLA/lytic gene combinations as described in Fig 1; note that HC7 (HLA-A*0301, A*1101, B*1501, B*3501, C*0303, C*0401) is used as an example in the diagram.