Fragments of pp65 were cloned in frame after a Kozak Initiation sequence in a modified version of pCINEO (see Materials and Methods). HLA A*0201 was also cloned as a genomic fragment in pCINEO. COS cells were transfected with and without HLA A*0201, and with fragments of pp65 in pCINEO as described in Materials and Methods. Ten thousand COS cells per well were incubated for 24 hours in a 96-well plate in triplicate with or without T-cell clone 3-3F4 at an E:T of 5. As controls, 3-3F4 T cells with and without PMA/PHA stimulation were also incubated in triplicate in a 96-well plate without COS cells. 50 μL of supernatant was introduced into wells containing the TNF-α sensitive WEHI-164 cells in triplicate for a 48-hour incubation.

Abbreviation: ND, not determined.