Determination of TNF-alpha production by ELISA. TNF-alpha production by purified naive CD8+ T cells (no additional MHC II depletion) after 4 days of stimulation with EpCAM-transfected CHO cells armed either with bscAb EpCAM x CD3 alone, thus providing only a TCR-like primary signal (hatched columns), or with bscAb EpCAM x CD3 and 500 ng/ml heterodimeric B7-1 construct providing an additional costimulatory or second signal (black columns). Controls (empty columns): C1, purified naive CD8+ T cells incubated with 250 ng/ml bscAb EpCAM x CD3 and 500 ng/ml heterodimeric B7-1 construct in the absence of EpCAM-transfected CHO cells. C2, purified naive CD8+ T cells incubated with 250 ng/ml bscAb EpCAM x CD3 alone in the absence of EpCAM-transfected CHO cells. C3, unseparated PBMC incubated with 250 ng/ml bscAb EpCAM x CD3 and 500 ng/ml heterodimeric B7-1 construct in the presence of EpCAM-transfected CHO cells. C4, unseparated PBMC incubated with 250 ng/ml bscAb EpCAM x CD3 alone in the presence of EpCAM-transfected CHO cells. C5, unseparated PBMC incubated in the presence of EpCAM-transfected CHO cells without bscAb EpCAM x CD3 and heterodimeric B7-1 construct. The OD₄₀₅ values for the TNF-alpha ELISA are shown.