An excess of uncomplexed native gp96 blocks the CTL activation induced by gp96/MART-1 complexes. (A) 40 µg/ml of the optimal gp96/MART-1 complex was coincubated with HLA-A2-positive APCs and CTLs (black bar) and with an excess of uncomplexed gp96 (purified from a MART-1 antigen negative B-cell line) at concentrations of 160 µg/ml and 320 µg/ml (diagonal-hatched and white bar, respectively). The presence of an 8-fold excess of uncomplexed gp96 (320 µg/ml) reduced the CTL stimulation by more than 50%. This competitive blockage is a strong indication that the binding of the gp96/MART-1 complexes to the APCs was necessary for the induction of the CTL stimulation. Binding to APCs and the subsequent endocytosis of HSP/antigen complexes is required for the cross-presentation of HSP-associated antigens by APCs. (B) Toxic side effects of the uncomplexed gp96 could be excluded. The activation of CTLs and APCs by 1 µg/ml phytohemagglutinin (PHA) (black bar) was not influenced by a 24-h coincubation with uncomplexed gp96 (diagonal-hatched bar 160 µg/ml, white bar 320 µg/ml).