Cross-presentation test with gp96/MART-1 complexes. Initially, 8x10⁴ HLA-A2-positive APCs were coincubated with the gp96/MART-1 complexes at the indicated concentrations for 1 h at 37°C. Then, 2x10⁴ CTLs were added to each well of the 96-well plate and incubated at 37°C for 24 h. The supernatant was collected and the CTL stimulation measured by IFN-gamma ELISA. The optimal gp96/MART-1 complexes (reconstituted at 50°C) activated the CTLs, a reaction which was highly dependent on the amount of added complex and on the presence of APCs (CTL stimulation with APCs versus without APCs, shown as black bars and thick diagonal-hatched bars respectively; paired t-test; P=0.0330). In contrast, the suboptimal complexes (reconstituted 4°C), which contained about one-tenth the complexed peptide, stimulated CTLs significantly less (paired t-test, P=0.03) and were practically APC-independent (CTL stimulation with APCs versus without APCs, shown as white bars and thin diagonal-hatched bars, respectively). These results indicate that the CTL stimulation was highly dependent on the amount of complexed peptides.