A system of overlapping/nonoverlapping defined class I and II peptides demonstrates that enhanced Th-1 responses mediated by DC are dependent upon sequence overlap of loaded class I and II antigenic epitopes. (A) Full class I/II sequence overlap. (B) Partial (5 residues) class I/II sequence overlap. (C) Peptides defined as B8- and DR3-restricted are somewhat promiscuous and can elicit responses in an HLA-B8⁻/HLA-DRβ1*03⁻ background (P < .004 in this representative experiment). For panels A, B, and C: equals IFN-γ release induced by DC loaded with overlapping class I and II peptides, □ equals IFN-γ release induced by DC loaded with nonoverlapping class I and II peptides or singly loaded DC; y-axis equals total IFN-γ release in (in μM²). (D) DCs loaded with overlapping class I and II peptide epitopes support enhanced production of activated CD8⁺ cells (P < .001). equals percent CD8⁺CD25⁺ cells induced by DC loaded with overlapping class I and II peptides. □ equals percentage of CD8⁺CD25⁺ cells induced by singly loaded DC. ■ equals average percentage of CD8⁺CD25⁺ cells induced by all populations of singly loaded DC; y-axis equals percentage of CD8⁺CD25⁺ cells.