To design side chain variants for modulation of immunogenicity, we modeled the complex of the HLA-A2 molecule with an immunodominant peptide, E75, from the HER-2/neu protooncogene protein recognized by CTL. We identified the side chain orientation of E75. We modified E75 at the central Ser(5) (E75 wild-type), which points upward, by removing successively the HO (variant S5A) and the CH2-OH (variant S5G). Replacement of the OH with an aminopropyl (CH2)3-NH3 (variant S5K) maintained a similar upward orientation of the side chain. S5A and S5G were stronger stimulators while S5K was a weaker stimulator than E75 for induction of lytic function, indicating that the OH group and its extension hindered TCR activation. S5K-CTL survived longer than did CTL induced by E75 and the variants S5A and S5G, which became apoptotic after restimulation with the inducer. S5K-CTL also recognized E75 endogenously presented by the tumor by IFN-gamma production and specific cytolysis. S5K-CTL expanded at stimulation with E75 or with E75 plus agonistic anti-Fas mAb. Compared with S5K-CTL that had been restimulated with the inducer S5K, S5K-CTL stimulated with wild-type E75 expressed higher levels of E75(+) TCR and BCL-2. Activation of human tumor-reactive CTL by weaker agonists than the nominal Ag, followed by expansion with the nominal Ag, is a novel approach to antitumor CTL development. Fine tuning of activation of tumor-reactive CTL by weak agonists, designed by molecular modeling, may circumvent cell death or tolerization induced by tumor Ag, and thus, may provide a novel approach to the rational design of human cancer vaccines.