FIG. 4.

KMP11 peptide-specific CTL response. (A) KMP11 deduced amino acid sequence and composition of synthesized peptides containing theoretically estimated A2 union motifs (17). The asterisk indicates the protein stop codon. The positions of the designed A2 peptides K1, K2, K3, and K4 are marked. (B) KMP11 peptide-specific CTLs elicited after immunization with the DNA vector carrying the KMP11-HSP70 fusion. Spleens from C57BL/6-A2.1/K^b mice immunized with saline solution (□) or the pCMV4 (∗), pCMV4.11 (▵), and pCMV4.11.70 (⧫) vectors were removed 2 weeks after the last immunization. Splenocytes were used as effector cells after being incubated for 6 days with EL4-A2.1/K^b cells treated with 50 μg of mitomycin/ml and loaded separately with each of the four KMP11 A2 peptides. CTL activity was measured against EL4-A2.1/K^b cells pulsed with or without each one of the respective peptides by a standard ⁵¹Cr release assay (19). Each panel corresponds to results with one A2 peptide. The level of lysis of EL4-A2.1/K^b cells in the absence of peptide was <5% for all groups (data not shown). Specific lysis was calculated using the following formula: percent specific lysis = (experimental release [cpm] − spontaneous release [cpm]/(total release [cpm] − spontaneous release [cpm] × 100), where cpm is counts per minute. Spontaneous release represents the counts obtained when the target cells were incubated in culture medium without effectors, and total release was obtained after treatment of target cells with 2.5% Triton X-100. Experiments with more than 20% spontaneous lysis were discarded. Data are representative of results with three mice per group.