(A) Tumor-free C57BL/6 mice were immunized with K^b-bound p53-derived peptide. Splenocytes were isolated, restimulated with the specific peptide for 6 days, and used as effectors in a CTL assay against EL-4 target cells loaded with specific (p53) or control peptides. EL-4 cells were pretreated overnight with 1.5 μg/ml DOX or 12.5 nM TAX. Standard 4-hour ⁵¹Cr-release assay was performed. For A–E, data are shown as mean ± SEM. Each experiment was performed 3 times with the same results. (B) Splenocytes from immunized mice were pretreated overnight with DOX or TAX at doses described above and used as effectors against EL-4 target cells loaded with specific or control peptides in a Cr⁵¹ release assay. (C) Results of a CTL assay wherein both effectors and targets were pretreated with TAX as described above. Spl, splenocytes. (D) T cells from mice immunized with Neu-derived p66 peptide were used as effectors against either nonmodified 4T1 cells or 4T1-Neu cells transfected with Neu-expressing plasmid. The targets were pretreated with TAX for 18 hours. (E) T cells from 2C-transgenic mice were used as effectors against EL-4 cells loaded with the specific (S.P.) or control (C.P.) peptides. The target cells were pretreated with TAX overnight. (F) Cytotoxicity against H332 SCLC cells was measured in duplicate in a standard 6-hour chromium release assay as detailed in Methods. PBMCs stimulated with SCLC tumor cells lysates were used as effectors. The viability of target cells and PBMCs in all tests was similar and greater than 85% at onset of assay. UNT, SCLC and PBMCs were not treated with TAX; SCLC+TAX, tumor cells were pretreated for 18 hours with 100 nM TAX; PBMC+TAX, PBMCs were pretreated for 18 hours with 100 nM TAX; SCLC/PBMC+TAX, tumor cells and PBMCs were treated with TAX. (G) Survivin-specific T cells were generated by 2 rounds of stimulation of mononuclear cells from HLA-A2⁺ healthy volunteers as described elsewhere (18). The T cells were column purified and added to the tumor cells at indicated ratios. Primary tumor from HLA-A2⁺ patient with non-SCLC cancer was used as target. The tumor cells were treated with 50 nM TAX 18 hours prior to ⁵¹Cr release assay.