Primary and secondary immune responses are reduced after immunization with BCR/ABL-expressing DCs. (A) Naive and memory p14 CD8⁺ T cells were restimulated in vitro for 4 days with titrated numbers of LPS-matured BCR/ABL-expressing (■) or control DCs (□, empty GFP vector–transduced) generated from H8 bone marrow cells. ³H-Thymidine incorporation was measured during the last 14 hours of culture. Results are mean plus or minus SEM of 3 to 4 samples per group. Pooled data from 2 independent experiments are shown. (B) Naive C57BL/6 mice were immunized intravenously with 2 × 10⁵ LPS-maturated BCR/ABL-expressing (■) or control DCs (□, empty GFP vector–transduced). Ten days later, the frequency and absolute number of gp33-specific CD8⁺ T cells in the spleen were determined by tetramer staining and by intracellular IFN-γ staining after in vitro restimulation with gp33. Results are mean plus or minus SEM of 4 mice per group. One representative experiment of 3 is shown. (C) C57BL/6 mice previously immunized with 2 × 10⁵ LPS-maturated BCR/ABL-expressing (■) or control DCs (□, empty GFP vector–transduced) and naive C57BL/6 mice (□ dotted) were immunized 14 days later with gp33- and np396-pulsed DCs generated from C57BL/6 mice. Ten days later, the frequency of gp33- and np396-specific CD8⁺ T cells was determined by intracellular IFN-γ staining after in vitro restimulation with gp33 and np396. (D) Splenocytes were isolated 10 days after challenge immunization and analyzed in a standard ⁵¹Cr-release assay. ● represents mice primarily immunized with BCR/ABL-expressing DCs; ■, mice with control DCs (empty GFP vector–transduced); ♦, mice receiving only challenge immunization; ▾, LCMV-immune mice; and ▴, naive C57BL/6 mice. Symbols represent ⁵¹Cr release of gp33-pulsed target cells. Specific ⁵¹Cr release of unpulsed target cells was less than 10%. CTL activity is given as mean plus or minus SEM of 4 mice per group, except for LCMV-immune and naive C57BL/6 mice; ns indicates not significant.