Increased IFN-γ–producing cells and augmented in vivo cytolytic activity elicited by targeting of MUC1 to B cells in MUC1 Tg mice. MUC1 Tg mice (n = 3 or 4) were immunized with isotype-MUC1 or anti–CD19-MUC1 conjugates in the presence or absence of CpG adjuvant. Mice were immunized twice at a 2-week interval and were killed at day 21. One group of mice was first injected with anti-CD4 depleting mAb as described in Figure 4B before immunization. (A) Splenocytes from immunized mice were cultured with 20 μg/mL MUC1 peptide overnight and then stained for intracellular IFN-γ production. Representative dot plots are shown. Cells were gated on CD8⁺ T cells. (B) The percentage of IFN-γ–producing CD8⁺ T cells. Mean plus or minus SE is shown. ns indicates not significant. (C) In vivo cytotoxicity. CFSE-labeled syngeneic B cells pulsed with anti–CD19-MUC1 conjugates (CFSE^high) or not (CFSE^low) were injected intravenously into immunized mice. Mice were killed 24 hours after transfer and splenocytes were harvested and assessed by flow cytometry. Cells were gated on the CFSE-positive cells. Error bars show SEM. (D) Cytolytic activity in different regimen immunized mice. Data are representative of at least 3 experiments.