(A) EL-4, 4T1, or 86M1 tumor cells were treated with 12.5 nM TAX, 25 ng/ml CIS, or 25 ng/ml DOX for 18 hours. Cells were washed, blocked with 10% mouse or human serum for 20 minutes at 4°C, and incubated with 1:100 vol/vol cation-independent MPR antibody (Abcam) followed by staining with goat anti-rabbit IgG Alexa Fluor 647. The cells were washed and acquired on a FACSCalibur flow cytometer. The MFI is shown. Data from 1 of 2 experiments with the same results are shown. (B and C) To block MPR, M6P (Sigma-Aldrich) was used at a concentration of 20 mM. 86M1 (B) or EL-4 (C) cells were treated with TAX, CIS, or DOX as described above. The cells were incubated with M6P for 15 minutes at room temperature. After 2 washes, the cells were incubated with recombinant human (B) or mouse (C) GrzB for 2 hours. The cells were permeabilized and labeled with anti–mouse GrzB–Alexa 647 or anti–human GrzB–PE. Two experiments with the same results were performed. (D) EL-4 cells were treated with TAX and loaded with control or specific peptides as described in Methods. In the last 15 minutes of peptide loading, one-half of the cells from each treatment group were incubated with 20 mM M6P. The cells were washed and incubated with DDAO-SE–labeled activated OT-1 T cells at a 1:15 ratio. After 1.5 hours, incubated cells were labeled with Annexin V–FITC and 7AAD. DDAO-SE–negative tumor cells were gated and apoptosis measured by flow cytometry. Three experiments with the same results were performed. (E) Neu-specific T cells were generated by immunization of mice as described in Methods and used as effector cells in CTL assay. 4T1 and 4T1-Neu tumor cells were treated with TAX and M6P as described above and used as targets in ⁵¹Cr release assay. Experiments were performed in duplicate. Data are presented as mean ± SEM.