ATRA- and ATRA + DNA–treated mice develop specific immune responses against APL cells and PML-RARα sequences. Cytotoxic CFSE-based assays were performed as previously described.¹⁴  APL cells and control FVB/N Con A blasts were resuspended at 2 × 10⁷/mL in Dulbecco phosphate saline buffer (Eurobio) and labeled with 10μM CFSE (Molecular Probes Europe) for 20 minutes at 37°C. The reaction was stopped by adding an equal volume of FCS, followed by incubation for 2 minutes at room temperature. After 2 washes, 10⁴ labeled targets were incubated at the following E:T ratio: 0:1, 25:1, 50:1, and 100:1, for 6 hours at 37°C in 200 μL of culture medium (RPMI); 10 000 Trucount beads were then added with propidium iodide (PI; 1 μg/mL) to quantify the number of viable cells. The samples were then immediately analyzed by flow cytometry. For each sample, 2000 microbeads were acquired. Only CFSE⁺ and PI⁻ cells were considered as good survivors. The percentage survival was calculated as follows: % survival (y-axis) = [absolute count of viable CFSE + PI⁻ targets with effector (t = 6 hours)]/[absolute count of viable CFSE + PI⁻ targets only (t = 6 hours)] × 100. Effector cells from ATRA- and ATRA + DNA–treated mice have a specific cytotoxicity against APL cells (●) compared with 48-hour induced syngeneic (FVB/N) Con A blasts (○) in a CFSE-based assay. (A) Effectors from 3 ATRA-treated mice assayed at day 57. (B) Effectors from 3 ATRA + DNA–treated mice assayed at days 34 and 48. (C) Effectors from 3 LTSs (ATRA + DNA–treated group), assayed at days 186, 193, and 215; E:T ratio, 25:1, 50:1, and 100:1. Assays were done in triplicate, and values are mean ± SD. (D) The cytotoxicity of effector cells from ATRA + DNA–treated mice is MHC restricted. CFSE-based cytotoxic assays were performed with a blocking anti–H-2D^q/H-2L^q monoclonal antibody (KH117 clone). After staining with CFSE, APL and CBF (○) targets were preincubated with saturating amounts (10 μg/mL) of either the anti–H-2^q mAb (▴) or the isotype control IgG2aκ antibody (●); E:T ratio, 25:1 and 50:1. Assays were done in triplicate, and values are mean ± SD. (E) CD107a⁺ activated T cells from ATRA + DNA–treated LTSs are endowed with an APL-specific cytotoxic activity. APL-activated T cells from LTSs (day 298) were separated by sorting CD3⁺CD8⁺CD107a⁺ cells, using the FACSVantage cell sorter and assessed in a CFSE assay using as targets FVB/N cells (○) and APL cells (●); E:T ratio, 1:1 and 5:1. Assays were done in triplicate, and values are mean ± SD. (F) Ex vivo detection of T-cell responses in APL mice after treatment. Splenocytes were harvested from individual ATRA- and ATRA + DNA–treated mice at day 22 after APL engraftment. A total of 10⁶ splenocytes were incubated with 0.25 × 10⁶ APL cells or with 1μM each of 3 different peptides (PML, RARΑα, and PML/RARα) for 18 hours at 37°C in 5% CO₂ to assess T-cell responses to the PML/RARα sequences of the vaccine. The peptide sequences are: PML, EVFLPNSNHVASGAGEAA (18-mer); RARα, AIETQSSSSEEIVPSPPS (18-mer); PML/RARα, ASGAGEAAIETQSSS (15-mer). The numbers of spot-forming cells (SFC) secreting IFN-γ were assessed ex vivo by ELISpot assay. Assays were done in triplicate; baseline responses without stimulation (none) are indicated. The mean values with SD of SFC per million splenocytes are shown on the y-axis. Data from individual mice are shown; representative data from 1 of 2 identical experiments are shown. ATRA (5 mg) was administered by subcutaneous implantation of 21-day release pellets.