Murine il 2

Suppressor activity of spleen and tumor-derived MDSCs was evaluated using CFSE proliferation assay. Spleen and tumor cells obtained from tumor-bearing mice were labeled with monoclonal anti-CD11b antibodies conjugated with magnetic nanoparticles (BD Imag). CD11b⁺ cells were magnetically sorted and cocultured in a 1:1 ratio with splenocytes from healthy mice labeled with CFSE dye (15 min, 37°C; Molecular Probes) for 3 days in the presence of concanavalin A (2 µg/ml; Sigma-Aldrich) and murine IL-2 (100 LU/ml). After that, cells were harvested and stained with anti-CD4 APC and anti-CD8 PE-Cy7 monoclonal antibodies (BD Biosciences). The intensity of CFSE fluorescence in CD4⁺ and CD8⁺ cells was measured with FACS Calibur apparatus with CellQuest software.

We collected viruses 48h (first portion) and 72h (second portion) after transfection. The day before transduction, splenocytes were isolated as described above and activated with ConA (3 μg/ml, Sigma, USA) and murine IL-2 (10 U/ml, Sigma) for 24h. Retroviral transduction was performed by two rounds of spinoculation using the first portion of the virus in the first round and using the second portion of the virus in the second round. The conditions for each spinoculation were the following: 2h, 2000g, 22°C. The efficiency of transduction was estimated 3 days after by measuring GFP fluorescence using flow cytometry, and on average it was 40-60%. The cells were immediately used in the experiments 3-4 days after transduction. Cells with transduction efficiency lower than 30% were not used in the experiments.