Magnetic activated cell sorter system

CD4⁺ T cells were purified by the magnetic activated cell sorter system (Miltenyl Biotec Inc., Sunnyvale, CA) as described previously [27 (link), 28 (link)]. Briefly, cells were incubated in a nylon wool column (Polysciences Inc., Warrington, PA) to remove B cells and macrophages. Enriched T cell populations were then incubated with biotinylated anti-CD4 (GK 1.5) mAb followed by streptavidin-conjugated microbeads and passed through a magnetized column. The purified T cell fractions were > 97% CD4⁺ and were > 99% viable. Cells were resuspended in complete medium and purified CD4⁺ T cells (4 x 106 cells/ml) were cultured with or without 1 mg/ml of OVA in the presence of T cell-depleted, irradiated (3000 Rads) splenic Ag-presenting cells (APCs). These APCs were derived from naïve mice and were placed in 96-well or 24-well tissue culture plates (Corning Glass Works, Corning, NY) for 5 days at 37°C in a moist atmosphere of 5% CO2 in air. In some experiments, culture supernatants were harvested after 2 or 5 days of incubation and were then subjected to a cytokine-specific ELISA.

Blood samples were collected from CML patients at chronic phase in Zhongda Hospital Southeast University, Nanjing, China. Leukopheresis samples were underway for CD34⁺ cells separation with CliniMACS (Miltenyi Biotech, Germany). CD34⁺ cells were selected by using anti-CD34 magnetic beads in a magnetic activated cell sorter system (Miltenyi Biotec). All subjects signed an informed consent form. The procedure of cell collection from patients conformed to guidelines in the Declaration of Helsinki, and was approved by the Institutional Review Board of Zhongda Hospital Southeast University.