Recombinant human il4

Effector cytotoxic lymphocytes (CTLs) were generated as described by Chu et al. [28 (link)], with some modifications. MEC-1 cells infected with either LV AS miR181b or LV AS CTRL were seeded on HeLa-CD40L (40:1, MEC-1/HeLa-CD40L) for 72 h in AIM-V (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 2.5% FBS (Sigma-Aldrich, Saint Louis, MO, USA) and recombinant human IL4 (10 ng/mL, BD Biosciences, San Jose, CA, USA). CD40-activated MEC-1 cells were placed in a new well to allow any contaminant HeLa cells to attach. After 3 h, floating cells were collected, treated with mitomycin C (80 µg/mL, Sigma-Aldrich, Saint Louis, MO, USA) for 1 h at 37 °C, and then washed 3 times with serum-free AIM-V. Cells were seeded at 0.5–2.3 × 10⁵/well and co-cultured with purified CD3+ T cells from healthy donors at an effector/target (E/T) ratio of 2:1 in serum-free AIM-V. After 4 days of B and T lymphocytes co-culture, cells were supplemented with IL-2 (50 U/mL, BD Biosciences, San Jose, CA, USA) and cultured for additional 4 days. On day 8, T cells were harvested and analyzed by flow cytometry, used as effector cells in CTL assays, or mixed with CLL cells for the second round of CTL generation.

Drugs, ligands and inhibitors used in this study include etoposide (Sigma Aldrich, St. Louis, MO, USA), SN-38 (Cayman Chemical, Ann Arbor, MI, USA), gemcitabine (Sigma Aldrich), carbonyl cyanide m-chlorophenylhydrazone (CCCP, Sigma Aldrich), phorbol 12-myristate 13-acetate (PMA, Sigma Aldrich), bryostatin 1 (Tocris, Bristol, UK), Ro 31-8220 (Tocris), TPCA-1 (Sigma Aldrich), recombinant human IL-4 (PharMingen, Franklin Lakes, NJ, USA) and paclitaxel (LC Labs, Woburn, MA, USA). All drugs and inhibitors were dissolved in DMSO (Fisher, Waltham, MA, USA) except recombinant human IL-4 which was dissolved in water and paclitaxel which was dissolved 50:50 Kolliphor/ethanol.