Nylon wool fiber column

All procedures involving mice were performed according to protocols approved by the Laboratory Animal Care and Use Committees of Wright State University, Kumamoto University and University of the Ryukyus according to the NIH guidelines. Mouse spleen dissection and T cell isolation was performed as previously described in detail⁵³ . Each spleen was weighed on a scale shortly after dissection. For splenocyte culture, the spleen was crushed between two glass slides to release splenocytes which were passed through 40 µm nylon cell strainer (Fisher Scientific, Fair Lawn, NJ) and cultured in RPMI-1640 (Lonza, Walkersville, MD) containing 2 mM L-glutamine, supplemented with 10% fetal bovine serum (Thermo Fisher Scientific, Waltham, MA), 1 mM 1,4-dithiothreitol (DTT; Research Products International, Mount Prospect, IL), 50 IU/ml penicillin and 50 µg/ml streptomycin (MP Biomedicals, Irvine, CA). T cells were purified from splenocytes using nylon wool fiber columns (Polysciences, Inc., Warrington, PA) and cultured in supplemented RPMI-1640. The purified T-cell preparation had approximately 42% CD4 and 35% CD8 cells as determined by flow cytometry (data not shown).

Human and mouse T cells were isolated, activated, and transduced as described previously (28 (link), 29 (link)). In brief, peripheral blood mononuclear cells were isolated from leukopacks (New York Blood Center, New York, New York, USA), from which T cells were isolated with a EasySep Human T Cell Isolation Kit (Stemcell Technologies). T cells were activated with CD3/CD28 Dynabeads (bead/cell ratio of 1:5) and 100 IU/mL IL-2 (Peprotech) and cultured in RPMI1640 supplemented with 10% FBS, 2 mM L-glutamine, and 1% penicillin/streptomycin. After 48 hours, CD3/CD28 beads were removed, and T cells were transduced by spinoculation on plates coated with retronectin (Takara Clontech) with retroviral supernatant from viral packaging cells. Mouse T cells were isolated from spleens, following red blood cell lysis, with a nylon wool fiber column (Polysciences). T cells were activated with CD3/CD28 Dynabeads (bead/cell ratio of 1:2) and 100 IU/mL IL-2 and cultured in RPMI1640 supplemented with 10% FBS, 2 mM L-glutamine, nonessential amino acids, sodium pyruvate, HEPES, 2-mercaptoethanol, and 1% penicillin/streptomycin. After 24 and 48 hours, T cells were transduced by spinoculation on retronectin-coated plates in viral supernatant from Phoenix ECO cells. T cells were rested for 1 day and then used for in vitro and in vivo assays.