Journal: Science translational medicine
Article Title: A human orthogonal IL-2 and IL-2Rβ system enhances CAR T cell expansion and antitumor activity in a murine model of leukemia
doi: 10.1126/scitranslmed.abg6986
Figure Lengend Snippet: (A) SeAx, an IL-2-dependent CTCL cell line, was transduced with a lentiviral vector encoding ortho-hIL-2Rβ-T2A-mCherry. After 24 hours of IL-2 starvation followed by a 2 hour additional serum starvation, wild-type SeAx (left panel) or human ortho-IL-2Rβ+ SeAx (right panel) cells were cultured with the indicated doses. Viable cells were enumerated by trypan blue exclusion using hemacytometer on day 4. (B) Primary human T cells were stimulated with anti-CD3/anti-CD28 T cell activator beads for 16 to 20 hours and transduced with a lentiviral vector encoding CAR19 followed by a secondary transduction 16 hours later with ortho-hIL-2Rβ-T2A-mCherry lentiviral particles. Nontransduced T cells (NTD), CAR19 transduced T cells, and ortho-hIL-2Rβ and CAR19 co-transduced T cells were prepared as in (A) 10 days post-transduction. The cells were stimulated with 100nM of wild-type hIL-2 or ortho-hIL-2 for 20 minutes followed by a western blotting with indicated antibodies. Actin served as the loading control. NTD, non-transduced control. (C) Primary human CD8+ T cells were stimulated and transduced as in (B). CD8+ CAR19 alone (left panel) and ortho-hIL-2Rβ+ CAR19 (right panel) T cells were cultured in culture media alone (Control), 20nM of wild-type hIL-2, or 20nM of ortho-hIL-2, and cells were enumerated every other day for 25 days. (D) Primary total T cells were stimulated as in (B) and transduced with a single lentiviral vector encoding CD19BBz and ortho-hIL-2Rβ. After 9 days post-transduction, CAR19-ortho-hIL-2Rβ CAR T cells were co-cultured with irritated Nalm6 cells at an Effector-to-Target (E:T) ratio of 2:1 in media, 20nM of wild- type hIL-2, or 20nM of ortho-hIL-2 for 4 weeks. Nalm6 cells were added every week and CAR T cells were counted by flow cytometry every other day. (E) Total cell yield of CAR T cell growth (3 donors) that received Nalm6 cells compared to CAR T cells without receiving Nalm6 cells is shown for the indicated culture conditions. Comparison of CAR T cells with and without Nalm6 in ortho-hIL-2 and wild-type IL-2 treated groups was performed by repeated measures (RM) one-way ANOVA. (F) ELISA detection of IFN-γ, TNF-α, and GM-CSF is shown for Nalm6 and CAR T co-cultures after 24 hours at an E:T ratio of 2:1. Data represent the mean ± standard deviation of 3 independent donors. (G) Cytolytic activity was measured at the indicated E:T ratios in culture media alone, 20nM of wild-type IL-2, or 20nM ortho-hIL-2 by a luminescence-based cytotoxicity assay using luciferase-expressing Nalm6 (Nalm6-LUC) tumor cells. Cytotoxicity is reported as the mean±SD of triplicate determinations. (H) Cytolytic activity under ortho-hIL-2 treatment is shown. Data were compared using a paired t test. One lytic unit (LU) is defined as 20% specific lysis. The lytic units were calculated using an exponential growth model.
Article Snippet: Lentivirus was produced by transfecting HEK293T cells with a combination of above transfer plasmid and three lentiviral package plasmids, pCI VSV-G, pRSV Rev, and pMDL gag/pol.RRE (Addgene).
Techniques: Transduction, Plasmid Preparation, Cell Culture, Western Blot, Control, Flow Cytometry, Comparison, Enzyme-linked Immunosorbent Assay, Standard Deviation, Activity Assay, Cytotoxicity Assay, Luciferase, Expressing, Lysis
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