Human il 2

To antagonize miR-21, we used the miRZip-21 lentiviral vector expressing anti-sense miR-21 (Systems Biosciences). miRZip-scrambled hairpin vector was used as a control (System Biosciences). The vector additionally contained a GFP reporter. Lentivirus was produced by transfection of a lentiviral vector, along with psPAX2 (Plasmid #12260; Addgene) and pMD2.G (Plasmid #12259; Add gene) expression vectors into HEK293T cells by using FuGENE (Promega). Lentiviral particles were collected 48 and 72 hours after transfection, filtered through a 0.45-mm syringe filter (Millipore), concentrated using Peg-it solution (System Biosciences) and titered on HEK293T cells. For lentiviral transduction, naive CD4⁺ or CD8⁺ T cells labeled with CellTrace Violet (Thermo Fisher Scientific) were activated with anti-CD3/anti-CD28 beads and transduced with a lentiviral vector expressing scrambled control RNA or anti-sense miR-21 at a multiplicity of infection of 10 in the presence of 8 mg/ml polybrene (Sigma) and 10 U/ml human IL-2 (Peprotech). After 36 hours, activated cells were washed and cultured on plates coated with 1 mg/ml anti-CD3 (CD3–2) plus 2 mg/ml soluble anti-CD28 Ab (CD28.2) and 10 U/ml human IL-2 (Peprotech).

Isolated lymphocytes were suspended in RPMI 1640 medium (Gibco, Gaithersburg, MD) supplemented with 10% fetal bovine serum (FBS; Biochrom, Berlin, Germany), 1% (v/v) of penicillin/streptomycin solution (10,000 units/mL of penicillin, 10,000 μg/mL of streptomycin), 50 μg/mL gentamicin, 2.5 μg/mL amphotericin B, 1 μg/mL phyto-hemagglutinin (PHA) (all from Gibco), and 25mM HEPES (Sigma-Aldrich), and incubated at 37°C with 5% CO₂ for 24 hours. After 24 hours, cells were washed by centrifugation at 250 × g for 10 minutes, re-suspended, and transferred to a new 6-well tissue culture plate. After 3 days, cells were washed and transferred to a new plate containing the same medium above, but with PHA replaced by 20 ng/mL human IL-2 (Gibco), and incubated for 7 days using standard proliferation techniques. Total numbers of viable cells were determined using an automated cell counter with Trypan Blue (Countess II, ThermoFisher).

MDA-MB-468 cells were maintained in DMEM (Corning) supplemented with 10% FBS (Corning) and 1% penicillin/streptomycin (ThermoFisher). Healthy human T cells were obtained from the Human Immunology Core (University of Pennsylvania) and expanded as previously described.^{26 (link)} Briefly, CD4 and CD8 T cells were incubated 1:1 and stimulated with CD3/CD28 Dynabeads (Gibco). Human IL-2 (Gibco) was maintained at a concentration of 50 IU/mL for 10 days. The Dynabeads were removed after 7 days of culturing, and the cells were maintained at 0.5–1 M/mL an additional 7 days. The cells were frozen down using a 1:1 mixture of X-VIVO media (Lonza) and 10% DMSO in FBS and allowed to rest in RPMI media (Corning) supplemented with 10% FBS and 1% penicillin/streptomycin 24 hours before cytolysis assays.
10,000 tumor cells were seeded per well 24 hours prior to adding BsAb treatments and T cells at an E:T ratio of 10:1. Controls included 3-fold serial dilutions starting at 100 nM or 10 nM of the monoclonal antibodies alone (Cetuximab and OKT3 separately) and an equimolar mixture of Cetuximab and OKT3. Tumor cytolysis was tracked up to 72 hrs post-treatment using xCelligence Real-Time Cell Analysis (ACEA Biosciences). Data were analyzed and plotted with GraphPad Prism8.

NK cells were isolated by negative selection with the EasySep Human NK Cell Isolation Kit (StemCell Technologies). After 24 hours of activation with 10 ng/mL of human IL15 (R&D Systems), NK cells were stimulated with unmodified K562 feeder cells previously irradiated at 200 Gy. Irradiated K562 cells were cultured with NK cells at a 2:1 K562:NK-cell ratio. NK cells were expanded in Stem Cell Growth Medium (CellGenix) supplemented with 200 IU/mL human IL2, 10 ng/mL human IL15, 10% FBS (Gibco, Thermo Fisher Scientific), and 1% Glutamax (Gibco, Thermo Fisher Scientific). NK cells were stimulated again on day 12 of culture at a 2:1 (K562:NK-cell) ratio. Sample sizes (number of donor-derived lines) used for each experiment are specified in each figure legend.