Human T cells expressing NY-ESO-1 receptor were prepared by retroviral transduction. In brief, PBMCs were stimulated using plates coated with anti-CD3 (5 μg/mL; OKT3, Janssen Pharmaceutica) and RetroNectin (25 μg/mL; Takara Bio) and were cultured with GT-T551 (Takara Bio) supplemented with 600 IU/mL human recombinant IL-2 (Novartis), 0.2% human serum albumin (CLC Behring), and 0.6% autologous human plasma. On days 4 and 5, these cells were transduced with the retroviral vector pMS3 containing NY-ESO-1 using the RetroNectin-bound virus infection method, wherein retroviral solutions were preloaded onto RetroNectin (Takara Bio)-coated plates and centrifuged at 2,000 × g for 2 h at 32°C, followed by expansion culture. The cells were used for experiments on day 11.
Retronectin
Manufactured by Takara Bio
Sourced in Japan, United States, France, Denmark, China
RetroNectin is a recombinant human fibronectin fragment that promotes efficient retroviral transduction. It facilitates the binding of retroviral particles to target cells, thereby enhancing the efficiency of gene delivery.
Automatically generated - may contain errors
Lab products found in correlation
Lipofectamine 2000, by Thermo Fisher Scientific (42 mentions)
Polybrene, by Merck Group (24 mentions)
Il 15, by Thermo Fisher Scientific (17 mentions)
Interleukin 2 (il 2), by Thermo Fisher Scientific (16 mentions)
Interleukin 3 (il 3), by Thermo Fisher Scientific (16 mentions)
Cd3 cd28 dynabead, by Thermo Fisher Scientific (15 mentions)
Interleukin 6 (il 6), by Thermo Fisher Scientific (15 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (14 mentions)
Stem cell factor (scf), by Thermo Fisher Scientific (14 mentions)
Recombinant human il 2, by Thermo Fisher Scientific (13 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (12 mentions)
Rpmi 1640, by Thermo Fisher Scientific (11 mentions)
Protamine sulfate, by Merck Group (10 mentions)
Streptomycin, by Thermo Fisher Scientific (10 mentions)
Glutamax, by Thermo Fisher Scientific (10 mentions)
Interleukin 7 (il 7), by Thermo Fisher Scientific (9 mentions)
Lenti x concentrator, by Takara Bio (9 mentions)
Anti cd3 cd28 beads, by Thermo Fisher Scientific (9 mentions)
Penicillin, by Thermo Fisher Scientific (9 mentions)
Facsaria 2, by BD (9 mentions)
590 protocols using retronectin
1
Preparation of NY-ESO-1-Expressing T Cells
2
Optimized EBVST Transduction Protocol
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In our previous clinical trials, EBVSTs or triVSTs were transduced on day 19 with a different method. Retroviral supernatant was adhered to retronectin-coated coated plates (0.5 mL for 30 minutes × 2), then 0.5 × 106 EBVSTs were resuspended in 1.5 mL of retroviral supernatant and 0.5 mL of fresh medium and centrifuged for 5 minutes on the coated plates before culture. In the current study, the transduction was performed either early (three days after the first stimulation) or late (three days after the third stimulation on day 19). In both cases, 1.5 ml of retroviral supernatant was added per well of a retronectin (Takara, Mountain View, CA) coated, non-tissue culture-treated plates and centrifuged at 2000 × G for 1 hour at 30°C to allow virus adherence of the retronectin and removal of the possible inhibitory factors that are present in retroviral supernatants. Following removal of the retroviral supernatant, 0.5 × 106 VSTs were added to each well in T-cell media containing 10 ng/ml IL-7 and 400 U/ml IL-4. The plates were centrifuged at 1000 × G for 5 minutes at room temperature and then incubated at 37°C/5%CO2 for an additional 7 days. Expression of the GD2.CAR was assessed by flow cytometry using the 1A7 antibody 4–5 days later and at various times during subsequent culture.
3
Lentiviral Transduction of LSK Cells
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Cultured LSK cells were transduced with lentiviruses on day 2 using a RetroNectin (T100B, Takara)-based method.56 (link) Briefly, lentivirus carrying pcl20-ANK1-GFP, pcl20-ANK1-GG38DD-IRES-GFP, or pcl20-ANK1-GG63DD-IRES-GFP were loaded into RetroNectin coated 12-well plates to aim for a transduction efficiency of 25%–30%. Cultured LSK cells were transferred to the lentivirus-loaded plates and incubated for 1 more day. On day 3, 285K LSK cells were mixed with 500,000 Sca1-depleted competitor BM cells and injected retro-orbitally into lethally irradiated (12 Gy, split dose, Orthovoltage Precision X-ray) recipient heterozygous BERK mice. GFP+ BM cells (constituting 5%–10% of transplanted mouse bone marrow) were isolated via FACSAria-based cell sorting.
4
T cell Transduction with Retronectin
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Transduction of T cells was performed with Retronectin (Takara) as previously described.22 (link),64 (link) Briefly, non-tissue cultured treated 24-well plates were coated overnight with 50 μg/mL Retronectin (Takara), washed once with 1 mL/well PBS prior to adding 300–500 μL/well viral supernatant. Plates were centrifuged for 30 min at 4 °C at 4500 rpm (2820 g). 1 × 106 T cells were added/well, spun for 5 min at 1000 rpm (180 g), and incubated overnight at 37°C. The following day, cells were harvested and cultured in T25/75 flasks at a concentration of 0.8 × 106 cells/mL for 6–8 days in presence of rhIL-2 and rhIL-15.
5
Generating CAR T Cells from PBMCs
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CAR T cells were generated from peripheral mononuclear blood cells (PBMCs) and cultured in RPMI medium supplemented with 10% fetal bovine serum and 1% Penicillin-Streptavidin, all purchased from Life Technologies. B cells were isolated using αCD20 magnetic beads (CD19+ cells post isolation; mean 96.4% ranging from 90.5 to 99.5%) and T cells were subsequently purified from the CD20 negative fraction using pan T magnetic beads (CD3+ cells post isolation; mean 99.3% ranging from 99 to 99.7%), both from Miltenyi Biotec GmbH (cat no 130-091-104 and 130-096-535, respectively). T cells were activated with 1μg OKT-3/mL (Biolegend, cat no 317315) at day 0. On day 2, T cells were stimulated with 100IU IL-2/mL (Proleukin®, Novartis) and the following day (day 3) transduced on RetroNectin-coated (RetroNectin; Takara Bio Inc, cat no T100B) plates pre-incubated with retroviral supernatant and kept in 100IU IL-2/ml.
6
Ectopic Protein Expression in Group1 ILCs
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Expression of ectopic proteins in group1 ILCs was performed by using RetroNectin (Takara) kit. In brief, RetroNectin (Takara) coating and washing were carried out according to the manufacturer's instruction. The retrovirus was added to wells coated with RetroNectin, followed by 4 h incubation at room temperature and removal of the retrovirus. After 24 h co-culture, sorted group1 ILCs from both cKO mice and their wild type compartments were directly placed on plates coated with RetroNectin and retrovirus. Trypsinized OP9-DL1 cells were added to the group1 ILCs cells. Forty-eight hour later, cells were stimulated with IL12 and IL18 and the secretion level of IFNγ was analyzed by ELISA.
7
Activation and Transduction of Primary T Cells
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Buffy coats were provided by the MD Anderson Cancer Center Blood Bank (Houston, USA) and were obtained from whole blood donations of healthy adult donors. PBMCs were isolated using Ficoll-Paque PLUS solution (Cytiva, Marlborough, USA) according to the manufacturer’s instructions. PBMCs were stimulated with an antibody cocktail (ImmunoCult human CD3/CD28 T-cell activator; STEMCELL Technologies, Cambridge, USA) in the presence of IL-2 (Peprotech, Cranbury, USA, 250 IU/mL), IL-2 (150 IU/mL) plus IL-21 (Peprotech, 15 ng/mL), or IL-7 (Peprotech, 5 ng/mL) plus IL-15 (Peprotech, 5 ng/mL) for 3 days. For transduction, 12-well tissue culture plates were coated with Retronectin (Takara Bio USA, Sam Jose, USA; 10 µL of 1 µg/mL Retronectin in PBS per well) overnight, loaded with 100 µL of concentrated LV particles, and centrifuged for 90 min at 1,500 × g, 32°C. Thereafter, 2.5 × 105 activated PBMCs were added to each well. Plates were centrifuged for 45 min at 800 × g, 32°C. On the following day, cells were washed with fresh Roswell Park Memorial Institute (RPMI) medium and incubated with the respective cytokine cocktails for up to 12 days. Transduction efficiency was analyzed by flow cytometry on days 3, 5, and 7 post-transduction.
8
Imaging PD-1 Blockade and IL-2 Signaling
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Human CD4+ T cells freshly purified from PBMCs were activated on anti-CD3/CD28-coated plates. Three days after activation, CD4+ T cells were collected and stained with 10 μM CellTracker Blue CMAC Dye (Invitrogen, C2110) for 15 min at 37 °C; 150,000 cells were seeded on RetroNectin-coated imaging slides and allowed to adhere for 30 min at 37 °C. For RetroNectin coating, imaging slides were treated with 1 μg μl–1 RetroNectin (Takara, T100B) for 40 min at room temperature. Subsequently, T cells were treated for 1 h or 3 h at 4 °C or 37 °C with 630 pM (0.1 μg ml–1) of in-house-produced FAP-IL2v AF647 or 630 pM (0.1 μg ml–1) of in-house-produced PD1-IL2v AF647. Where indicated, cells were pretreated with 10 μg ml–1 anti-PD-1 to saturate all PD-1 binding sites. Afterwards, samples were fixed and permeabilized (BD Cytofix/Cytoperm, 554714) for 20 min at 4 °C and then stained with a non-competing unconjugated anti-PD-1 antibody (1:100; D4W2J, Cell Signaling Technology) for 45 min at 4 °C, followed by staining with goat anti-rabbit IgG (H+L), F(ab′)2 Fragment AF488 (Cell Signaling Technology, 4412, lot 18; 2 mg ml–1; 1:1,000). All samples were then imaged with an inverted confocal microscope (Leica Sp8), adopting a ×40 objective. For each image, one optical section was acquired with a resolution of 1,024 × 1,024 at a pixel size of 0.379 μm.
9
Lentiviral Transduction of Activated T Cells
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Example 9
Highly purified T cells (AllCells) were pelleted at 200 xg for 5 min at RT and resuspended at 1×106 cells/mL in fresh media (RPMI1640 containing 10% fetal calf serum and 1% penicillin/streptomycin). Add CD3/CD28 activator Dynabeads (Thermo Fisher, 25 uL for 1×106 cells) and seed 24 well plate with 1 mL of cells and add IL2 (Thermo Fisher). Monitor cells daily and split cells if needed. The day before the transduction coat a plate with Retronectin (Takara) and store it overnight at 4° C. The next, remove the Retronectin solution add add a blocking solution (2% BSA in PBS) and incubate 30 min at RT. Remove BSA solution add add PBS until cells are ready. Collect activated T cells and resuspend them at 0.5×106 cells/mL in fresh media. Add 1 mL of cells to the Retronectin treated plate, 1 mL of lentivirus solution and IL2. Cells were spinoculated by centrifugation of the pate at 1000 xg for 90 min at RT. The plate was return to the incubator overnight. Next, remove 1 mL of media, add 1 mL of virus and repeat spinoculation. Monitor cells and split them if necessarry at a density of 0.5-1×106 cells/mL. T cells can be used for cytokine release assay or cytotoxicity assay 48h post transduction.
10
Lentiviral Transduction of Mouse and Human T Cells
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Mouse T cells were activated by adding CD3/CD28 Dynabeads (Thermo Fisher Scientific) according to the manufacturer’s instructions and then transduced with lentiviral supernatants. Lentivirus was then added to retronectin (Takara)-coated non-tissue culture–treated plates to reach a total volume of 400 µL and mixed gently. T cells and virus were centrifuged at 1000 g for 2 hours and then incubated at 37°C. After 3 days of culture, the medium was changed to complete RPMI-1640 medium (10% FBS, 2 mM GlutaMAX, 100 µM β-mercaptoethanol, 1% penicillin/streptomycin, 50 U/mL rmIL-2).
Human T cells were stimulated by CD3/CD28 Dynabeads (Thermo Fisher Scientific) according to the manufacturer’s instructions. On day 2, T cells were plated in non-tissue culture-coated 24-well plates, and polybrene (8 µg/mL) was added to the medium. Lentiviral supernatant was first centrifuged at 1500 g for 2 hours on retronectin (Takara)-coated non-tissue culture-treated plates. T cells were then plated and centrifuged at 1000 g for 20 min and incubated at 37°C. After 3 days, the medium was changed to 45% RPMI-1640% and 45% Click’s medium containing 10% FBS and supplemented with rhIL-2 (50 U/mL), rhIL-7 (10 ng/mL), and rhIL-15 (5 ng/mL).
Human T cells were stimulated by CD3/CD28 Dynabeads (Thermo Fisher Scientific) according to the manufacturer’s instructions. On day 2, T cells were plated in non-tissue culture-coated 24-well plates, and polybrene (8 µg/mL) was added to the medium. Lentiviral supernatant was first centrifuged at 1500 g for 2 hours on retronectin (Takara)-coated non-tissue culture-treated plates. T cells were then plated and centrifuged at 1000 g for 20 min and incubated at 37°C. After 3 days, the medium was changed to 45% RPMI-1640% and 45% Click’s medium containing 10% FBS and supplemented with rhIL-2 (50 U/mL), rhIL-7 (10 ng/mL), and rhIL-15 (5 ng/mL).
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