From a leukapheresis preparation from healthy humans with the HLA-A2 haplotype (Biological Specialty Corporation, 213-15-04), human PBMCs were obtained by Ficoll-Hypaque (Sigma-Aldrich, GE17-1440-02) density gradient centrifugation.24 The PBMCs isolated from uninfected human donors or naïve sheep were cultured in 100 mm culture dishes (BD Biosciences, 353003) for 1 h at 37°C in AIM V media (Thermo Fisher Scientific, 12055–091) with 2.5% autologous heat inactivated plasma. Following culture, the non-adherent cells were removed and adherent cells harvested and seeded into 96 well v-bottom plates (Corning, 3894) at 2 × 105 cells/well. All remaining steps were performed at 4°C. Cells were incubated for 1 h with 1, 5, 20, 50 µg/mL C-HBV diluted with PBSB (Dulbecco’s phosphate buffered saline, Thermo Fisher Scientific, 14190–250 containing 0.1% (w/v) bovine serum albumin (BSA)). Protein binding was detected by incubating cells with rat anti-mouse IgG1-biotin (BD Biosciences, 553441) in PBSB for 20 min, followed by streptavidin phycoerythrin cyanine-5 (SA-PE-Cy5;BD Biosciences, 554062) for 20 min. Cells were re-suspended in PBSB containing 2% paraformaldehyde (PF) and surface binding of C-HBV assessed by flow cytometry.
Aim 5 media
Manufactured by Thermo Fisher Scientific
Sourced in United States, Canada
AIM-V media is a serum-free, protein-free cell culture medium designed for the growth and maintenance of a variety of cell types, including lymphocytes and other immune cells. It provides essential nutrients and growth factors to support cell proliferation and viability.
Automatically generated - may contain errors
Lab products found in correlation
Rpmi 1640, by Thermo Fisher Scientific (5 mentions)
Interleukin 2 (il 2), by Thermo Fisher Scientific (4 mentions)
Human ab serum, by Merck Group (4 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (4 mentions)
Il 15, by Thermo Fisher Scientific (3 mentions)
Cell dissociation buffer, by Thermo Fisher Scientific (3 mentions)
Dnase 1, by Roche (3 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (3 mentions)
Interleukin 4 (il 4), by Thermo Fisher Scientific (3 mentions)
Anti cd3 cd28 monoclonal antibodies, by Miltenyi Biotec (3 mentions)
Recombinant m csf, by BioLegend (2 mentions)
Retronectin coated plate, by Takara Bio (2 mentions)
Lympholyte h density separation, by Cedarlane (2 mentions)
Anti cd3 antibody okt3, by BioLegend (2 mentions)
Human il 2, by BioLegend (2 mentions)
Facsaria 2 cell sorter, by BD (2 mentions)
Ficoll paque, by GE Healthcare (2 mentions)
Non essential amino acids (neaa), by Thermo Fisher Scientific (2 mentions)
Glutamine, by Thermo Fisher Scientific (2 mentions)
Glutamax, by Thermo Fisher Scientific (2 mentions)
105 protocols using aim 5 media
1
Measuring HBV Protein Binding to PBMCs
2
Cryopreserved PBMC Stimulation Assay
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Cryopreserved human PBMCs from the Montreal HIV cohort were thawed, washed with AIM-V media (ThermoFisher Scientific), and rested at 37°C for 3 hours. Cells were aliquoted into wells of a 96-well plate, at a total of 1 x 106 cells per well. For each assay, 5 conditions were used: no exogenous stimulation (“UN”), three antigen stimulations, or SEB (1 μg/ml). The antigen stimulations were overlapping peptide pools corresponding to one of HCMVA pp65 (human cytomegalovirus, JPT, PM-PP65), HIV-1 Gag ULTRA (JPT, PM-HIV-Gag), or HIV-ENV ULTRA (JPT, PM-HIV-ENV); the peptide pools were used to stimulate at a final concentration of 0.5μg/ml of total peptide. Following an 18 hr stimulation, the cells were stained for 1hr at 4°C according to the panel in S1 Table . The CXCR5 antibody was included during both the stimulation incubation and also included with the rest of the panel. The cells were washed, fixed with 1% formaldehyde, washed, and acquired the same day.
3
Ex vivo TIL Expansion for Cell Therapy
4
PBMC and Plasma Isolation from Venous Blood
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Venous blood (up to 9 mL) was collected in acid citrate dextrose tubes (BD Vacutainer). Peripheral blood mononuclear cells (PBMC) (n = 95 children with enough cells for analysis) and plasma (n = 103) were isolated by Ficoll-Paque density gradient centrifugation (Ficoll-Paque Premium 1.077; GE Healthcare) using SepMate 50-mL tubes (Stemcell Technologies) following the manufacturer’s instructions. Cells were cryopreserved at −150°C in 25% fetal calf serum/10% dimethyl sulfoxide (DMSO)/65% AIM-V media (Gibco; Thermo Fisher Scientific, Waltham, MA), and plasma was stored at −80°C until used.
5
Thawing and Stimulating Immune Cells
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Cells were thawed by briefly incubating them in a 37°C water bath and were added dropwise to room temperature AIM-V media (Thermo Fisher) containing 20 units/ml Benzonase (Novagen). Cells were washed extensively and rested overnight at 37°C in AIM-V media supplemented with 10% fetal bovine serum (FBS) (Gibco) for flow cytometry and Luminex experiments or were stimulated for 5 days in media (0.25 × 106 cells/ml) supplemented with 10% FCS, 2.5 µg/ml R848 (InvivoGen), and 1,000 U/ml IL-2 (PeproTech) for microengraving experiments (58 (link)).
6
Phenotypic Profiling of Tumor-Infiltrating Immune Cells
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Ex vivo tumor single cell suspensions were cultured at 3 × 105 viable cells in triplicates in a 96-well plate (U-bottom) using serum-free AIM-V media (ThermoFisher, Massachusetts, USA) and treated with either Ad5/3-E2F-d24 or Ad5/3-E2F-d24-hIL7 as described above. On day 3, supernatants were collected and plated in the bottom chamber of Transwell (Corning, New York, USA) with a 5 μM pore membrane. 5 × 105 PBMCs were added in the upper chamber and left for migration for 24 h.
After migration, the cells in the bottom chamber were stained with following antibodies: PE anti-human CD45, Alexa Fluor 700 anti-human CD3, V500 anti-human CD4, FITC anti-human CD8, PerCP-Cy5.5 anti-human CD56, APC anti-human CD14, APC-Cy7 anti-human CD11b, BV421 anti-human CD83 and PE-Texas Red anti-human CD206 (Supplementary table S2). Absolute cell count was performed using 123count eBeads (Invitrogen, Massachusetts, USA) according to the manufacturer’s recommendations. Samples were acquired in triplicates using FACS Aria II cell sorter (BD Biosciences, New Jersey, USA) collecting at least 50000 events per sample. Data analysis was performed using Flowjo software v10 (Flowjo LLC, BD Biosciences, New Jersey, USA).
After migration, the cells in the bottom chamber were stained with following antibodies: PE anti-human CD45, Alexa Fluor 700 anti-human CD3, V500 anti-human CD4, FITC anti-human CD8, PerCP-Cy5.5 anti-human CD56, APC anti-human CD14, APC-Cy7 anti-human CD11b, BV421 anti-human CD83 and PE-Texas Red anti-human CD206 (Supplementary table S2). Absolute cell count was performed using 123count eBeads (Invitrogen, Massachusetts, USA) according to the manufacturer’s recommendations. Samples were acquired in triplicates using FACS Aria II cell sorter (BD Biosciences, New Jersey, USA) collecting at least 50000 events per sample. Data analysis was performed using Flowjo software v10 (Flowjo LLC, BD Biosciences, New Jersey, USA).
7
Culturing Bladder Tumor Tissues for Analysis
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The bladder tumor tissues were cut into 2 mm3 cubes using a sharp 4 mm sterile scalpel and cultured in AIM-V media (#12055091, Thermo Fisher Scientific, Cambridge, MA, USA) (around 5–7 cubes per well, 48 well plates) for 24 to 48 h under different conditions as described previously [7 (link),33 (link),34 (link)]. Each condition/treatment was set up at least in triplicates. Tissues were harvested for mRNA analysis, while supernatants were harvested for chemokine release analyses by ELISA and chemotactic assay [7 (link),33 (link),34 (link)].
8
CFSE-based T cell proliferation assay
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Carboxyfluorescein succinimidyl ester (CFSE) labeling was performed as described previously [11 (link)]. CFSE-labeled PBMCs (1 × 105/well) were stimulated with TT (10 Lf/mL; Statens Serum Institut, Copenhagen, Denmark) in AIM V™ media (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 1.5% human serum. Blocking antibodies to immune checkpoints were used at a final concentration of 8 µg/mL. After 6–7 days, percentage of CFSElow CD4+ T lymphocytes was analyzed by flow cytometry. A single data point represents the triplicate mean of a donor. Responses with a stimulation index of 1.5 (at least 1.5-fold increase in the percentage of CFSElow CD4+ T cells in TT-stimulated cultures with respect to the CFSElow CD4+ T cells in control cultures) were considered reactive and used for further analysis.
9
Rapid Expansion Protocol for TIL Cells
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REP expansion was performed as previously described (51 (link)). The REP used anti-CD3 antibody (Miltenyi Biotec, clone OKT3, 130-093-387) and rhIL-2 in the presence of irradiated, allogeneic feeder cells at a 200:1 ratio of feeder cells to TIL cells. Frozen PBMC feeder cells were obtained each time from 3 different healthy donors, thawed, washed, resuspended in CM, and irradiated (50 Gy). A total of 2 × 108 PBMC, OKT3 antibody (30 ng/mL), CM (75 mL), AIM V media (Thermo Fisher Scientific, 75 mL), and 1 × 106 TIL cells were combined into a 175 cm2 tissue culture flask. Flasks were incubated upright at 37°C in 5% CO2, and rhIL-2 was added at 3000 IU/mL on day 2. On day 5 and day 10, 120 mL of culture supernatant was removed by aspiration, and media was replaced with a 1:1 mixture of CM/AIM V containing 3000 IU/mL IL-2. REP typically was allowed to proceed for 2 weeks, and cell expansion was monitored throughout. At the end of REP, cells were analyzed phenotypically by FACS, tested functionally by ELISA or ICS, and finally cryopreserved.
10
Cytotoxicity and Cytokine Analysis of Novel Immunotherapies
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LoVo, HT29 and SCC25 cells were purchased from ATCC, engineered to express luciferase via lentiviral transduction (Biosettia), and cultured per manufacturer’s recommendation. Human PBMC were isolated from leukopak utilizing EasySep technology (Stem Cell Technologies). Cells were plated at an E:T ratio of 5:1, combined with NCL-186 and cTAK-186 and incubated for 48 hours at 37° in AIM-V media (Thermo Fisher). To assess T-cell-dependent cellular cytotoxicity (TDCC), SteadyGlo (Promega) was added to quantitate the amount of luciferase labeled target cells remaining after the duration of the assay. RAJI cells engineered to express luciferase as well as human EGFR were also tested in this manner. Cytokine release was assessed by combining tumor cells, PBMC and MVC-NCL and pcTAK-186 as above; supernatants were collected after 24 hours. Respective cytokine concentrations were determined using MSD Proinflammatory panel 1 (human) kit for detection (MesoScale Discovery). Data were analyzed using SoftMax Pro (Molecular Devices) and Prism (GraphPad).
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