WT BV173 cells were co-cultured with either sTCR+ or sTCR+CoCAR+ ATCs at an E:T ratio of 1:5. Four days later, flow cytometry was performed to measure tumor elimination, and 5×105 BV173 cells were added back per well of culture. 24 hours later, the co-cultured cells were harvested. In conditions in which BV173 cells were still present, CD33 MACS beads (130–045-501, Miltenyi Biotech, Germany) were used to deplete residual tumor cells. Total RNA was collected from ATCs using the RNeasy Micro Kit (Qiagen, Germantown, MD) following kit instructions for extraction from cells. Gene expression analysis (NanoString, Seattle, WA) was performed by the Baylor College of Medicine Genomic and RNA Profiling Core using the nCounter CAR-T Characterization Panel. Submitted samples were >250 ng and <5 μL. Data were analyzed using the nSolver 3.0 software (NanoString). See statistical section for additional information on NanoString analysis.
Ncounter car t characterization panel
Manufactured by NanoString
Sourced in United States
The NCounter CAR-T Characterization Panel is a laboratory equipment product developed by NanoString. The panel is designed to provide a comprehensive analysis of key features and markers associated with chimeric antigen receptor (CAR) T-cell samples. The product enables the simultaneous measurement of multiple gene expression targets related to CAR-T cell function and phenotype.
Automatically generated - may contain errors
Lab products found in correlation
Nanodrop spectrophotometer, by Thermo Fisher Scientific (2 mentions)
Cd33 macs beads, by Miltenyi Biotec (1 mentions)
Nsolver 3, by NanoString (1 mentions)
Rneasy micro kit, by Qiagen (1 mentions)
2100 bioanalyzer, by Agilent Technologies (1 mentions)
Mirneasy mini kit, by Qiagen (1 mentions)
Rlt buffer, by Qiagen (1 mentions)
Proteinase k, by Thermo Fisher Scientific (1 mentions)
Ncounter digital analyzer, by NanoString (1 mentions)
Mastercycler ep gradient, by Eppendorf (1 mentions)
96 well microplates, by Starlab (1 mentions)
Ncounter sprint profiler, by NanoString (1 mentions)
Quick rna microprep kit, by Zymo Research (1 mentions)
Tissue homogenizer, by Thermo Fisher Scientific (1 mentions)
Nsolver 4, by NanoString (1 mentions)
Fragment analyzer, by Agilent Technologies (1 mentions)
Ncounter human immunology v2 panel, by NanoString (1 mentions)
Ncounter platform, by NanoString (1 mentions)
Nanodrop spectrophotometer, by Agilent Technologies (1 mentions)
Nsolver analysis software v4, by NanoString (1 mentions)
9 protocols using ncounter car t characterization panel
1
Evaluating Tumor Elimination by CAR-T Cells
2
nCounter CAR-T Characterization Workflow
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Hundred ng of total RNA was hybridized in solution with the nCounter CAR-T Characterization panel (human codeset) at 65 ℃ for 17 h. The hybridized samples were loaded into the nCounter CAR-T cartridge (NanoString Technologies), which was then sealed and placed in the instrument for processing and analysis. RCC files containing raw counts for 780 genes were generated and loaded into nSolver Analysis Software 4.0 for normalization by housekeeping genes and positive controls. The normalized data were imported to Partek Genomics Suite 7.0 to remove any batch effects. Downstream analysis was performed in a Rstudio environment with custom code.
3
RNA Profiling of CAR T-Cells
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Total RNA was isolated from frozen CAR T-cell samples using an miRNeasy mini kit (QIAGEN) and quantified by 2100 Bioanalyzer (Agilent). 100 ng of total RNA was hybridized in solution with the nCounter CAR-T Characterization panel (Nanostring Technologies) at 65 °C for 17 h. The hybridized samples were loaded into the nCounter CAR-T cartridge, which was then sealed and placed in the instrument for processing. RCC files containing raw counts for 780 genes were generated and imported into nSolver Analysis Software 4.0 to normalize. Downstream analysis was performed in a Rstudio environment or Partek Genomic Suite 7.0 (Partek Inc., St. Louis, MO, USA) data visualization and hierarchical cluster analysis.
4
Characterizing CAR T Cell Transcriptome
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2E5 T cells from the final cell product (run #3) were lysed for 20 min in a 1:3 mix of RLT buffer (Qiagen, Hilden, Germany) and water. Native CD4 T cells and CD8 peripheral blood T cells were used as controls. Lysed samples were processed for gene expression analysis using the nCounter CAR T characterization panel from Nanostring (Nanostring, Seattle, Washington, USA.), following the manufacturer’s instructions. For hybridization, 200 µg/mL Proteinase K (Invitrogen, Waltham, Massachusetts, USA) was added to the master mix. Mastercycler epgradient (Eppendorf, Hamburg, Germany), nCounter Pro Prep Station and nCounter Digital Analyzer (Nanostring) were used for further sample processing. Counting was performed using the setting 555 fields of view.
Data were first analyzed with nSolver analysis software V.4.0 using default settings for quality control and for normalization on positive control, housekeeping genes (without UBB) and sample references. Data were analyzed with a HyperScale architecture developed by Rosalind Inc (San Diego, California, USA) using default settings for fold changes and p values. The threshold for the log2 fold change was set between 1.5 and −1.5 and the significance threshold (p-adj.) was set at 0.01.
Data were first analyzed with nSolver analysis software V.4.0 using default settings for quality control and for normalization on positive control, housekeeping genes (without UBB) and sample references. Data were analyzed with a HyperScale architecture developed by Rosalind Inc (San Diego, California, USA) using default settings for fold changes and p values. The threshold for the log2 fold change was set between 1.5 and −1.5 and the significance threshold (p-adj.) was set at 0.01.
5
CAR T Cell Characterization Protocol
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Recombinant FasL (2 μg) (PeproTech, 310-03H) was immobilized onto 96-well microplates (Starlab, CC7672-7596) overnight at 4°C and the plate was washed several times with PBS. CAR T cells (2 × 105) were then seeded onto the FasL-immobilized microplate and incubated at 37°C, 5% CO2 for 3 days. RNA was extracted from the microplate using the RNAspin Mini Kit (Merck, GE25-0500-71) and quantified using a NanoDrop Spectrophotometer (Thermo Fisher Scientific). Extracted RNA (50 ng) was sequenced using the nCounter CAR-T Characterization Panel (NanoString) and analyzed on the nCounter SPRINT Profiler (NanoString) according to the manufacturer’s instructions.
6
Profiling CAR-T Cell Gene Expression
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CD4+CD127+CD25lowCXCR5+ T cells were sorted using FACSAria II. Total RNA was extracted using Quick-RNA Microprep kit (Zymo Research) per the manufacturer’s protocol. The gene expression profile was determined using the nCounter CAR-T Characterization Panel (NanoString). NanoString assay was performed using 10 ng RNA. Data analysis was performed using NanoString nSolver software v4.0.
7
Transcriptomic Profiling of Skin Biopsies
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Skin biopsies from patients were immediately frozen in liquid nitrogen and stored at −80 °C until processing. RNA was isolated using the TRIzol/chloroform method and a tissue homogenizer (Thermo Fisher Scientific). The quality and quantity of RNA was measured using a NanoDrop spectrophotometer and RNA integrity was analyzed on a Fragment Analyzer (Agilent). mRNA expression was analyzed with the nCounter CAR-T characterization panel and the nCounter Human Immunology V2 panel on the nCounter platform (NanoString Technologies, Seattle, WA, USA) using 100 ng of RNA per skin sample. A quality check was done for each sample and data were normalized and analyzed using nSolver 4.0 (NanoString Technologies). The expression of selected genes was used to generate the heatmap using R. The full NanoString dataset is provided in the Source data file.
8
Profiling CAR-T Cell Transcriptome via NanoString
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The multiplexed NanoString nCounter™CAR-T Characterization panel was used as expression assay for profiling 780 human genes (NanoString Technologies, Inc., Seattle, WA, USA). The assay was performed according to the manufacturer's protocol (see Supplemental Methods for more details). Pre-processing and normalization of the raw counts was performed with nSolver Analysis Software v4.0 (www.nanostring.com ). Gene expression data were normalized by using all the 10 housekeeping genes present in the CAR-T Characterization panel. The 6 spiked-in RNA Positive Control and the 8 Negative controls present in the panel were used to confirm the quality of the run. The heatmap was generated using the package “ComplexHeatmap” within R version 3.5.1. Prior to clustering, data were log10 transformed and mean centered and genes related to TCR were removed from the data. The heatmap data was clustered by selecting “clustering_distance_columns = canberra” and “clustering_method_columns = ward.D" with all the rest of parameters left as default [19 (link),20 ].
9
CAR T Cell Transcriptional Profiling
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CAR T cells were sorted using anti-CD34 magnetic beads (Miltenyi) and activated for 24 h with 50 ng per ml CAR-specific anti-idiotype. After stimulation, RNA was extracted from CAR T cells using the Quick RNA kit (Zymo Research). Samples were prepared according to the manufacturer’s protocols for the Nanostring nCounter CAR-T Characterization Panel (Nanostring). A list of genes and target probe sequences can be found at www.nanostring.com . Cartridges were run on the nCounter Sprint Profiler. Data analysis was conducted using the nSolver software and R studio v3.6.
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