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| Status |
Public on May 11, 2023 |
| Title |
B16_Tumor_aCD4_DT_CD8_2 |
| Sample type |
SRA |
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| Source name |
tumor on day 14 after innoculation of B16
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| Organism |
Mus musculus |
| Characteristics |
strain: C57BL/6
genotype: Bone marrow chimera of Fscn1 KI
treatment: anti-CD4 mAbs with DT
cell type: Intravenous (i.v.) staining-negative, lineage-negative (CD11b, B220, NK1.1 and Ter119), TCRbeta+ and CD4- CD8+ T cells
age: 10 weeks post bone marrow transplantation
Sex: female
tumour cell line: B16F10 melanoma
organ: B16F10
date: 0
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| Treatment protocol |
B16F10 cells (5 × 105 cells) were inoculated subcutaneously (s.c.) into the right flanks of the Fscn1-KI BMC mice. Anti-PD-L1 mAb (clone 10F.9G2, BioLegend) or Anti-CD4 mAb (clone GK1.5, BioLegend) was injected intraperitoneally (i.p.) at a dose of 200 μg per mouse on days 5 and 9 after tumor inoculation.
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| Extracted molecule |
total RNA |
| Extraction protocol |
Intravascular leukocytes were stained by i.v. injection of FITC-conjugated anti-CD45.2 mAb (3 mg/mouse, clone 104, BioLegend) three minutes before sacrifice.
dLN was digested for 45 minutes at 37°C with 0.1% collagenase (FUJIFILM Wako) then subjected to 25% Percoll PLUS (Cytiva) gradient and leukocytes were recovered from the pellet.
Tumors were cut into small fragments and digested for 45 minutes at 37°C with 0.1% collagenase (FUJIFILM Wako), then subjected to 40% Percoll PLUS (Cytiva) and Histopaque-1083 (Sigma-Aldrich) gradient, and leukocytes were recovered from the interphase. The cell concentration of the suspensions was determined using Flow-Count fluorospheres (Beckman Coulter) and a Cytoflex flow cytometer (Beckman Coulter).
Cells were stained with a mix of Fc Block (anti-mouse CD16/CD32 mAb; clone 93, BioLegend) and fluorophore-conjugated anti-mouse mAbs. For dLN samples, Lineage+ (CD11b, B220, NK1.1, Ter119) cells were depleted using BD IMag Streptavidin Particles Plus-DM (BD Biosciences). For tumor samples, TCRb+ T cells were enriched magnetically using Anti-APC MicroBeads (Miltenyi) and LS columns (Miltenyi).
CD8+ T cells from tumor, and CD8+ CD44high T cells from dLN were sorted using FACS Aria II or Aria III (BD Biosciences). Nonviable cells were excluded from the analysis based on forward and side scatter profiles and propidium iodide staining. Intravascular leukocytes were also excluded. Purity of sorted cells was always over 95%.
Bulk TCR sequencing library was prepared according to the previous report (Tsunoda et al., 2021). In brief, to perform reverse transcription and template-switching, mRNA-trapped oligo-dT-immobilized Dynabeads M270-streptavidin (Thermo Fisher Scientific) were suspended in 10 µL of RT mix [1× First Strand buffer (Thermo Fisher Scientific), 1 mM dNTP, 2.5 mM DTT (Thermo Fisher Scientific), 1 M betaine (Sigma-Aldrich), 9 mM MgCl2 (NIPPON GENE), 1 U/µL RNaseIn Plus RNase Inhibitor (Promega), 10 U/µL Superscript II (Thermo Fisher Scientific), and 1 µM of i5-TSO], and incubated for 60 min at 42ºC and immediately cooled on ice. To amplify the TCR cDNA containing complementarity determining region 3 (CDR3), nested PCR of the TCR locus was performed following the purification of PCR product by an Agencort AM Pure XP kit (Beckman Coulter) at a 0.7:1 ratio of beads to sample and eluted with 20 µL of 10 mM Tris-HCl (pH 8.0). To amplify TCR libraries and add adaptor sequences for the next-generation sequencer, the third PCR was performed. The third-PCR products were purified as second PCR. The products were pooled and then purified and subjected to dual size selection using ProNex size-selective purification system (Promega) and eluted with 25 μL of 10 mM Tris-HCl (pH 8.5). Final TCR libraries, whose lengths were about 600 base pairs were sequenced using an Illumina Novaseq 6000 S4 flowcell (67 bp read 1 and 140 bp read 2) (Illumina). Only read2 contained the sequence regarding the definition of T cell clones.
Bulk TCR-seq
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| Library strategy |
OTHER |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
Illumina NovaSeq 6000 |
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| Description |
The Fscn1 knockin (Fscn1-KI) mouse (Accession No. CDB0154E: http://www2.clst.riken.jp/arg/mutant%20mice%20list.html) was generated by CRISPR/Cas9-mediated knockin in zygotes as previously described (Abe et al., 2020). For the homologous recombination-mediated knockin, the donor vector consisting of homology arms and P2A-DTR-P2A-Cre (insert gene) was generated to insert the P2A-DTR-P2A-Cre cassette at the 3-base upstream of the PAM sequence. For microinjection, the mixture of crRNA (CRISPR RNA) (50ng/ul), tracrRNA (trans-activating crRNA) (100ng/ul), donor vector (10ng/ul) and Cas9 protein (100ng/ul) were injected into the pronucleus of one-cell stage zygote. From 233 zygotes, 57 F0 mice were obtained and 3 of them were knockin mice confirmed by PCR, in which the PCR fragments of KI allele with 5’FW and 5’REV primers (1301 bp), and 3’FW and 3’REV primers (587 bp) were detected. The germline transmissions of knockin F0 mice were confirmed by genotyping of F1 mice. gRNA target sequences and primers used for genotyping are shown in Supplementary Table 1.
Recipient B6 mice were lethally irradiated (8.5 Gy, split into 2 doses given 3 hours apart) the day before transplantation with 3 × 106 Fscn1-KI BM cells. Mice were used for the experiments at least 8 weeks after transplantation.
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| Data processing |
Data processing of Bulk TCRseq was performed as described in Aoki et al. (Aoki et al., 2021).
Adapter trimming and quality filtering of sequencing data were performed by using Cutadapt-3.2 (Martin et al., 2011) and PRINSEQ-0.20.4 (Schmieder et al., 2011). Sequencing data were processed by MiXCR-3.0.5 (Bolotin et al., 2015). In MiXCR, filtered reads were aligned to reference mouse TCR V/D/J sequences registered in the international ImMunoGeneTics (IMGT) information system: -vParameters.geneFeatureToAlign = VTranscript -vjAlignmentOrder = JThenV, then identical sequences were assembled and grouped in clones with PCR and sequencing error correlation with the following parameters: -badQualityThreshold=10, –separateByV=true, --only-productive=true, –region-of-interest=CDR3. The Variable (V) and Joining (J) segment of TCRs were represented in IMGT gene nomenclature.
List of final clones were analyzed by VDJtools-1.2.1 (Shugay et al., 2015). Then, the sequencing reads of sample was normalized to the cell count in each sample by “DownSample” command of VDJtools. T-cell clones were determined as TCR reads with the same TCR V segment, J segment and CDR3 nucleotide sequence.
Assembly: IMGT reference sequences of mouse TRA and TRB
Supplementary files format and content: Text files in VDJtools format include read count and frequency, complementarity determining region 3 (CDR3) nucleotide and amino acid sequences, Variable(V)/ Diversity(D)/ Joining(J) segment names, and V, D and J segment boundaries within CDR3 nucleotide sequence of individual clones
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| Submission date |
Mar 09, 2022 |
| Last update date |
May 11, 2023 |
| Contact name |
Hiroyasu Aoki |
| E-mail(s) |
haoki-tky@rs.tus.ac.jp
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| Phone |
08013746493
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| Organization name |
Tokyo University of Science
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| Department |
Research Institute for Biomedical Sciences
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| Lab |
Division of Molecular Regulation of Inflammatory and Immune Diseases
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| Street address |
2669 Yamazaki
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| City |
Noda |
| State/province |
Chiba |
| ZIP/Postal code |
278-0022 |
| Country |
Japan |
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| Platform ID |
GPL24247 |
| Series (2) |
| GSE198207 |
Bulk TCRseq analysis on CD8+ T cells in FSCN1-KI bone marrow chimera mice following anti-CD4 and anti-PDL1 mAb treatment |
| GSE198211 |
Clonal spreading of tumor-infiltrating T cells underlies the durable antitumor effects of PD-1 blockade and anti-CD4 monoclonal antibody |
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| Relations |
| BioSample |
SAMN26539921 |
| SRA |
SRX14415082 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM5940725_B16_Tumor_aCD4_DT_CD8_2.txt.gz |
44.2 Kb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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