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| Status |
Public on Aug 06, 2019 |
| Title |
post2_Blood_CD8_0006 |
| Sample type |
SRA |
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| Source name |
CD8+ peripheral blood T cells
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| Organism |
Homo sapiens |
| Characteristics |
cell type: CD8+ CD4- T cells
age: 35
Sex: female
cancer type: colorectal
dose level: 1.0 mg/kg
agent: IT1208
organ: peripheral blood
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| Treatment protocol |
Patients with advanced solid tumors for whom no standard therapy was available, were treated with intravenous infusion of IT1208 at dose of 0.1 or 1.0 mg/kg. First patient in each cohort was treated as a single administration and other patients received two administrations of IT1208 on day 1 and 8 followed by safety and efficacy assessment with DLT evaluation period as 4 weeks.
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| Extracted molecule |
total RNA |
| Extraction protocol |
For bulk biopsy samples, total RNA was extracted using a RNA easy mini extraction kit (Qiagen) according to standard protocol, and diluted with Lysis buffer (100 mM Tris-HCl pH7.5, 500 mM LiCl, 10 mM EDTA, 5 mM DTT). For sorted CD4+ and CD8+ T cells, cells were collected directry into the Lysis buffer.
TCR sequencing libraries for next generation sequencing were prepared according to the previous report (Aoki, et al, GSE115425) with some modifications. PolyA RNAs were isolated and amplified from sorted T cells according to a previous report (Shichino, et al, GSE110711). To amplify the TCR cDNA containing complementarity determining region 3 (CDR3), nested PCR of the TCR locus was performed as follows. The first PCR mixture comprised 0.4 μL of 10 μM primer mix (5′ WTA and Trbc_ex), 4.6 μL of template, and 5 μL of KAPA Hifi Hotstart ReadyMix (KAPA Biosystems). The thermal cycling conditions were programmed as follows: denaturation at 95°C for 3 min, 10 cycles of denaturation for 20 sec at 98°C, annealing for 15 sec at 58°C and extension for 30 sec at 72°C, followed by final extension at 72°C for 2 min. Next, 10 μL of the first-PCR products was used for purification with an Agencort AM Pure XP kit (Beckman Coulter) at a 0.7:1 ratio of beads to sample, and eluted with 12 μL of DW. The second PCR mixture consisted of 1.25 μL of 10 μM primer mix (5′ WTA and Trbc_in-Bio), 11.25 μL of template and 12.5 μL of KAPA Hifi Hotstart ReadyMix. The thermal cycling conditions were same as the first PCR except the cycle number; 13 cycles. Next, 25 μL of the second-PCR products were purified using Agencort AM Pure XP kit (Beckman-Coulter) at a 0.8:1 ratio of beads to sample, and eluted in 15 μL of DW. The purified PCR products were sheared randomly using NEBNext dsDNA fragmentase (New England Biolabs). The fragmentation reaction mix consisted of 6 μL of DW, 2 μL of 10X Fragmentase Reaction Buffer v2, 10 μL of PCR product, and 2 μL of fragmentase. The fragmentation reaction was incubated at 37°C for 30 min, and then 5 μL of 0.5 M EDTA was added to stop the reaction on ice. The sheared PCR product was then purified and subjected to size selection using the Agencort AM Pure XP kit (Beckman Coulter) at a 0.8:1 ratio of beads to sample to remove large fragments, a 0.8:1 ratio of beads to sample to remove the smaller fragments, and eluted with 20 μL of Tris-HCl (pH 8.0). To capture the TCR cDNA containing the end of Constant region, the purified PCR products were incubated with 10 μL of Dynabeads M-270 Streptavidin (Thermo Fisher Scientific) for 30 min at room temperature (r.t.), washed 3 times with B&W-T buffer [5 mM Tris-HCl (pH 7.5), 1 M NaCl, 0.5 mM EDTA, and 0.1% Tween-20], and once with Tris-HCl (pH 8.0), and DW. The captured TCR cDNA was repaired using NEBNext Ultra II End Repair/dA-Tailing Module (New England Biolabs). The repair reaction mix contained 1.2 μL of reaction buffer, 8.3 μL of beads re-suspended with DW, and 0.5 μL of enzyme mix. The repair reaction was incubated at 20°C for 30 min, then washed once with lysis buffer [1% LiDS, 100 mM Tris-HCl (pH 7.5), 500 mM LiCl, 10 mM EDTA, and 5 mM DTT], 3 times with B&W-T buffer, and once with Tris-HCl (pH 8.0). The repaired TCR cDNA was attached to the sequencing adaptor using the DNA Ligation Kit ＜Mighty Mix＞ (TaKaRa Bio). The ligation reaction mix consisted of 1 μL of 10 μM P1 adaptor, 6.5 μL of beads re-suspended in Tris-HCl (pH 8.0), and 15 μL of enzyme mix. The ligation reaction was incubated at 16°C for 60 min using a thermal cycler with the cover open, washed once with lysis buffer, 3 times with B&W-T buffer, and once with Tris-HCl (pH 8.0). The third PCR were carried out using barcoded primers to enrich the TCR cDNA flanked with sequencing adapters. The third PCR mixture consisted of 0.35 μL of 10 μM trP1 primer, 1 μL of 3.5 μM IonA-BC-Trbc primer, 3.65 μL of beads resuspended in DW and 5 μL of KAPA Hifi Hotstart ReadyMix. The thermal cycling conditions were same as the first PCR except the cycle number; 12 cycles. The PCR product was purified and subjected to size selection using Agencort AM Pure XP kit (Beckman Coulter,) at a 0.75:1 ratio of beads to sample to remove large fragments, a 0.65:1 ratio of beads to sample to remove smaller fragments, and eluted with 20 μL of Tris-HCl (pH 8.0). Amplified TCR libraries were quantified using a KAPA Library Quantification Kit ((KAPA Biosystems) and size distribution was analyzed by agarose electrophoresis and SYBR Gold staining (Thermo Fisher Scientific). Final TCR libraries, whose lengths were 200–300 base pairs, were pooled and sequenced using an Ion Hi-Q Chef kit, an Ion PI v3 Chip kit, and an Ion Proton Sequencer (Thermo Fisher Scientific) according to the manufacturer’s instructions, except the input library concentration (100 pM) and flow number (550).
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Ion Torrent S5 |
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| Data processing |
Adapter sequences and low-quality reads were trimmed using cutadapt-v1.11 and PRINSEQ-0.20.4. Filtered reads were processed using “analyze amplicon” command of MiXCR-3.0.2 with following high-level options: -s = hsa, -starting-material = rna, -5-end = no-v-primers, -3-end = c-primers, -adapters = no-adapters, -receptor-type = trb, -region-of-interest = CDR3. In alignment to ImMunoGeneTics (IMGT) reference human TCR V/D/J sequences, we used the following parameters: -OvParameters.geneFeatureToAlign = VTranscript -OvjAlignmentOrder = JThenV. In assembling identical sequences into clones with PCR and sequencing error correlations, we used the following parameters: -ObadQualityThreshold=10, -OseparateByV=true. Only clones with productive CDR3 sequences were analyzed using VDJtools (ver 1.2.1.). Then, the sequencing coverage of samples, which was defined as the ratio of total reads to the starting number of T cells, was normalized to ×5 using the “DownSample” command in VDJtools. After coverage normalization, clones with lead count less than sequencing coverage were excluded as sequencing noise. Finally, for PBMC sample, we identified clones shared between CD4+ and CD8+ T cell repertoire as contamination, and separated these shared clones into CD4+ or CD8+ repertoire by their frequency in CD4+ or CD8+, because these shared clones were very few and their frequencies were biased to either CD4+ or CD8+ (Data not shown)
Genome_build: IMGT reference sequences of human 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 |
Sep 18, 2018 |
| Last update date |
Aug 06, 2019 |
| Contact name |
Shigeyuki Shichino |
| E-mail(s) |
s_shichino@rs.tus.ac.jp
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| Organization name |
Tokyo University of Science
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| Department |
Department of Molecular regulation of Inflammatory and Immune Diseases
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| Street address |
2641, Yamasaki, Noda-shi
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| City |
Chiba |
| ZIP/Postal code |
278-0022 |
| Country |
Japan |
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| Platform ID |
GPL23934 |
| Series (2) |
| GSE120101 |
T-cell receptor repertoire analysis of advanced solid tumors in first-in-human phase 1 study of IT1208, a defucosylated humanized anti-CD4 depleting antibody [TCR-seq] |
| GSE120102 |
TCR repertoire and transcriptomic analyses of advanced solid tumors in first-in-human phase 1 study of IT1208, a defucosylated humanized anti-CD4 depleting antibody |
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| Relations |
| BioSample |
SAMN10081702 |
| SRA |
SRX4707766 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM3393412_post2_Blood_CD8pure_0006.txt.gz |
378.1 Kb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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