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SRX11436694: GSM5449991: Group B - Patient 2-TCR; Homo sapiens; OTHER
1 ILLUMINA (Illumina NovaSeq 6000) run: 30M spots, 6G bases, 2Gb downloads

Submitted by: NCBI (GEO)
Study: Identification of Pathogenic Immune Cell Subsets in Checkpoint Inhibitor-induced Myocarditis
show Abstracthide Abstract
Immune checkpoint inhibitors (ICIs) are monoclonal antibodies used widely to activate the immune system against tumor cells. Despite their therapeutic benefits, ICIs are known to cause immune-related adverse events (irAE) such as myocarditis, a rare but serious side effect with up to 50% mortality. To identify pathogenic immune subset(s) responsible for ICI myocarditis and other irAE, we performed single cell mass cytometry (CyTOF) on peripheral blood mononuclear cells from 40 patients and controls with irAE including four patients with ICI myocarditis. We also profiled 15 patients/controls using single cell RNA sequencing (scRNA-seq) with feature barcoding for surface marker expression confirmation. In both CyTOF/scRNAseq analyses, we found expansions of cytotoxic CD8+ T effector cells re-expressing CD45RA (Temra CD8+ cells) in ICI myocarditis patients compared to controls. Using T cell receptor sequencing, we demonstrated a significant clonal expansion of CD8+ Temra cells in myocarditis patients. Transcriptomic profiling of these Temra CD8+ clones confirmed their highly activated and cytotoxic phenotype with expression of granzyme/perforin. Longitudinal data revealed the progression of these Temra CD8+ cells into an exhausted phenotype two months after diagnosis of myocarditis and after treatment with glucocorticoids. Differential expression of several proinflammatory chemokines (CCL4/CCL4L2/CCL5) was uncovered in the clonally expanded Temra CD8+ cells and ligand-receptor analysis implicated their regulation of other inflammatory cells such as monocytes and NK cells. These data suggest that the therapeutic modulation of these chemokines may serve as an attractive strategy for reducing life-threatening irAE in ICI-treated cancer patients. Overall design: PBMCs were collected and isolated from three main patient groups: Group A - patients on immune checkpoint inhibitors without any known immune adverse events (irAEs), Group B - patients on immune checkpoint inhibitors with non-myocarditis irAEs, Group C - patients on immune checkpoint inhibitors with myocarditis. High-throughput sequencing in the form of scRNA-seq, single cell TCR sequencing, and feature barcoding were then performed. CyTOF (mass cytometry) data was also obtained on samples (not included on this record). The processed data for the CITE-seq component are included in the single-cell RNA-seq sample data matrices..
Sample: Group B - Patient 2-TCR
SAMN20203638 • SRS9479541 • All experiments • All runs
Organism: Homo sapiens
Library:
Instrument: Illumina NovaSeq 6000
Strategy: OTHER
Source: TRANSCRIPTOMIC
Selection: other
Layout: PAIRED
Construction protocol: Frozen PBMCs were removed from liquid nitrogen and resuspended in warm RPMI media + 10% FBS (@ 37ºC) by adding 1 mL, 3 mL, 4 mL, 8 mL 16 mL media at 30-60 second intervals. Cells were washed and passed through a 40 um filter, centrifuged @ 300 rcf x 5 min and resuspended in PBS with 50 ul 1% BSA and Biolegend Fc Receptor Blocking Solution (Human TrueStain FcX (Cat. No. 422301) for 20 minutes. Afterwards, another 50 uL of antibody mastermix containing Biolegend TotalSeq-C antibodies for staining (Biolegend TotalSeq™-C0063 anti-human CD45RA Antibody Cat. No. 304163; Biolegend TotalSeq™-C0072 anti-human CD4 Antibody Cat. No. 300567; Biolegend TotalSeq™-C0046 anti-human CD8 Antibody Cat. No. 344753) for final manufacture-recommended concentration of 1.0 ug of antibody in 100 ul of staining buffer for every 1 million cells for a total of 10-15 minutes incubation. After incubation, cells were washed 2x by resuspending in 1.5 mL PBS + 1% RNase-free BSA (MACS BSA Stock Solution No. 130-091-376) and then 1 mL PBS + 0.04% BSA. 10 uL of cell suspension was then taken for counting and appropriate volume of PSA + 0.04% BSA was added (200-500 uL) to aim for final target concentration of 1 million cells/mL). Cells were then loaded into Chromium Next GEM Chip G (10x PN 2000177) to achieve 6000-8000 target cell recovery per sample according to the manufacture's protocol. All steps from cDNA isolation to library preparation were completed according to 10x's manufacturing protocols for the Chromium Next GEM Single Cell V(D)J Reagent Kits v1.1. Isolated cDNA was amplified (14 cycles), and then cDNA was allocated for TCR enrichment, feature barcode library generation, and 5' cDNA library generation. cDNA and library quality was evaluated using the high sensitivity DNA kit on the Agilent 2100 Bioanalyzer.
Experiment attributes:
GEO Accession: GSM5449991
Links:
Runs: 1 run, 30M spots, 6G bases, 2Gb
Run# of Spots# of BasesSizePublished
SRR1512798630,000,0006G2Gb2022-06-30

ID:
15296826

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