Gene Expression
Nebulas
Gene Expression NebulasSummary: COVID-19 has posed a significant threat to global health. Early data has revealed that IL-6, a key regulatory cytokine, plays an important role in the cytokine storm of COVID-19. Multiple trials are therefore looking at the effects of Tocilizumab, an IL-6 receptor antibody that inhibits IL-6 activity, on treatment of COVID-19, with promising findings. As part of a clinical trial looking at the effects of Tocilizumab treatment on kidney transplant recipients with subclinical rejection, we performed single-cell RNA sequencing of comparing stimulated PBMCs before and after Tocilizumab treatment. We leveraged this data to create an in vitro cytokine storm model, to better understand the effects of Tocilizumab in the presence of inflammation. Tocilizumab-treated cells had reduced expression of inflammatory-mediated genes and biologic pathways, particularly amongst monocytes. These results support the hypothesis that Tocilizumab may hinder the cytokine storm of COVID-19, through a demonstration of biologic impact at the single-cell level
Overall Design: Examination of peripheral blood mononuclear cells (PBMCs) from 15 human kidney transplant recipients, diagnosed with subclinical rejection on their 6-month post-transplant protocol biopsy and randomized to either continue standard of care (Tacrolimus, mycophenolate, and steroid) immunosuppression (control arm, 8 patients) or standard of care plus Tocilizumab (Tocilizumab treatment arm, 7 patients)
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| Growth Protocol: | PBMCs were isolated from blood samples by Ficoll-PaqueTM PLUS density gradient centrifugation and frozen in fetal bovine serum containing 10% (vol/vol) dimethyl sulfoxide. Cells were frozen and not thawed until the day of the experiment when they were used directly for in vitro stimulation. |
| Treatment Protocol: | Cells were stimulated with soluble anti-CD3 and anti-CD28 antibodies at 37 Celsius, 5% CO2 for 24 hours. Unstimulated PBMCs were incubated under identical conditions to reduce any confounding from incubation conditions other than stimulation. |
| Extract Protocol: | Cells were multiplexed into pools of cells from different patients with the intent of using in silico genetic demultiplexing tools to recover more single cells and reduce batch effect. No samples from the same person were multiplexed into the same pool. Pools were loaded onto the 10x Genomics controller using several lanes per pool, and processed using standard protocols, including cell lysis, reverse transcription, amplification and library generation. |
| Library Construction Protocol: | Libraries were constructed using standard protocols from 10x Genomics. |
| Molecule Type: | Poly(A)+ RNA |
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| Library Layout: | PAIRED |
| Library Strand: | - |
| Platform: | ILLUMINA |
| Instrument Model: | Illumina NovaSeq 6000 |
| Strand-Specific: | - |
| Data Resource | GEN Sample ID | GEN Dataset ID | Project ID | BioProject ID | Sample ID | Sample Name | BioSample ID | Sample Accession | Experiment Accession | Release Date | Submission Date | Update Date | Species | Race | Ethnicity | Age | Age Unit | Gender | Source Name | Tissue | Cell Type | Cell Subtype | Cell Line | Disease | Disease State | Development Stage | Mutation | Phenotype | Case Detail | Control Detail | Growth Protocol | Treatment Protocol | Extract Protocol | Library Construction Protocol | Molecule Type | Library Layout | Strand-Specific | Library Strand | Spike-In | Strategy | Platform | Instrument Model | Cell Number | Reads Number | Gbases | AvgSpotLen1 | AvgSpotLen2 | Uniq Mapping Rate | Multiple Mapping Rate | Coverage Rate |
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