Gene Expression
Nebulas
Gene Expression NebulasSummary: Since the beginning of the SARS-CoV-2 pandemic, COVID-19 has appeared as a unique disease with unconventional tissue and systemic immune features. While COVID-19 severe forms share clinical and laboratory aspects with various pathologies such as hemophagocytic lymphohistiocytosis, sepsis or cytokine release syndrome, their exact nature remains unknown. This is severely impeding the ability to treat patients facing severe stages of the disease. To this aim, we performed an in-depth, single-cell RNA-seq analysis of more than 150.000 immune cells isolated from matched blood samples and broncho-alveolar lavage fluids of COVID-19 patients and healthy controls, and integrated it with clinical, immunological and functional ex vivo data. We unveiled an immune signature of disease severity that correlated with the accumulation of naïve lymphoid cells in the lung and an expansion and activation of myeloid cells in the periphery. Moreover, we demonstrated that myeloid-driven immune suppression is a hallmark of COVID-19 evolution and arginase 1 expression is significantly associated with monocyte immune regulatory features. Noteworthy, we found monocyte and neutro-phil immune suppression loss associated with fatal clinical outcome in severe patients. Additionally, our analysis discovered that the strongest association of the patients clinical outcome and immune phenotype is the lung T cell response. We found that patients with a robust CXCR6+ effector memory T cell response have better outcomes. This result is line with the rs11385942 COVID-19 risk allele, which is in proximity to the CXCR6 gene and suggest effector memory T cell are a primary feature in COVID-19 patients. By systemically quantifying the viral landscape in the lung of severe patients, we indeed identified Herpes-Simplex-Virus 1 (HSV-1) as a potential opportunistic virus in COVID-19 patients. Lastly, we observed an unexpectedly high SARS-CoV-2 viral load in an immuno-compromised patient, allowing us to study the SARS-CoV-2 in-vivo life cycle. The development of my-eloid dysfunctions and the impairment of lymphoid arm establish a condition of immune paralysis that supports secondary bacteria and virus infection and can progress to “immune silence” in patients facing death.
Overall Design: Cells from blood or bronchoalveloar lavage samples from COVID-19 patients or healthy donors were sequenced using the Chromium 10X scRNA-seq technology (NextGEM Single Cell 3′ GEM, Library& Gel Bead kit v3.1)
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| Treatment Protocol: | For each patient approximately 20 ml of BAL fluid was obtained, stored at room temperature and processed within 2 hours in a BSL-3 laboratory. An unprocessed aliquot was used for bacterial cul-ture. The BAL fluid was filtered 2 times through a nylon gauze and a 100-μm nylon cell strainer to remove clumps and debris. The supernatant was then washed with PBS 1x and centrifuged. RBCs were lysed with 4 mL of 0.2% NaCl solution (3 minutes, RT) and the reaction was blocked by adding 9 mL of 1.2% NaCl solution. The cells were washed with PBS 1x, re-suspended in RPMI 1640 me-dium supplemented with 5% bovine serum albumin and counted. Cell viability was determined by Trypan blue exclusion. BAL fluids of patients with COVID-19 infection contained a heterogeneous number of cells ranging from 0.83 x106 to 22 x106 of cells. Cells were re-suspended at a concentration of 1 × 106 /ml for single cell analysis. Peripheral blood (PB) from COVID-19 patients and HDs was collected in EDTA-coated tubes. 2 ml of PB was washed once with PBS 1x and the RBCs lysis was performed twice adding 15 mL of 0.2% NaCl solution (3 minutes, RT) and the reaction was blocked by adding 35 mL of 1.2% NaCl solution. The cells were washed with PBS 1x, re-suspended in RPMI 1640 medium supplemented with 5% bovine serum albumin, filtered through a 100-μm nylon cell strainer and counted. Cell viability was determined by Trypan blue exclusion. Cells were re-sus-pended at a concentration of 1 × 106 /ml for single cell analysis |
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| Library Construction Protocol: | After synthesis of cDNA, sequencing libraries were generated. Final 10X library quality was assessed using the Fragment Analyzer High Sensitivity NGS kit (Agilent Technologies, Santa Clara, CA, USA) and then sequenced on the Illu-mina NextSeq500 (Illumina, San Diego CA, USA) generating 75 base pair paired-end reads (28bp read1 and 91bp read2) at a depth of 50,000 reads/cell. |
| Molecule Type: | Poly(A)+ RNA |
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| Library Layout: | PAIRED |
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| Platform: | ILLUMINA |
| Instrument Model: | Illumina NextSeq 500 |
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| 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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