Coronavirus disease 2019 (COVID-19) is a mild to moderate respiratory tract infection, however, a subset of patients progress to severe disease and respiratory failure. The mechanism of protective immunity in mild forms and the pathogenesis of severe COVID-19 associated with increased neutrophil counts and dysregulated immune responses remain unclear. In a dual-center, two-cohort study, we combined single-cell RNA-sequencing and single-cell proteomics of whole-blood and peripheral-blood mononuclear cells to determine changes in immune cell composition and activation in mild versus severe COVID-19 (242 samples from 109 individuals) over time. HLA-DR hi CD11c hi inflammatory monocytes with an interferon-stimulated gene signature were elevated in mild COVID-19. Severe COVID-19 was marked by occurrence of neutrophil precursors, as evidence of emergency myelopoiesis, dysfunctional mature neutrophils, and HLA-DR lo monocytes. Our study provides detailed insights into the systemic immune response to SARS-CoV-2 infection and reveals profound alterations in the myeloid cell compartment associated with severe COVID-19.

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'Severe Acute Respiratory Syndrome - Coronavirus-2' (SARS-CoV-2) infection causes Coronavirus Disease 2019 (COVID-19), a mild to moderate respiratory tract infection in the majority of patients. A subset of patients, however, progresses to severe disease and respiratory failure with acute respiratory distress syndrome (ARDS). Severe COVID-19 has been associated with increased neutrophil counts and dysregulated immune responses. The mechanisms of protective immunity in mild forms and the pathogenesis of dysregulated inflammation in severe courses of COVID-19 remain largely unclear. Here, we combined two single-cell RNA-sequencing technologies and single-cell proteomics in whole blood and peripheral blood mononuclear cells (PBMC) to determine changes in immune cell composition and activation in two independent dual-center patient cohorts (n=46 + n=54 COVID-19 samples), each with mild and severe cases of COVID-19. We observed a specific increase of HLA-DR high CD11c high inflammatory monocytes that displayed a strong interferon (IFN)-stimulated gene signature in patients with mild COVID-19, which was absent in severe disease. Instead, we found evidence of emergency myelopoiesis, marked by the occurrence of immunosuppressive pre-neutrophils and immature neutrophils and populations of dysfunctional and suppressive mature neutrophils, as well as suppressive HLA-DR low monocytes in severe COVID-19. Our study provides detailed insights into systemic immune response to SARS-CoV-2 infection and it reveals profound alterations in the peripheral myeloid cell compartment associated with severe courses of COVID-19.

25Current single-cell RNA sequencing approaches gives a snapshot of a cellular phenotype but convey no information on the temporal dynamics of transcription. Moreover, the stochastic nature of transcription at molecular level is not recovered. Here, we present single-cell SLAM-seq (scSLAM-seq), which integrates metabolic RNA labeling, biochemical nucleoside conversion and single-cell RNA-seq to directly measure total transcript levels and transcriptional activity by 30 differentiating newly synthesized from pre-existing RNA for thousands of genes per single cell. scSLAM-seq recovers the earliest virus-induced changes in cytomegalovirus infection and reveals a so far hidden phase of viral gene expression comprising promiscuous transcription of all kinetic classes. It depicts the stochastic nature of transcription and demonstrates extensive gene-specific differences. These range from stable transcription rates to on-off dynamics which coincide with 35 gene-/promoter-intrinsic features (Tbp-TATA-box interactions and DNA methylation). Gene but not cell-specific features thus explain the heterogeneity in transcriptomes between individual cells and the transcriptional response to perturbations. 3 Main 40Regulation of gene expression is a fine-tuned process, which allows cells to maintain homeostasis and respond to changing environmental conditions. Single-cell RNA sequencing (scRNA-seq) allows to quantify transcript levels for thousands of genes in individual cells. This revealed that intercellular heterogeneity plays an important role in phenotype variability in health and disease 1-4 . However, current scRNA-seq approaches only quantify total cellular RNA profiles rather than transcriptional activities. More 45 importantly, the RNA profile of each individual cell can only be analyzed once at a given time point. This precludes direct monitoring of transcriptional changes in individual cells due to perturbations. Accordingly, changes can only be inferred from differences in transcript levels between distinct cell populations or conditions 5 . Furthermore, gene expression is a stochastic process, with intrinsic and extrinsic noise in transcription and translation leading to intercellular heterogeneity in mRNA and protein levels 6 . These 50 inherent characteristics of transcription are not resolved by current scRNA-seq approaches. Single-cell SLAM-seqHere, we introduce single-cell SLAM-seq (scSLAM-seq) to directly quantify transcriptional activity in single cells and resolve the involved regulatory elements. We validate scSLAM-seq by profiling changes 55 occurring after challenge with a virus. scSLAM-seq is based on thiol(SH)-linked alkylation for the metabolic sequencing of RNA (SLAM-seq) 7,8 , which involves a brief exposure of cells to the nucleoside analog 4-thiouridine (4sU). 4sU is incorporated into RNA during transcription and converted to a cytosine analog using iodoacetamide (IAA) prior to RNA-seq. Sequencing reads originating from "new" RNA, which has been transcribed during the time of 4sU labeling, can be id...

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