Human SARS-CoV-2 challenge resolves local and systemic response dynamics (preprint)

The COVID-19 pandemic is an ongoing global health threat, yet our understanding of the cellular disease dynamics remains limited. In our unique COVID-19 human challenge study we used single cell genomics of nasopharyngeal swabs and blood to temporally resolve abortive, transient and sustained infections in 16 seronegative individuals challenged with preAlpha-SARS-CoV-2. Our analyses revealed rapid changes in cell type proportions and dozens of highly dynamic cellular response states in epithelial and immune cells associated with specific timepoints or infection status. We observed that the interferon response in blood precedes the nasopharynx, and that nasopharyngeal immune infiltration occurred early in transient but later in sustained infection, and thus correlated with preventing sustained infection. Ciliated cells showed an acute response phase, upregulated MHC class II while infected, and were most permissive for viral replication, whilst nasal T cells and macrophages were infected non-productively. We resolve 54 T cell states, including acutely activated T cells that clonally expanded while carrying convergent SARS-CoV-2 motifs. Our novel computational pipeline (Cell2TCR) identifies activated antigen-responding clonotype groups and motifs in any dataset. Together, we show that our detailed time series data (covid19cellatlas.org) can serve as a 'Rosetta stone' for the epithelial and immune cell responses, and reveals early dynamic responses associated with protection from infection.

Single-cell analysis of bronchoalveolar cells in inflammatory and fibrotic post-COVID lung disease (preprint)

Rationale: Persistent pulmonary sequelae are evident in many survivors of acute coronavirus disease 2019 (COVID-19) but the molecular mechanisms responsible are incompletely understood. Post-COVID radiological lung abnormalities comprise two broad categories, organising pneumonia and reticulation, interpreted as indicative of subacute inflammation and fibrosis, respectively. Whether these two patterns represent distinct pathologies, likely to require different treatment strategies is not known. Objectives: We sought to identify differences at molecular and cellular level, in the local immunopathology of post-COVID inflammation and fibrosis. Methods: We compared single-cell transcriptomic profiles and T cell receptor (TCR) repertoires of bronchoalveolar cells obtained from convalescent individuals with each radiological pattern of post-COVID lung disease (PCLD). Measurements and Main Results: Inflammatory and fibrotic PCLD single-cell transcriptomes closely resembled each other across all cell types. However, CD4 central memory T cells (TCM) and CD8 effector memory T cells (TEM) were significantly more abundant in inflammatory PCLD. A greater proportion of CD4 TCM also exhibited clonal expansion in inflammatory PCLD. High levels of clustering of similar TCRs from multiple donors was a striking feature of both PCLD phenotypes, consistent with tissue localised antigen-specific immune responses, but there was no enrichment for known SARS-CoV-2 reactive TCRs. Conclusions: There is no evidence that radiographic organising pneumonia and reticulation in PCLD are associated with differential immmunopathological pathways. Inflammatory radiology is characterised by greater bronchoalveolar T cell accumulation. Both groups show evidence of shared antigen-specific T cell responses, but the antigenic target for these T cells remains to be identified.

The emergence of goblet inflammatory or ITGB6hi nasal progenitor cells determines age-associated SARS-CoV-2 pathogenesis (preprint)

Children infected with SARS-CoV-2 rarely progress to respiratory failure, but the risk of mortality in infected people over 85 years of age remains high, despite vaccination and improving treatment options. Here, we take a comprehensive, multidisciplinary approach to investigate differences in the cellular landscape and function of paediatric (<11y), adult (30-50y) and elderly (>70y) nasal epithelial cells experimentally infected with SARS-CoV-2. Our data reveal that nasal epithelial cell subtypes show different tropism to SARS-CoV-2, correlating with age, ACE2 and TMPRSS2 expression. Ciliated cells are a viral replication centre across all age groups, but a distinct goblet inflammatory subtype emerges in infected paediatric cultures, identifiable by high expression of interferon stimulated genes and truncated viral genomes. In contrast, infected elderly cultures show a proportional increase in ITGB6hi progenitors, which facilitate viral spread and are associated with dysfunctional epithelial repair pathways. A video explaining this work can be found here - https://youtu.be/uExP4bx6D_A .

Mapping interindividual dynamics of innate immune response at single-cell resolution (preprint)

Common genetic variants modulate the cellular response to viruses and are implicated in a range of immune pathologies, including infectious and autoimmune diseases. The transcriptional antiviral response is known to vary between infected cells from a single individual, yet how genetic variants across individuals modulate the antiviral response (and its cell-to-cell variability) is not well understood. Here, we triggered the antiviral response in human fibroblasts from 68 healthy donors, and profiled tens of thousands of cells using single-cell RNA-seq. We developed GASPACHO (GAuSsian Processes for Association mapping leveraging Cell HeterOgeneity), the first statistical approach designed to identify dynamic eQTLs across a transcriptional trajectory of cell populations, without aggregating single-cell data into pseudo-bulk. This allows us to uncover the underlying architecture and variability of antiviral response across responding cells, and to identify more than two thousands eQTLs modulating the dynamic changes during this response. Many of these eQTLs colocalise with risk loci identified in GWAS of infectious and autoimmune diseases. As a case study, we focus on a COVID-19 susceptibility locus, colocalised with the antiviral OAS1 splicing QTL. We validated it in blood cells from a patient cohort and in the infected nasal cells of a patient with the risk allele, demonstrating the utility of GASPACHO to fine-map and functionally characterise a genetic locus. In summary, our novel analytical approach provides a new framework for delineation of the genetic variants that shape a wide spectrum of transcriptional responses at single-cell resolution.

The local and systemic response to SARS-CoV-2 infection in children and adults (preprint)

While a substantial proportion of adults infected with SARS-CoV-2 progress to develop severe disease, children rarely manifest respiratory complications. Therefore, understanding differences in the local and systemic response to SARS-CoV-2 infection between children and adults may provide important clues about the pathogenesis of SARS-CoV-2 infection. To address this, we first generated a healthy reference multi-omics single cell data set from children (n=30) in whom we have profiled triple matched samples: nasal and tracheal brushings and PBMCs, where we track the developmental changes for 42 airway and 31 blood cell populations from infancy, through childhood to adolescence. This has revealed the presence of naive B and T lymphocytes in neonates and infants with a unique gene expression signature bearing hallmarks of innate immunity. We then contrast the healthy reference with equivalent data from severe paediatric and adult COVID-19 patients (total n=27), from the same three types of samples: upper and lower airways and blood. We found striking differences: children with COVID-19 as opposed to adults had a higher proportion of innate lymphoid and non-clonally expanded naive T cells in peripheral blood, and a limited interferon-response signature. In the airway epithelium, we found the highest viral load in goblet and ciliated cells and describe a novel inflammatory epithelial cell population. These cells represent a transitional regenerative state between secretory and ciliated cells; they were found in healthy children and were enriched in pediatric and adult COVID-19 patients. Epithelial cells display an antiviral and neutrophil-recruiting gene signature that is weaker in severe paediatric versus adult COVID-19. Our matched blood and airway samples allowed us to study the spatial dynamics of infection. Lastly, we provide a user-friendly interface for this data as a highly granular reference for the study of immune responses in airways and blood in children.

The cellular immune response to COVID-19 deciphered by single cell multi-omics across three UK centres (preprint)

The COVID-19 pandemic, caused by SARS coronavirus 2 (SARS-CoV-2), has resulted in excess morbidity and mortality as well as economic decline. To characterise the systemic host immune response to SARS-CoV-2, we performed single-cell RNA-sequencing coupled with analysis of cell surface proteins, providing molecular profiling of over 800,000 peripheral blood mononuclear cells from a cohort of 130 patients with COVID-19. Our cohort, from three UK centres, spans the spectrum of clinical presentations and disease severities ranging from asymptomatic to critical. Three control groups were included: healthy volunteers, patients suffering from a non-COVID-19 severe respiratory illness and healthy individuals administered with intravenous lipopolysaccharide to model an acute inflammatory response. Full single cell transcriptomes coupled with quantification of 188 cell surface proteins, and T and B lymphocyte antigen receptor repertoires have provided several insights into COVID-19: 1. a new non-classical monocyte state that sequesters platelets and replenishes the alveolar macrophage pool; 2. platelet activation accompanied by early priming towards megakaryopoiesis in immature haematopoietic stem/progenitor cells and expansion of megakaryocyte-primed progenitors; 3. increased clonally expanded CD8+ effector:effector memory T cells, and proliferating CD4+ and CD8+ T cells in patients with more severe disease; and 4. relative increase of IgA plasmablasts in asymptomatic stages that switches to expansion of IgG plasmablasts and plasma cells, accompanied with higher incidence of BCR sharing, as disease severity increases. All data and analysis results are available for interrogation and data mining through an intuitive web portal. Together, these data detail the cellular processes present in peripheral blood during an acute immune response to COVID-19, and serve as a template for multi-omic single cell data integration across multiple centers to rapidly build powerful resources to help combat diseases such as COVID-19.