Using Flow Cytometry and Single-Cell Multiomics for Analyzing COVID-19 Immune Responses

The human immune system exerts coordinated responses capable of overcoming a broad spectrum of pathogenic challenges, including viral infections. Understanding the nature and sequence of these responses paves the way for targeting specific cells for further translational research. Several approaches have been used for investigating immune responses toward SARS COVID-19, and single-cell multiomics analyses have the potential to provide a multi-dimensional angle to immune responses, which is highly valuable given their complex nature.

In the publication “Single-cell multi-omics analysis of the immune responses in COVID-19”, Stephenson et al. use single-cell transcriptomic analysis, surface proteomic analysis and T and B lymphocyte antigen receptor analysis to analyze over 780,000 peripheral blood mononuclear cells (PBMC) from 130 patients with varying severities to COVID-19 infection. The study was performed in three UK centers (New Castle, Cambridge and London) and the results from all three were integrated to get a better understanding of the coordinated systemic immune responses from COVID-19 patients. Controls included healthy volunteers, individuals with non-COVID-19 severe respiratory illness and healthy volunteers administered with intravenous lipopolysaccharide as a surrogate for an acute systemic inflammatory response.

Methods included sequencing, cluster analysis, single-cell transcriptome analysis, surface proteome analysis and flow cytometry analysis.

• The study reveals several patterns in immune responses and differences in responses with increasing severity of disease:
  
• Single-cell transcriptome analysis revealed that relative expression of proliferating lymphocytes, proliferating monocytes, platelets and mobilized hematopoietic stem cell and progenitor cells (HSPCs) in varying stages of disease progression, and an expansion of B-cells and plasmablasts was observed in the most severe and critical cases.
• Expression of type I/III interferon response genes in monocytes, dendritic cells (DCs) and HSPCs across all spectra of disease severity was observed. IFNAR2, the type I/III interferon response gene was upregulated in COVID-19 patients compared to healthy individuals. It was also highly expressed by plasmoblasts, monocytes and DCs.
• Multiplex analysis showed that CCL4, CXCL10, IL-7 and IL-1α were associated with severe and critical disease, suggesting monocyte and NK lymphocyte recruitment and T cell activity.
• Transcriptome analysis of blood mononuclear phagocytes (MPs) identified DC subsets and three monocyte states (proliferating, classical CD14⁺ and activated CD83⁺). Transcriptome analysis also showed that proliferating monocytes population changed significantly with symptom duration.
• Surface protein analysis showed increased expression of B cells (CD19⁺/CD20⁺), plasma cells (CD38⁺) and HSPCs (CD34⁺).
• Partition-based graph abstraction analysis comparing circulating CD14⁺ monocytes and branchoalveolar lavage (BAL) macrophages indicated transcriptional similarity between them in health. On the other hand, in COVID-19, a greater similarity was observed between BAL macrophages and C1QA/B/C⁺CD16⁺ monocytes, suggesting that the origin of alveolar macrophages may be different between healthy individuals and COVID-19 patients.
• Migratory DCs contained in BAL macrophages express IL-10 in health and TNF, IL-12 and IL-23 subunit IL12B in COVID-19, suggesting an altered capacity for T cell polarization.
• Interrogation of 3297 CD34⁺ HSPCs using Leiden clustering and uniform manifold and projection (UMAP) visualization identified six transcriptional clusters (CD34⁺CD38^- HSPCs, CD34⁺CD38⁺ early progenitor HSPCs and CD34⁺CD38⁺ erythroid, megakaryotic and myeloid progenitors.) The megakaryote progenitors comprised about 5% of CD34⁺ cells in all symptomatic individuals, while they were absent in healthy and asymptomatic individuals, suggesting perturbation of normal homeostatic functioning of the bone marrow HSPC compartment by COVID-19.
• Differential gene expression analysis between megakaryocyte, myeloid and erhythroid progenitor clusters showed enrichment of megakaryocyte progenitor signature in the CD38⁺  HSPC populations in moderate COVID-19 compared to health. However, no enrichment of myeloid or erythroid signatures were observed in CD38⁺  or CD38^- HSPCs.
• Flow cytometry analysis showed T cell compartment cellular composition with activated CD4⁺  T cells expressing IL22, circulating follicular helper T (T_FH) cells, type 1 helper T (T_H1) cells, CD8 effector memory T (T_EM) cells and MAIR cells in asymptomatic or mildly infected individuals, and NKT, proliferating CD8⁺  and CD4⁺, and CD8⁺ terminal effector T (T_TE) cells in individuals with severe infection.
• T cell receptor (TCR) clonality analysis revealed maximum clonal expansion of effector CD8 T cells, with increased relative proportion with disease severity. There was a decrease in the relative proportion of clonally expanded effector memory CD8⁺ T_EM cells  as well in individuals with severe disease.
• The clonal alterations followed the infection trajectory, as CD8⁺ T_EM clonal expansion was observed in individuals with longer symptom duration, suggesting that the outcome of CD8⁺ T cell differentiation may contribute toward antiviral protection and immunopathology.
• Symptomatic individuals showed a significant expansion of plasmablasts and plasma cells, with an increased magnitude of expansion from mild to moderate disease and an attenuated expansion in severe to critical disease.
• There was also a decrease in IgA cells in symptomatic individuals due to a significant decrease of the IgA2 subclass, suggesting that there is an effective mucosal humoral response is maintained in asymptomatic individuals.
• In asymptomatic individuals a strong positive correlation between cT_FH cells and plasma cells, which was lost with increasing disease severity.
• These results suggest that a coordinated T cell and B cell response that generates effective antiviral humoral immunity is present in asymptomatic individuals and those with mild disease, which is uncoupled in those with severe and critical disease.
• A marked increase in IFNγ response was observed in all B cells subsets in individuals with asymptomatic or mild disease, which decreased with disease severity. TNF signaling via NF-ƙB was enriched in immature, naïve and switched memory B cells in asymptomatic individuals, but NF-ƙB expression decreased in immature B cells.
• B cell analysis showed that B cells exhibit a more pronounced response to interferons, increased NF-ƙB activation and increased expression of BCR activation signaling genes in asymptomatic individuals and those with mild disease, suggesting a higher BCR activation in these individuals.
• BCR clonality analysis showed expanded clonality, primarily in plasmablast/plasma cell clusters, in symptomatic individuals.
• Interestingly, a significant increase in clonotype distribution and BCR mutations was observed in women with COVID-19 compared to men.
• Taken together, the study reveals a coordinated immune response that contributes toward COVID-19 pathogenesis and identifies distinct cell subsets that could potentially be targeted in translational research.

The study provides a wealth of information for translational studies and an extensive background and template for meta-analysis of single-cell multiomics data sets for further analysis.  Read the Nature Medicine paper for further details.

Reference

Stephenson E, Reynolds G, Botting RA., et al. Single-cell multi-omics analysis of the immune response in COVID-19. Nature Medicine, May 2021; 27:904-916.