Fig 1.
Overall procedure of the study and transcriptional estimated infiltration of 22 types of immune cells in the lungs of COVID-19 patients.
(A) Diagram showing the comprehensive procedure of the study; (B) CIBERSORT abs score of 22 immune cells, colors represent the classification of the following: T cell lineage, B cell lineage, Myeloblast lineage, and other immune cells; (C) Similarity matrix representing the correlation among 22 immune cells in GSE150316.
Fig 2.
In silico simulated T-cell lineage infiltration in lung tissue between COVID-19 and IPF patients.
*P<0.05, **P<0.01 and ***P<0.001, with comparisons indicated by brackets.
Fig 3.
In silico simulated B-cell lineage infiltration in lung tissue between COVID-19 and IPF patients.
*P<0.05, **P<0.01 and ***P<0.001, with comparisons indicated by brackets.
Fig 4.
In silico simulated myeloblast lineage infiltration in lung tissue between COVID-19 and IPF patients.
*P<0.05, **P<0.01 and ***P<0.001, with comparisons indicated by brackets.
Fig 5.
GO:BP functional enrichment map summary of GSEA in the lung tissues of COVID-19 and IPF patients.
(A) The top NES-ranked enrichment results of biological processing by gene ontology (GO) functional enrichment analysis in IPF or COVID-19 (NES > 2; FDR < 0.05). The normalized enrichment score (NES) was presented in the X-axis, and the names of enriched functional gene sets were shown in the Y-axis. The color gradation on the right side indicates false discovery rate (FDR). Size represents the number of genes in the gene set. Color of the text represents whether the gene set was enriched in both (purple) or in individual type of disease (COVID-19: Red, IPF: Green). (B) Venn diagram: two circles represent the intersection status of the top 100 genes from the Naive B cell (Red) and Memory B cell (Green) subtypes extracted from the LM22 immune cell gene signatures, respectively. The detailed list of genes in the intersection is shown in the table below. Enrichment map: Commonality of positive enrichments in the GO:BP gene sets after GSEA assessment using enrichment map visualization in COVID-19 (GSE150316, Red) and IPF (GSE124685 and GSE53845, Green). Single color: the node is positively correlated in only one disease condition; Mixed: the node is positively correlated in two or three databases. Diamond: Naïve or Memory B cell specific signature, genes in the signature were shown in the table below. Lines connecting nodes or diamonds represent the degree of overlapping between two gene sets. Criteria for enrichment significance screening: P-value <0.05, FDR <0.05.
Fig 6.
Validation of the phenotypes of B-cell populations in single-cell RNA sequencing of lung-associated body fluid samples from COVID-19 and IPF patients.
UMAP shows the visualized distribution of whole cells in single-cell RNA sequencing data from two COVID-19 databases (A) and IPF database (B). the “B-cell clusters” annotated by the original authors are shown in red, the unassigned cells are shown in gray. B-cell clusters are further colored red and blue to represent those from disease or healthy donors (HC) respectively. The proportion and average expression of genes related to Naive or Memory B cells in the B-cell population are shown as dot plots. Relative gene expression is shown in color and the overall B-cell expression gene percentage is shown in dot size (%). The mean Z scores of all genes’ expression in B cell populations from HC or disease groups are shown in Violin plot. Statistical significance was performed using Welch’s t-test approach. *: P < 0.05, **: P < 0.01, ***: P < 0.001. The actual P-values are shown under the asterisks as well.
Fig 7.
Assessing the phenotypic and clinical status differences of B-cell populations between mild and severe symptoms of COVID-19.
(A) Schematic diagram of the GSEA analyzing process for B cells of mild and severe COVID-19 patients in different databases. GSEA results were cross-referenced and the overlapping enriched data sets of the two databases were listed. The NES scores of each gene set are additionally represented by shades of red to represent the relative enrichment level. Those considered relevant to the B-cell population of patients with severe COVID-19 in the two immune cell comparisons are marked in red. Criteria for enrichment significance screening: P-value <0.05, FDR <0.25. (B) The dot plots represent the overall expression ratio and average expression of genes thought to be related to antibody-secreting cells and DN2 B cells. The overall expression percentage is represented by the size of the dots and the average expression is represented by the color. Welch’s t-test was performed to analyze the significance of gene expression in two conditions. **: P < 0.01, ***: P < 0.001. (C) Comparison of B-cell infiltration ratio and clinical status of patients with COVID-19. Box plots are shown for patients with B cell infiltration ratio < 1% (Black box) or > 1% (Red box) for duration of hospitalization and ICU (Days). Pie charts show the severity, recovery and complications of multi-organ failure in COVID-19 patients with different levels of B-cell infiltration.
Fig 8.
Diagrammatic representation illustrate the postulated role of naïve B cell in triggering humoral immune response in lung tissues of COVID-19 patients and the locations where B cell targeted therapeutic strategies function.
SARS-CoV-2 infection may activate and promote the adhesion and accumulation of naïve B cells to the mediastinal lymph node with β2 integrin (LFA-1) and α4β1 integrin. The increased infiltration of naïve B cells activated by spike proteins from SARS-CoV-2 secretes large amounts of IgM to promote extrafollicular response and humoral immune response. Monocytes are recruited and differentiate to macrophage in response to the robust humoral immune response. Secreted IgM simultaneously activates the complement system and Fc receptor in dendritic cells and macrophage to increase antigen presentation and phagocytosis to facilitate innate and adaptive immunity. On the other hand, high affinity or abundant SARS-CoV-2 presented by antigen-presenting cell (APC) such as dendritic cell may stimulate IL-12-dependent plasma cell differentiation in naïve B cells to produce more IgM, instead of promoting B cell proliferation and differentiation by CD40/CD40L signaling mediated germinal center formation. Ibrutinib inhibits B-cell growth by specifically inhibiting Bruton kinase, which is thought to be critical for the BCR signaling pathway; Natalizumab reduces B-cell migration by blocking α4β1 integrin. Fingolimod reduces B-cell egress out of the lymph node by stimulating S1PR1/3 to internalize the receptors.