B cell analysis in SARS-CoV-2 versus malaria: Increased frequencies of plasmablasts and atypical memory B cells in COVID-19

Abstract

B cells play a central role in antiviral and antiparasitic immunity, not only as producers of antibodies, but also as APCs and mediators of inflammation. In this study, we used 16-color flow cytometry analysis to investigate the frequency, differentiation, and activation status of peripheral B cells of patients with SARS-CoV-2 infection or acute Plasmodium falciparum malaria compared with the healthy individuals. As a main result, we observed an increase of the frequency of (CD27^-, CD21^-) atypical memory B cells and (CD19⁺, CD27⁺, CD38⁺) plasmablasts in malaria and COVID-19 patients. Additionally, CD86, PD-1, CXCR3, and CD39 expression was up-regulated, whereas CD73 was down-regulated on plasmablasts of COVID-19 and malaria patients compared with the bulk B cell population. In particular, there was a more pronounced loss of CD73⁺ B cells in malaria. The frequency of plasmablasts positively correlated with serum levels of CRP, IL-6, and LDH of COVID-19 patients. In the longitudinal course of COVID-19, a rapid normalization of the frequency of atypical memory B cells was observed. The role and function of plasmablasts and atypical memory B cells in COVID-19 and other acute infections remain to be further investigated. The role of B cells as either "driver or passenger" of hyperinflammation during COVID-19 needs to be clarified.

Keywords: CD19; CD39; CD73; hyperinflammation.

Graphical abstract

FIGURE 1

Frequencies of CD27^–, CD21^– B cells and plasmablasts are elevated in acute COVID-19 and malaria compared with the healthy donors (HDs). (A) Representative FACS plots showing the frequency of transitional B cells (left panel) and the distribution of mature B cell subsets defined by CD21/CD27 (right panel) in HDs, COVID-19, and malaria patients. The panels are gated on CD19⁺, CD20⁺ cells, in the right panel transitional cells have been excluded (see Supplemental Fig. S3 for the detailed gating strategy). (B) Comparison of B cell subset distribution in HDs, COVID-19, and malaria patients. The pie charts show the mean frequency of transitional B cells (defined as CD24⁺, CD38⁺), naïve (CD21⁺, CD27^–), CD21+ memory (CD21⁺, CD27⁺), CD21^–, CD27⁺ memory(CD21^–, CD27⁺) and atypical memory (CD21^–, CD27^–) populations in HDs, COVID-19, and malaria patients. (C) Representative FACS plots showing the frequency of plasmablasts (CD19⁺, CD27⁺, CD38⁺) in HDs, COVID-19, and malaria patients (see Supplemental Fig. S4 for the detailed gating strategy). (D) Frequency of plasmablasts in HDs and COVID-19 and malaria patients. (E and F) Comparison of the frequencies of atypical memory B cells (E) and plasmablasts (F) between mild, moderate, and severe COVID-19 patients. Data are shown as mean ± sd, where *, **, ***, and **** indicate P values <0.05, <0.01, <0.001, and <0.0001

FIGURE 2

Frequency of plasmablasts and atypical memory B cells correlates with inflammation parameters. (A–D) Correlations between the frequency of plasmablasts and serum levels of IL-6 (A), CRP (B), and LDH (C), as well as the frequency of atypical memory B cells and serum levels of ferritin (D) in COVID-19 patients. Pearson correlation coefficients were applied for parametric data, respectively, and P values < 0.05 were considered significant

FIGURE 3

Phenotypic analysis of B cells in COVID-19 and malaria compared with the healthy donors. (A) A 2-dimensional map of CD19⁺ B cells concatenated from all samples (gray) was generated by t-SNE analysis. Samples from healthy donors (green), COVID-19 (blue), and malaria (red) patients were projected onto the total sample map. (B) Plasmablasts were identified by Boolean gating of concatenated CD19⁺ B cells from all samples and projected onto the t-SNE map (black). (C) Projection of subpopulations associated with B cell differentiation onto the t-SNE map: transitional (CD24⁺, CD38⁺; black), naïve (CD21⁺, CD27^–; green), CD21⁺ memory (CD21⁺, CD27⁺; purple), classical memory (CD21^–, CD27⁺, blue), and atypical memory (CD21^–, CD27^–, red) B cells were identified by Boolean gating of concatenated CD19+ B cells from all samples and projected onto the t-SNE map. (D) The expression levels (measured as mean fluorescence intensities) of the indicated surface molecules were projected onto the t-SNE map. (E) Frequency of cells expressing PD-1, CD86, CXCR3, CD11c, CD39, or CD73 among total CD19⁺, CD20⁺ B cells and atypical memory B cells (CD21^–, CD27^–) in healthy donors, COVID-19, and malaria patients. (F) Frequency of cells expressing PD-1, CD86, CXCR3, CD11c, CD39, or CD73 among total CD19⁺ B cells and plasmablasts in healthy donors, COVID-19, and malaria patients. Data are shown as mean ± sd, where *, **, ***, and **** indicate P values <0.05, <0.01, <0.001, and <0.0001

FIGURE 4

Longitudinal analysis of B cells in COVID-19. (A) Representative FACS plots showing the frequencies of B cell subsets and plasmablasts during acute infection (T0) or at follow-up (T1). The plots were gated on CD19⁺, CD20⁺ B cells excluding transitional B cells and plasmablasts (left panel) or on CD19⁺ B cells (right panel, see gating strategy in Supplemental Figs. 3 and 4). (B) Frequency of atypical memory B cells (CD27^–, CD21^–) and plasmablasts of 5 individual COVID-19 patients during acute infection (T0) or at follow-up (T1). (C) Correlation between the frequency of atypical memory B cells and the number of days since the begin of symptoms. (D) Distribution of B cell subsets in COVID-19 patients during acute infection (T0) or at follow-up (T1). The pie charts show the mean frequencies of the indicated subsets in 5 patients. (E and F) Comparison of frequencies of plasmablasts and atypical memory B cells between COVID-19 patients who produced SARS-CoV-2 IgM or IgG antibodies at the time of sampling during acute infection. Data are shown as mean ± sd, ** indicates P < 0.01

FIGURE 5

B cell differentiation and phenotype in COVID-19 and malaria patients. Plasmablasts and atypical memory B cells are more frequent in patients with COVID-19 and malaria than in healthy subjects, and severe courses of COVID-19 are associated with a higher frequency of atypical memory B cells. Illustration created with the online software BioRender (San Francisco, CA)