Fig 2: In vitro plasmablast differentiation of primary human B cells. (A–E) Primary B cells were purified from PBMC of healthy donors and differentiated into plasmablasts in vitro. The cells were stained for indicated surface markers and intracellular IgA and analyzed with FACS. Representative CD138/CD38 and CD27/IgA plots at indicated time points are shown in (A). The percentages of IgA+CD27+ cells are shown in (B). The levels of IgA and IgG in the supernatant were quantified with ELISA (C). The transcript levels of PADs (D) and MZB1 [(E), the top panel] were measured with qPCR. Whole cell extract was analyzed in western blotting using anti-MZB1 and anti-tubulin [(E), the bottom two panels]. (F) Primary human B cells were differentiated in the presence of DMSO or AFM-30a (20 uM). Cell extract from two experiments was pooled together; the levels of total and PG-tagged MZB, total IgM, and total IgA were examined with western blotting.

Fig 3: SARS-CoV-2 infection alters the immune phenotype and function of B cells and reduces CD19 expression on B cells from recovered COVID-19 patients. Flow cytometry analysis of the frequencies of naive B cells (naive, CD19+ IgD+CD27−), class-switched B cells (switched, CD19+ IgD−CD27+), unswitched memory B cells (unswitched, CD19+IgD+CD27+), transitional B cells (transitional, CD19+CD24+CD38+), plasma blasts (PBC, CD19+CD38+CD24−) and atypical memory B cells (atypical, CD19+IgD−CD27−) in resting and stimulated PBMCs from COVID-19 recovered patients (n = 14–15) and healthy controls (n = 15). Shown are representative dot plots (a, d) and the average percentages (±SEM) of B-cell subpopulations (b, c, e). Flow cytometry analysis of the MFI of CD19 on total B cells (B, CD19+), naive B cells, class-switched B cells, unswitched memory B cells, transitional B cells, plasma blasts, and atypical memory B cells in resting PBMCs from COVID-19 recovered patients (n = 15) and healthy controls (n = 15). Resting = cells before stimulation, Stimulated = cells after stimulation with 10 µg/ml biotin-F(ab′)2-anti-Ig(M + G) for 24 h (f). RT-PCR analysis of CD19 mRNA levels of B cells from recovered patients (n = 4) and healthy controls (n = 4) (g). Immunofluorescence analysis of CD19 expression in nephritic sections from SARS-CoV-2 infected mice (n = 3) and mock mice (n = 3). Shown are representative images (h, i). Statistical evaluation was performed using the two-tailed Student’s t-test. Statistical significance is indicated using asterisks: *p < 0.05,**p < 0.01, ***p < 0.001, and ****p < 0.0001

Fig 4: Dysregulated peripheral B cell subsets and functions. (A-L) Flow cytometry data on CD19+ B cells (A, F), transitional B cells (CD19+CD24+CD38+), plasmablasts (CD19+CD24−CD38+) (B, G), switched memory B cells (CD19+CD27+IgD−), unswitched memory B cells (CD19+CD27+IgD+), naïve B cells (CD19+CD27−IgD+) (C, H), double-negative B cells (DNs, CD19+CD27−IgD−) (I), DN1 (IgD−CD27−CXCR5+CD11c−), DN2 (IgD−CD27−CXCR5−CD11c+), DN3 (IgD−CD27−CXCR5−CD11c−), and DN4 (IgD−CD27−CXCR5+CD11c+) (J) in PBMCs from 3 age-matched HC and patient. (D) Flow cytometry analysis of CD19 MFI on CD19+ B cells and switched memory B cells (CD19+CD27+IgD−). (E) Flow cytometry analysis of CD80 and CD62L expression on CD19+ B cells and switched memory B cells (CD19+CD27+IgD−). (K) Flow cytometry analysis of percentage of Annexin V+ on CD19+ B cells. (L) Flow cytometry analysis of percentage of Ki67+ on CD19+ B cells. The percentage of each B cell subset is represented graphically for the HC (blue bars) and the patient (orange bars). (A-E) Samples were collected in May 2023. (F-L) Samples were collected in April 2024.