Figure legends
Figure 1: Conceptual overview of the protocol for SARS-CoV-2
spike specific Memory B-cell detection . *Detailed B-cell staining panel
is provided in Table 2. Abbreviations: EDTA = Ethylenediaminetetraacetic
acid, PBMC = Peripheral blood mononuclear cell.
Figure 2: Detection of SARS-CoV-2 specific B cell subsets. (a)
Gating strategy for the detection of B cell subsets. (b) Representative
example for the detection of dual-labelled SARS-CoV-2 S-protein binding
B-cells and quantification of antigen-specific B cell subsets.
Comparison of samples without fluorochrome-coupled SARS-CoV-2 protein
(“No S-protein”) and SARS-CoV-2 S-protein in concentrations of 1:500
and 1:1000 with and without excess unlabelled protein to block B cell
receptors.
Figure 3: Detection of
COVID-19 specific BMEMORY cells using spike (S)-protein. Peripheral
blood mononuclear cells of convalescent COVID-19 patients (n=26) and
healthy donors (n=14) were incubated with fluorochrome labeled
SARS-CoV-2 S-protein with or without excess unlabelled protein to block
labeling. (a) Percentage of IgD- CD27+ BMEMORY, IgD+
CD27+ unswitched BMEMORY, IgD+ CD27-
BNAIVE and CD27++ CD38++ plasmablasts within the entire
B-cell population analysed for COVID-19 disease and healthy cohorts.
Bars show median with interquartile range. (b) Correlation of
fluorochrome labelled SARS-CoV-2 S-protein binding B cells and
anti-S1/S2-IgG. n=21 samples of 19 COVID-19 patients (two patients with
two samples collected at different time points) and 14 healthy donors.
Analysis was performed with Spearman’s rank coefficient, rho=0.42,
P=0.011. (c)-(f) Frequencies of S-protein binding
BMEMORY (c), BNAIVE (d), plasmablasts
(e) and unswitched BMEMORY cells (f) after staining with
preincubation of unlabelled Covid-19 antigen (blocked, left) and without
preincubation (not blocked staining, right) samples. n=26 COVID-19
patients and n=14 healthy donors. Statistical comparison was done with
two-way repeated measurements ANOVA and Sidak’s multiple comparisons
test. (g) Correlation of fluorochrome labelled SARS-CoV-2 S-protein
binding B cells and days after COVID-19 diagnosis. n=25 samples of 23
COVID-19 patients (two patients with two samples collected at different
time points). The analysis was performed with Spearman’s rank
coefficient, rho= -0.34, P=0.095.