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.