Figure 2Phenotypes of blood B cells. B cells from 15 ml of peripheral blood from each of three convalescent donors were isolated by negative selection with magnetic beads, exposed to adherent cells expressing spike-mCherry for 3 hours, then retrieved and labeled with fluorescent antibodies and measured by flow cytometry. (A) UMAP algorithm (5000 randomly selected cells/sample) was used to depict the major B cell subsets clustered according to marker expression.Phenotypes of blood B cells. B cells from 15 ml of peripheral blood from each of three convalescent donors were isolated by negative selection with magnetic beads, exposed to adherent cells expressing spike-mCherry for 3 hours, then retrieved and labeled with fluorescent antibodies and measured by flow cytometry. FlowSOM-based B cell subpopulations are overlaid as a color dimension, and the colors of the clusters are shown on the left of heatmap (B). B. Heatmap showing mean population expression levels of markers used for UMAP visualization and FlowSOM-clustering. Colors shown in the legend on the left are also used in the UMAP representation in (A).Phenotypes of blood B cells. B cells from 15 ml of peripheral blood from each of three convalescent donors were isolated by negative selection with magnetic beads, exposed to adherent cells expressing spike-mCherry for 3 hours, then retrieved and labeled with fluorescent antibodies and measured by flow cytometry. C. Gating strategy to define CD69-high, mCherry-high, spike-capturing B cells (i.e., membrane antigen capture activated cells, MACAC, red box). Dot plot shows CD69 and mCherry fluorescence for B cells from one of three donors gated by forward and side scatter, and negative for the dye used to mark the antigen donor cells.Phenotypes of blood B cells. B cells from 15 ml of peripheral blood from each of three convalescent donors were isolated by negative selection with magnetic beads, exposed to adherent cells expressing spike-mCherry for 3 hours, then retrieved and labeled with fluorescent antibodies and measured by flow cytometry. D. Relative fractions of different B cell subpopulations, from three donors, as shown in A-B, in all B cells compared to within MACAC B cells. P values are based on two-tailed t-tests between the groups. Correlation coefficients (r) were calculated from the z-statistic of the Wilcoxon-Mann-Whitney test. A black horizontal line represents the median. Boxplots represent the interquartile range (IQR). Whiskers extend to the farthest data point within a maximum of 1.5× IQR. Every point represents one donor.E. Gating strategy in MACAC sorting. Single cells are selected based on scatter, antigen-expressing TE spike-mCherry cells excluded on Cell Trace Violet label, and the spike-capturing (mCherry-high), activated (CD69-high) B cells (population labeled "MACAC" in red on the right-most plot) are sorted. The middle plot shows B cells that did not adhere to the TE-spike-cherry antigen-expressing cells (putatively antigen-irrelevant), and the right plot those that did (putatively antigen-recognizing). Plots show data from one of five convalescent donors Cells in the MACAC gate were singly distributed into wells of 384-well plates and cultured for 9 days with IL-21 and CD40L, and then the single well culture supernatants were screened for anti-spike antibody binding and virus neutralization as described below. F. Results of single well supernatant screening for antibody binding to SARS-CoV-2 spike protein. Results from 3266 wells from 5 donors are shown for IgM (left), IgG (middle), and IgA (right).