Figure 1. Characterization of anti-IsdB antibodies in the human memory B cell repertoire.

(a) A persistent population of IsdB+ memory B cells from the peripheral blood mononuclear cells (PBMC) of a donor (D3) was observed over a 15-month period. By FACS, ∼0.06% of the IgM− CD19+ CD27+ memory B cells in the total memory B cell repertoire of this donor bind IsdB. (b) Most of the cloned BCR transcripts of the IsdB+ memory B cells collected at month 1, 3 and 15 are clonally related. BCR sequences from single-cell cloning of IsdB+ memory B cells were clustered based on heavy chain V-gene usage and CDR-H3 sequences. In total, we identified 31 unique clusters from donor D3 over the three collection time points. The Venn diagram shows that sibling clones within a cluster can be isolated at multiple time points. (c) Two distinct sets (bin C and bin P) of function-blocking antibodies specifically target NEAT1 and NEAT2, respectively. Single-cell cloning was performed at three different time points for donor D3 and one time each for donors D1, D2, and D4. In total, 75 unique antibodies targeting IsdB were identified and characterized. Shown here are the results of a comprehensive epitope binning analysis of 67 antibodies. Each reformatted clone is shown as a box and coloured according to its VH germline usage. The height of the box indicates the number of clustered BCR transcripts represented for each reformatted clone. There are in total 9, 34, 327, and 68 anti-IsdB single-cell BCR transcripts for D1, D2, D3 and D4, respectively. Each column of clones represents an epitope bin and this is overlaid on top of a linear representation of the IsdB molecule with NEAT1 in orange, and NEAT2 in blue. Clones that are able to fully block haemoglobin binding are outlined with a red box.