Extended Data Fig. 3: GC B cell response to influenza virus vaccine is clonally diverse.

a, Schematic of single-cell monoclonal antibody cloning and expression. Paired heavy and light chain genes were amplified from singly sorted PBs or GC B cells. Variable portions of heavy chains were cloned into a Cγ1 expression vector and variable portions of κ and λ light chains were cloned into respective expression vectors. Paired heavy and light chain expression vectors were co-transfected into 293F cells, and monoclonal antibodies were purified from culture supernatant by protein A affinity chromatography, then screened for QIV specificity by ELISA. b, c, Minimum positive concentrations of clonally unique monoclonal antibodies generated from singly sorted PBs (b) and GC B cells (c) from the indicated participants as determined by QIV ELISA; positive binding defined as greater than 3× background. d,Distance-to-nearest-neighbour plots for choosing a distance threshold for inferring clones via hierarchical clustering. After partitioning sequences based on common V and J genes and junction length, the nucleotide Hamming distance of a junction to its nearest non-identical neighbour from the same participant within its partition was calculated and normalized by junction length (blue histogram). For reference, the distance to the nearest non-identical neighbour from other participants was calculated (green histogram). A clustering threshold of 0.1 (dashed black line) was chosen via manual inspection and kernel density estimate (dashed purple line) to separate the two modes of the within-participant distance distribution representing, respectively, sequences that were probably clonally related and unrelated. e, Clonal overlap of sequences from monoclonal antibody cloning and bulk repertoire analysis between PBs sorted from PBMCs 1 week after vaccination and GC B cells from the indicated time point among total (top) and only QIV-binding (bottom) sequences. Purple chords link overlapping GC and PB clones; black chords link GC clones found at multiple time points that did not participate in the early PB response. Chord width corresponds to clonal population size. Percentages are of GC sequences overlapping with PBs. f, Clonal rank-abundance distributions of GC B cells from indicated time points (left) and of early blood PBs (right). The number of GC B cells or early blood PBs in a clone as a percentage of the total GC or early blood PB repertoire (y axis) is plotted against the abundance rank of that clone (x axis). Solid lines represent the estimated clonal abundance curves, with shaded bands representing the 95% confidence intervals from 200 bootstraps. g, tSNE clusters of B cells from FNA scRNA-seq samples from participant 05. Each dot represents a cell, coloured by phenotype as defined by gene expression profile. Total numbers of cells are given below clusters. GC percentages are indicated in blue. h, IGHV mutation frequency of naive B cells pooled from all time points (left) and the indicated populations at the indicated time point (right) from scRNA-seq of whole and memory B cell-enriched PBMC and FNA samples from participant 05. Horizontal lines represent medians. P values were determined by two-sided Dunn’s multiple comparisons test.