Two distinct durable human class-switched memory B cell populations are induced by vaccination and infection

Abstract

Memory lymphocytes are durable cells that persist in the absence of antigen, but few human B cell subsets have been characterized in terms of durability. The relative durability of eight non-overlapping human B cell sub-populations covering 100% of all human class-switched B cells was interrogated. Only two long-lived B cell populations persisted in the relative absence of antigen. In addition to canonical germinal center-derived switched-memory B cells with an IgD^-CD27⁺CXCR5⁺ phenotype, a second, non-canonical, but distinct memory population of IgD^-CD27^-CXCR5⁺ DN1 B cells was also durable, exhibited a unique TP63-linked transcriptional and anti-apoptotic signature, had low levels of somatic hypermutation, but was more clonally expanded than canonical switched-memory B cells. DN1 B cells likely evolved to preserve immunological breadth and may represent the human counterparts of rodent extrafollicular memory B cells that, unlike canonical memory B cells, can enter germinal centers and facilitate B cell and antibody evolution.

Keywords: CP: Immunology; TP63 in lymphocytes; clonal diversity; extrafollicular memory B cells; germinal center derived memory B cells; human B cells; human class-switched memory B cells; humoral immune durability; lymphocyte durability.

Figure 1.. Two durable antigen-specific class switched B cell populations emerge after vaccination.

A) Pie charts displaying spike-specific B cells profiled from the blood of previously uninfected vaccinees at baseline (n=11), 1–2 weeks post-dose 1 (n=9), time of dose 2 (n=19), 1 week post-dose 2 (n=19), 4–8 weeks post-dose 2 (n=12), and 24 weeks post-dose 2 (n=10), as well as from patients with acute (n=13) or convalescent from COVID-19 (n=66). The median of each B cell subset (as a proportion of all spike⁺ IgD⁻ B cells) from each time point are plotted as parts of a whole. Key: DN = double negative, IgD⁻ CD27⁻; ABC = age-associated B cells; CoV = COVID-19; Conv = convalescent. B) Scatter plots displaying the proportion of spike-specific B cell subset and total spike-specific B cells across the same cohorts depicted in part A. p-values were calculated using the Mann-Whitney test. Asterisks indicate p-values with values as follows: * 0.05 to 0.01; ** <0.01 to >0.001; *** <0.001 to >0.0001; and **** <0.0001.

Figure 2.. DN1 memory B cells and CD27⁺ SWM B cells accumulate at steady state and during convalescence

A-B) Dot plots with connecting lines displaying proportions of A) CD27⁺ SWM and B) DN1 B cells among all spike-specific B cells measured longitudinally across 11 patients during convalescence from COVID-19. Each color represents a different patient. C) Dot plot displaying proportions of all 8 non-overlapping, non-plasmablast, B cell populations among CD19⁺ IgD⁻ B cells across 24 pre-pandemic healthy donors. Lo/Lo and Hi/Hi refer to expression levels of CXCR5 and CD11c within the two CD27⁺ B cell subsets. D) Pie chart displaying the median values for each B cell subset as parts of a whole representing all non-plasmablast CD19⁺ IgD⁻ B cells profiled across 24 pre-pandemic healthy donors. E) Dot plot displaying the proportions of DN1 B cells expressing IgG, IgA, or IgM at the protein level among healthy donor PBMC samples (n=10). F) Dot plots with connecting lines comparing frequencies of IgG, IgA, and IgM surface expression between DN1 and SWM B cells. G) Dot plots with connecting lines showing pre- and post-boost proportions of RBD-specific B cells among all CD19⁺ B cells (first panel) or of each B cell subset among RBD-specific B cells (subsequent 5 panels) profiled across paired PBMC samples (n=11 from each time point). Each color represents a different donor. p-values were calculated using the Mann-Whitney test. NS = not significant. Asterisks indicate p-values with values as follows: * 0.05 to 0.01; ** <0.01 to >0.001; *** <0.001 to >0.0001; and **** <0.0001.

Fig 3.. DN1 B cells exhibit lower SHM frequency, less positive selection pressure, and reduced repertoire diversity relative to canonical CD27+ SWM

A-C) Dot plots with connected lines displaying somatic hypermutation frequency, proportions of BCRs with unmutated germline sequences, and average CDR3 length among flow purified follicular naïve, DN1, and canonical SWM B cells across 6 healthy donor biologic replicates analyzed on the Adaptive platform. D-H) Dot plots with connected lines displaying (D) somatic hypermutation frequency, (E) proportions of BCRs with unmutated germline sequences, (F) average CDR3 length, (G) CDR replacement/silent (R/S) mutation ratio, and (H) FWR R/S mutation ratio among flow purified follicular naïve, DN1, and canonical SWM B cells across 4 different healthy donor biologic replicates analyzed by sequencing of the full length rearranged VH gene. I) FWR3 region R/S ratio displayed across the same biologic replicates in parts A-C using the Adaptive platform. J) Rarefaction curves of follicular, canonical SWM, and DN1 B cells displaying the BCR repertoire diversity (number of unique clones) relative to sequences analyzed for each population. Follicular naïve populations are depicted in tones of red, canonical SWM in tones of blue, and DN1 in tones of green. Each rarefaction curve is label with the cell population and associated biologic replicate the cells were sorted from. Key: CDR = complementarity determining region; nt = nucleotide; FWR = framework region; FO = follicular. Full length VH refers to sequencing of the complete VH gene (see Methods section).

Figure 4.. DN1 memory B cells are transcriptionally distinct from canonical SWM B cells and can be defined by a TP63 linked signature

A) PCA plot of bulk transcriptomes shows strong separation with respect to sorted B cell subpopulations, including separation between switched memory (green) and DN1 (blue) B cells along PC2 (N=6/group). (B) Plot showing the number of differentially expressed genes (DEG) between DN1 and naïve (y-axis) or SWM B cells (x-axis), where each dot represents a gene. Colored dots represent genes with either the same (red) or opposite (green) direction of change in DN1 relative to both naïve and SWM. (C) Venn diagrams showing the number of significantly differentially expressed genes (DEG) that are increased (top) or decreased (bottom) in DN1 B cells compared to naïve and/or SWM B cells identified via bulk RNAseq. (D) Heatmaps showing the topmost DEG either increased (left) or decreased (right) in DN1 compared to the other B cell populations. (E) Plots showing VSD-normalized counts (log-scale) of gene regulators broadly associated with B cell differentiation comparing transcript expression levels across the three B cell populations. (F) Heatmap showing the expression of a TP63-dependent gene signature across the three B cell populations, where “cellDeath” indicates whether the indicated gene is found in the Gene Ontology (GO) annotation of negative (green) or positive (red) regulation of cell death, or both (gray). (G) Validation of TP63 expression in DN1 and SWM B cells using qPCR.