Antibody repertoire and gene expression dynamics of diverse human B cell states during affinity maturation

Background:

The production of antibodies by B cells is a key feature of adaptive immunity, is often critical for long-lasting protection from re-infection, and underlies the protective effect of most vaccine strategies. Upon exposure to an antigen challenge, B cells undergo three main processes which underly the efficacy and long-term potential of antibody responses. Through a process largely coordinated in and around a special structure known as a germinal center (GC), B cells modify their genome through the processes of somatic hypermutation (SHM) and class switch recombination (CSR), and differentiate into several lineages, including long-lived memory B cells and plasma cells (PCs).

SHM and CSR are two distinct processes that alter the potential function of an antibody through modification of the encoding immunoglobulin gene. SHM involves the random mutation of the gene, which is then selected for improved affinity towards the antigen in a complex process dependent on help from T cells and occurring in a spatially defined pattern in the GC. CSR involves excision of components of the immunoglobulin gene locus from the genome, which changes the constant region of the resulting antibody and endows the antibody with its effector potential – such as the complement-activating potential of some IgG subclasses or the dimerization of particular IgAs. The precise coordination of these two processes with commitment to memory or PC lineages is an open area of investigation.

In this preprint, King et al undertake a deep investigation of human B cells from the tonsils, a secondary lymphoid organ containing germinal centers as well as naïve B cells. This offers a very rich dataset for understanding of human B cell dynamics and maturation through the memory B cell compartment, as well as insights into the timing and effects of CSR and SHM.

 

Key Findings:

• Integrated bulk and single-cell repertoire analysis improves mapping of B cell responses

The authors performed multiple analyses on the same tonsil samples, including deep bulk repertoire sequencing (obtaining up to hundreds of thousands of immunoglobulin sequences) along with scRNA-seq including Ig region VDJ sequencing. The bulk repertoires were used to improve mapping of clonally related B cells in the single-cell data – B cells descended from the same original cell that have undergone differential SHM or CSR. The bulk repertoire data allow for more powerful analysis of clonal trees and connections between related cells, while the single-cell data allows detailed characterization of every cell along with clonal information – so combining the two is an important step to fully understand B cell development through multiple stages of antibody gene alteration and differentiation.

• Discovery of a pre-GC B cell population primed for CSR

The single-cell data revealed a population of “pre-GC” B cells that had to undergo clonal expansion, CSR, or SHM, but had begun to acquire gene expression signatures similar to some GC B cells – including those necessary for cell-cell interactions with antigen-presenting cells and T cells. This population showed the highest expression of germ-line transcripts (GLTs), important indicators of potential for CSR, as well as components of DNA repair machinery necessary for CSR (e.g. APE1). While in contrast with the “textbook” description of CSR as occurring in the GC, it is consistent with recent work suggesting that most CSR occurs early on in B cell activation, before entry into the GC (Roco et al 2019)

• Impact of CSR on subsequent B cell development in the GC

Deep repertoire coverage also enabled comparison of “similar” GC B cells that have class-switched to different isotypes. After controlling for level of SHM (a proxy of affinity), the authors identified differences in genes a variety of critical pathways, including those regulating cell survival and BCR signaling. Further sampling and scRNA-seq of memory B cells isolated from the same samples revealed broad differences between switched and unswitched memory B cells, including differences that suggested increased potential for the unswitched population to re-enter GCs and undergo CSR.

 

Importance:

This study is a deep exploration of B cell progression through three key functional processes – CSR, SHM, and lineage commitment. It will serve as a rich resource for further investigation of B cell development through a GC response in humans. Additionally, it offers critical insights into the timing and location of CSR, supporting the notion that CSR may occur prior to GC entry and could therefore influence subsequent SHM and differentiation pathways.

 

Moving Forward / Questions for Authors:

• The FCRL2/3+ population is quite interesting, but somewhat mysterious. Do the FCRL2/3+ GC B cells show clonal relationships with the FCRL2/3+ MBCs? That is, are they likely to commit to that fate and maintain it after leaving the GC? Is FCRL2/3 expression driven by IgM-specific signaling, such that failure to undergo CSR early on in activation biases a B cell towards an inhibited fate that remains more “quiescent” (no subsequent CSR, and lower SHM and proliferation) in preparation for a secondary response? The clear existence and delineation of this subset should certainly prompt new detailed investigations!
• What are the eventual fates of the pre-GC B cells? The clonal relationships from Figure 2M don’t show strong evidence of eventual entry into the GC, but that may be because they are pre-expansion and the numbers are insufficient to detect their eventual progeny. They also don’t appear to express AID – how quickly upon induction of AID can they undergo CSR from this “poised” state?
• On the theme of pre-GC cells, how does the pre-GC memory B cell population form? Are they continuously poised for re-entry and CSR in a secondary response, or does their phenotype depend on reactivation by antigen exposure (or some other signal)?

 

References:

• Roco JA et al, “Class-Switch Recombination Occurs Infrequently in Germinal Centers”, Immunity (2019) 51(2) 337-350

 

doi: https://doi.org/10.1242/prelights.21958

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