Phenotypic determinism and stochasticity in antibody repertoires of clonally expanded plasma cells

Abstract

Significance B cell clonal selection and expansion from a genetically diverse antibody repertoire guides the immune response to a target antigen. It remains unclear if clonal selection and expansion follow any deterministic rules or are stochastic with regards to phenotypic antibody properties such as antigen-binding, affinity, and epitope specificity. We perform the in-depth genotypic and phenotypic characterization of antibody repertoires following immunization in mice. We identify the degree to which clonal expansion is driven by antibody binding, affinity, and epitope specificity and as such may provide greater insight into vaccine-induced immunity.

Generation of a single-cell B cell atlas of antibody repertoires and transcriptomes to identify signatures associated with antigen-specificity

“…To profile B cell selection following serial protein immunizations, we immunized a cohort of 3-month-old (3m) male C57BL6 mice (n=5) with five successive injections of 10 μg of the extracellular domain of human TNFR2 mixed with 20 μg of the adjuvant monophosphoryl lipid A (MPLA). Our previous findings demonstrated that immunization of young mice with an immunogenic protein antigen (ovalbumin) results in highly expanded plasma cells within the bone marrow (Neumeier, Yermanos, et al 2021). For this study, we selected an antigen that has potentially reduced immunogenicity given its high sequence similarity (~61.3%) to the murine homologue of TNFR2.…”

“…Nevertheless, clonally expanded cells of IgM, IgG and IgA isotypes were still present in both organs (Figures S5, S6, S7). Previous single-cell antibody repertoire sequencing studies suggest a strong correlation between class-switched clones and the number of amino acid sequence variants existing within an individual clone (Neumeier, Pedrioli, et al 2021; Neumeier, Yermanos, et al 2021). Here, we observed that the number of cells for the most expanded IgG and IgA clones demonstrated a minor correlation with the number of amino acid variants, whereas some IgM clones contained a considerable number of amino acid variants (Figure 1F).…”

“…We next determined the extent to which clonal expansion is correlated with antigen-specific binding. We focused on the most expanded clones that possessed cells mostly with an IgG subtype, as these have been previously shown to be preferentially associated with antigen specificity (Neumeier, Yermanos, et al 2021). We used a previously established system for rapid antibody cloning and expression in mammalian cells by CRISPR-Cas9 genome editing (Cristina Parola et al 2019; Pogson et al 2016).…”

“…Additionally, the emergence of single-cell sequencing (scSeq) technologies has made it possible to identify endogenously paired variable heavy (VH) and variable light chain (VL) regions from single B cells at high-throughput, which can be used to subsequently reconstruct antibodies for experimental testing (DeKosky et al 2013; DeKosky et al 2015) . For example, our group recently performed scSeq of plasma cells following immunization or viral infection in mice, and subsequent expression and screening of antibodies from highly expanded clones was shown to correlate with antigen-specificity (Neumeier et al 2022; Neumeier, Yermanos, et al 2021). A similar approach was applied to plasma cells isolated from convalescent Covid-19 individuals and led to the discovery of a panel of antibodies, including potent neutralizing antibodies against SARS-CoV-2 (Ehling et al 2022).…”

Understanding repertoire sequencing data through a multiscale computational model of the germinal center

“…Both these observations are in line with our simulations, as they show a large variability in subclone a nity. Recently, it has been reported in an OVA-immunized mouse model that a nities measured for plasma cells from blood samples do not correlate with clonal abundance on both a clone and subclone level 43 .…”