Profiling the specificity of clonally expanded plasma cells during chronic viral infection by single‐cell analysis

Abstract

Plasma cells and their secreted antibodies play a central role in the long-term protection against chronic viral infection. However, due to experimental limitations, a comprehensive description of linked genotypic, phenotypic, and antibody repertoire features of plasma cells (gene expression, clonal frequency, virus specificity, and affinity) has been challenging to obtain. To address this, we performed single-cell transcriptome and antibody repertoire sequencing of the murine BM plasma cell population following chronic lymphocytic choriomeningitis virus infection. Our single-cell sequencing approach recovered full-length and paired heavy-and light-chain sequence information for thousands of plasma cells and enabled us to perform recombinant antibody expression and specificity screening. Antibody repertoire analysis revealed that, relative to protein immunization, chronic infection led to increased levels of clonal expansion, class-switching, and somatic variants. Furthermore, antibodies from the highly expanded and class-switched (IgG) plasma cells were found to be specific for multiple viral antigens and a subset of clones exhibited cross-reactivity to nonviral and autoantigens. Integrating single-cell transcriptome data with antibody specificity suggested that plasma cell transcriptional phenotype was correlated to viral antigen specificity. Our findings demonstrate that chronic viral infection can induce and sustain plasma cell clonal expansion, combined with significant somatic hypermutation, and can generate cross-reactive antibodies.

Thymus Antibody-Secreting Cells Represent a Tissue Specific Population That Possess an Activated Cellular Phenotype

“…As a result, understanding ASC biology has become of growing importance as the repertoire of ASC functions seems to depend on the context in which ASCs exist. Along these lines, recent studies have utilized RNA-seq at both bulk (Lino et al, 2018; Pioli et al, 2019; Price et al, 2019; Wilmore et al, 2021) and single cell (scRNA-seq) (Lam et al, 2018; Neumeier et al, 2022) levels in an attempt to define different ASC subsets or functional states. In general, these datasets have provided valuable resources that have led to the determination of critical ASC regulatory mechanisms.…”

Persistent virus-specific and clonally expanded antibody secreting cells respond to induced self antigen in the CNS

“…Selected antibodies were produced in PnP-mRuby hybridoma cells as previously described (Parola et al, 2019) and validated using normalized supernatant ELISAs against purified LCMV clone 13 NP, LCMV clone 13 GP, DNP-OVA, insulin (Sigma, I5500), mouse genomic DNA (Sigma, 692339), MOG (Sigma, 692339) as previously described (Neumeier et al 2021). An anti-mouse IgG-HRP (Sigma, A2554) was employed at 1:1500 and used for detection.…”

“…For single-cell sequencing, brain B cells (CD19+ CD3-CD45.1+) were FACS-sorted from STOP:GP, aCD8-GFAP:GP and GFAP:GP mice 7 days after start of TAM, and from MOG:GP mice 47 days after i.c infection. Combined single-cell transcriptome and immune repertoire sequencing was performed using the 10x Genomics Chromium Single Cell V(D)J Reagents Kit (CG000166 Rev A) according to established methods (Neumeier et al 2021). Single cells together with gel beads (10x Genomics, 1000006) were encapsulated in gel emulsion microdroplets using 4 lanes of one Chromium Single Cell A Chip (10x Genomics, 1000009) with a target loading of 13,000 cells per reaction.…”

“…Recent advancements in sequencing and microfluidic techniques have enabled comprehensive profiling of B cells and their corresponding antibody repertoires (Yermanos, Agrafiotis, et al 2021; Neumeier et al 2021; Agrafiotis et al 2021; Horns, Dekker, and Quake 2020). Current iterations of this technology provide both single-cell antibody repertoires (full-length, paired heavy and light chain antibody sequences) and single-cell transcriptomes (Csepregi et al 2020).…”

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

“…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).…”