Single-Cell Technologies for the Study of Antibody-Secreting Cells

Abstract

Antibody-secreting cells (ASC), plasmablasts and plasma cells, are terminally differentiated B cells responsible for large-scale production and secretion of antibodies. ASC are derived from activated B cells, which may differentiate extrafollicularly or form germinal center (GC) reactions within secondary lymphoid organs. ASC therefore consist of short-lived, poorly matured plasmablasts that generally secrete lower-affinity antibodies, or long-lived, highly matured plasma cells that generally secrete higher-affinity antibodies. The ASC population is responsible for producing an immediate humoral B cell response, the polyclonal antibody repertoire, as well as in parallel building effective humoral memory and immunity, or potentially driving pathology in the case of autoimmunity. ASC are phenotypically and transcriptionally distinct from other B cells and further distinguishable by morphology, varied lifespans, and anatomical localization. Single cell analyses are required to interrogate the functional and transcriptional diversity of ASC and their secreted antibody repertoire and understand the contribution of individual ASC responses to the polyclonal humoral response. Here we summarize the current and emerging functional and molecular techniques for high-throughput characterization of ASC with single cell resolution, including flow and mass cytometry, spot-based and microfluidic-based assays, focusing on functional approaches of the secreted antibodies: specificity, affinity, and secretion rate.

Keywords: B cells; antibodies; antibody secreting cell; droplet microfluidics; functional bioassay; high-throuput technique; plasma cell (PC).

Figure 1

Overview of common techniques for single ASC characterization. Antibody secreting cells (ASC) may be characterized functionally (red region) or molecularly (green region). Functional methods include microfluidic approaches (top), including stationary and flowed droplet-based systems and microwell systems (far right: Berkeley Lights Beacon setup), cytometry-based approaches, and spot-based assays. The red streak represents a laser beam; the yellow and dark red bulb shapes indicate a positive microwell). Molecular methods (bottom) may assess the V_H (red band) and V_L (blue band) antibody-encoding mRNA transcripts or non-antibody related mRNA transcripts (green bands). Molecular approaches commonly amplify VH chains only (e.g., VH-seq), V_H and V_L chains with the addition of barcodes (grey bands) or linkage (e.g., BCR-seq), or all mRNA within the cell (e.g., scRNAseq).

Figure 2

Comparison of DropMap vs a classical pipeline to define affinity repertoires. The affinity repertoire of ASC towards a given antigen is an important metric for the quality of the ASC response. ASC are commonly isolated from the spleen, bone marrow, or blood of donor/patients or experimental animals. The DropMap assay (top) offers a platform that within 1 hour can return the affinity repertoire in a single assay, as well as the IgG secretion rate and frequency of ASC according to direct ex vivo measurement (refer to main text for details); this approach is limited by requiring ASC to secrete antibodies at the time of data acquisition, data can only be acquired once, and afterwards cells of interest are lost. An alternative strategy (bottom) to yield a similar affinity repertoire first requires cell isolation by FACS, followed by V_H and V_L targeted RT-PCR (& BCR-seq if required) and cloning into an expression vector, transfection of the vector into an expression system and cell culture, purification and finally assessment of the recombinant expressed antibody by BLI or SPR; this pipeline requires 1½ -2 weeks to complete, is significantly more complex and costly, and can only assess V_H-V_L pairs that could be successfully amplified, cloned and expressed as recombinant antibodies.