SEC-seq: association of molecular signatures with antibody secretion in thousands of single human plasma cells

Abstract

The secreted products of cells drive many functions in vivo; however, methods to link this functional information to surface markers and transcriptomes have been lacking. By accumulating secretions close to secreting cells held within cavity-containing hydrogel nanovials, we demonstrate workflows to analyze the amount of IgG secreted from single human B cells and link this information to surface markers and transcriptomes from the same cells. Measurements using flow cytometry and imaging flow cytometry corroborate the association between IgG secretion and CD38/CD138. By using oligonucleotide-labeled antibodies we find that upregulation of pathways for protein localization to the endoplasmic reticulum and mitochondrial oxidative phosphorylation are most associated with high IgG secretion, and uncover surrogate plasma cell surface markers (e.g., CD59) defined by the ability to secrete IgG. Altogether, this method links quantity of secretion with single-cell sequencing (SEC-seq) and enables researchers to fully explore the links between genome and function, laying the foundation for discoveries in immunology, stem cell biology, and beyond.

Single-Cell Secretion Analysis via Microfluidic Cell Membrane Immunosorbent Assay for Immune Profiling

“…23 For example, immune probes with dual antibodies were anchored to cell-surface proteins, such as CD45, to capture secretions for high-throughput single-cell secretion screening through flow cytometry, 24−27 while dual antibodies proved difficult to extend to other secreted cells, such as plasma B cells, that do not highly express CD45. 28 Recently, droplet-based phenotyping sequencing technology was developed to capture secreted proteins on cell surfaces, which were then labeled with gene barcodes for extensive analysis of single-cell secretion over time. 29 However, massive single-cell sequencing and de-barcoding were time-consuming and costly for diagnosis.…”

“…To extend the capacity of droplet screening, cell membrane-anchored technology, which captures targeted secretions on the cell surface through antibodies or aptamers, has been investigated . For example, immune probes with dual antibodies were anchored to cell-surface proteins, such as CD45, to capture secretions for high-throughput single-cell secretion screening through flow cytometry, − while dual antibodies proved difficult to extend to other secreted cells, such as plasma B cells, that do not highly express CD45 . Recently, droplet-based phenotyping sequencing technology was developed to capture secreted proteins on cell surfaces, which were then labeled with gene barcodes for extensive analysis of single-cell secretion over time .…”

A Functional Hydrogel Bead-Based High-Throughput Screening System for Mammalian Cells with Enhanced Secretion of Therapeutic Antibodies

“…15 In other studies, polyethylene glycol (PEG)-based hydrogel nanovials have been generated to analyze and sort single human plasma cells (human primary B cells, hybridoma cells, and Raji cells) based on their immunoglobulin (IgG) secretion. 16 PEG-based HB nanovials have also been used to select single viable cells of CHO, HEK 293T, and hybridoma cells based on their secreted products as well. 17 Koo and colleagues employed a combination of PEG and gelatin-based hydrogel nanovials to sort individual T cells based on cytokine secretion.…”

“…Gel beads have also been applied to coencapsulated yeast cells with murine IL-3 reporter cells to enrich the production of IL-3 . In other studies, polyethylene glycol (PEG)-based hydrogel nanovials have been generated to analyze and sort single human plasma cells (human primary B cells, hybridoma cells, and Raji cells) based on their immunoglobulin (IgG) secretion . PEG-based HB nanovials have also been used to select single viable cells of CHO, HEK 293T, and hybridoma cells based on their secreted products as well .…”

“…However, each method has its own limitations in throughput, sensitivity, or accuracy . To address these challenges, alternative approaches involving droplet and HBs microfluidic technology have been explored. ,− A drawback of most of these systems was that cells cannot be recovered and recultured after screening and selection processes.…”