SEC-seq: Association of molecular signatures with antibody secretion in thousands of single human plasma cells
Rene Yu-Hong Cheng, Joseph de Rutte, Cade Ellis K. Ito, Andee R. Ott, Lucienne Bosler, Wei-Ying Kuo, Jesse Liang, Brian E Hall, David J Rawlings, Dino Di Carlo, Richard G. James
Nature Communication, June 2023
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.
Workflow to link IgG secretion to surface markers and transcriptomes at the single-cell level. Human B cells are isolated from donors and expanded in a differentiation cocktail to promote differentiation into antibody-secreting cells. Cells are then loaded into a slurry of nanovials in a tube where they bind to antibodies on the nanovials for cell surface markers (CD27 or CD45). The loaded nanovials are incubated to accumulate secreted IgG on the surface via anti-IgG capture antibodies. Nanovials are then stained with fluorescent or oligo-barcoded anti-IgG, as well as viability dyes and other surface marker stains. Stained nanovials and associated cells are analyzed by flow cytometry (LSR II flow cytometer), imaging flow cytometry (ImageStream), or sorted (Nanocellect WOLF) for single-cell transcriptomics using the 10X Chromium system. Data linking IgG secretion with surface markers/functional dyes and transcriptomes at the single-cell level is acquired and analyzed.
Topics
Antibody Discovery, SEC-Seq
Cell Types
Human B Cells, Hybridomas
Instruments
Amnis ImageStream, BD LSR, WOLF Cell Sorter
10X Chromium
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