Time-resolved assessment of single-cell protein secretion by sequencing

Wu, Tongjin; Womersley, Howard John; Wang, Jiehao; Scolnick, Jonathan; Cheow, Lih Feng

doi:10.1101/2021.12.19.473400

Cited by 2 publications

(2 citation statements)

References 58 publications

(91 reference statements)

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“…The second limitation is that during this limited assay duration, the assay more prominently identifies cells with early and high functional capabilities (e.g., cells strongly secreting IFNγ within 1-2 h of antigen encounter). Notably, sixteen-hour ICS assays were found to identify more functionally responsive antigen specific T cells than 0, 1 or 6 h assays [48].…”

Section: Discussionmentioning

confidence: 97%

A Highly Sensitive Flow Cytometric Approach to Detect Rare Antigen-Specific T Cells: Development and Comparison to Standard Monitoring Tools

Levinsky

Brenner

Yalon

et al. 2023

Cancers

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Personalized vaccines against patient-unique tumor-associated antigens represent a promising new approach for cancer immunotherapy. Vaccine efficacy is assessed by quantification of changes in the frequency and/or the activity of antigen-specific T cells. Enzyme-linked immunosorbent spot (ELISpot) and flow cytometry (FCM) are methodologies frequently used for assessing vaccine efficacy. We tested these methodologies and found that both ELISpot and standard FCM [monitoring CD3/CD4/CD8/IFNγ/Viability+CD14+CD19 (dump)] demonstrate background IFNγ secretion, which, in many cases, was higher than the antigen-specific signal measured by the respective methodology (frequently ranging around 0.05–0.2%). To detect such weak T-cell responses, we developed an FCM panel that included two early activation markers, 4-1BB (CD137) and CD40L (CD154), in addition to the above-cited markers. These two activation markers have a close to zero background expression and are rapidly upregulated following antigen-specific activation. They enabled the quantification of rare T cells responding to antigens within the assay well. Background IFNγ-positive CD4 T cell frequencies decreased to 0.019% ± 0.028% and CD8 T cells to 0.009% ± 0.013%, which are 19 and 13 times lower, respectively, than without the use of these markers. The presented methodology enables highly sensitive monitoring of T-cell responses to tumor-associated antigens in the very low, but clinically relevant, frequencies.

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Section: Discussionmentioning

confidence: 97%

A Highly Sensitive Flow Cytometric Approach to Detect Rare Antigen-Specific T Cells: Development and Comparison to Standard Monitoring Tools

Levinsky

Brenner

Yalon

et al. 2023

Cancers

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“…Because of the laborious nature of the protocol only a few dozens of cells could be analyzed. Concurrently with the development of SEC-seq, another secretion and transcriptome method called Time Resolved Assessment of single cell Protein secretion (TRAP-seq) 15 was published that similarly uses an oligo-barcoded antibody to detect secretions in a typical scRNAseq workflow. This method uses a custom hybrid bi-specific antibody, anchoring the primary secretion capture antibody to the cell using covalent linkage to a second antibody targeting a highly-expressed surface protein on the cell of interest.…”

Section: Comparison With Other Methodsmentioning

confidence: 99%

Linking single-cell transcriptomes with secretion using secretion-encoded single-cell sequencing (SEC-seq)

Langerman,

Baghdasarian,

Cheng

et al. 2024

Preprint

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Cells secrete numerous proteins and other biomolecules into their surroundings to achieve critical functions - from communicating with other cells to blocking the activity of pathogens. Secretion of cytokines, growth factors, extracellular vesicles, and even recombinant biologic drugs defines the therapeutic potency of many cell therapies. However, gene expression states that drive specific secretory phenotypes are largely unknown. We provide a protocol that enables linking the Secretion amount of a target protein EnCoded (SEC) by thousands of single cells with transcriptional sequencing (seq). SEC-seq leverages microscale hydrogel particles called Nanovials to isolate cells and capture their secretions in close proximity, oligonucleotide-labeled antibodies to tag secretions on Nanovials, and flow cytometry and single-cell RNA-sequencing platforms for readout. Cells on Nanovials can be sorted based on viability, secretion amount, or other surface markers without fixation or permeabilization, and cell and secretion-containing Nanovials are directly introduced into microfluidic droplets-in-oil emulsions for single-cell barcoding of cell transcriptomes and secretions. We have used SEC-seq to link T-cell receptor sequences to the relative amount of associated cytokine secretions, surface marker gene expression with a highly secreting and potential regenerative population of mesenchymal stromal cells, and the transcriptome with high immunoglobulin secretion from plasma cells. Nanovial modification and cell loading takes under 4 hours, and once the desired incubation time is over, staining, cell sorting, and emulsion generation for scRNA-seq can also be completed in under 4 hours. By linking gene expression and secretory strength, SEC-seq can expand our understanding of cell secretion, how it is regulated, and how it can be engineered to make better therapies.

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Time-resolved assessment of single-cell protein secretion by sequencing

Cited by 2 publications

References 58 publications

A Highly Sensitive Flow Cytometric Approach to Detect Rare Antigen-Specific T Cells: Development and Comparison to Standard Monitoring Tools

A Highly Sensitive Flow Cytometric Approach to Detect Rare Antigen-Specific T Cells: Development and Comparison to Standard Monitoring Tools

Linking single-cell transcriptomes with secretion using secretion-encoded single-cell sequencing (SEC-seq)

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