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Review
.2021 Jun;51(6):1334-1347.
doi: 10.1002/eji.202048976. Epub 2021 Apr 1.

Measuring single-cell protein secretion in immunology: Technologies, advances, and applications

Affiliations
Review

Measuring single-cell protein secretion in immunology: Technologies, advances, and applications

Olivia T M Bucheli et al. Eur J Immunol.2021 Jun.

Abstract

The dynamics, nature, strength, and ultimately protective capabilities of an active immune response are determined by the extracellular constitution and concentration of various soluble factors. Generated effector cells secrete such mediators, including antibodies, chemo- and cytokines to achieve functionality. These secreted factors organize the individual immune cells into functional tissues, initiate, orchestrate, and regulate the immune response. Therefore, a single-cell resolved analysis of protein secretion is a valuable tool for studying the heterogeneity and functionality of immune cells. This review aims to provide a comparative overview of various methods to characterize immune reactions by measuring single-cell protein secretion. Spot-based and cytometry-based assays, such as ELISpot and flow cytometry, respectively, are well-established methods applied in basic research and clinical settings. Emerging novel technologies, such as microfluidic platforms, offer new ways to measure and exploit protein secretion in immune reactions. Further technological advances will allow the deciphering of protein secretion in immunological responses with unprecedented detail, linking secretion to functionality. Here, we summarize the development and recent advances of tools that allow the analysis of protein secretion at the single-cell level, and discuss and contrast their applications within immunology.

Keywords: Single-cell analysis; functional deep-phenotyping; microfluidic platforms; protein secretion; spot- and cytometry-based assays.

© 2021 The Authors. European Journal of Immunology published by Wiley-VCH GmbH.

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Conflict of interest statement

K.E. is a co‐inventor on patent applications based on stationary droplet arrays and may receive financial compensation via their employer's rewards to inventors’ scheme. O.T.M.B. and I.S. declare no competing interest.

Figures

Figure 1
Figure 1
Schematic representation of the different technologies enabling analysis of protein secretion at the single‐cell level, ranging from spot‐, cytometry‐, well‐, chamber‐ and droplet‐based assays, of which the last three are summarized as microfluidic methods in this review. Common features of all described technologies are spatial separation of the individual cells, and their reliance on labeled reporters, especially antibodies and variants thereof. However, all the described technologies differ in several respects, e.g., in the read‐out obtained, ease of application, multiplexing potential, cellular throughput, temporal resolution, and other characteristics that need consideration when a specific technique is used to analyze a sample. Therefore, all technologies have different applications and limitations (see also Table 1). The spot‐based assays ELISpot and FluoroSpot, based on the seeding of a heterogeneous population of cells on a protein‐binding membrane, allow for the rapid and simple determination of the frequency of secreting cells based on an antibody‐based immunoassay. The frequency of cells stained positively with a detection reagent can be assessed using cytometry‐based methods such as flow cytometry and mass cytometry. With this technology, the heterogeneous population of cells is measured temporally and spatially separated from each other, and positive events are gated and counted as a frequency of total input cells. The three microfluidic methods shown on the right are all based on the same concept of individually trapping and analyzing cells in small volumes. Here, the secreted molecule rapidly reaches a detectable concentration due to the small volume, allowing more accurate and rapid quantification of protein secretion. The microfluidic methods can be further divided based on the enclosing structure used. Well‐based assays encapsulate cells in small circular or square wells, whereas chamber‐based use elongated chambers that fit a higher volume and allow spatially separating the detection of various secreted proteins. Lastly, droplet‐based assays use two immiscible fluids to generate an emulsion to encapsulate individual cells in small volumes, and enable a surface‐free, volume‐based analysis of protein secretion in high‐throughput and with high precision.
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References

    1. Mousset, C. M. , Hobo, W. , Woestenenk, R. , Preijers, F. , Dolstra, H. and van der Waart, A. B. , Comprehensive phenotyping of t cells using flow cytometry. Cytometry A. 2019. 95: 647–654. - PubMed
    1. Dewhurst, J. A. , Lea, S. , Hardaker, E. , Dungwa, J. V. , Ravi, A. K. and Singh, D. , Characterisation of lung macrophage subpopulations in COPD patients and controls. Sci. Rep. 2017. 7: 7143. - PMC - PubMed
    1. Chokkalingam, V. , Tel, J. , Wimmers, F. , Liu, X. , Semenov, S. , Thiele, J. et al., Probing cellular heterogeneity in cytokine‐secreting immune cells using droplet‐based microfluidics. Lab Chip. 2013. 13: 4740–4744. - PubMed
    1. Ma, C. , Fan, R. , Ahmad, H. , Shi, Q. , Comin‐Anduix, B. , Chodon, T. et al., A clinical microchip for evaluation of single immune cells reveals high functional heterogeneity in phenotypically similar T cells. Nat. Med. 2011. 17: 738–743. - PMC - PubMed
    1. Lu, Y. , Xue, Q. , Eisele, M. R. , Sulistijo, E. S. , Brower, K. , Han, L. et al., Highly multiplexed profiling of single‐cell effector functions reveals deep functional heterogeneity in response to pathogenic ligands. Proc. Natl. Acad. Sci. USA. 2015. 112: E607–615. - PMC - PubMed

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