Time-resolved microwell cell-pairing array reveals multiple T cell activation profiles
Time-resolved microwell cell-pairing array reveals multiple T cell activation profiles
The differences in behaviour between individual cells in a large population are often important, yet are masked in bulk analyses where only average parameters are measured. One unresolved question in the field of immunology is the extent to which important immunological phenomena such as immunodominance to cancer antigens correlates with the average activity of a population of antigen-specific T lymphocytes, or with the activity of individual "outlier" cells. Despite progress in single cell technologies, few platforms are available that can deliver time-resolved, functional analysis at single cell resolution, for these investigations. We have developed an accessible high-throughput platform to measure single T cell signalling in real time following time-controlled stimulation by live antigen presenting cells. The cell-trap array consists of thousands of individual microwells cast in an agarose block, which is biocompatible and permeable to nutrients. Single T cells are isolated in wells via passive sedimentation and size exclusion, achieving up to 90% occupancy. The device enables simultaneous activation of thousands of single CD8+ cells. Stimulation with soluble reagents (ionomycin, anti-CD3 antibodies) or antigen presenting cells leads to changes in intracellular calcium concentrations which were measured using calcium-chelating fluorophore dyes. The platform was used to demonstrate a range of activation profiles among individual cells of a cloned, antigen specific CD8+ T cell hybridoma in response to both nonspecific stimuli and specific, physiologically relevant antigen stimulation. The presence of two different activation profiles was demonstrated, together with rare outlier behaviour among cells that are essentially clonal.
3772-3783
Desalvo, Anna
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Bateman, Faith
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James, Edward
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Morgan, Hywel
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Elliott, Tim
16670fa8-c2f9-477a-91df-7c9e5b453e0e
21 October 2020
Desalvo, Anna
45bbaca1-d004-4a5c-a9cd-3c58f5e2ee0d
Bateman, Faith
8d8813b8-25ee-4cd3-a84b-2ecfbad1d448
James, Edward
7dc1afb7-d326-4050-89fc-1f4e2a1a19a4
Morgan, Hywel
de00d59f-a5a2-48c4-a99a-1d5dd7854174
Elliott, Tim
16670fa8-c2f9-477a-91df-7c9e5b453e0e
Desalvo, Anna, Bateman, Faith, James, Edward, Morgan, Hywel and Elliott, Tim
(2020)
Time-resolved microwell cell-pairing array reveals multiple T cell activation profiles.
Lab on a Chip, 20 (20), .
(doi:10.1039/d0lc00628a).
Abstract
The differences in behaviour between individual cells in a large population are often important, yet are masked in bulk analyses where only average parameters are measured. One unresolved question in the field of immunology is the extent to which important immunological phenomena such as immunodominance to cancer antigens correlates with the average activity of a population of antigen-specific T lymphocytes, or with the activity of individual "outlier" cells. Despite progress in single cell technologies, few platforms are available that can deliver time-resolved, functional analysis at single cell resolution, for these investigations. We have developed an accessible high-throughput platform to measure single T cell signalling in real time following time-controlled stimulation by live antigen presenting cells. The cell-trap array consists of thousands of individual microwells cast in an agarose block, which is biocompatible and permeable to nutrients. Single T cells are isolated in wells via passive sedimentation and size exclusion, achieving up to 90% occupancy. The device enables simultaneous activation of thousands of single CD8+ cells. Stimulation with soluble reagents (ionomycin, anti-CD3 antibodies) or antigen presenting cells leads to changes in intracellular calcium concentrations which were measured using calcium-chelating fluorophore dyes. The platform was used to demonstrate a range of activation profiles among individual cells of a cloned, antigen specific CD8+ T cell hybridoma in response to both nonspecific stimuli and specific, physiologically relevant antigen stimulation. The presence of two different activation profiles was demonstrated, together with rare outlier behaviour among cells that are essentially clonal.
Text
d0lc00628a
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More information
Accepted/In Press date: 28 July 2020
e-pub ahead of print date: 31 August 2020
Published date: 21 October 2020
Additional Information:
Funding Information:
The authors wish to thank Dr Fabrizio Siracusa for technical help. This work was funded by CRUK Programme Grant A28279, and generous donations from Dr Norman Godinho.
Publisher Copyright:
© The Royal Society of Chemistry.
Identifiers
Local EPrints ID: 444824
URI: http://eprints.soton.ac.uk/id/eprint/444824
ISSN: 1473-0197
PURE UUID: 6acc25b8-8bbb-4238-94bb-aa711f15d1d0
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Date deposited: 05 Nov 2020 17:34
Last modified: 17 Mar 2024 03:06
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Author:
Anna Desalvo
Author:
Faith Bateman
Author:
Hywel Morgan
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