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Peer-reviewedCC BY-NC-NDCell Reports Methods

Reproducible detection of antigen-specific T cells and Tregs via standardized and automated activation-induced marker assay workflows

  • Torin Halvorson1,2,3
  • Gabrielle Boucher4
  • Daniel Yokosawa5
  • Jinqing Huang6,2,7
  • Rosa García6,2,7
  • Lieke Sanderink6,2,7
  • Lan Chen8
  • Lorraine Liu9,2,10
  • J. Ernesto Fajardo-Despaigne11
  • Jasper Halvorson12
  • Ryan R. Brinkman13,5,14
  • Suzanne Vercauteren9,2,10
5 more authors
  • Sylvie Lesage15,16,11
  • Jonathan L. Bramson8
  • John David Rioux17,18,4
  • Sabine M. Ivison6,2,7
  • Megan K. Levings19,2,7
19 affiliations
  1. 1BC Children's Hospital Research Institute, University of British Columbia, Vancouver, BC, Canada; Department of Surgery, University of British Columbia, Vancouver, BC, Canada; Department of Medicine, University of British Columbia, Vancouver, BC, Canada
  2. 2BC Children's Hospital Research Institute, University of British Columbia, Vancouver, BC, Canada
  3. 3Department of Medicine, University of British Columbia, Vancouver, BC, Canada
  4. 4Research Centre, Montreal Heart Institute, Montréal, QC, Canada
  5. 5Department of Basic and Translational Research, BC Cancer, Vancouver, BC, Canada
  6. 6BC Children's Hospital Research Institute, University of British Columbia, Vancouver, BC, Canada; Department of Surgery, University of British Columbia, Vancouver, BC, Canada
  7. 7Department of Surgery, University of British Columbia, Vancouver, BC, Canada
  8. 8Human Immune Testing Suite, McMaster University, Hamilton, ON, Canada
  9. 9BC Children's Hospital Research Institute, University of British Columbia, Vancouver, BC, Canada; Department of Pathology and Laboratory Medicine, BC Children's Hospital, Vancouver, BC, Canada
  10. 10Department of Pathology and Laboratory Medicine, BC Children's Hospital, Vancouver, BC, Canada
  11. 11Immunology-Oncology, Centre de recherche de l'Hôpital Maisonneuve-Rosemont, Montréal, QC, Canada
  12. 12Department of Software Engineering, University of Victoria, Victoria, BC, Canada
  13. 13Department of Basic and Translational Research, BC Cancer, Vancouver, BC, Canada; Faculty of Science, University of British Columbia, Vancouver, BC, Canada
  14. 14Faculty of Science, University of British Columbia, Vancouver, BC, Canada
  15. 15Immunology-Oncology, Centre de recherche de l'Hôpital Maisonneuve-Rosemont, Montréal, QC, Canada; Département de microbiologie, infectiologie et immunologie, Université de Montréal, Montréal, QC, Canada
  16. 16Département de microbiologie, infectiologie et immunologie, Université de Montréal, Montréal, QC, Canada
  17. 17Research Centre, Montreal Heart Institute, Montréal, QC, Canada; Department of Medicine, Université de Montréal, Montréal, QC, Canada
  18. 18Department of Medicine, Université de Montréal, Montréal, QC, Canada
  19. 19BC Children's Hospital Research Institute, University of British Columbia, Vancouver, BC, Canada; Department of Surgery, University of British Columbia, Vancouver, BC, Canada; School of Biomedical Engineering, University of British Columbia, Vancouver, BC, Canada. Electronic address: [email protected]
Open at sourceDOICite

Abstract

Activation-induced marker (AIM) assays are a promising tool to track antigen-specific T cells, but methodological heterogeneity between research groups hinders their clinical utility. To evaluate AIM assay reproducibility, we conducted a multi-site study of SARS-CoV-2 and cytomegalovirus AIMs. We found inherent variability in AIM assays and optimized approaches to enhance reproducibility, including a standardized workflow to minimize technical variability and a generalizable Box-Cox transformation-based statistical method to optimize calculation of AIM stimulation responses. We further standardized AIM data analysis through the development of automated flow cytometric gating software and demonstrated its superior reproducibility compared to manual analysis. We also characterized antigen-responsive regulatory T cells (Tregs) as CD134 + CD137 + cells among CD4 + FOXP3 + HELIOS + cells. The combined methodology results in a high degree of reproducibility within and between research groups, providing a comprehensive foundation from which standardized AIM assays can be implemented across diverse scientific and clinical settings.

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Cite

BibTeXRIS
BibTeX
@article{halvorson2026reproducible,
  title = {{Reproducible detection of antigen-specific T cells and Tregs via standardized and automated activation-induced marker assay workflows}},
  author = {Torin Halvorson and Gabrielle Boucher and Daniel Yokosawa and Jinqing Huang and Rosa García and Lieke Sanderink and Lan Chen and Lorraine Liu and J. Ernesto Fajardo-Despaigne and Jasper Halvorson and Ryan R. Brinkman and Suzanne Vercauteren and Sylvie Lesage and Jonathan L. Bramson and John David Rioux and Sabine M. Ivison and Megan K. Levings},
  journal = {Cell Reports Methods},
  year = {2026},
  month = may,
  doi = {10.1016/j.crmeth.2026.101458},
  url = {https://doi.org/10.1016/j.crmeth.2026.101458},
  note = {CC BY-NC-ND},
}
RIS
TY  - JOUR
TI  - Reproducible detection of antigen-specific T cells and Tregs via standardized and automated activation-induced marker assay workflows
AU  - Torin Halvorson
AU  - Gabrielle Boucher
AU  - Daniel Yokosawa
AU  - Jinqing Huang
AU  - Rosa García
AU  - Lieke Sanderink
AU  - Lan Chen
AU  - Lorraine Liu
AU  - J. Ernesto Fajardo-Despaigne
AU  - Jasper Halvorson
AU  - Ryan R. Brinkman
AU  - Suzanne Vercauteren
AU  - Sylvie Lesage
AU  - Jonathan L. Bramson
AU  - John David Rioux
AU  - Sabine M. Ivison
AU  - Megan K. Levings
PY  - 2026
DA  - 2026/05/19
JO  - Cell Reports Methods
DO  - 10.1016/j.crmeth.2026.101458
UR  - https://doi.org/10.1016/j.crmeth.2026.101458
AB  - Activation-induced marker (AIM) assays are a promising tool to track antigen-specific T cells, but methodological heterogeneity between research groups hinders their clinical utility. To evaluate AIM assay reproducibility, we conducted a multi-site study of SARS-CoV-2 and cytomegalovirus AIMs. We found inherent variability in AIM assays and optimized approaches to enhance reproducibility, including a standardized workflow to minimize technical variability and a generalizable Box-Cox transformation-based statistical method to optimize calculation of AIM stimulation responses. We further standardized AIM data analysis through the development of automated flow cytometric gating software and demonstrated its superior reproducibility compared to manual analysis. We also characterized antigen-responsive regulatory T cells (Tregs) as CD134 + CD137 + cells among CD4 + FOXP3 + HELIOS + cells. The combined methodology results in a high degree of reproducibility within and between research groups, providing a comprehensive foundation from which standardized AIM assays can be implemented across diverse scientific and clinical settings.
SN  - 2667-2375
ER  -