Journal
FRONTIERS IN IMMUNOLOGY
Volume 11, Issue -, Pages -Publisher
FRONTIERS MEDIA SA
DOI: 10.3389/fimmu.2020.587469
Keywords
type 1 diabetes; proliferation; proinsulin; CD4+T-cells; 5; 6-carboxylfluorescein diacetate succinimidyl ester
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Funding
- Leona M. and Harry B. Helmsley Charitable Trust
- Juvenile Diabetes Research Foundation Australia
- Arthritis Queensland
- NHMRC Research Fellowship
- University of Queensland Post-Graduate Award
- Pfizer Australasian Paediatric Endocrine Care Grant
- Butta Clinician Researcher Bursary
- Children's Health Foundation PhD Top up Scholarship
- Royal Australasian College of Physicians Foundation Development
- Research Entry Scholarship
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The development of tolerizing therapies aiming to inactivate autoreactive effector T-cells is a promising therapeutic approach to control undesired autoimmune responses in human diseases such as Type 1 Diabetes (T1D). A critical issue is a lack of sensitive and reproducible methods to analyze antigen-specific T-cell responses, despite various attempts. We refined a proliferation assay using the fluorescent dye 5,6-carboxylfluorescein diacetate succinimidyl ester (CFSE) to detect responding T-cells, highlighting the fundamental issues to be taken into consideration to monitor antigen-specific responses in patients with T1D. The critical elements that maximize detection of antigen-specific responses in T1D are reduction of blood storage time, standardization of gating parameters, titration of CFSE concentration, selecting the optimal CFSE staining duration and the duration of T-cell stimulation, and freezing in medium containing human serum. Optimization of these elements enables robust, reproducible application to longitudinal cohort studies or clinical trial samples in which antigen-specific T-cell responses are relevant, and adaptation to other autoimmune diseases.
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