Abstract
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.
Highlights
Type 1 Diabetes (T1D) is a chronic, incurable autoimmune disorder in which insulin-producing b-cells are selectively destroyed by islet-infiltrating T-cells [1, 2]
To optimize a carboxyfluorescein diacetate succinimidyl ester (CFSE)-based T-cell proliferation assay for detection of PI33-63 specific CD4+ T cells, CFSE-labeled freshly isolated PBMC from subjects with recent-onset T1D were stimulated for 7 days with or without PI33-63 or anti-CD3 as a positive control
After setting a gate around the CFSE(undivided) population to include all undivided cells, parameters for the borders of the CFSE(divided) gate were calculated mathematically based on the Mean Fluorescence Intensity (MFI) of the CFSE(undivided) population
Summary
Type 1 Diabetes (T1D) is a chronic, incurable autoimmune disorder in which insulin-producing b-cells are selectively destroyed by islet-infiltrating T-cells [1, 2]. The diagnosis of T1D is currently made at the onset of clinical symptoms. A long prodrome of autoimmune T and B cell activation leading to immune-mediated b-cell destruction precedes the onset of clinical diabetes. Human islet-infiltrating CD4+ T-cell clones were shown to recognize epitopes from proinsulin-derived C-peptide, as well as neoantigens such as hybrid insulin peptides (HIPs) [1, 5, 6]. There is currently an unmet need for a robust assay capable of tracking islet antigen-specific autoreactive
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