AUTHOR=Musthaffa Yassmin , Nel Hendrik J. , Ramnoruth Nishta , Patel Swati , Hamilton-Williams Emma E. , Harris Mark , Thomas Ranjeny TITLE=Optimization of a Method to Detect Autoantigen-Specific T-Cell Responses in Type 1 Diabetes JOURNAL=Frontiers in Immunology VOLUME=11 YEAR=2020 URL=https://www.frontiersin.org/journals/immunology/articles/10.3389/fimmu.2020.587469 DOI=10.3389/fimmu.2020.587469 ISSN=1664-3224 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.