Optimization of a Method to Detect Autoantigen-Specific T-Cell Responses in Type 1 Diabetes

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.

Proinsulin‐specific T‐cell responses correlate with estimated c‐peptide and predict partial remission duration in type 1 diabetes

“…A key strength of this study is that we quantified PI-specific responses in PB using an optimised, fitfor-purpose assay, validated for intra-assay and inter-assay repeatability. 31 However, our study has limitations. While the at-risk and T1D participant groups comprised adults and children, the healthy control group was significantly older.…”

“…A key strength of this study is that we quantified PI‐specific responses in PB using an optimised, fit‐for‐purpose assay, validated for intra‐assay and inter‐assay repeatability 31 . However, our study has limitations.…”

“…Tetanus toxoid (AJ Vaccines, 10 LfU mL −1 ), ultra-LEAFpurified anti-human CD3 (Biolegend, San Diego USA, Clone OKT3, 0.1 µg mL −1 ) and proinsulin (PI) or HIP epitopes (GL Biochem, Shanghai, China) were included in CD4 + T-cell proliferation assays at a final concentration of 10 µM. 31 Proinsulin epitopes included the following sequences: PI 33…”

“…Peripheral blood mononuclear cells (PBMC) were freshly isolated and labelled with carboxyfluorescein diacetate succinimidyl ester (CFSE, CellTrace ™ CFSE Cell Proliferation Kit for flow cytometry, Invitrogen, Thermo Fisher Scientific, Waltham, Massachusetts, USA) as described. 31,54 CFSE-stained cells (2 × 10 6 PBMC mL −1 , 200 μL per well) were cultured for 7 days with medium alone (negative control), peptides or with positive controls, tetanus toxoid and/or anti-CD3 to obtain a cell division index (CDI). 54 Three replicates were tested for each experiment.…”

“…Since epitopes of proinsulin and hybrid islet peptides containing proinsulin sequences have been described, 1,11,12,30 we measured autoreactive CD4 + T-cell proliferation towards a panel of proinsulin peptides. We used our optimised fit-for-purpose CFSE-based T-cell proliferation assay, 31 to evaluate the frequency of positive responses to each epitope, and to determine the relationship between the level of proliferation and the clinical features of the patients with T1D. We found that CD4 + T-cell responses to proinsulin 33-63 were the most frequent of the islet peptides tested, and that the level of proinsulin 33-63 -specific CD4 + T-cell proliferation was positively associated with β-cell function and predicted survival in remission, suggesting that proinsulin 33-63 -specific CD4 + T-cell proliferation is a novel immune biomarker of predicted β-cell function.…”