Current approaches to measuring human islet-antigen specific T cell function in type 1 diabetes

Abstract

Type 1 diabetes (T1D) is an autoimmune disease caused by the T cell-mediated destruction of the pancreatic insulin-producing beta cells. Currently there are no widely accepted and standardized assays available to analyse the function of autoreactive T cells involved in T1D. The development of such an assay would greatly aid efforts to understand the pathogenesis of T1D and is also urgently required to guide the development of antigen-based therapies intended to prevent, or cure, T1D. Here we describe some of the assays used currently to detect autoreactive T cells in human blood and review critically their strengths and weaknesses. The challenges and future prospects for the T cell assays are discussed.

Fig. 1

Examples of wells from an enzyme-linked immunospot (ELISPOT) assay. Spots indicate cells, or groups of cells, that have produced interferon-γ after stimulation with either: tetanus toxoid, a recall antigen used as a positive control, an islet antigen-derived peptide or dimethylsulphoxide (peptide solvent), which serves as a negative control. The number of spots in each well are counted to determine the frequency of responding cells. A response is considered to be positive when it is significantly greater than the peptide solvent control (cut-off determined for each ELISPOT format).

Fig. 2

Flow cytometry plots of 5,6-carboxylfluorescein diacetate succinimidyl ester (CFSE) and CD4-labelled human peripheral blood mononuclear cells after 7 days' culture with either: no antigen, recombinant proinsulin or tetanus toxoid. The number of cells that proliferated (CFSE^dim events, in the top left box) in each treatment is determined. The magnitude of the response is expressed as a ratio of the number of CFSE^dim cells with antigen: CFSE^dim cells without antigen, per 5000 CFSE^bright, CD4⁺ cells. Cells shown are gated to exclude dead, propidium iodide-positive, cells.