Detection of Autoreactive T Cells in Type 1 Diabetes Using Coded Autoantigens and an Immunoglobulin‐Free Cytokine ELISPOT Assay: Report from the Fourth Immunology of Diabetes Society T Cell Workshop

Abstract

The "gold standard" for evaluation of immunoassays is blinded testing, using coded samples and antigens. Although assays for autoreactive T cells are no exception to this rule, it is nonetheless rarely applied in this context. To facilitate such investigations, we coded a panel of 10 peptides representing T cell epitopes reported to be of relevance to islet autoimmunity. These were deployed in a novel cytokine ELISPOT assay, in which the use of immunoglobulin-free medium reduces background reactivity and thus potentially enhances the specificity and sensitivity of detection of autoreactive T cells. Significant IFN-gamma production against GAD65 (554-575), insulin (B9-23), and IA-2 (709-736) peptides were observed in type 1 diabetic patients, whereas no significant changes from background were detected in healthy controls. These results confirm the utility of the blinded performance of T cell assays as the most robust platform for assessing technologies to T cell autoreactivity.

Do MHCII-Presented Neoantigens Drive Type 1 Diabetes and Other Autoimmune Diseases?

“…this idea. On the other hand, anti-insulin autoantibodies are nearly universal in human T1D (Zhang et al 2008) and some data suggest that the B:9-23 peptide may contain a CD4 þ T-cell epitope (Nagata et al 2004;Higashide et al 2006). Alternately, HLA-DR4 presentation of an insulin a-chain peptide has been reported (Kent et al 2005;Mannering et al 2005), but the details of the MHCII-peptide structure are not known.…”

Combination Therapy Using IL-2/IL-2 Monoclonal Antibody Complexes, Rapamycin, and Islet Autoantigen Peptides Increases Regulatory T Cell Frequency and Protects against Spontaneous and Induced Type 1 Diabetes in Nonobese Diabetic Mice

“…T cells reactive to islet Ags such as insulin, the 65-kDa isoform of glutamic acid decarboxylase (GAD65), and islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP) play pivotal pathogenic roles during the development of T1D in NOD mice (2,3) and in humans (4)(5)(6)(7)(8).…”

Methyldopa blocks MHC class II binding to disease-specific antigens in autoimmune diabetes

“…ELISPOT assays were conducted as previously described using human IFN-γ ELISPOT kits (U-CyTech Biosciences) (61). Briefly, cryopreserved PBMCs were thawed, and 10 6 cells were cultured in 250 μl serum-free AIM-V Medium (Invitrogen, Thermo Fisher Scientific) with 10 μg/ml peptide.…”