|dc.description.abstract||Uncovering the primary antigen targets in type 1 diabetes (T1D) is essential to our understanding of disease pathophysiology. Despite the clear role of CD4+ T cells in orchestrating the immune destruction of the pancreatic cells, what they are targeting in human T1D has remained poorly defined. Most knowledge of in vivo T-cell responses in T1D derives from studies in mouse models, and translating results to humans has been limited to analysis of peripheral blood. However, only 3% of the total T cells in the body reside in the peripheral blood. Prior work at this institute by Mannering and colleagues on islet-infiltrating CD4+ T cells in humans, complemented by other similar studies, provided insight into the resident T-cell population of the target organ in subjects with T1D. These studies have concurred that a cleavage product of proinsulin, C-peptide, is a target antigen of islet-infiltrating CD4+ T cells. Because these studies were done in just a handful of deceased organ donors with T1D, they led to the question how relevant is C-peptide as an autoantigen in T1D more generally?
Given the pancreas is not routinely accessible, to address this question, evidence of C-peptide as a target of CD4+ T cells was sought from the peripheral blood in subjects with T1D. The main obstacle to assessing T-cell targets in the peripheral blood is the lack of a sufficiently sensitive and reproducible T-cell assay. In Chapter 3, the CFSE-based proliferation assay was optimised for detection of C-peptide-specific CD4+ T-cell responses. The CFSE-based proliferation assay was demonstrated to have comparable reproducibility as compared to other currently available T-cell assays, and greater sensitivity than the commonly used ELISpot assay. In Chapter 4, using the CFSE-based proliferation assay, >60% of people with recent-onset T1D were shown to have a detectable peripheral C-peptide-specific CD4+ T-cell response. The response was disease specific because few control subjects were positive. Analysis of cloned C-peptide-specific CD4+ T cells revealed they were restricted by HLA alleles strongly associated with T1D risk, namely HLA-DQ8, -DQ2, -DQ8trans, -DQ2trans and HLA-DR4. This added further support to the notion that they were pathogenic. In Chapter 5, the hypothesis that autoantibodies to C-peptide may be detected in the serum of people with T1D, was tested. Using solid-phase ELISA, it was found, unlike C-peptide-specific CD4+ T cells, C-peptide autoantibodies are not detectable in the serum of subjects with T1D in a disease-specific manner.
Together, these findings indicate that proinsulin C-peptide is commonly a target of autoreactive CD4+ T cells in newly-diagnosed T1D. Hence, C-peptide is a promising candidate for biomarker development and antigen-specific immunotherapy.||en_US