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Title: Processing and presentation of hybrid polypeptides in type 1 diabetes
Author: Harbige, James
ISNI:       0000 0004 9357 3157
Awarding Body: King's College London
Current Institution: King's College London (University of London)
Date of Award: 2020
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Alleles at the HLA-DRB1, -DQA1 and -DQB1 loci confer the greatest genetic predisposition to type 1 diabetes (T1D). However, details linking HLA-DQ-mediated predisposition and T cell autoreactivity in T1D are scant. An attractive explanation lies in the discovery of a novel form of modified peptide generated by covalent crosslinking of proinsulin peptides to other pancreatic β-cell peptides, termed hybrid insulin peptides (HIPs). To date, HIPs have been identified in b-cells by mass spectrometry (MS) and HIP-reactive CD4⁺ T cells detected in the islets and peripheral blood mononuclear cells (PBMCs) of patients with T1D. I hypothesise that the hybrid peptides that have been reported derive from antigen-presenting cell (APC)- dependent processing of an immunogenic hybrid polypeptide. To address this, I optimised an antigen delivery system by conjugating synthetic peptides to carrier proteins, permitting peptide targeting to the APC surface and internalisation. Pulsing of Epstein-Barr virus-transformed B (EBV B) cell lines homozygous for high-risk HLA alleles with a pokeweed mitogen (PWM)- peptide conjugate results in HLA-restricted presentation of a GAD65 epitope and T cell activation. This effect was not observed using peptide-conjugated anti-DEC205 antibody despite evidence of efficient internalisation. Large-scale EBV B cell cultures were pulsed with PWM conjugated to a C-peptide:WE14 hybrid polypeptide and HLA-DR and -DQ peptide complexes isolated for immunoaffinity purification. MS analysis of eluted peptides from HLADR4, -DR3, -DQ8 and -DQ2 molecules did not identify hybrid epitopes although evidence of class II processing was observed. Using FluoroSpot to investigate immunogenicity in T1D patient-derived PBMCs, I present data to suggest that immunological processing of hybrid polypeptides to generate a cognate hybrid epitope is limited. Together these findings indicate hybrid epitopes may not be generated through conventional processing and presentation of a long hybrid polypeptide. This project provides novel insight into the mechanism(s) of hybrid peptide generation, highlighting unconventional antigen presentation pathways in T1D.
Supervisor: Peakman, Mark ; Tree, Timothy Ian Martin ; Mishto, Michele Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available