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Analysis of the repertoire of insulin-reactive CD8+ T cells

Pearson, James 2014. Analysis of the repertoire of insulin-reactive CD8+ T cells. PhD Thesis, Cardiff University.
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Abstract

Proinsulin is an auto-antigen in type 1 diabetes in the Non-obese Diabetic (NOD) mouse. The CD8+ T cell clone, G9C8, which utilises a T cell receptor (TCR) comprising TRAV8-1*01TRAJ9 and TRBV19*01TRBD1TRBJ2-3, recognises insulin B15-23 presented by H-2Kd, escapes negative selection and rapidly causes diabetes upon adoptive transfer into immunocompromised NODscid mice. To understand how these insulin B15-23 reactive CD8+ T cells develop, G9C8-derived single TCR TRAV8-1*01TRAJ9 chain transgenic NOD mice were generated. These mice were bred with different proinsulin-expressing NOD mice to generate proinsulin1 deficient, proinsulin2 deficient, proinsulin2 over-expressing and proinsulin1 and 2 deficient mice with a mutated proinsulin transgene, preventing G9C8 antigen recognition. Although proinsulin-specific CD8+ TCR repertoire changes in TRBV19 were seen in these mice, the proportion of insulin B15-23 reactive CD8+ T cells was unaffected by proinsulin expression. Interestingly, TCR clonotyping of these insulin B15-23 reactive T cells revealed minimal shared sequences between the strains, with mice exhibiting individual clonal expansions. However, by isolating TRBV19-expressing insulin B15-23-responsive T cells, shared sequences across the different proinsulin-expressing mice were identified, with a requirement of TRBJ2-3 for insulin recognition (used by the original G9C8 T cell clone). Furthermore, male proinsulin2 deficient TRAV8-1*01TRAJ9 mice developed diabetes, with a higher incidence seen upon antibiotic administration. Although the TCR repertoire was unaffected by antibiotics, the gut microbial diversity was greatly reduced in all the mice with age, independent of antibiotic use, with firmicutes bacteria comprising 90-97% of the microbiota. In summary, proinsulin expression and antibiotics modify diabetes susceptibility; however, insulin B15-23 reactive T cells develop independently of antigen expression and are not directly affected by antibiotics. This data suggests that iii non-antigen dependent mechanisms exist in controlling the development of auto-reactive T cells, but T cell activation and pathogenicity is influenced indirectly by a combination of antigen expression and gut microbiota.

Item Type: Thesis (PhD)
Status: Unpublished
Schools: Medicine
Last Modified: 01 Jun 2020 10:35
URI: http://orca-mwe.cf.ac.uk/id/eprint/68395

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