Poster Presentation The 13th International Congress of the Immunology of Diabetes Society 2013

Analysis of the repertoire of insulin-reactive CD8+ T cells (#106)

James A Pearson 1 , Terri C Thayer 1 , Evy DeLeenheer 1 , Joanne Davies 1 , James E McLaren 1 , Kristin Ladell 1 , Kelly Miners 1 , David A Price 1 , F. Susan Wong 1
  1. Cardiff University, Cardiff, UK, United Kingdom

Insulin is a major autoantigen in type 1 diabetes. The insulin-reactive CD8+ T cell clone, G9C8, isolated from the pancreatic islets of a non-obese diabetic (NOD) mouse, recognizes the insulin B chain epitope 15-23 with low avidity and utilizes T cell receptor (TCR) chains TRAV8-1*01TRAJ9*01 and TRBV19*01TRBJ2-3*01. G9C8 cells rapidly induce diabetes in immunocompromised NOD.scid mice after adoptive transfer.

We generated single chain TCR transgenic mice using TRAV8-1*01TRAJ9*01 from the G9C8 clone crossed with TCRCα-/- mice (designated A22Cα-/- mice) to investigate selection of endogenous TRBV chains in the contexts of insulin2 over-expression (A22Cα-/-Ins2tg), insulin2 deficiency (A22Cα-/-Ins2-/-), and replacement of native insulin1 and insulin2 with a mutated form of insulin (A22Cα-/-Y16Atg), both within the thymus and the periphery.

Flow cytometric analysis revealed a proportional decrease in CD8+ T cells in mice with insulin2 deficiency compared to mice with normal insulin expression. Insulin2-specific effects on TCRVβ19, the same chain utilized by G9C8, were apparent in the total CD8+ T cell population. In addition, TCRVβ16 and TCRVβ29 were proportionally increased when insulin2 was deficient, while insulin2 over-expression increased the proportion of TCRVβ15-expressing CD8+ T cells.

Using peptide-MHC tetramers specific for insulin B chain 15-23, we identified insulin-reactive CD8+ T cells in mice expressing different levels of insulin2. We found that more tetramer+ CD8+ T cells were present when insulin2 was either deficient or over-expressed. Molecular analysis of TRB gene rearrangements within these insulin-specific CD8+ T cell populations revealed comparatively polyclonal repertoires constrained by preferential usage of TCRVβ19 regardless of insulin2 expression levels.

These findings suggest that insulin2 expression modulates selection of the insulin-reactive CD8+ T cell repertoire and potentially provides new insights into the process of diabetogenesis.