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

TCR repertoire and antigen-specificity of human islet-infiltrating CD8+ T cells (#105)

Vimukthi Pathiraja 1 , Janine Kuehlich 1 , Peter Campbell 1 , Bala Murthy 1 , Helen Thomas 1 , Tom Kay 1 , Stuart Mannering 1
  1. St. Vincents Institute of Medical Research, Melbourne, Vic, Australia

Type 1 diabetes (T1D) is caused by the  T-cell mediated destruction of the pancreatic insulin-producing beta cells. Some progress has been made in identifying the epitopes seen by CD8+ T cells implicated in the pathogenesis of human T1D. However, these studies have been hindered by technical problems associated with detecting rare, autoantigen-specific, CD8+ T cells in the peripheral blood. To study human T-cell responses at the site of beta-cell destruction we cloned CD8+ T cells from the residual pancreatic islets of a deceased organ donor who suffered from T1D for three years.

We isolated 27 CD8+ T-cell clones from this donor. Analysis of the TRAV and TRBV genes showed no evidence of strong TCR repertoire skewing. However, some TRAV and TRBV genes were expresses by more than one clone.  Of the 27 clones studied: 4 (14.8%) expressed  TRAV 19*01, three expressed (11.1%) TRAV 21*01/02 and two (7.4%) expressed TRAV 6*03. Analysis of the TRBV from these 27 clones revealed that three (11.1%) expressed TRBV 5-6*01 and another three expressed TRBV 28*01 or 30*01-05. None of the clones analyzed expressed identical TCR genes.

To start investigating the clones’ antigen specificity we selected 12 published HLA A2 restricted epitopes derived from: preproinsulin (6x), GAD (2x), IA-2 (1x), IGRP (2x) and ZnT8 (1x). Peptides mimicking these epitopes were synthesized and tested for their capacity to stimulate IFNγ secretion by the clones when presented by HLA A2 positive antigen presenting cells. All clones produced IFNγ in response to PMA/ionomcyin, but none responded to any of the peptide epitopes tested. We are currently generating the reagents to test a broader array of beta-cell antigens. Nonetheless, our results suggest that a diverse repertoire of CD8+ T cells, that respond to a diverse array of epitopes, infiltrate the islets of people with established T1D.