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

  Assessing insulin-specific T-cell tolerance in the NOD mouse model tolerant to proinsulin (#70)

Jonathan Chee 1 , Hyun-Ja Ko 2 , Prerak Trivedi 1 , Gaurang Jhala 1 , Bala Murthy 1 , Helen Thomas 1 , Andrew Lew 2 , Tom Kay 1
  1. St Vincents Institute, Fitzroy, VIC, Australia
  2. Walter and Eliza Hall Institute, Parkville, VIC, Australia

T-cell responses to insulin are important in type 1 diabetes (T1D) development. We studied INSB15-23-specific CD8+ T-cells and INSB9-23-specific CD4+ T-cells in NOD mice, and in NOD mice tolerant to proinsulin II (NOD-PI), which do not develop diabetes or insulitis.

Insulin-specific T-cells were enumerated using the magnetic bead and tetramer enrichment method. CD8+ function was assessed by peptide stimulation and in-vivo cytotoxicity assay. Transferring CD4+ T-cells into NOD8.3/RAG-/- assessed CD4+ help. NOD8.3/RAG-/- have CD8 TCR transgenic T-cells specific for IGRP but have reduced diabetes incidence because CD4+ T-cell help is absent.

There was no significant difference in the absolute number of INSB15-23-specific CD8+ T-cells in the thymus of NOD and NOD-PI mice. INSB15-23 T-cells significantly decreased in the peripheral lymphoid tissue (PLO) of old NOD-PI compared to NOD mice. INSB15-23 CD8+ T-cells in NOD mice expanded significantly more in response to stimulation by INSB15-23 peptide compared to NOD-PI. In-vivo cytotoxic activity in NOD-PI was reduced compared to NOD. The absolute number of INSB9-23 specific CD4+ T-cells in the thymus and PLO of NOD and NOD-PI was similar. The proportion of INSB9-23 T-cells that were FoxP3+ was also similar. INSB9-23 CD4+ T-cells in the NOD and NOD-PI were tested on whether they could help CD8+ T-cells in NOD8.3/RAG-/- mediate diabetes. NOD8.3/RAG-/- developed diabetes rapidly after NOD CD4+ T-cells were transferred (median=32days). When we transferred CD4+ cells from NOD mice lacking INSB9-23 T-cells into NOD8.3/RAG-/-, the diabetes was delayed (median=112days) and reduced, indicating the presence of functional INSB9-23 T-cells. 7/8 of recipients did not develop diabetes when NOD-PI CD4+ T-cells were transferred.

In NOD-PI mice, insulin-specific CD4+ and CD8+ T-cells were detected, suggesting that the main mechanism of tolerance is not deletion. It is more likely that these cells could be rendered anergic.