CD4+ islet-infiltrating T cells from a recent onset donor with type 1 diabetes (T1D) recognize DQ8-restricted deamidated islet-associated epitopes

Background We reported that T cells, isolated directly from the islets of donors with T1D, recognized a broad repertoire of islet-associated targets, including post-translationally modified epitopes. From a study examining islet-associated proteins for potential sites of deamidation by transglutaminase, it was found that deamidated epitopes, from several islet proteins, were capable of binding to HLA-DQ8cis/trans. Peripheral CD4+ T cell autoreactivity from individuals with T1D to these peptides was demonstrated (van Lummel et al., 2014). We investigated if CD4+ T cells derived directly from the islets of a DQ8+ donor with 2 years T1D duration, recognized deamidated epitopes of islet-associated targets.

Methods Isolated islets were received from nPOD case 6342, a 14-year-old female with 2 years of T1D duration (HLA: DQA1*01:01, DQA1*03:01, DQB1*05:01, DQB1*03:02). One-hundred islets were hand-picked to increase purity and plated on a gel matrix with anti-CD3, anti-CD28, anti-Fas, mifepristone, IL-2, IL-4, IL-7, and IL-15. Cellular outgrowth from individual islets were collected and expanded with growth factors. Irradiated, autologous EBV-transformed splenic B cells were pulsed with pools or de-convoluted pools of native and deamidated peptides for 2 hours, washed and co-cultured with CD4+ T cell lines for 48 hours. Reactivity of T cell lines was determined by up-regulation of T cell activation markers and characterized by intracellular staining for IFNγ and TNFα.

Results All six CD4+ T cell lines from nPOD 6342 recognized deamidated peptides from IA-2, GAD65 or ZnT8. Further studies will de-convolute these reactivities. 6342 CD4+ Line 2.3 reacted with deamidated IA-2_144-156, but not the native peptide or deamidated IA-2_412-424. Upon polyclonal stimulation and intracellular staining, these lines expressed TNFα, but not IFNγ. These studies will be expanded to other islet donors bearing DQ2/DQ8. These studies extend the importance of examining islet-derived T cells from donors with T1D and post-translationally modified epitopes in the etiology of T1D.