Citrullinated GRP78: an autoantigen in human T1D

Background: The beta-cell itself has become recognized as a central player in the pathogenesis of type 1 diabetes (T1D), with the generation of neo-antigens in response to inflammation as potential trigger for breaking immune tolerance. Given the heterogeneity of T1D, the identification of new antigens is critical to better understand disease pathology, to discover new markers for patient stratification, and to develop novel disease interventions, especially in the view of more personalized therapies. Based on our previous findings in non-obese diabetic mice, we now demonstrate that inflammation-induced citrullinated glucose-regulated protein 78 (citGRP78) is a neo-antigen in human T1D.

Methods: We performed proteome analysis by 2-dimensional gel electrophoresis (2DGE) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) on cytokine-exposed human islets to identify PTMs in GRP78, with a specific focus on the conversion of arginine (R) into citrulline (citrullination). CD4+ T-cell and B-cell autoreactivity against citGRP78 in the circulation of T1D patients was studied using HLA-DRB1*04:01 class II tetramers and ELISA, respectively.

Results:  2DGE revealed increased PTM of GRP78 in 3 out of 5 human islet preparations after cytokine exposure. The LC-MS/MS spectrum of GRP78 identified 2 out of 28 R-residues as potential citrullination sites. Citrullination of GRP78 led to the generation of neo-epitopes that could effectively be presented by HLA-DRB1*04:01 molecules. Significantly elevated levels of CD4+ T-cells directed against citGRP78 epitopes were detected in the circulation of T1D patients compared to healthy controls (p=0.013). These responding T-cells were derived to a large extent from the central memory T-cell population. In 33% of the T1D patients autoantibodies against citGRP78 were detected, whereas only 5.6% of the healthy control subjects were autoantibody positive (p<0.001). These results strongly suggest that PTM of beta-cell proteins, exemplified here by citGRP78, contributes to loss of self-tolerance towards beta-cells in human T1D.