The effect of diabetes-associated autoantigens on cell processes in human PBMCs and their relevance to autoimmune diabetes development

Abstract

Type 1 Diabetes (T1D) is considered to be a T-helper- (Th-) 1 autoimmune disease; however, T1D pathogenesis likely involves many factors, and sufficient tools for autoreactive T cell detection for the study of this disease are currently lacking. In this study, using gene expression microarrays, we analysed the effect of diabetes-associated autoantigens on peripheral blood mononuclear cells (PBMCs) with the purpose of identifying (pre)diabetes-associated cell processes. Twelve patients with recent onset T1D, 18 first-degree relatives of the TD1 patients (DRL; 9/18 autoantibody positive), and 13 healthy controls (DV) were tested. PBMCs from these individuals were stimulated with a cocktail of diabetes-associated autoantigens (proinsulin, IA-2, and GAD65-derived peptides). After 72 hours, gene expression was evaluated by high-density gene microarray. The greatest number of functional differences was observed between relatives and controls (69 pathways), from which 15% of the pathways belonged to "immune response-related" processes. In the T1D versus controls comparison, more pathways (24%) were classified as "immune response-related." Important pathways that were identified using data from the T1D versus controls comparison were pathways involving antigen presentation by MHCII, the activation of Th17 and Th22 responses, and cytoskeleton rearrangement-related processes. Genes involved in Th17 and TGF-beta cascades may represent novel, promising (pre)diabetes biomarkers.

Figure 1

Verification of gene microarray data. Relative expression of TGF beta1, STAT3, and CD4 by qRT-PCR (data were obtained from an independent cohort of 14 newly diagnosed patients with T1D and 12 healthy volunteers).

Figure 2

Genes differentially activated in each group (cluster formation). The enhanced gene expression heatmap was constructed using probe signal intensities that had a log fold change that was greater than +1 or less than −1. Genes that were significantly altered in the relatives group clustered into specific gene families.

Figure 3

Immune response and Th17 cell differentiation. Differences in Th17 polarisation were observed when controls were compared with T1D patients using microarray data. Genes of interest were analysed by qRT-PCR and were found to be upregulated in T1D patients. STAT3 and TGF-beta were chosen as representatives of Th17 cell differentiation. Microarray data demonstrated that CD4 was one of the most significantly upregulated molecules in T1D patients.