Type 1 diabetes (T1D) is a tissue specific autoimmune disease that results in destruction of insulin producing β-cells. A hallmark of T1D is insulitis, characterized by the presence of infiltrating T cells, predominantly CD8+ T cells, surrounding β-cells within the pancreatic islets. Human leukocyte antigen (HLA) alleles have been strongly implicated in the pathoetiology of T1D, with both HLA class II and class I genes independently associated with disease outcome. The class I HLA encoded molecules present antigenic peptide fragments derived from endogenous autoantigens to incoming CD8+ T cells. To date, well established autoantigens associated with T1D are predominantly β cell secretory proteins. Although several T1D autoantigens have been established, few T cell epitopes derived from these antigens have been reported.
In this study we have identified novel autoantigenic epitopes restricted to T1D associated HLA alleles via an in vitro reversed immunology approach. To accomplish this, MHC bound peptides were isolated from a panel of engineered surrogate β cell lines enriched for an endogenous islet specific autoantigen (such as pre-(pro)insulin and chromogranin A) and overexpressing a T1D-associated class I HLA allele. Peptide identities are then identified by mass spectrometry. Along with this several novel autoantigenic peptides restricted to HLA-A*0201 were also identified. Furthermore, using a targeted mass spectrometry approach, multiple reaction monitoring, expression level of these specific HLA A2-peptide complexes on the surface of human beta cells was determined. Parallel to in vitro study, we used the targeted mass spectrometry approach to quantitate the surface density of these identified novel peptides and known epitopes on the surface of human islet β cells ex vivo.
This work will ultimately allow the correlation of antigen expression, antigen presentation and T cell autoreactivity, leading to a better understanding of the nature of autoreactive T cell response in T1D.