Presentation of insulin granule-derived peptides on HLA in Enterovirus-infected beta cells and type 1 diabetes

PhD student: Zuzana Marinicova
Supervisor at TUD: Michele Solimena
Supervisor at KCL: Mark Peakman
Start date: 04.09.2017  Date of defense: 11.11.2021   PhD

T1 Type 1 diabetes (T1D) is a chronic autoimmune disease characterized by loss of insulin-producing beta cells resulting in life-long insulin deficiency. Beta cell destruction by autoreactive CD8⁺ effector T-cells is thought to be the main cause of loss of insulin output. Autoreactive T-cells are similarly to autoantibodies, which have been established as markers of risk and progression of the disease, directed towards autoantigens of T1D. These are most notably, insulin, 65 kDa glutamic acid decarboxylase (GAD65, also known as GAD2), insulinoma-associated protein 2 (IA-2, also known as PTPRN or ICA512) or zinc transporter 8 (ZNT8). Most of the known T1D autoantigens are components of insulin secretory granules (SGs). T1D arises from an interplay of genetic and environmental factors, which are thought to act as triggers in susceptible individuals. Predisposing alleles in genetic loci for human leukocyte antigen (HLA) account for by far the highest contribution to the risk of disease development, followed by an array of polymorphisms thought to play a role in either immune cells or beta cells. Of environmental factors that potentially add to the risk of disease progression, the most evidence-supported are Enteroviruses (EVs). Most notably, their genome and viral proteins, as well as higher expression of cellular proteins involved in viral response were detected more often in blood and pancreata of patients with T1D than in healthy population. In addition, recent evidence from a large long-term observational study has implicated prolonged shedding of specifically species Enterovirus B in the stools of children as a risk factor in development of beta cell autoimmunity in children with high genetic risk of T1D. For these reasons, many researchers have studied the potential mechanisms of EV involvement in T1D pathogenesis. In our laboratory, we have investigated the effects of coxsackievirus B5 (CVB5) infection on murine insulinoma MIN6 cells. Previously, we have reported that glucose-stimulated translation of SG proteins can be carried out in a cap-independent manner and is not shut down as part of the early effects of CVB5 infection on MIN6 cells. We have also observed that mature forms of SG proteins are being degraded during viral infection. As intracellular protein degradation is one of the major pathways to supply peptides for presentation on HLA I for immune recognition, we hypothesized that concomitant production and degradation of SG proteins upon viral infection could lead to altered presentation of mainly peptides derived from insulin SG component proteins and potentially drive the response of autoreactive T-cells.

To address this hypothesis, we aimed to identify appropriate conditions to study the impact of EV infection on antigen presentation of ECN90 cells. To that end, we established a panel of markers examined by SDS-PAGE and immunoblotting. Stage of viral infection was assessed based on the detection of the viral protein VP1 and cleavage of cellular factors such as eukaryotic translation initiation factor 4 G (eIF4G), poly(A)-binding protein (PABP1), polypyrimidine tract-binding protein 1 (PTBP1), poly (ADP-ribose) polymerase (PARP) and caspase 3, which is mediated by viral proteases. Furthermore, we assessed the levels of ICA512 and chromogranin A and their pro-forms to estimate the size of insulin SG stores, and the expression of HLA I and β2 microglobulin to confirm sufficient antigen presentation. Peptides presented on both HLA I and II were isolated by immunoaffinity purification and identified by liquid chromatography-tandem mass spectrometry analysis. About 500 unique HLA I‑presented peptides were found on average per replicate and condition with purity of 89% (peptides predicted to bind HLA alleles expressed by ECN90 cells). The distribution of unique peptides presented by infected ECN90 cells significantly differed from those presented by control cells as 54 unique peptides were present only in all infected samples and none of uninfected and 13 peptides were only found in uninfected cells. In total, we identified 26 unique peptides from known T1D autoantigens associated with SGs (e.g. insulin, chromogranin A, ICA512) in both conditions. The majority of them were predicted to bind HLA I alleles B*40:01 and A*02:01, while two identified viral peptides were found to bind B*40:01 and A*03:01 alleles. Both of the viral peptides and almost half of the peptides originating from known T1D autoantigens have not been described before. In addition, on average 300 unique HLA II peptides were found per replicate and condition. Similarly to HLA I peptides, the distribution of unique peptides across infected and control cells differed as well, showing that antigen presentation was altered in infected cells. We identified two viral HLA II-eluted peptides and peptides originating from only two known T1D autoantigens, 35 originated from insulin and 157 from chromogranin A. As most of the newly identified HLA I peptides originating from T1D autoantigens and one peptide from viral proteins were restricted by the allele HLA-B*40:01, our further efforts were invested in the development of a recombinant disulfide-stabilized biotinylated peptide-receptive HLA molecule of this allele. This technology has been extensively validated, and will allow us to test the wide array of novel peptides identified by us for the ability to bind this allele, as well as asses frequencies and responses of specific T-cells in subject populations relevant for T1D.

Publication:

The Major Capsid Protein, VP1, of the Mouse Polyomavirus Stimulates the Activity of Tubulin Acetyltransferase 1 by Microtubule Stabilization.  L. Hornikova, K. Brustikova, B. Ryabchenko, I. Zhernov, M. Fraiberk, Z. Marinicova, Z. Lansky, J. Forstova. Viruses. 2020;12:227.