Mapping of a hybrid insulin peptide in the inflamed islet β-cells from NOD mice

Abstract

There is accumulating evidence that pathogenic T cells in T1D recognize epitopes formed by post-translational modifications of β-cell antigens, including hybrid insulin peptides (HIPs). The ligands for several CD4 T-cell clones derived from the NOD mouse are HIPs composed of a fragment of proinsulin joined to peptides from endogenous β-cell granule proteins. The diabetogenic T-cell clone BDC-6.9 reacts to a fragment of C-peptide fused to a cleavage product of pro-islet amyloid polypeptide (6.9HIP). In this study, we used a monoclonal antibody (MAb) to the 6.9HIP to determine when and where HIP antigens are present in NOD islets during disease progression and with which immune cells they associate. Immunogold labeling of the 6.9HIP MAb and organelle-specific markers for electron microscopy were employed to map the subcellular compartment(s) in which the HIP is localized within β-cells. While the insulin B9-23 peptide was present in nearly all islets, the 6.9HIP MAb stained infiltrated islets only in NOD mice at advanced stages of T1D development. Islets co-stained with the 6.9HIP MAb and antibodies to mark insulin, macrophages, and dendritic cells indicate that 6.9HIP co-localizes within insulin-positive β-cells as well as intra-islet antigen-presenting cells (APCs). In electron micrographs, the 6.9HIP co-localized with granule structures containing insulin alone or both insulin and LAMP1 within β-cells. Exposing NOD islets to the endoplasmic reticulum (ER) stress inducer tunicamycin significantly increased levels of 6.9HIP in subcellular fractions containing crinosomes and dense-core granules (DCGs). This work demonstrates that the 6.9HIP can be visualized in the infiltrated islets and suggests that intra-islet APCs may acquire and present HIP antigens within islets.

Keywords: NOD; antigen; autoimmune diabetes; hybrid insulin peptide; islets; post-translational modification; type 1 diabetes; β-cells.

Figure 1

Characterization of 6.9HIP MAb. (A) Western blot (with 6.9MAb) and Coomassie blue-stained gel with titration of recombinant NUS-6.9HIP (5.0–0.1 mg) and NUS-2.5HIP (5 mg) fusion proteins. HIP sequences are aligned on the right. (B) Western blots with recombinant BALB/c 6.9HIP, NOD 6.9HIP, and HuC-pepG3/IAPP2 HIP proteins probed with HIS and 6.9HIP primary antibodies and goat anti-mouse-HRP secondary antibodies. HIP sequences are aligned below. (C) ELISA showing the binding of the 6.9HIPMAb to plate-bound 6.9HIP peptide. (D) Competitive ELISA showing the inhibition of the 6.9MAb:6.9HIP binding by indicated competitor peptides. The peptide sequences related to the 6.9HIP are underlined.

Figure 2

Visualization of 6.9HIP in inflamed islets by confocal microscopy. (A–E) Intact islets isolated from NOD mice of indicated sex and age were stained with unconjugated AIP (A, B) or 6.9HIP MAbs (C–E) followed by a secondary fluorochrome-conjugated anti-mouse IgG F(ab)2 antibody. (A) A representative islet (left) from 6-week-old male NOD mice stained with the AIP antibody. A selected region (rectangle) is enlarged in inset (right), showing punctate peptide (B9-23) staining (red arrow), and staining of intra-islet phagocytes by secondary antibody (blue arrow). (B) A representative islet from NOD.Rag1^-/- mice stained with the AIP antibody. (C) A representative islet from 6-week-old male NOD mice stained with the 6.9HIP MAb. (D) Two representative islets from 27- and 30-week-old female NOD mice stained with the 6.9HIP MAb. Enlarged images on the right show the punctate staining of the 6.9HIP distributed within the islets. (E) A representative islet from 30-week-old female NOD.IAPP^-/- mice stained with the 6.9HIP MAb. (F–I) Intact islets isolated from 25- to 30-week-old female NOD mice were stained with fluorochrome-conjugated antibodies to 6.9HIP (red), CD4 (white), F4/80 (blue), and Sirpα (green). (F, G) Representative islets showing the co-localization of 6.9HIP with F4/80+ macrophages (F) or Sirpα+ cDC2 (G). (H, I) Representative islets showing the presence of 6.9HIP when CD4+ T cells interact with Sirpα+ cDC2 (H) or F4/80+ macrophages (I). White scale bars in all images are 50 µm.

Figure 3

Immunogold electron microscopy reveals that 6.9HIP is localized in two sets of β-cell granules. Islets from 30-week-old (A–C) or 5-week-old (D) NOD mice were triple labeled with primary rabbit anti-insulin, rat anti-LAMP1, and mouse anti-6.9HIP antibodies, followed by staining with secondary antibodies conjugated to colloidal gold, including anti-rabbit 6-nm (insulin, black), anti-rat 12-nm (LAMP1, red), and anti-mouse 18-nm (6.9HIP, purple). Each image in (A–D) shows representative β-cells. The selected regions are illustrated in the enlarged inserts depicting granules containing 6.9HIP in the presence of insulin alone or insulin and LAMP1. ER = endoplasmic reticulum, m = mitochondria. (E) The table summarizes the numbers of β-cells containing at least one 6.9HIP spot and the total numbers of 6.9HIP spots quantified from 100 β-cells from either 30- or 5-week-old NOD mice. The violin plot shows the number of 6.9HIP spots from 100 individual β-cells from each condition. Data summarize results from two independent experiments. Each experiment used 200–300 islets isolated from two 5-week-old or five 30-week-old NOD mice. **p < 0.01; Mann–Whitney test.

Figure 4

ER stress promotes the production of 6.9HIP in β-cell granules. (A) Schematic for assessing the level of 6.9HIP in crinosome and DCG fractions isolated from islets of 6-week-old male NOD mice in the presence or absence of tunicamycin and TUDCA. (B) Responses of three 6.9HIP-reactive CD4 T-cell hybridomas to C3.g7 APCs pulsed with indicated concentrations of the 6.9HIP peptide. (C) Responses of three 6.9HIP-reactive T-cell hybridomas that were untreated or exposed to tunicamycin for 2 h, in the absence or presence of exogenous pulse with the 6.9HIP peptide (10 µM). (D) Responses of the three 6.9HIP-reactive T cells to either the crinosome or the DCG subcellular fractions isolated from islets that were untreated, treated with tunicamycin alone, or treated with both tunicamycin and TUDCA. Data summarize results from three independent experiments; each point represents a biological replicate of a granule fraction. ns, not significant; ****p < 0.001; two-way ANOVA with Sidak’s multiple comparisons test.