A TCR-like antibody against a proinsulin-containing fusion peptide ameliorates type 1 diabetes in NOD mice

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Highlights

  • Generation of a TCR-like monoclonal antibody specific to the hybrid insulin peptide presented on MHC class II molecules.

  • Development of a novel NFAT-GFP TCR reporter system to detect antigen-specific T cell response.

  • Diabetogenic T cell response was blocked by the hybrid insulin peptide-specific TCR-like monoclonal antibody specific to the hybrid insulin peptide presented on MHC class II molecules.

  • Type 1 diabetes in pre-diabetic NOD mice was ameliorated by the novel hybrid insulin peptide-specific monoclonal antibody.

Abstract

Type 1 diabetes (T1D) is an autoimmune disease caused by destruction of insulin-producing β cells. The response of autoreactive T cells to β cell antigens plays a central role in the development of T1D. Recently, fusion peptides composed by insulin C-peptide fragments and other proteins were reported as β cell target antigens for diabetogenic CD4+ T cells in non-obese diabetic (NOD) mice. In this study, we generated a T cell-receptor (TCR)-like monoclonal antibody (mAb) against a fusion peptide bound to major histocompatibility complex (MHC) class II component to elucidate the function of the fusion peptides in T1D. In addition, we developed a novel NFAT-GFP TCR reporter system to evaluate the TCR-like mAb. The NFAT-GFP reporter T cells expressing the diabetogenic TCR were specifically activated by the fusion peptide presented on the MHC class II molecules. By using the NFAT-GFP reporter T cells, we showed that the TCR-like mAb blocks the diabetogenic T cell response against the fusion peptide presented on the MHC class II molecules. Furthermore, the development of T1D was ameliorated when pre-diabetic NOD mice were treated with this mAb. These findings suggest that NFAT-GFP reporter T cells are useful to assess the function of specific TCR and the recognition of fusion peptides by T cells is crucial for the pathogenesis of T1D.

Introduction

Type 1 diabetes (T1D) is a T cell-mediated autoimmune disorder [[1], [2], [3]]. Using the non-obese diabetic (NOD) mouse model, previous studies have shown that the autoreactive response of CD4+ T cell to several self-antigens expressed in pancreatic β cells plays an important role in the pathogenesis of T1D [[4], [5], [6]]. NOD mice exhibit a unique major histocompatibility complex (MHC) class II allele, I–Ag7, which is essential for the development of T1D [[7], [8], [9]]. This suggests that certain peptide-antigens that are specifically presented on I–Ag7 are involved in the T1D development in NOD mice. Among several β cell antigens that are suggested to be targets of diabetogenic CD4+ T cells in T1D, insulin is considered to be a key autoantigen [1,5,10]. However, it is unclear how self-antigens expressed in β cells, such as insulin, become autoantigens for pathogenic T cells. To address this issue, CD4+ T cell clones that have the ablity to induce T1D were established from NOD mice and their target antigens were extensively studied [11,12]. Among these CD4+ T cell clones targeting peptide-antigens expressed in pancreatic β cells [13,14], some diabetogenic T cell clones recognize fusion peptides composed by the insulin C-peptide fragment and fragments of other proteins such as chromogranin A (ChgA) or islet amyloid polypeptide; these fusion peptides were named hybrid insulin peptides (HIPs) [15]. For example, BDC2.5 and BDC10.1 T cell clones recognize a fusion peptide composed by insulin C-peptide and ChgA fragments [15]. Furthermore, CD4+ T cell clones isolated from human T1D patients recognized similar fusion peptides [15,16]. This suggests that some diabetogenic CD4+ T cells are activated by fusion peptides bound to MHC class II molecules not only in NOD mice but also in human T1D patients. However, it remains unclear whether specific fusion peptides are directly involved in the pathogenesis of T1D.

In the present study, we elucidate the function of insulin C-peptide-containing fusion peptides in the pathogenesis of T1D in NOD mice using a Tcell-receptor (TCR)-like antibody that specifically recognizes peptide-MHC complexes. We successively generated a TCR-like monoclonal antibody (mAb) that specifically recognizes a fusion peptide presented on I–Ag7. The TCR-like mAb not only blocked the diabetogenic T cell response to the fusion peptide-I-Ag7 complex in vitro but also inhibited the development of T1D in NOD mice. These findings suggest that insulin C-peptide-containing fusion peptides play an important role in the pathogenesis of T1D.

Section snippets

Mice

NOD/SHiJcl (NOD) mice were purchased from CLEA JAPAN, housed under specific pathogen-free conditions, and treated with 0.5 mg/ml Vancomycin (Pfizer, M037AB6) to remove segmented filamentous bacteria that prevent the onset of T1D in NOD mice [17]. NOD mice were immunized subcutaneously (s.c.) with 50 nmol of the hybrid insulin peptide 2.5HIP in incomplete Freund’s adjuvant (Beckton Dickinson, 263910). The primary immunization was performed at 4 weeks of age and a booster injection was

Induction of antibodies against a fusion peptide presented on I–Ag7

2.5HIP is a fusion peptide composed by insulin C-peptide and ChgA fragments [15]. This fusion peptide is specifically recognized by pathogenic T cell clones BDC2.5 and BDC10.1, which induce T1D upon adoptive transfer [23,26]. We immunized NOD mice with 2.5HIP and found that this induces the production of antibodies against microbeads coated with 2.5HIP alone (Fig. 1A). In addition, we found that 2.5HIP immunization also induces the production of antibodies against 2.5HIP-pulsed I–Ag7

Discussion

Certain HIPs and HIP-reactive T cells are present in the islets of NOD mice [29]. In addition, HIP-reactive T cells are also present in the peripheral blood of NOD mice [15,30]. Recent studies have shown that HIP-reactive T cells play an important role in the pathogenesis [30] and progression of T1D [31,32]. However, it was so far not clear how fusion peptides like HIPs are involved in T1D development. Here, we addressed how 2.5HIP-containing peptides, formed after the fusion of insulin and

Declaration of competing interest

The authors declare that they have no conflicts of interest.

Acknowledgments

We thank W. Nakai and N. Arase for many helpful discussions, K. Shida for providing technical assistance, and C. Kita for providing secretarial assistance. This work was supported by JSPS KAKENHI Grant Numbers JP18H05279, JP18K19450, JP18K07174, MEXT KAKENHI Grant Numbers JP19H04808, JP16H06501, Japan Agency for Medical Research and Development (AMED) under Grant Number J20ek041005h0003.

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