Elsevier

Journal of Autoimmunity

Volume 37, Issue 3, November 2011, Pages 151-159
Journal of Autoimmunity

Discovery of low-affinity preproinsulin epitopes and detection of autoreactive CD8 T-cells using combinatorial MHC multimers

https://doi.org/10.1016/j.jaut.2011.05.012Get rights and content

Abstract

Autoreactive cytotoxic CD8 T-cells (CTLs) play a key pathogenic role in the destruction of insulin-producing beta-cells resulting in type 1 diabetes. However, knowledge regarding their targets is limited, restricting the ability to monitor the course of the disease and immune interventions. In a multi-step discovery process to identify novel CTL epitopes in human preproinsulin (PPI), PPI was digested with purified human proteasomes, and resulting COOH-fragments aligned with algorithm-predicted HLA-binding peptides to yield nine potential HLA-A1, -A2, -A3 or -B7-restricted candidates. An UV-exchange method allowed the generation of a repertoire of multimers including low-affinity HLA-binding peptides. These were labeled with quantum dot-fluorochromes and encoded in a combinatorial fashion, allowing parallel and sensitive detection of specific, low-avidity T-cells. Significantly increased frequencies of T-cells against four novel PPI epitopes (PPI4–13/B7, PPI29–38/A2, PPI76–84/A3 and PPI79–88/A3) were detected in stored blood of patients with recent onset diabetes but not in controls. Changes in frequencies of circulating CD8 T-cells against these novel epitopes were detected in blood of islet graft recipients at different time points after transplantation, which correlated with clinical outcome. In conclusion, our novel strategy involving a sensitive multiplex detection technology and requiring minimal volumes of stored blood represents a major improvement in the direct ex-vivo characterization and enumeration of immune cells in the pathogenesis of type 1 diabetes.

Introduction

Type 1 diabetes (T1D) is an auto-immune disease resulting from progressive destruction of the insulin-producing beta-cells in the pancreas by autoreactive T-cells. In recent years, it has become increasingly clear from studies in mice and men that autoreactive CD8 T-cells are involved in both the initiation of the disease and the destruction of beta-cells [1], [2], [3], [4], [5], [6]. Thus, detecting and monitoring specific CD8 T-cells in humans may provide a valuable tool to assess disease activity. Most of the currently known epitopes have been identified by a process referred to as “reverse” immunology requiring immunization of HLA-A2 transgenic mice [7], [8], [9]. This strategy may introduce false-positive results as the majority of epitopes identified display at least one amino acid difference with the endogenous murine sequence, rendering them xeno-antigens to the HLA-A2 transgenic mice. Nonetheless, several of these epitopes are recognized by CD8 T-cells of type 1 diabetic patients [7], [10]. Alternatively, islet-specific autoreactive CD8 T-cell reactivity has been detected against an insulin epitope identified by high HLA-binding affinity using HLA-A2 tetramers and cytokine secretion assays [11]. The amount of insulin expression in the thymus is an important risk factor in T1D. Indeed, a recent study showed an association between low insulin expression in the thymus, controlled by a polymorphism in the variable number of tandem repeat (VNTR) elements in the human insulin promoter and high frequencies of insulin-specific CD4 T-cells in peripheral blood [12]. More recently, the identification of novel beta-cell epitopes was approached by eluting peptides from surface HLA-A2 molecules of surrogate beta-cells, expressing preproinsulin (PPI) [13]. An epitope that lies in the signal peptide (i.e. PPI15–24) was identified as a major target of CD8 T-cell responses in HLA-A2+ T1D patients. Moreover, expanded clones of PPI15–24-specific CD8 T-cells were shown to lyse human HLA-A2+ beta-cells in-vitro. In contrast to the “reverse” immunology approach, the PPI15–24 epitope could not have been revealed by immunization of HLA-A2 transgenic mice, since murine and human sequences are divergent in this region. However, this approach is HLA-A2-specific, did not identify any other preproinsulin epitopes, and may lack sensitivity. There remains a need, therefore, for approaches of autoreactive CTL epitope discovery that are both sensitive and unaffected by HLA or species differences. In addition, these should be complemented by detection technologies suited to enumerate rare autoreactive T-cells within the limitations imposed by the blood volume obtained from children with, or at-risk of disease. Moreover, low-affinity HLA-binding peptides have been shown to play a role in autoimmunity [14]. As the avidity of the auto-immune TCR can be low [11], both factors may contribute to an increase in background, as observed in many of the current detection methods.

With these considerations in mind, we set out to establish a multi-step approach to epitope discovery linked to an optimized detection platform. We focused on PPI as an important target for autoreactivity in mice and humans and combined proteasome digestion with HLA-binding prediction algorithms. Subsequently peptide-MHC (pMHC) monomers were generated by UV-light-induced exchange reactions [15] and encoded with different quantum dot (Qdot)-fluorochromes in a combinatorial fashion for each putative epitope [16]. This new multimer production method allows the generation of a new repertoire of multimers presenting both high and low-affinity HLA-binding peptides compared to conventional tetramers. Using this combination of methods, we uncovered five novel PPI-derived epitopes (PPI4–13/B7, PPI29–38/A2, PPI9–18/A3, PPI76–84/A3 and PPI79–88/A3). We show that T-cells specific for PPI4–13, PPI29–38, PPI76–84 and PPI79–88 are selectively present in the blood of recent onset diabetic patients and also in patients receiving islet cell grafts, in whom their presence correlated with clinical outcome of transplantation.

Section snippets

Patients

After informed consent, T1D patients were retrieved from the Kolibri T1D cohort, which includes 350 patients with juvenile-onset T1D. The cohort was collected consecutively after diagnosis by pediatricians in the southwestern part of the Netherlands between 1995 and 1999. The diagnosis was made according to International Society of Pediatric and Adolescent Diabetes and World Health Organization criteria. This study was approved by the institutional medical ethical committee.

Twelve patients were

Proteasome processing of preproinsulin

Proteasome cleavage pattern analysis allows accurate and selective prediction of CTL epitopes [20]. Therefore, we incubated overlapping 30-mer polypeptides, spanning whole PPI, with purified proteasome complexes. Human 20S proteasomes isolated from an EBV-transformed B cell line (expressing mainly immunoproteasome) and a HeLa cell line (expressing constitutive proteasome, also the dominant proteasome type expressed in pancreatic beta-cells) were used for digestions of 1-, 4-, and 24-h

Discussion

Our systematic search for new CTL epitopes reflecting antigen processing, and addressing peculiarities regarding epitope binding affinity and TCR avidity that may distinguish autoreactive CTLs from other specificities, has successfully led to the identification of several epitopes associated with the pathogenesis of T1D. We uncovered five novel PPI-derived epitopes. Four of these are restricted by other HLA class I alleles than HLA-A2, thereby broadening the patient populations that can be

Acknowledgments

We thank Mrs. W. Benckhuijsen and Mr. P. de Koning for synthesis of peptides. This work has been supported in part by the JDRF Program Grant (7-2005-877 to M.P, B.O.R, W.W.J.U and S.L) and the Dutch Diabetes Research Foundation DFN (to J.V. and J.R.F.A.). MP was supported by the UK Department of Health via the National Institute for Health Research (NIHR) comprehensive Biomedical Research Centre award to Guy’s & St Thomas’ NHS Foundation Trust in partnership with King’s College London.

References (51)

  • P. Santamaria et al.

    Beta-cell-cytotoxic CD8+ T cells from nonobese diabetic mice use highly homologous T cell receptor alpha-chain CDR3 sequences

    J Immunol

    (1995)
  • F.S. Wong et al.

    CD8 T cell clones from young nonobese diabetic (NOD) islets can transfer rapid onset of diabetes in NOD mice in the absence of CD4 cells

    J Exp Med

    (1996)
  • R.T. Graser et al.

    Identification of a CD8 T cell that can independently mediate autoimmune diabetes development in the complete absence of CD4 T cell helper functions

    J Immunol

    (2000)
  • Y. Hassainya et al.

    Identification of naturally processed HLA-A2 – restricted proinsulin epitopes by reverse immunology

    Diabetes

    (2005)
  • Q. Ouyang et al.

    Recognition of HLA class I-restricted {beta}-cell epitopes in type 1 diabetes

    Diabetes

    (2006)
  • N.E. Standifer et al.

    Identification of novel HLA-A∗0201-restricted epitopes in recent-onset type 1 diabetic subjects and antibody-positive relatives

    Diabetes

    (2006)
  • A. Toma et al.

    Recognition of a subregion of human proinsulin by class I-restricted T cells in type 1 diabetic patients

    PNAS

    (2005)
  • G.G. Pinkse et al.

    Autoreactive CD8 T cells associated with beta cell destruction in type 1 diabetes

    PNAS

    (2005)
  • I. Durinovic-Bello et al.

    Insulin gene VNTR genotype associates with frequency and phenotype of the autoimmune response to proinsulin

    Genes Immun

    (2010)
  • A. Skowera et al.

    Glucose-regulated cytotoxic T-cell epitope of preproinsulin targets killing of beta-cells in human type 1 diabetes

    J Clin Invest

    (2008)
  • M.G. Levisetti et al.

    Weak proinsulin peptide-major histocompatibility complexes are targeted in autoimmune diabetes in mice

    Diabetes

    (2008)
  • M. Toebes et al.

    Design and use of conditional MHC class I ligands

    Nat Med

    (2006)
  • S.R. Hadrup et al.

    Parallel detection of antigen-specific T-cell responses by multidimensional encoding of MHC multimers

    Nat Methods

    (2009)
  • B. Keymeulen et al.

    Correlation between beta cell mass and glycemic control in type 1 diabetic recipients of islet cell graft

    PNAS

    (2006)
  • N.J. Beekman et al.

    Abrogation of CTL epitope processing by single amino acid substitution flanking the C-terminal proteasome cleavage site

    J Immunol

    (2000)
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    1

    Present address: Department of Cell Biology and Immunology, VUmc, Amsterdam, The Netherlands.

    2

    Present address: Center for Cancer Immunotherapy, Department of Hematology, Herlev University Hospital, Herlev, Denmark.

    3

    Present address: Division of Immunology and Pathogenesis, Department of Molecular and Cell Biology, University of California, Berkeley, CA.

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