Discovery of low-affinity preproinsulin epitopes and detection of autoreactive CD8 T-cells using combinatorial MHC multimers
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.
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Cited by (51)
Noncontiguous T cell epitopes in autoimmune diabetes: From mice to men and back again
2021, Journal of Biological ChemistryCitation Excerpt :A proportion of beta cell–reactive T cells evade the central tolerance mechanism of negative selection despite the thymic expression of several important beta cell antigens, including insulin (92). This has been explained in part by the finding that the cognate peptides for these T cells in humans are sometimes characterized by weak MHC binding (93–95). Poor peptide binding to MHC would lead to a low-avidity interaction between the T cell receptor (TCR) of the developing T cell and peptide/MHC in the thymus, allowing cognate T cells to escape negative selection.
Interleukin-15 in autoimmunity
2020, CytokineCitation Excerpt :The protective effect of the anti-γc treatment suggests that inhibiting the effector cells that mediate the destruction of islets is more effective than increasing the activity of Tregs as the anti-γc treatment would also inhibit IL-2 signaling in Tregs. In diabetic patients, allogenic response to the islet grafts during transplantation and the islet-specific T cell response by low avidity and memory CD8+ T cells contribute to the graft rejection despite immunosuppression [142–143], indicating that antigen specific CD8+ T cell responses are critical. Additionally, the immunosuppressive T cell depletion regimens used in transplantation increase the production of homeostatic cytokines such as IL-7 and IL-15 to permit the expansion of residual T cells [144].
Post-Translational Peptide Splicing and T Cell Responses
2017, Trends in ImmunologyCitation Excerpt :At present, we can only speculate that PCPS might be involved in the CD8+ T cell-mediated immune response against self antigens in those diseases (e.g., myelin in multiple sclerosis or insulin in T1DM) where infections have been hypothesized to be involved in autoimmune pathogenesis [55,56]. In multiple sclerosis and T1DM, both CD4+ and CD8+ T cells can target their response toward self epitopes presented by either MHC-I or MHC-II molecules [20,56–59]. In particular, in T1DM, MHC-II-restricted and post-translationally spliced epitopes have been recently described.
A roadmap of the generation of neoantigens as targets of the immune system in type 1 diabetes
2016, Current Opinion in ImmunologyCitation Excerpt :Activated islet-autoreactive T cells occur in both circulation and insulitic lesions [1,2••,3], but it remains poorly understood how these cells avoid thymic education, central and peripheral tolerance and immune regulation, and how they are primed. Improper presentation of self-antigens by antigen-presenting cells due to low-affinity native antigens may trigger these T cells to escape thymic deletion [4–6]. Besides failures in central tolerance, it is conceivable that beta cells are key-players in their own destruction by generating neoantigens avoiding central tolerance [7].
Neoantigens and Microenvironment in Type 1 Diabetes: Lessons from Antitumor Immunity
2016, Trends in Endocrinology and MetabolismT cells in type 1 diabetes: Instructors, regulators and effectors: A comprehensive review
2016, Journal of AutoimmunityCitation Excerpt :Our own data highlight the fact that HLA molecules do not selectively present the highest affinity binding peptides alone; rather they can sample epitopes from the same protein across a range of binding-affinities [58]. Furthermore, although it may seem counter-intuitive, the strength of binding affinity between peptide and MHC does not predict the strength of the T cell response to peptide-HLA and CD8+ T cells reactive against low affinity peptide-MHC appear to be abundant in type 1 diabetes [62]. Again, with the “Streetlight effect” in our minds, we should be aware that the available techniques to detect naturally processed and presented peptide ligands (elution from immunoaffinity purified peptide-HLA and mass spectrometry [3]) and CD8 T cell reactivity (peptide-HLA multimer technology) might limit our ability to detect very low affinity epitopes which might have disease relevance [63].
- 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.