CTLs are targeted to kill β cells in patients with type 1 diabetes through recognition of a glucose-regulated preproinsulin epitope
Ania Skowera, … , Bart O. Roep, Mark Peakman
Ania Skowera, … , Bart O. Roep, Mark Peakman
Published September 18, 2008
Citation Information: J Clin Invest. 2008;118(10):3390-3402. https://doi.org/10.1172/JCI35449.
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Research Article

CTLs are targeted to kill β cells in patients with type 1 diabetes through recognition of a glucose-regulated preproinsulin epitope

Abstract

The final pathway of β cell destruction leading to insulin deficiency, hyperglycemia, and clinical type 1 diabetes is unknown. Here we show that circulating CTLs can kill β cells via recognition of a glucose-regulated epitope. First, we identified 2 naturally processed epitopes from the human preproinsulin signal peptide by elution from HLA-A2 (specifically, the protein encoded by the A*0201 allele) molecules. Processing of these was unconventional, requiring neither the proteasome nor transporter associated with processing (TAP). However, both epitopes were major targets for circulating effector CD8+ T cells from HLA-A2+ patients with type 1 diabetes. Moreover, cloned preproinsulin signal peptide–specific CD8+ T cells killed human β cells in vitro. Critically, at high glucose concentration, β cell presentation of preproinsulin signal epitope increased, as did CTL killing. This study provides direct evidence that autoreactive CTLs are present in the circulation of patients with type 1 diabetes and that they can kill human β cells. These results also identify a mechanism of self-antigen presentation that is under pathophysiological regulation and could expose insulin-producing β cells to increasing cytotoxicity at the later stages of the development of clinical diabetes. Our findings suggest that autoreactive CTLs are important targets for immune-based interventions in type 1 diabetes and argue for early, aggressive insulin therapy to preserve remaining β cells.

Authors

Ania Skowera, Richard J. Ellis, Ruben Varela-Calviño, Sefina Arif, Guo Cai Huang, Cassie Van-Krinks, Anna Zaremba, Chloe Rackham, Jennifer S. Allen, Timothy I.M. Tree, Min Zhao, Colin M. Dayan, Andrew K. Sewell, Wendy Unger, Jan W. Drijfhout, Ferry Ossendorp, Bart O. Roep, Mark Peakman

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Figure 6

Generation of SP epitopes of PPI is independent of TAP.

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Generation of SP epitopes of PPI is independent of TAP. (A) To examine T...
(A) To examine TAP dependency of PPI15–24 presentation by K562 cells, the CTL clone (1E6) recognizing PPI15–24 restricted by HLA-A2 was used in cytotoxicity assays in the presence of the varicellovirus-derived TAP inhibitor UL49.5 (19). As a control, we used the HA-2 CTL clone 5H17 (53), specific for an endogenously expressed, TAP-dependent minor histocompatibility antigenic epitope (sequence YIGEVLVSV derived from a diallelic gene encoding a novel human class I myosin protein) presented by HLA-A2 (54). Target cells for clone 1E6 were K562-PPI-A2 cells or K562-PPI-A2 cells additionally transfected to express UL49.5. Target cells for clone 5H17 were K562-A2 cells or K562-A2 cells additionally transfected to express UL49.5. Data are expressed as percent killing (see Methods for calculation of specific cytotoxicity using DELFIA assay); bars represent means and error bars, SEMs. In the presence of the 1E6 PPI15–24–specific CTLs, killing of K562-PPI-A2 cells cotransfected with UL49.5 (black bars) was comparable to that of untransfected K562-PPI-A2 cells (white bars), indicating that TAP inhibition by UL49.5 does not reduce PPI15–24 presentation. In contrast, killing of target cells by the HA-2–specific CTL 5H17 that recognizes a TAP-dependent epitope is markedly inhibited in the presence of UL49.5 (black bars). (B) To examine proteasome dependency of PPI15–24 generation, MS analyses of immuno- and constitutive proteasome digests of PPI1–30 were performed. Results are shown in Table 2. In the same experiment, several other fragments of PPI1–30 were generated by proteasome digestion. Sequences marked with an asterisk were generated by the constitutive proteasome only. For reference, the 10-mer PPI15–24 peptide eluted from surrogate β cells is indicated by the black rectangle.