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 8

High ambient glucose increases the efficiency of human β cell killing.

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High ambient glucose increases the efficiency of human β cell killing. (...
(A) Specific lysis of human islet cells after 14 hours preculture at different glucose concentrations (5.6 mM, 11 mM, and 20 mM; filled squares, filled circles, and open squares, respectively). Higher glucose results in significantly enhanced CTL killing by 1E6 (1-way ANOVA, P < 0.005; significant linear trend, P < 0.005). Representative agarose gel of RT-PCR amplification using insulin- and glucagon-specific primers of total RNA from human islet cells after 14 hours at different glucose concentrations (B) and band density in the same gel (C). Islet cells at 5.6, 11, and 20 mM glucose exposed to killing by 1E6 clone cells show increasing loss of insulin mRNA, declining from approximately 50,000 to approximately 0 counts. Glucagon expression is identical to that at 20 mM glucose in the absence of 1E6 clone cells (see Figure 7A). (D) In contrast, human islet cells pulsed with PPI15–24 show no β cell specificity of killing and no additive killing at higher glucose concentrations (symbols as above). (E) Agarose gel of RT-PCR using insulin- and glucagon-specific primers after human islet cells were cultured for 14 hours at 20 mM glucose, either with PPI15–24 peptide and exposed to killing by 1E6 (lane 1) or cultured alone (lane 2) and (F) graphical representation of band density. The results show loss of both insulin and glucagon expression, demonstrating a lack of β cell–specific killing of islets prepulsed with PPI15–24. Cytotoxicity assays were performed in triplicate; symbols represent means, and error bars represent SEMs.