A distinct immunogenic region of glutamic acid decarboxylase 65 is naturally processed and presented by human islet cells to cytotoxic CD8 T cells

Abstract

CD8 T cells specific for islet autoantigens are major effectors of β cell damage in type 1 diabetes, and measurement of their number and functional characteristics in blood represent potentially important disease biomarkers. CD8 T cell reactivity against glutamic acid decarboxylase 65 (GAD65) in HLA-A*0201 subjects has been reported to focus on an immunogenic region 114-123 (VMNILLQYVV), with studies demonstrating both 114-123 and 114-122 epitopes being targeted. However, the fine specificity of this response is unclear and the key question as to which epitope(s) β cells naturally process and present and, therefore, the pathogenic potential of CD8 T cells with different specificities within this region has not been addressed. We generated human leucocyte antigen (HLA)-A*0201-restricted CD8 T cell clones recognizing either 114-122 alone or both 114-122 and 114-123. Both clone types show potent and comparable effector functions (cytokine and chemokine secretion) and killing of indicator target cells externally pulsed with cognate peptide. However, only clones recognizing 114-123 kill target cells transfected with HLA-A*0201 and GAD2 and HLA-A*0201(+) human islet cells. We conclude that the endogenous pathway of antigen processing by HLA-A*0201-expressing cells generates GAD65114-123 as the predominant epitope in this region. These studies highlight the importance of understanding β cell epitope presentation in the design of immune monitoring for potentially pathogenic CD8 T cells.

Keywords: CD8 T cell clones; GAD65; autoimmunity; peptide-processing; type 1 diabetes.

Fig. 1

CD8 T cell clones display differential staining with tetramer loaded with either glutamic acid decarboxylase 9 (GAD9)-mer or GAD10-mer peptide. CD3⁺CD8⁺ clone cells raised against the immunogenic 114–123 region of GAD65 were stained with an irrelevant human leucocyte antigen (HLA)-A*0201 tetramer (loaded with preproinsulin 15–24), GAD9-mer tetramer (GAD_114–122) or GAD10-mer tetramer (GAD_114–123). (a) Clone RK9P9-3 stains with the GAD9-mer tetramer only. (b) Clone RK9C10-1 stains with both GAD9-mer and GAD10-mer tetramers. Histograms are displayed as % of maximum for 10 000 live events, the line indicative of positive staining represents the fluorescence-minus-one control.

Fig. 2

Differential recognition of peptide-pulsed human leucocyte antigen (HLA)-A*0201⁺ cell lines by CD8 T cell clones. Representative intracellular staining for tumour necrosis factor (TNF)-α and surface staining of CD107a (degranulation marker) of clone cells after 18 h co-culture with peptide-pulsed target cell lines. (a) RK9P9-3 shows a high percentage of events positive for TNF-α production and degranulation following co-culture with glutamic acid decarboxylase 9 (GAD9)-mer-pulsed target cells, but minimal positivity using GAD10-mer-pulsed target cells. In contrast, RK9C10-1 displays a cross-reactive profile to both GAD9-mer- and GAD10-mer peptide-pulsed cell lines. (b) These responses contrasted against a negative control peptide stimulation (IGRP_265–273) and the positive control of phorbol myristate acetate/ionomycin stimulation. (c) Specific lysis of GAD9-mer-pulsed K562-A2 target cells by both RK9P9-3 and RK9C10-1 and killing of GAD10-mer-pulsed target cells by RK9C10-1 only; 10 000 live CD3⁺CD8⁺ events were recorded for flow cytometry analyses; cytotoxicity and functional assays are representative data performed at an effector : target ratio of 25:1 for 4 h. Data shown are representative single experiments.

Fig. 3

CD8 T cell clone recognition of endogenously presented glutamic acid decarboxylase 65 (GAD65) by transduced target cells. CD8 T cell clones were co-cultured with GAD2 transduced human leucocyte antigen (HLA)-A2 expressing K562 cells (K562-A2-GAD65) for 18 h, and supernatants assessed for macrophage inflammatory protein (MIP)-1α and MIP-1β production. Both RK9P9-3 and RK9C10-1 respond similarly to stimulation with GAD9-mer peptide-pulsed cells. Similarly, neither respond to the empty vector control cell line. RK9C10-1 produced high levels of chemokines when cultured with K562-A2-GAD65 target cells, but RK9P9-3 did not respond. These data indicate that only RK9C10-1 recognizes GAD65 after endogenous processing, and that the naturally processed and presented epitope is GAD65_114–123. Cells were incubated at an effector : target ratio of 1:1. Bars show triplicate means and error bars standard errors of the mean of representative experiments.

Fig. 4

CD8 T cell clone recognizing glutamic acid decarboxylase 65 (GAD65)_114–123 recognizes and kills human islet cells. (a) Cytotoxicity assays were performed using RK9P9-3 and RK9C10-1 clone cells and human leucocyte antigen (HLA)-A*0201⁺ human islet cells as targets. As shown in (a) RK9C10-1 robustly kills islet cells [comparable to the positive control (b), namely killing of islet cells from the same preparation by the preproinsulin (PPI)-specific CD8 T cell clone 3F2 specific for PPI_15–24], whereas RK9P9-3 displays background levels of specific lysis in the absence of cognate peptide. (b) Negative control, cytomegalovirus (CMV)-specific clone A2-CMVpp65_495–503, which does not kill islet cells in the absence of cognate peptide. (c) Analysis of supernatants of 18-h co-cultures of clone cells and islet cells for secretion of macrophage inflammatory protein (MIP)-1β. RK9C10-1 showed robust production of MIP-1β in response to islet cells, whereas RK9P9-3 production of MIP-1β is similar to background. (d) Negative and positive control responses to the same islet cell preparations. Cytotoxicity assays were performed at an effector : target ratio of 25:1. Bars show means of triplicates and error bars the standard errors of the mean (data are representative of n = 2 experiments using the same islet donor).