Demonstration of islet-autoreactive CD8 T cells in insulitic lesions from recent onset and long-term type 1 diabetes patients - PubMed (original) (raw)

Demonstration of islet-autoreactive CD8 T cells in insulitic lesions from recent onset and long-term type 1 diabetes patients

Ken T Coppieters et al. J Exp Med. 2012.

Abstract

A direct association of islet-autoreactive T cells with β cell destruction in human pancreatic islets from type 1 diabetes (T1D) patients has never been demonstrated, and little is known about disease progression after diagnosis. Frozen pancreas samples were obtained from 45 cadaveric T1D donors with disease durations ranging from 1 wk to >50 yr, 14 nondiabetic controls, 5 nondiabetics with islet autoantibodies, 2 cases of gestational diabetes, and 6 T2D patients. Sections were systematically analyzed for the presence of insulin-sufficient β cells, CD8(+) insulitic lesions, and HLA class I hyperexpression. Finally, consecutive sections from HLA-A2-expressing individuals were probed for CD8 T cell reactivity against six defined islet autoantigens associated with T1D by in situ tetramer staining. Both single and multiple CD8 T cell autoreactivities were detected within individual islets in a subset of patients up to 8 yr after clinical diagnosis. Pathological features such as HLA class I hyperexpression and insulitis were specific for T1D and persisted in a small portion of the patients with longstanding disease. Insulitic lesions consistently presented in a multifocal pattern with varying degrees of infiltration and β cell loss across affected organs. Our observations provide the first direct proof for islet autoreactivity within human islets and underscore the heterogeneous and chronic disease course.

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Figures

Figure 1.

Figure 1.

Histopathological survey of diabetic donors and controls for insulin (green) and MHC class I (red) expression. (A and B) Positive control staining for HLA-ABC on human spleen section and parallel isotype-matched negative control staining. (C) Nondiabetic individual with autoantibodies against ZnT8. (D) Case of undefined diabetes. (E and F) Nondiabetic donors. (G and H) Type 2 diabetics. (I and L) Longstanding T1D cases without HLA class I hyperexpression. All case numbers correspond to

Table S1

. Bars, 100 µm.

Figure 2.

Figure 2.

Histological analysis of longstanding cases shows protracted pathology. Frozen pancreas sections from patient 2 (A–C), patient 4 (D–F), patient 5 (G–I), and patient 6 (J–L) were stained for insulin, CD8, and with antibodies that recognize a common epitope from the HLA-A, -B, and -C gene products. Bars, 100 µm.

Figure 3.

Figure 3.

Immunohistochemical detection of islet antigen-reactive CD8 T cells within insulitic lesions of T1D patients. (A–C) Typical tetramer staining as observed on pancreas sections from recent-onset patients. (A and B) Reactivity against insulin and IGRP in two distinct islets from patient B. (C) insulin reactivity in patient A. (D) IGRP reactivity in longstanding patient 1. (E–H) In longstanding patient 2, tetramers loaded with IGRP and IA-2 yielded distinct signal. (E–H) Analysis of the same islet within consecutive sections showed that both these specificities could be detected within a single islet (G and H), whereas others contained only a single specificity (E and F). (I) Negative controls included HLA-mismatched pancreas tissue from patient C, which contained CD8+ insulitis but did not show tetramer signal. Arrows indicate tetramer-positive cells. Dashed lines are islet contours. Bars, 100 µm.

Figure 4.

Figure 4.

Immunofluorescent detection of islet–reactive CD8 T cells in patient 5. (A) Representative examples of positive signal from distinct CD8+ inflamed islets within eight consecutive pancreas sections. Background threshold was set on multiple islets from the section stained with irrelevant control tetramer and tetramer-associated signal was then collected using these instrument settings. (B) Reconstructed 3D image from a distinct pancreatic region, representing a single islet captured within consecutively cut sections (not by z-stacking, see Materials and methods). Magnified region shows tetramer positivity within distinct sections of the stack. Staining with ppIAPP in this patient’s pancreatic tissue exhibited high background, as indicated by its apparent lack of colocalization with CD8, and was therefore not interpreted. Bars, 20 µm.

Figure 5.

Figure 5.

Graphical overview of results from systematic staining and in situ tetramer assay. The graph represents combined data from

Table S1

, Table 1, and Table 2, according to presence of autoantibodies and disease duration. The number of samples within each category is shown at the top. Tetramer staining was performed only on HLA-0201–positive samples with insulitis. A total of 14 nondiabetic controls (

Table S1

), 5 auto-antibody positive subjects (

Table S1

), and 34 (

Table S1

) + 11 (Table I) Type 1 diabetics were included in the graph.

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