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Insulin-dependent diabetes mellitus and its animal models
F Susan Wong and Charles A Janeway Jr*
Major questions are still unanswered in the understanding of
the pathogenesis of type 1 diabetes, including the important
question of the nature of the autoantigen(s) recognised in the
development of disease. In the nonobese diabetic mouse
model, there is new evidence that insulin plays an important
role: not only is it an antigen for pathogenic CD4+ T cells but
also it is recognised by highly diabetogenic CD8+ T cells.
Further studies using transgenic mice have also highlighted the
role of glutamic acid decarboxylase as an autoantigen. It
remains to be seen whether one or both of these autoantigens
can be used in strategies to prevent human diabetes.
Addresses
Section of Immunobiology and *Howard Hughes Medical Institute,
Yale School of Medicine, LH 416, 310 Cedar Street, PO Box 208011,
New Haven, CT 06520-8011, USA
*e-mail: Charles.janeway@yale.edu
Current Opinion in Immunology 1999, 11:643–647
0952-7915/99/$ — see front matter © 1999 Elsevier Science Ltd.
All rights reserved.
Abbreviations
GAD glutamic acid decarboxylase
NOD nonobese diabetic
Introduction
Over the past 25 years since the recognition that type 1 dia-
betes has an immune basis and that T cells play an
important role in its pathogenesis, the question of the tar-
gets recognised by these T cells has been of major interest.
The earliest clues to the nature of the autoantigenic targets
originally came from the detection of autoantibodies in pre-
diabetic patients and newly diagnosed patients with
diabetes. Proteins recognised by these autoantibodies
include insulin [1], the 64K protein — also known as glu-
tamic acid decarboxylase (GAD) [2,3] — and the tyrosine
phosphatase ICA512 (also known as IA-2) [4–6]. Indeed, the
presence of a combination of antibodies that includes those
reactive to these three autoantigens can predict subsequent
development of diabetes in prediabetic relatives with a sen-
sitivity of >80% [7] and may also be useful in the general
population [8]. However, it is considerably more difficult to
test for T cell reactivities in diabetic or prediabetic patients
and studies in this area have yielded conflicting results. The
results of the recent work of the first T cell workshop have
highlighted these problems [9]. Nevertheless T cells from
diabetic patients have been cloned that respond to peptides
of insulin, GAD and ICA512 although their role in patho-
genesis has yet to be established.
Parallel with this, the role of T cells has also been exten-
sively studied in the nonobese diabetic (NOD) mouse
model of diabetes and also in the BB rat model. Unlike
humans, however, it has sometimes been difficult to detect
643
antibodies to putative autoantigens in NOD mice [10,11].
Nevertheless, as T cells are thought to play a major role in
the pathogenesis of diabetes, many studies have been per-
formed to try to identify the islet-specific autoantigens to
which T cells respond and the antigens identified in the
human antibody studies have been investigated. It is
important to bear in mind that, in these studies, many dif-
ferent substances (at least 125) have been able to prevent
diabetes in NOD mice and these may not all have rele-
vance in humans [12•].
Most work to date has focused on insulin and its precursors
as well as on the enzyme GAD and, more recently, on
ICA512. To examine the roles of these antigens, a number
of approaches have been used which investigate the spon-
taneous responses to the antigens, the ability to obtain
immune responses and the use of these various antigens in
tolerization protocols to prevent disease. In addition, in the
NOD mouse model, it has been possible to use transgenic
technology to try to examine the role of important putative
autoantigens to the development of diabetes. We will
review recent work on these antigens. There are other
antigens, such as heat shock protein 60 (HSP60), for which
a role in the NOD mouse has been shown but, as there is
not known to be a parallel in humans at present, these will
not be discussed in this review.
Insulin as an autoantigen
Insulin or its precursors are obvious target autoantigens.
Insulin is abundantly expressed in the pancreatic islets. Its
expression was thought to be islet specific but recently it
has been shown that there are cells within the thymus that
express peripheral antigens, such as insulin and GAD [13],
in humans [14,15] and in rats and mice [16–18]. Within the
islets of NOD mice there are clearly documented CD4+
T cells that recognise insulin [19], specifically a peptide
consisting of amino acids 9–23 of the B chain, and when
cloned these cells are capable of causing diabetes on adop-
tive transfer [20]. Most recently, in the identification of the
first autoantigen for pathogenic CD8+ T cells in the NOD
mouse, we have shown that a highly diabetogenic CD8+
T cell clone recognises a peptide consisting of amino acids
15–23 of the B chain of insulin [21••]. Thus, the insulin B
chain is clearly a target for both pathogenic CD4+ and
CD8+ T cells in the NOD mouse.
If insulin-expressing antigen-presenting cells are found in
the thymus, an obvious question is how autoreactive
T cells responding to this antigen escape from negative
selection in the thymus. It is possible that, under normal
circumstances, the peptides recognised by the autoreactive
T cells are not present at a sufficient concentration to
induce negative selection. When proinsulin II is expressed
as a transgene under an MHC class II promoter such that
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644 Autoimmunity
it is expressed in the thymus as well as peripherally, spon-
taneous diabetes was prevented in the NOD mouse [22].
Similarly, the intrathymic injection of insulin B chain but
not A chain inhibited the development of disease [23].
This implies that if proinsulin or insulin is expressed at a
high enough level in the thymus, tolerance can occur.
The NOD mouse has a genetic susceptibility to diabetes,
the main locus being found within the MHC region on
chromosome 17. Recent studies have suggested that the
NOD MHC class II molecule, I-Ag7, is a poor peptide-
binder and it has been shown that peptides form loose
complexes with it [24]. In addition it appears that side-
chains have limited influence on binding to I-Ag7 and
many peptides can therefore bind weakly [25], which
could allow potentially autoreactive T cells to enter the
periphery. This could be one of the reasons that mice bear-
ing this MHC may have an increased complement of cells
that could be involved in diabetes [26].
An analogous situation may exist for autoreactive CD8+
T cells selected on relatively common MHC class I mole-
cules, Kd and Db. Of the few pathogenic CD8+ T cell
clones that have been isolated, most are restricted to Kd
[27–30]. Although motifs have been defined for Kd, it is
clear that the insulin peptide recognised by the highly
pathogenic CD8+ T cell clone G9C8 [21••] does not con-
form to the suggested motif (X–Y–X–X–X–X–X–X–L/I;
single-letter code is used for amino acids, with X repre-
senting any amino acid) [31]. Instead, there is a small
glycine residue at position 9 giving rise to relatively weak
binding of the peptide (FS Wong, CA Janeway Jr, unpub-
lished data). This may explain why these cells have
escaped from negative selection in the thymus and hence
are available for reactivity in the periphery in NOD mice.
The existence of pathogenic CD4+ and CD8+ T cells in
the NOD mouse that can recognise peptides of insulin,
remarkably from the same region in the B chain, clearly has
important implications for immunoprevention.
In the attempt to cross the bridge from mouse investiga-
tions to human studies, several groups have used transgenic
mice bearing human MHC class II HLA-DR and -DQ mol-
ecules and lacking mouse class II — on the C57BL/6
(non-diabetes-prone) genetic background as well as in the
NOD mouse — to investigate epitopes of preproinsulin
[32,33] and GAD (as will be discussed later). The insulin B
chain peptide shown to be important in the NOD mouse
was not identified by either of these investigations but
these were not in vivo studies and it will be interesting to
discover, in further development of these models, whether
cells can be induced to react to this region of the B chain
and cause insulitis/diabetes. A CD4+ T cell clone isolated
from a recently diagnosed diabetic patient was shown to
react to insulin B chain 11–27, restricted by HLA-DR [34].
Previous studies using the NOD mouse have shown that
insulin or insulin B-chain peptide 9–23 given orally [20,35],
subcutaneously [36] or intranasally [37], protect from dia-
betes. Insulin that has been metabolically inactivated by
changing one amino acid in the B chain is also effective at
diabetes prevention [38]. Certainly, CD4+ T cells that have
a protective function and recognise insulin as their target
antigen have also been isolated [39]. In addition, it has
been shown that regulatory γδ CD8+ T cells are induced
after nasal administration of insulin [37]. It remains to be
seen whether success in modulating diabetes in the NOD
mouse using various modes of delivery of insulin/proin-
sulin or non-metabolically active insulin can be translated
similarly to humans.
GAD as an autoantigen
The other major putative autoantigen in humans, GAD, is
somewhat different. GAD exists in two independently
encoded forms, GAD-65 and GAD-67, differing mainly in
the amino acid sequence at the amino-terminal end. GAD is
of major importance in the central nervous system where it
catalyzes the formation of the inhibitory neurotransmitter,
γ-amino butyric acid (GABA). GAD is mainly found in neu-
roendocrine tissues and the distribution of the isoforms of
GAD differs between man and the mouse animal models of
diabetes. GAD-65 is more predominant in the pancreas in
humans whereas GAD-67 more highly expressed in the
islets of mice [40,41]. The physiological role of GAD and
GABA within the pancreas is still uncertain.
Two groups initially reported that T cell reactivity to GAD
may be important in the NOD mouse, the earliest respons-
es to this antigen being found in young NOD mice at a time
when reactivity to other antigens was not detectable [42,43].
Subsequently, a number of studies have been carried out in
the NOD mouse that show that it is possible, by adminis-
tration of GAD in a number of ways [23,43–45], to induce
tolerance to GAD and prevent diabetes. However, this was
not true of all peptides of GAD used [23]. Although there is
a report of CD4+ T cells that are responsive to GAD and are
diabetogenic in NOD mice [46], to date no CD8+ T cells
have been isolated that respond to GAD in mice although
they have been found in humans [47].
GAD has a region of similarity to the Coxsackie virus
P-2C protein. One suggestion for its role in the patho-
genesis of diabetes is via the phenomenon of molecular
mimicry. T cells in humans that may respond in a viral
infection could cross-react with this endogenous
autoantigen [48]. Using the BDC2.5 TCR-transgenic
mouse model of diabetes [49], where the T cells respond
to an islet granule antigen whose nature is not yet known
[50], it was shown that infection with Coxsackie B4 virus
accelerated diabetes [51•]. Although it was not shown in
this report, it was inferred from previous studies that the
transgenic T cells are not responsive to the viral proteins
and it was suggested that diabetes following viral infec-
tion in the TCR-transgenic mice occurs more in
response to bystander activation of inflammatory cells
than by molecular mimicry.
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Insulin-dependent diabetes mellitus and its animal models Wong and Janeway
Several transgenic models have now been developed in
NOD mice in order to attempt to further clarify the role of
GAD in diabetes. Under normal circumstances in the
NOD mouse, GAD-65 is expressed at very low levels in
the pancreas. One of the engineered models had a mutat-
ed GAD expressed on an MHC class I promoter such that
GAD-65 was expressed highly in all tissues [52]. The rea-
son for mutating the GAD at a site that destroyed catalytic
activity, but was presumed not to affect immunoreactivity,
was that previous attempts to generate transgenic mice had
failed. This was thought perhaps to be due to toxicity of
widespread expression of functional GAD. It was found
that the expression of mutated GAD in this manner in one
out of three transgenic lines resulted in a greater degree of
insulitis in the males and a greater final incidence of dia-
betes. This widespread expression had the effect of
neither centrally deleting GAD-responsive cells nor caus-
ing peripheral tolerance.
Another transgenic model expressed GAD-65 on the
RIP-7 promoter, such that the GAD-65 was expressed at
high levels in the pancreatic islets [53]; this would be
more analogous to the level of expression in humans and
in rats. Two lines were generated, one of which —
expressing higher levels of GAD — was protected from
diabetes; it was suggested that this was due to an increase
in the production of IL-10. There was no apparent differ-
ence in the incidence of diabetes in the other line (with
the transgene inserted into the Y chromosome). It is
clearly important to observe more than one transgenic
line and the site of insertion of the transgene may signif-
icantly affect the phenotype.
The third and most recently reported transgenic model
investigating GAD as an autoantigen used an antisense con-
struct to prevent the expression of both GAD-65 and
GAD-67 [54••]. In one of three transgenic lines, with appar-
ently the greatest reduction in GAD-65 and GAD-67
expression, not only were diabetes and insulitis abolished
but also the cells developing in these transgenic mice were
unable to transfer diabetes. In addition, the islets were pro-
tected from adoptive transfer of diabetic spleen cells. These
data are interesting and perhaps might have relevance for
use in engineered islets for use in transplants. However, it
raises a number of questions as to exactly how the antisense
construct is working because the results presented make it
unlikely that protection is solely due to the immunological
effects of suppression of GAD-65 and GAD-67.
MHC-class-II-binding peptides of GAD-65 have been
mapped in HLA-DRB1*0401-transgenic mice [55,56] and
HLA-DQA1*0301/DQB1*0302-transgenic mice [55,57,58].
It is clear that the best MHC-binding peptides are not
necessarily those that are important in stimulating T cells
in vivo and this is in keeping with the idea that autoreac-
tive T cells, both in humans and in rodents, may be
selected because of poor MHC-binding. These mice also
have the potential to be used to investigate in vivo disease
645
processes. In HLA-DQA1*0301/DQB1*0302-transgenic
C57BL/6 mice, lacking mouse class II, T cell lines
responding to GAD peptides 247–266 and 509–528 were
adoptively transferred. There was no effect when the
T cells were transferred alone but the cells caused insuli-
tis when a mild islet insult in the form of low-dose
streptozotocin, insufficient to cause disease itself, was
given [59•]. As more experience is gained with these mod-
els, it will be interesting to see whether they will be able
to allow discoveries related to CD4+ T cells reacting to
peptides in the context of I-Ag7 in the NOD mouse to be
translated also to the human class II molecules known to
be important in susceptibility to diabetes.
Although GAD is clearly important as an autoantigen,
questions still remain as to the precise role it plays and if it
can be used in a strategy to prevent diabetes.
ICA512 as an autoantigen
Although this tyrosine phosphatase has been considered
to be important in the prediction of type 1 diabetes in
humans, there is little known about its importance in the
rodent. Anti-ICA512 antibodies have been found in the
NOD mouse; the antibodies appeared close to the onset
of hyperglycaemia and were usually transient [10].
Nondiabetic NOD mice also produced anti-ICA512 but
at a later age and at lower levels than the diabetic NOD
mice. There are no published studies on T cell reactivi-
ties to this antigen in rodents and only time will tell
whether rodent models will be of assistance in investigat-
ing this autoantigen.
Conclusions
Insulin and GAD clearly are important autoantigens in dia-
betes. A considerable amount of evidence now exists for
the role of insulin as a major autoantigen and indeed trials
are currently underway (i.e. DPT-1 [60] and EPP-SCIT
[61]) to attempt to use insulin to prevent diabetes.
Whether GAD, or peptides of GAD, can be used in a sim-
ilar manner remains to be determined as some of the
evidence is still controversial and, unlike insulin, GAD
autoreactivity has not been demonstrated in the BB rat
model [62]. Not enough, as yet, is known about ICA512.
There is still considerable activity to try to identify other
autoantigens and to assign primacy of one antigen over
another. It is possible that more than one antigen may be
involved in the earliest phases of disease, especially if
autoreactive T cells recognising a number of antigens may
exist in genetically predisposed individuals. It is clear that
further studies are required to firmly establish the role of
both GAD and ICA512 and a major focus of future
research activity should be on how to therapeutically mod-
ulate the immune response to known autoantigens.
Acknowledgements
FSW is a recipient of a Juvenile Diabetes Foundation International (JDFI)
career development award and CAJ is an Investigator of the Howard
Hughes Medical Institute. The work is supported by the National Institutes
of Health and the JDFI.
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646 Autoimmunity
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