Hindawi Publishing Corporation
Anatomy Research International
Volume 2012, Article ID 457546, 20 pages
Norio Kanatsuna,1George K. Papadopoulos,2AntonisK. Moustakas,3and˚ Ake Lenmark1
1Department of Clinical Sciences, Sk˚ ane University Hospital (SUS), Lund University, CRC Ing 72 Building 91:10,
205 02 Malm¨ o, Sweden
2Laboratory of Biochemistry and Biophysics, Faculty of Agricultural Technology, Technological Educational Institute of Epirus,
47100 Arta, Greece
3Department of Organic Farming, Technological Educational Institute of Ionian Islands, 27100 Argostoli, Greece
Correspondence should be addressed to Norio Kanatsuna, email@example.com
Received 2 November 2011; Accepted 12 January 2012
Academic Editor: Ruijin Huang
Copyright © 2012 Norio Kanatsuna et al. This is an open access article distributed under the Creative Commons Attribution
License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly
Autoimmunity against pancreatic islet beta cells is strongly associated with proinsulin, insulin, or both. The insulin autoreactivity
is particularly pronounced in children with young age at onset of type 1 diabetes. Possible mechanisms for (pro)insulin auto-
immunity may involve beta-cell destruction resulting in proinsulin peptide presentation on HLA-DR-DQ Class II molecules in
pancreatic draining lymphnodes. Recent data on proinsulin peptide binding to type 1 diabetes-associated HLA-DQ2 and -DQ8 is
the proinsulin autoantigen over and over again through insulin-carrying insulin autoantibodies. In contrast to autoantibodies
against other islet autoantigens such as GAD65, IA-2, and ZnT8 transporters, it has not been possible yet to standardize the
insulin autoantibody test. As islet autoantibodies predict type 1 diabetes, it is imperative to clarify the mechanisms of insulin auto-
The pancreatic islets constitute about 2-3% of the pancreas
weight that is about 100 grams in adults . The islets repre-
sent the endocrine portion of the pancreas and are present as
the pancreas [2, 3]. Each pancreatic islet (Figure 1) is com-
posed of about 54% beta cells, 35% alpha cells, and 11%
delta cells in addition to connective tissue and capillary cells
. Proinsulin, converted to insulin (Figure 2), is the major
1 are produced by the alpha cells, somatostatin by the delta
cells, and pancreatic polypeptide by the PP cells. Pancreatic
islet cells are also reported to produce ghrelin , apelin [6,
7], and CART [8–10]. These polypeptide hormones may be
coexpressed with insulin in the beta cells or with other hor-
mone-producing cells . PP cells are more often seen in the
head of the pancreas, while alpha cells dominate the tail [11,
12]. Insulin is the life-saving hormone for people suffering
from type 1 and at times type 2 diabetes (see what follows).
More beta cells are available than necessary to main blood
glucose at normal levels. However, loss of insulin has catas-
trophic consequences. It has been estimated that 50% of the
pancreas may be removed by surgery without a development
disease leading to a progressive loss of beta cells as they are
attacked by the patients’ own immune system (for reviews
see [15–18]). T1D has a prodromal stage of islet autoim-
four autoantigens: insulin, GAD65, IA-2, or ZnT8 (Table 1),
to the clinical onset than older children, young adults, or
adults . These individuals may have multiple islet auto-
antibodies for years before the clinical onset of the dis-
ease . GAD65, not insulin, autoantibodies characterize
patients with latent autoimmune diabetes in adults (LADA)
[15–18]. It has been estimated that although an individual
may be positive for islet autoantibodies for months to years,
2Anatomy Research International
Figure 1: Immunocytochemistry of human islet cell subtypes.
An isolated human islet is shown after immunocytochemical
staining with antibodies against insulin (red), glucagon (blue), and
somatostatin (green). Note that the architecture of human islet
endocrine cells are distinctly different from that of rats and mice
where glucagon cells occupy the mantel of the islets. Published with
courtesy of Erik Renstr¨ om.
the clinical onset does not occur until 80–90% of the beta
cells have been killed . Hence, T1D appears due to the
selective autoimmune destruction of the pancreatic beta cells
[16, 22]. The major genetic factor for T1D is the HLA-
DQ locus on chromosome 6p21 . Recent reviews can be
found in [24, 25]. The association between the HLA Class
II genes and T1D is well established and several HLA-DQ
genotypes have been used to randomize newborn children
to follow up investigations of the development of islet
90%) of newly diagnosed T1D children do not have a first-
degree relative (father, mother, or sibling) already affected by
the disease. The presence of certain HLA-DQ already at birth
confers the genetic risk for T1D (Table 2). The highest risk
is conferred by the HLA-DQ2/8 genotype. The risk for T1D
with this genotype is highest in the young but is markedly
decreasing with increasing age [31, 32]. Affected sib-pairs
with T1D share HLA alleles more often than expected, and
alleles at the Class II DR and DQ loci are not only associated
with susceptibility to but also negatively associated with T1D
and therefore offer at least partial protection . In a large
population-based study the HLA DQ A1∗01:02-B1∗06:02
of 10; however, the negative association was decreased with
increasing age and lost at 30 years of age . It is noted that
other HLA genotypes, often with somewhat similar physic-
ochemical properties confer T1D risk in other populations
such as in Japan and China (Table 2) [35–39]. As indicated
the risk for T1D conferred by HLA-DQ is dependent on age.
It is therefore important that autoantibodies against insulin
are not only present particularly in young children at the
time of clinical diagnosis of T1D but also prior to the clinical
Table 1: Islet autoantigens in type 1 diabetes.
Present before clinical
onset [17, 40, 41]. As will be reviewed the autoimmune
reaction against insulin in T1D has been mapped in terms
of both cellular [42, 43] and humoral [17, 44] recognition.
However, insulin is a target not only in T1D but also in other
autoimmune conditions. In Hirata’s disease insulin autoan-
tibodies are detected in association with hypoglycemia in
the patient . This disease is also associated with HLA
Class II (Table 2) [46, 47]. The detailed mechanisms by
which patients recognize their own insulin as an autoan-
tigen may therefore have vastly different consequences for
the patient and these differences will be discussed in the
present paper. The reader is referred to the following reviews
where insulin autoimmunity in T1D [17, 48, 49] or in the
insulin autoimmune syndrome [47, 50, 51] has previously
Note. The first crystal structure of a human pathogenic TCR
in complex with HLA-A2—InsS15-23 has been determined,
and the TCR orientation with respect to the HLA-A2—
peptide complex is diagonal [52, 53].
2.Insulininthe Etiology of Type 1 Diabetes
T1D may be viewed as a two-step disease. The first step is the
initiation of islet autoimmunity; the second step is precipita-
tion of diabetes when islet autoimmunity has caused a major
β-cell loss (>80%), and insulin deficiency becomes clinically
manifest. The pancreatic beta cells are destroyed in an
aggressive autoimmune process. The immunopathogenesis
of T1D is associated with T-lymphocyte autoimmunity,
and the disease is often referred to as a T-cell-mediated
disease [79–81]. This is somewhat self-evident as an immune
response cannot be initiated without the help from CD4+
positive T-helper cells. Also it is rare that an immune
response does not engage all cells in the immune system as
cytotoxic CD8+ T cells are not able to develop without the
the activation of B cells to differentiate into autoantibody-
in the pathogenesis of T1D is illustrated in recent clinical
trials [82, 83]. Monoclonal antibody therapeutics, depleting
T cells (CD3 antibodies) or B cells (CD20 antibodies;
Rituximab), had similar effects to transiently inhibit the
Anatomy Research International3
Figure 2: The primary structure of human preproinsulin. The B1–B23 peptide bound to HLA-DQ8 (A1∗03:01-B1∗03:02) is shown in red
and can be found in Figure 3(a). The peptides bound to HLA-A2 as illustrated in (b), (c), and (d) are shown in blue for C6–C14, C6–C15 as
well as for B5–B14.
progression of beta-cell loss after the clinical onset of T1D
measured as residual beta-cell function [82–86].
Studies of children who have been followed from birth
indicate that autoantibodies against insulin often appear
before GAD65, IA-2, or ZnT8 autoantibodies [87, 88]. So
far, it is not known whether CD4+ or CD8+ T cells specific
for insulin can be detected in the peripheral blood prior to
and CD8+ T cells towards insulin as well as preproinsulin
(Figure 2) epitopes was reported both in newly diagnosed
and in long-term patients [62, 69, 70, 89–91]. The reactivity
of CD4+ T cells has been recorded mostly in connection
with the susceptibility alleles HLA-DR3, HLA-DR4, or both,
and only rarely in connection with HLA-DQ alleles [43, 65–
67, 92–97]. A wide variety of reactivities to the preproinsulin
(Figure 2) molecule have been reported. It is remarkable that
even CD4+ T cells specific for posttranslationally A6Cys-
A7Cys disulfide-linked insulin have been detected . This
also extends to the mouse reactivities where most H2-A/E
alleles appear to have very strongly binding epitopes to
the proinsulin molecule . By contrast, the reactivity of
CD8+ T cells is not linked to any particular HLA-A/B/C
allele. A large variety Class I molecules bind insulin epitopes
presented in T1D patients to sensitized T cells leading in vivo
to proinflammatory cytokine secretion (IFNγ, TNFα) and
cytotoxicity to beta cells [69, 70, 89–91]. It is remarkable
that a protein of only 110 amino acids contains so many
epitopes for a large spectrum of HLA Class I and Class II
alleles. It should be noted that insulin autoantibodies may
also react with proinsulin. Hence, it cannot be excluded that
the triggering autoantigen is proinsulin rather than insulin.
Still, it remains to be clarified why IAA is strongly associated
with islet autoimmunity in the very young.
In laboratory mice attempts were made to answer this
question. NOD mice unable to express the insulin 1 and
insulin 2 genes were given a mutated proinsulin transgene
in which tyrosine on residue 16 in the B chain was changed
to alanine. This mutation abrogated the T-cell stimulation
of insulin autoreactive T-cell clones. Female mice with only
the altered proinsulin did not develop insulin autoanti-
bodies, insulitis, or diabetes. It was suggested that the proin-
sulin/insulin molecules have a sequence that is a primary
target of the insulin autoreactivity in the spontaneously
diabetic NOD mouse. The conclusion was that the insulin
peptide B(9–23) might be an essential target of the immune
destruction of the NOD mouse . It is important to note
that the InsB9–23 sequence (identical in mouse I, mouse II,
epitopes: the H2-Kd-specific InsB15–23  and the I-Ag7-
first case, B16 tyrosine is the anchor at pocket B of this very
weakly binding peptide, and the alanine substitution leads
to no binding and no recognition by the respective T-cell
clone; hence, such cells are not even selected in the thymus
or the periphery. In the second case, the tyrosine residue on
position B16 would be the prime TCR contact residue p5, so
its substitution with alanine would most likely result in no
recognition at all.
4Anatomy Research International
Table 2: HLA haplotypes in the general population conferring sig-
nificant risk for islet autoimmunity and type 1 diabetes.
(a) Type 1 diabetes
Sub-Saharan Africa (haplotypes)
Latin America (haplotypes)
(b) Insulin autoimmune syndrome (haplotypes)
The observation in gene-manipulated laboratory mice
may be relevant to human T1D as B(9–23)-specific T cells
could be demonstrated in freshly isolated lymphocytes from
patients with recent-onset T1D as well as from subjects at
high risk for the disease . In humans the register for
binding of the InsB9–23 peptide to the four major HLA-DQ
alleles conferring susceptibility to T1D is identical and repre-
InsB12–20 in NOD mice [63, 101]. It was speculated that
these insulin autoreactive cells may contribute to the T1D
disease process as the T cells produced the proinflammatory
cytokine IFN-γ . This observation led to a clinical trial
B16Tyr and B18Cys were changed to Ala . The trial
was unsuccessful and one of the reasons may be found in
subsequent studies of another autoimmune disease, multiple
sclerosis: the orientation of the cognate T-cell recept-
or from pathogenic myelin basic protein-specific and
HLA-DRB1∗15:01-, HLA-DRB5∗01:01-, or -DQB1∗05:02/
A1∗01:02-restricted CD4+ T cells was strikingly different
from the canonical diagonal one seen in the case of
recognition of complexes of microbial peptides with MHC II
molecules [103–105]. Essentially, in all three cases the TCR
was tilted to recognize elements of the MHC II molecule and
the N-terminus of the bound peptide (mostly up to position
5); also, TCR recognition was sensitive to the alanine-sub-
stitution of a very limited set of residues and surprisingly
tions, indicating perhaps remarkable flexibility by the self-
reactive TCR in adjusting to the substitutions in order to
maintain productive binding to the complex. Any future
trials with altered peptide ligands must take these facts into
Current investigations in humans at risk for T1D still
do not answer the question what factor may trigger the in-
sulin autoimmunity. Numerous studies during more than
100 years suggest that virus infection of beta cells may ex-
plain the induction of islet autoimmunity. It has been sug-
gested based on experiments in laboratory mice that virus
infection and replication in beta cells will eventually lyses
these cells. Following the lytic event, virus as well as beta-
cell debris will be engulfed by local dendritic cells or by
during development) express on the outer leaflet of their cell
membrane phosphatidyl serine that is specifically recognized
by dendritic cells (the most effective of APC) and apoptotic
cells are engulfed in a way leading to tolerance. In case of an
as Toll-like receptors, TLR) present in all nucleated cells, are
activated upon contact with specific microbial components;
hence, the engulfment of virally infected cells under aberrant
conditions may not lead to immunological tolerance [107–
APC engulfing virally infected pancreatic islet beta cells
will be activated as they process the cell debris. The activat-
ed APC will move through the lymphatics to the draining
lymph nodes of the pancreas. Hence, presentation of beta
cell autoantigen (as well as of the virus antigens) will take
place in the lymph nodes rather than in the pancreatic islets
Anatomy Research International5
themselves. In the lymph nodes the APC will present islet
autoantigen including insulin to the T-cell receptor (TCR)
of CD4+ T-helper cells. Insulin is by far the most plentiful
protein in the beta cell, as a single human beta cell contains
about 12pg of this protein. Once such a CD4+ T-helper cell
munological reaction against insulin by recruiting both
CD8+ cytotoxic T cells and also antibody-producing B
cells. Pancreatic tissue from six T1D donors revealed that
Coxsackie B4 enterovirus could be demonstrated in the islets
in three of the six diabetic patients. The infection was indeed
specific to beta cells. However, the data indicted a nondes-
tructive islet inflammation mediated mainly by natural killer
cells . It is possible therefore that the destruction of
mune system. The NK-cell-mediated destruction may in
turn stimulate the regulated immune system to develop islet
autoimmunity but only in subjects with certain HLA-DQ
genotypes (Table 2).
An alternative hypothesis is that dietary cow’s milk
insulin could trigger beta-cell autoimmunity . A pri-
mary immune reaction against bovine insulin would be the
trigger of an immune reaction towards human insulin. How-
ever, when analyzing enterovirus infections in relation to the
consumption of cow’s milk formula, there seemed to be an
interaction between these two factors in inducing islet auto-
3.Type1 DiabeteswithIslet Autoantibodies
Most patients with T1D have islet autoantibodies at the
time of clinical diagnosis. Autoimmune diabetes rather than
T1D would therefore be a more appropriate designation of
the disorder. Several authors have reported that about 10–
15% of newly diagnosed patients with diabetes classified
with T1D have no islet autoantibodies at the time of clinical
onset. The question will then arise if patients have had islet
The use of autoantibody tests against ICA, insulin, GAD65,
IA-2, and the three variants of ZnT8 as well as islet cell anti-
bodies (ICAs) by indirect immunofluorescence, in more 600
newly diagnosed T1D children, indicates that only 5% did
not have any of the seven different types of autoantibodies
. It was not possible to determine if these children have
had autoantibodies and lost them prior to diagnosis. How-
ever, in children born to mothers with islet autoantibodies
during pregnancy, it was found that such children tended to
autoantibodies at birth may explain why some T1D children
are islet autoantibody negative at clinical diagnosis .
In Japan, a distinct subtype of T1D characterized by a
rapid clinical onset (duration of symptoms before presen-
tation and insulin treatment may be days and usually no
longer than two weeks) and without islet autoantibodies
has been established as fulminant type 1 diabetes mellitus
(FT1DM) . FT1DM, recently reviewed in [116, 117],
is considered to have the following three diagnostic criteria:
(1) rapid occurrence (within 7 days) of diabetic ketosis or
ketoacidosis after the onset of hyperglycemia symptoms (po-
lydipsia, polyuria and fatigue). Often patients have elevated
ketone bodies in the urine and serum at presentation;
(2) plasma glucose levels would be ≥16.0mmol/L but
glycated hemoglobin level < 8.5% at presentation; (3)
urinary C-peptide excretion <10μg/day or fasting serum C-
peptide level < 0.3ng/mL (<0.10nmol/L) and <0.5ng/mL
(<0.17nmol/L) after intravenous glucagon (or after meal)
tested within 1-2 weeks after presentation. Ancillary criteria
include the absence of autoantibodies against islet auto-
antigens such as insulin, GAD65, and IA-2 [116, 118].
Serum pancreatic enzyme levels (amylase, lipase, or elastase-
1) were found to be elevated in 98% of the patients. Flu-like
symptoms (fever, upper respiratory symptoms, etc.) or gas-
trointestinal symptoms (upper abdominal pain, nausea,
vomiting, or both, etc.) precede disease presentation in 70%
of the patients . The disease may also occur during
pregnancy or just after delivery [116, 120]. As a part of the
Japanese nationwide survey of FT1DM, it was found that the
Class II HLA-DR4-DQ4 (DRB1∗04:05-DQB1∗04:01) haplo-
. Interestingly enough, the HLA immunogenetics of
pregnancy-associated FT1DM may differ. In a recent study
it was reported that the haplotype frequency of HLA-
DRB1∗09:01-DQB1∗03:03 was significantly higher in preg-
nancy-associated FT1DM compared to both FT1DM not
associated with pregnancy as well as to controls .
Both biopsy and postmortem immunocytochemical inves-
tigations at onset revealed an infiltration of T lympho-
cytes and macrophages  in and around pancreatic
islets. Mononuclear cell infiltrations were also found in the
exocrine portion of the pancreas [123, 124]. A detailed post-
mortem histopathological investigation of three patients
revealed that macrophages and T cells but no natural killer
In addition, enterovirus may be present in beta cells in
association with several markers of innate system activation
and cytokine expression [126, 127]. It was speculated that a
strong inflammatory reaction may explain the rapid loss of
beta cells. Although islet autoantibodies were negative at the
time of clinical diagnosis, GAD65 autoantibodies appeared
tigating the possible appearance of insulin autoantibodies
or insulin antibodies (developing after insulin treatment).
FT1DM was also described in patients in Korea , China
, and France . Further studies of possible FT1DM
patients in other countries are needed.
4.Antigen Presentationof Insulinby HLA-DQ
Insulin autoantibodies are primarily detected in children
below the age of 5 years [15–17]. In Kappa statistics of
agreement there was a moderate to fair agreement between
any pairs of autoantibodies against GAD65, IA-2, or ZnT8
a slight agreement with any combination . It is often
observed that insulin autoantibodies are the first to appear,
at least in children younger than 3–5 years of age. How-
ever, it has been difficult to dissect the sequence of events
6Anatomy Research International
that leads to the formation of insulin autoantibodies in
very young children. One could envisage the following scen-
ario. Beta cells would be killed, perhaps lysed by a virus
infection. The dead beta cells or remnants thereof would
be engulfed by APC. These cells are activated and migrate
through the lymphatic system to the lymph nodes that
drain the pancreas. Antigen presentation to CD4+ T cells
would take place in the lymph node. It is possible that the
antigen presentation is particularly effective in small child-
ren leading to an early insulin autoantibody response .
This hypothetical mechanism is consistent with studies in
experimental animals [132, 133].
The APCs are expected to process preproinsulin (asso-
ciated with remnants of the endoplasmic reticulum), proin-
sulin, or insulin to peptides, which may be picked up by
HLA-DR, DQ, or both, heterodimers in the small lysosomal-
like transition vesicles. The higher affinity proinsulin/insulin
peptides will replace the invariant chain peptide (CLIP) that
“protects” the groove, and the resulting trimolecular com-
plex is eventually presented on the APC surface. The appear-
ance of insulin autoantibodies was found to be associated
with HLA-DQ8 as well as with the regulatory region of the
insulin gene (INS VNTR) on chromosome 11 . Mole-
cular studies have aimed at identifying which insulin pep-
tides might possibly be presented by HLA-DQ and -DR het-
erodimers on APC [134, 135]. In fact, insulin peptides from
the A chain have been identified as high affinity binders to
HLA-DRB1∗04:03, an allele associated with protection from
autoimmune diabetes in the high-risk HLA-DQ2/8 hetero-
zygotes . T cells oligoclonally expanded from pancre-
atic draining lymph nodes obtained from long-term T1D
patients recognized the insulin A1-15 epitope and were
restricted by DR4 . Yet these clones required high
amounts of insulin peptides to proliferate, so it is not clear
what stage of the pathogenesis they represent.
As previously noted, the insulin B13–B21 nonamer core
binds in the same register to all of the four HLA-DQ haplo-
types in the HLA-DQ2/8 heterozygote: A1∗05:01-B1∗02:01,
A1∗03:01-B1∗03:02, A1∗03:01-B1∗02:01, and A1∗05:01-
er these four particular trimolecular complex epitopes would
allow degenerate recognition by a single TCR on CD4+ T
cells (Figure 3(a)). One could easily note, however, that as far
asreactivity to insulin is concerned,the dominant protection
conferred by HLA-DQB1∗06:02 concerns its high affinity
binding in the register of InsB6-14 . The high affinity
binding would result in denying (stealing) this epitope from
any of the lower affinity binding diabetes-susceptible alleles.
Such high affinity binding may induce regulatory T cells
that could prevent the initiation of autoimmunity by diabe-
togenic T cells . It is important to note that the recog-
nition of the trimolecular complex (DQA1 chain—insulin
peptide—DQB1 chain) by CD4+ T cell TCRs is likely to
represent the very initiation of an autoimmune response
to (pro)insulin as an autoantigen. The presentation of the
insulin peptide in a DQ8 trimolecular complex would repre-
sent the very first initiation of an immune response to in-
sulin. The first responder cells are expected to be CD4+ T-
helper cells . Such cells have already been found in the
peripheral blood of newly diagnosed patients reactive with
the InsB9–23 peptide . Insulin-specific CD4+ T-helper
cells would in turn help both CD8+ T-cytotoxic cells as
well as B cells expressing autoantibodies recognizing insulin.
CD8+ T-cytotoxic cells would be expected to express a TCR
recognizing an insulin peptide presented on HLA Class I
molecules on the beta-cell surface (Figures 3(b)–3(d)).
5.Antigen Presentationof Insulinby
ClassI HLA-A,B,or C
CD8+ T-cytotoxic cells directed against insulin peptides
expressed on MHC Class I have been described both in the
NOD mouse and in man [100, 101, 141, 142]. Remarkably,
the two NOD mouse epitopes InsB15-23 and InsB25-C34,
respectively, bind either very weakly or very strongly to the
restriction element, H2-Kd[70, 142]. The preproinsulin epi-
allele in the Caucasian population, -A2) (Figures 3(b)–3(d))
span the entire molecule, and the frequency of reactivity to
the different epitopes varies [69, 70, 89–91]. Remarkably,
occasional high responses to certain peptides are also seen in
controls (SI > 4), with no other sign of autoimmunity .
In a pioneering study on in situ reactivity of persons at onset
of type 1 diabetes and patients with long-standing disease, it
has been shown that CTLs in HLA-A2+individuals showed
reactivity to single epitopes from 6 different autoantigens
(preproinsulin included, epitope 15–23). There was an
inversely proportional staining of pancreases with HLA-A2
tetramers with respect to age from diagnosis. In fact, no such
from the date of onset of type 1 diabetes .
An alternative pathway to the formation of IAA is illustrated
in Figure 4. This pathway remains to be fully explored in
humans. The clinical trial with Rituximab (CD20 mono-
clonal antibody) in newly diagnosed T1D children demon-
strates that depletion of B lymphocytes was associated with a
significant preservation of mixed meal-stimulated C-peptide
. The contribution of B lymphocytes to T1D pathogen-
esis may have been overlooked. As illustrated in Figure 4,
B lymphocytes with an antigen receptor recognizing insulin
would take up the insulin and process it to be presented
on HLA-DR, -DQ, or both. The trimolecular complex with
insulin would next be recognized by a TCR on the surface
of a mature, matching CD4+ T-helper cell. Upon the cell-to-
cell contact the CD4+ T-helper cell is activated to produce
cytokines (such as IL-4 or IL-10). These cytokines would
help the B cell to differentiate, replicate, and mature into an
IAA producing B lymphocyte, and eventually turning into a
plasma cell. It is important to note that Rituximab treatment
appeared to reduce antibody formation to new antigens such
as the bacteriophage PhiX174 . It was suggested that
Rituximab decreased both antibody production and isotype
switching . However, at the same time as residual C-
Anatomy Research International7
Figure 3: (a) Insulin B1–B23 epitope modeled into the α1β1 domain of the HLA-DQ8 heterodimer. It has been shown that CD4+ T cells
from type 1 diabetes patients are sensitized to this complex . The insulin peptide is in space-filling form with its atoms colored as
follows: carbon, green; oxygen, red; nitrogen, blue; hydrogen, white; sulfur, yellow. The HLA-DQ8 (A1∗03:01-B1∗03:02) heterodimer is
in van der Waals surface representation, colored according to atom charge (red, negative; blue, positive; gray, neutral; partial charges in
shades in-between). A few residues from the HLA-DQ molecule in contact with the antigenic peptide are shown via a transparency function
in stick form (same color notation as in the peptide with the exception of carbon that is in orange). Modeling and binding studies have
shown that the insulin peptide binds to the other three HLA-DQ diabetes-susceptible haplotypes (A1∗05:01-B1∗02:01, A1∗05:01-B1∗03:02,
A1∗03:01-B1∗02:01) in an identical register . This view is as seen from the T-cell receptor, which might fit with its symmetry axis in
an approximate diagonal fashion with respect to the peptide axis (fitting of TCRs specific for microbial peptides). The few examples of
structures of autoimmune TCR in complex with cognate MHC II-peptide complexes reveal an off-diagonal recognition involving mostly
the N-terminal half of the peptide and more selective contacts with the MHC II molecule. Molecule drawn from coordinates provided in
. (b) TCR view of the complex of HLA-A2, the most frequent Class I allele among Caucasians, with the proinsulin peptide C6–C14
(DLQVGQVEL; anchors in bold). Color code and conventions are as in (a). The peptide shown is part of epitope pool 60 (AEDLQVGQVEL,
EDLQVGQVEL, DLQVGQVEL, and LQVGQVEL). All four epitopes should bind well to HLA-A2, with SI < 3 in controls and SI > 3 in
3/6 T1D patients . The epitope depicted was first identified, though not tested on PBMCs of T1D patients in . (c) TCR view of the
complex of HLA-A2, with the insulin peptide C6–C15 (DLQVGQVELG, anchors in bold). Color code and conventions are as in (a). This is
part of epitope pool 61 (EDLQVGQVELG, DLQVGQVELG, LQVGQVELG, and QVGQVELG). The first three of the epitopes should bind
weakly to HLA-A2 and the fourth one hardly at all. It is also possible to have a different register altogether, especially for the last two peptides
(LQVGQVELGand QVGQVELG), with SI < 2 in all controls and SI > 3 in 2/6 T1D patients . (d) TCR view of the complex of HLA-A2
with the insulin peptide B5–B14 (HLCGSHLVEA), recently identified as an epitope for HLA-A2 in type 1 diabetes patients of recent onset,
with the very sensitive tetramer labeling using the quantum dot technique . This peptide belongs to pool 30  (QHLCGSHLVEA,
HLCGSHLVEA, LCGSHLVEA), where it has also shown reactivity. Note that this peptide will also bind very strongly to the protective allele
HLA-DQB1∗06:02, as well as to the slightly susceptible allele HLA-DQB1∗06:04, in the same core nonamer register B6–B14 [139, 140].
8Anatomy Research International
A B cell is triggered when it encounters
This combination of antigen
and MHC attracts the help of
a mature, matching T cell.
Cytokines secreted by the T cell
help the B cell to multiply
and mature into antibody
producing plasma cells.
Then it displays antigen fragments
bound to its unique MHC molecules.
Released into the blood,
antibodies lock onto matching
complexes are then cleared by
the complement cascade or
by the liver and spleen.
its matching antigen (insulin).
The B cell engulfs the antigen and digests it.
antigens. The antigen-antibody
Figure 4: Schematic representation of possible mechanisms by which insulin (or proinsulin) may be processed by B lymphocytes. The B
lymphocyte as viewed as a professional antigen-presenting cell (APC).
the levels of postdiagnosis GADA, IA-2A, and ZnT8A .
In the European-Canadian cyclosporine trial, it was demon-
strated that cyclosporine reduced the formation of insulin
antibodies in response to the regular insulin therapy given
to all the participating T1D patients . It is of interest
in this regard that Rituximab-treated patients were thought
to be able to develop immunological tolerance to bacteri-
ophage PhiX174 . In Stiff Person Syndrome, Rituximab
was reducing GADA in some  but not in all 
patients. Further studies are warranted to determine the in-
teraction between APC, T-helper cells, and B lymphocytes.
It needs to be established to what extent B lymphocytes may
be acting as APC that either initiate, maintain, or both, the
autoimmune response to insulin in children.
7.Analysisof IAAand Standardizationof
Measurement of IAA was initially limited by the large serum
volume required for the early immunoprecipitation as-
says, which used polyethylene glycol to separate immune
complexes . The first IAA assay required one milliliter
serum or plasma . Insulin was labeled by125I in an
approach similar to that which had been used for both
regular insulin radioimmunoassays as well as for insulin-
receptor-binding experiments . Later it was found that
labeling of multiple tyrosine residues compromised both
antibody—as well as receptor binding . These obser-
vations resulted in the now established use of only insulin
Anatomy Research International9
that is monoiodinated at position A14 . The improve-
ment in insulin iodination procedures [154, 155] made it
possible to develop alternative radiobinding assays that re-
quired less serum. This type of microassay allowed a major
say specificity [156, 157].
The development of alternative assays continues especi-
ally as the international standardization workshops demon-
strate significant interlaboratory variability [158, 159]. In
the first workshops it was found that IAA could not be
detected in an ELISA type of assay . The IDW 
and DASP [158, 159] international workshops to standardize
IAA continued to demonstrate that there was a poor inter-
laboratory consistency in the IAA assay [149, 156, 157, 162].
IAA determination varies more between laboratories com-
pared to other diabetes autoantibodies such GADA and
IA-2A. The Diabetes Antibody Standardization Program
associated with T1D .
8.IAA before the ClinicalOnset of Diabetes
The IAA radioimmunoassay was first tested in serum or
plasma samples from siblings to first-degree relatives with
were followed longitudinally for the appearance of IAA and
other islet autoantibodies [163, 164], in larger prospective
studies such as BABYDIAB [87, 165], DIPP [29, 166], and
DAISY [167, 168]. IAA was reported to show an association
between levels and risk for T1D, which was not observed for
GADA or IA-2A . These observations seemed also to be
corroborated in studies of children at genetic risk for T1D
based on HLA typing rather than having a first-degree rela-
tive with the disease . In the Diabetes Prevention Trial-
1 (DPT-1) , GADA, IA-2A were measured along with
ICA and IAA . No subjects with IAA as single autoanti-
bodies developed T1D . When a second autoantibody
appeared, any other autoantibody except IAA was added
significantly to the prediction of T1D . In the DIPP
study of children born with high-risk HLA, IAA tended to be
the first autoantibody to appear . It is therefore possible
that the initiation of the T1D disease process may involve
insulin itself or proinsulin, perhaps also preproinsulin [20,
41, 87, 171]. However, most authors suggest that the number
of islet autoantibodies is the strongest predictor of clinical
onset of T1D . It can however not be excluded that IAA
affinity may be a better predictor for T1D in children with
multiple autoantibodies [74, 172]. Indeed, high-affinity cell
surface antibody on B lymphocytes readily promotes their
differentiation and proliferation upon antigen binding, in
contrast to low-affinity antibody .
9.AreIAAEpitopes Related to
HLAClassI or IIHeterodimers?
There is a paucity of detailed investigations to clarify IAA
epitopes of proinsulin and insulin (Table 3). There is a lack
of information to what extent HLA-DQ or DR are associated
with IAA binding to either A chain, B chain, or proinsulin
autoantibody epitopes. Similar to HLA Class I peptide
binding (Figures 3(b)–3(d)), IAA was reported to recognize
the A8–A10 (13) epitope [74, 76, 77]. It is not clear why IAA
would recognize the same epitope as might be presented on
HLA Class I molecules. The B1–B3  as well as the B3
position [72, 73], both presented on HLA Class II molecules
(Table 2) may also be recognized by IAA. Studies with
systematic site-directed mutagenesis of the preproinsulin
cDNA may prove useful to map the IAA binding site more
carefully in relation to the HLA-DQ and DR genotypes of
newly diagnosed, non-insulin-treated T1D patients, or IAA-
positive nondiabetic subjects. Such studies are also impor-
tant as it has been suggested that the IAA levels may be the
as in children born to mothers with T1D in the BABY DIAB
study . Further studies are also needed to determine
epitope specificity in relation to the apparent polyclonal
nature of IAA and their similarity to the insulin antibod-
ies (IA) detected after insulin therapy has been initiated
the Diagnosisof Type1 Diabetes
The diagnostic sensitivity of IAA for T1D is on the average
only about 30%  but varies with the age at onset. In
children below the age of 3 years the diagnostic sensitivity
may be as high as 50–60%  but decreases to about
10% in T1D patients diagnosed after 20 years of age. It
was estimated that IAs produced in response to the insulin
treatment appear after about 7 days . When comparing
the two antibody types were comparable in several affinity
tests . The authors therefore concluded that both IAA
and IA were polyclonal in nature and that both developed in
response to insulin as the antigen . In some individuals,
it is therefore possible that insulin itself is able to break the
immunological tolerance to allow the formation of IAA. It
to type 2 diabetes patients in Japan was thought to induce
T1D . In these patients insulin antibodies (IAs) of high
ciency. These IAs were characterized by an extremely high-
affinity and a very low-binding capacity. The characteristics
of these insulin-treatment-induced IA were thought to be
similar to the IA found in the insulin autoimmune syndrome
. The insulin aspart had comparable immunogenicity
to human insulin, and antibodies developing in response
to either insulin seemed to cross-react [177–180]. High
titer insulin antibodies requiring immunosuppression have
been reported . Epitope-specific insulin antibodies may
develop in some patients who showed benefit when one
insulin analogue was replaced by another [181–183]. Similar
to animal insulins, also insulin analogues such as Lispro
insulin may cause insulin allergy .
10Anatomy Research International
Table 3: Proinsulin and insulin peptides recognized by HLA-DQ, HLA-DR, and HLA-A2 compared to the epitopes recognized by IAA.
Proinsulin/insulin sequence Reference
B11-C24, C28-A21, B20-C4,
B1–B16, B11–B27, C13–C29
Insulin, not proinsulin
c is cis and t is trans in transcription. S is signal peptide.
11.The InsulinGeneinType 1 Diabetesand
bility locus. The variable nucleotide tandem repeat (VNTR)
in the promoter region of the insulin gene may contribute
to T1D possibly by mechanisms of central tolerance .
The INS VNTR is composed of 14 to 15 bp variant repeats.
The shortest (Class I) variable number of tandem repeat
(VNTR) alleles was found to increase, whereas the longest
(Class III) alleles were observed to decrease in the patients
in comparison to the controls . The possible role of
central tolerance is illustrated by the observation that IAAs
in newly diagnosed T1D patients was found to be associated
with the INS VNTR polymorphism in some [34, 186] but
not all studies . In children born to mothers with T1D,
it was reported that the combination of genotyping for high-
risk HLA-DQ (e.g., HLA-DQ2/8 and 8/8) and INS VNTR
identified a minority of children with an increased T1D risk
. One study compared the INS VNTR polymorphism
between Finland and Sweden . The T1D risk genotypes
(Class I/I and I/III) were significantly more common in
Finland than in Sweden, both among patients and controls.
Class III homozygous genotypes showed varying degrees
of protective effect due to polymorphisms within Class III.
These observations suggest that heterogeneity between pro-
tective Class III lineages could exist.
However, it is important to note that the frequency
of disease-associated Class I haplotype is markedly high
(>90%) in the Japanese general population . While
comparisons of risk may be evaluated in high incidence
countries such as Finland and Sweden, this will be difficult in
the Japanese population. However, a meta-analysis suggested
that the Class I haplotype as such was significantly associated
with T1D in Japanese .
Anatomy Research International11
It was reported that most Class III alleles are associated
with higher levels of INS transcription than Class I alleles in
the thymus [191, 192]. Higher levels of INS expression in the
thymus may promote negative selection of insulin-specific T
lymphocytes, which may play a critical role in the pathogen-
esis of T1D . Studies of the human thymus is compli-
cated by the relative inaccessibility of this tissue as further
studies are needed to establish a possible relation between
INS VNTR genotypes, thymic expression of preproinsulin,
and a negative selection of (prepro)insulin-specific T lym-
phocytes. It is interesting to note that in one of the few
studies on the subject thus far the INS VNTR Class III allele,
in a homozygous or heterozygous state, has been shown to
promote regulatory type IL-10-producing CD4+ T-cell re-
Recent research has indicated that tolerance to insulin
and other tissue-specific proteins is affected via a number
of different mechanisms. For one, such proteins are contin-
uously produced and presented via MHC I and II proteins
in the thymus as well as in peripheral lymphoid organs
(spleen and lymph nodes) [195–197]. One of the important
transcription factors promoting the expression of many such
dism, Addison’s disease, and candidiasis, and invariably to
in the thymus and in peripheral organs and controls to vari-
able extents the expression of several tissue-specific proteins
in the thymus and in the lymph nodes. It seems that in the
latter organ in mice, both endothelial cells as well as fibro-
blastic reticular cells express in subsets a number of differ-
ent such proteins (e.g., tyrosinase, GAD67, and retinal S-an-
tigen) [197, 200]. The impaired expression of alpha-myosin
in the thymus in mice and humans was shown to be in-
ntial that all T1D autoantigens, especially insulin, be tested
in humans, for expression both in the thymus and in the ly-
mph nodes, in order to better understand the possible mech-
anisms of immune tolerance. The second important advance
in the field of immune tolerance has been the “rediscovery”
of the former suppressor T cells, now called regulatory T
as well [202, 203]. These cells are characterized by contact-
obligatory inhibition of proliferation of T-effector cells, and
their development and maintenance is dependent on TGFβ
and the transcription factor FoxP3. In fact, mutations in
the FoxP3 gene (located on the X-chromosome) lead to the
IPEX (immune dysfunction, polyendocrinopathy, entero-
pathy, X-liked) syndrome, characterized by stillbirth or food
allergy, diarrhea, and several endocrine autoimmune dis-
orders, with neonatal T1D as most prominent [204, 205].
Remarkably, in one such case with IPEX syndrome and
neonatal diabetes, bone marrow transplant at 18 months of
age resolved the immune deficiency and reduced the daily
insulin requirement . T-regulatory cells have received a
lot of attention in connection with T1D. It has been shown
that patients with the disease (newly diagnosed or of long
standing) have Tregs with insufficient regulatory capacities,
while there has been a disagreement regarding their absolute
deficiency, owing mostly to the different ways of defining the
phenotype of these cells [207–209]. The inability of Tregs
from T1D patients to regulate the activity of diabetogenic T
cells has been attributed to the nonsusceptibility of the latter
cells to regulation [210, 211]. It will be interesting to see if in
any way the induction and maintenance of insulin-specific
Tregs can alter the antigen-specific Th1-prone cytokine
response to a regulatory type, avoiding thus the outcome of
T1D . Remarkably, CD8+ T-regulatory cells specific
for preventing GAD65 autoreactivity at the T-cell level have
already been shown to exist in control subjects and be de-
ficient in diabetic patients . In a retrospective study of
pancreatic tissue from patients who died soon after clinical
presentation of T1D, it was noted that no FoxP3+ Tregs
could be detected, while other immunocytes were plentiful
in insulin-bearing islets , a finding whose significance
has yet to be assessed.
The insulin autoimmune syndrome (IAS, Hirata disease) is
characterized by a combination of fasting and sometimes
postprandial hypoglycemia, high serum concentrations of
total immunoreactive insulin, and presence in the serum
of polyclonal autoantibodies against native human insulin
It is noted that IAS has a strong genetic predisposition
and the majority of the IAS patients were reported from
Japan, where it is the third leading cause of hypoglycemia.
[46, 47]. It is also known that some drugs with sulphydryl
groups in their chemical structures can induce the formation
of insulin autoantibodies in predisposed individuals . A
patient with Graves’ disease who has the haplotype HLA-
Bw62/Cw4/DR4 with a specificity for DRB1∗04:06 may be at
risk of developing IAS after administration of methimazole
. Since 2003, a rapidly increasing number of patients
with alpha-lipoic-acid-induced IAS have been reported [51,
217–219]. Generally in Japan, alpha-lipoic acid has gained
popularity as a supplement for dieting and antiaging since
2004 . Although critical amino acid residue(s) for in-
sulin antigen presentation on the DRB1∗04:06 molecules
have been identified , the mechanisms that trigger this
presentation and the subsequent chronic autoimmune re-
sponse that generates both polyclonal and monoclonal in-
sulin autoantibodies remain to be clarified.
Humoral autoimmunity against insulin, first described in
1983 is established but needs to be better defined. The cur-
rent IAA radioimmunoassay continues to perform poorly in
simply too large to allow valuable comparisons between lab-
oratories throughout the world. The autoreactivity against
(pro)insulin also needs to be better defined. It will be neces-
dead or damaged beta cells is taken up by APC, processed
12Anatomy Research International
and finally presented on HLA-DR and -DQ molecules. Is
antigen-presentation by—DQ more critical than—DR to
induce autoreactivity? Once IAAs have been formed, it will
be critical to define the role of IAA-producing B cells and
plasma cells. What is the role of B cells as APC in the disease
process? Both CD4+ T-helper and regulatory T cells specific
for (pro)insulin need also to be identified in humans at
risk for T1D; the former have been shown to exist at the
population but not at the clonal level. Insulin-specific CD8+
T cells may be critical to identify in children at increased risk
for T1D as such children tend to develop IAA early, while
self-reactive immunocytes. It will be important to compare
the insulin autoantibodies IAS with the IAA in T1D. The IAS
is characterized by a combination of fasting hypoglycemia,
high concentration of total serum immunoreactive insulin,
and presence of autoantibodies to native human insulin in
serum . The release of insulin from the IAS insulin
autoantibodies may cause hypoglycemia, and further studies
are needed to explain why this type of autoantibodies may be
related to hypoglycemia with no apparent loss of beta cells.
The view of insulitis has been revised: it is no longer
considered an initiating phenomenon but rather the end
stage of prolonged subclinical presence of islet autoantibod-
ies including IAA. To what extent do insulin-specific CD8+
T cells contribute to insulitis? The possible effect of insulin
treatment as a trigger or accelerator of autoimmune (type
1) diabetes needs further exploration. Why is it that insulin
treatment may induce insulin dependency in Japanese type 2
diabetes patients, but not in others? Efforts need to be made
to answer the nagging question whether insulin admini-
stration accelerates the loss of beta cells in Caucasian T1D
Autoimmune polyendocrine syndrome
Cocaine and amphetamine regulated
Cluster of differentiation
Class-II-associated invariant chain
Diabetes association in support of youth
Diabetes antibody standardization
Diabetes prediction and prevention
Enzyme-linked immunosorbent assay
Forkhead box P3
FT1DM: Fulminant type 1 diabetes mellitus
GAD: Glutamic acid decarboxylase
HLA: Human leukocyte antigen
IA: Insulin antibodies
IAA: Insulin autoantibodies
IAS: Insulin autoimmune syndrome
IA-2: Insulinoma-associated antigen-2
ICA: Islet cell antibody
IPEX syndrome: Immune dysregulation,
MHC:Major histocompatibility complex
TGF:Transforming growth factor
TNF:Tumor necrosis factor
T1D:Type 1 diabetes
VNTR:Variable number of tandem repeats
ZnT8:Zinc transporter isotype 8.
International diabetes workshop
The research in the authors laboratory was supported in part
by the Swedish Research Council, the National Institutes of
Health (NIH), the Swedish Diabetes Association, the UMAS
Fund, the Knut and Alice Wallenberg Foundation, and the
Sk˚ ane County Council for Research and Development. The
research of G. K. Papadopoulos and A. K. Moustakas report-
ed here has been supported by a grant from the EPEAEK
II scheme of the 3rd Community Support Framework for
Greece(programArchimedes). TheSilicon GraphicsFuelin-
strument and the accompanying software used for molecular
simulations depicted here were obtained via an equipment
grant to Epirus Institute of Technology from the Epirus Re-
gional Development Program of the 3rd Community Sup-
port Framework (80% EU funds, 20% Hellenic State funds).
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