Curr Diab Rep (2016) 16: 53
DOI 10.1007/s11892-016-0738-2
IMMUNOLOGY AND TRANSPLANTATION (L PIEMONTI AND V SORDI, SECTION EDITORS)
Islet Autoantibodies
Vito Lampasona 1,2 & Daniela Liberati 1,2
Published online: 25 April 2016
# Springer Science+Business Media New York 2016
Abstract Islet autoantibodies are the main markers of
pancreatic autoimmunity in type 1 diabetes (T1D). Islet
autoantibodies recognize insulin (IAA), glutamic acid
decarboxylase (GADA), protein phosphatase-like IA-2
(IA-2A), and ZnT8 (ZnT8A), all antigens that are found
on secretory granules within pancreatic beta cells. Islet
antibodies, measured by sensitive and specific liquid
phase assays, are the key parameters of the autoimmune
response monitored for diagnostics or prognostics in
patients with T1D or for disease prediction in at-risk
individuals before T1D onset. Islet autoantibodies have
been the main tool used to explore the natural history of
T1D; this review summarizes the current knowledge
about the autoantigens and the phenotype of islets auto-
antibodies acquired in large prospective studies from
birth in children at risk of developing T1D.
Keywords Autoantibodies . T1D . GAD65 . Insulin . IA-2 .
ZnT8
This article is part of the Topical Collection on Immunology and
Transplantation
* Vito Lampasona
lampasona.vito@hsr.it
Daniela Liberati
liberati.daniela@hsr.it
1
Division of Genetics and Cell Biology, IRCCS San Raffaele
Scientific Institute, via Olgettina 60, 20132 Milano, Italy
2
Diabetes Research Institute, IRCCS San Raffaele Scientific Institute,
via Olgettina 60, 20132 Milano, Italy
Introduction
Islet autoantibodies have been fundamental in the recognition
of type 1 diabetes (T1D) as an autoimmune disease. A clear
role of the immune system in the pathogenesis of insulin-
dependent diabetes was widely recognized after Willy
Gepts’ systematic description of insulitis in pancreatic islets
at onset of diabetes [1]. However, the autoimmune nature of
this lymphocytic infiltration became fully established only in
1974, when Franco Bottazzo and Deborah Doniach [2] report-
ed the presence of islets cell antibodies (ICA) against pancreatic
islets in patients with insulin-dependent diabetes.
The detection of ICA, in combination with metabolic
studies of islet function in the pre-clinical phase of diabetes,
was key in laying the foundations of the current conceptual mod-
el of progressive disease stages in T1D pathogenesis, that was
elegantly formulated by George Eisenbarth in his 1986 seminal
paper [3].
Today, islet autoantibodies are key markers of T1D associ-
ated autoimmunity and are used in variety of clinical and re-
search settings that include the diagnostics of T1D [4], the
prediction of future T1D development [5], the use as
prognostic markers in the context of pancreas [6] or islet trans-
plantation [7], and as surrogate markers of efficacy in the con-
text of experimental therapies aimed at T1D prevention [8].
Islet Autoantibody Standardization Program
After the discovery of ICA, the discrepancies that soon
emerged across results from laboratories using different pro-
tocols for ICA testing, led to the establishment of the immu-
nology of diabetes workshops, a grassroots effort by the sci-
entific community aimed at the standardization of islet auto-
antibody assays and the evaluation of laboratory performance.
53 Page 2 of 10
The workshops proved remarkably successful for solving sci-
entific controversy and fostered the achievement of high qual-
itative standards in islet autoantibody measurement [9].
Today, the program continues under the name Islet
Autoantibody Standardization Program, previously known
as DASP [10], supervised by the Immunology of Diabetes
Society, supported by the NIDDK branch of the NIH, and
organized by the TrialNet Islet Cell Autoantibody Core
Laboratory at the Department of Pathology of the University
of Florida.
Overall, the major contribution of workshops has been the
clarification that assays in which antibody binding occurs at
the interface of a solid surface coated with antigen, like direct
ELISA, do not allow for sensitive and specific detection of
islet autoantibodies [11–15]. Consequently, the de facto gold
standard formats for islet autoantibodies measurement
are based on radio binding, bridge-ELISA, and ECL
assays [12, 16, 17], in which the interaction of antibodies with
antigen happens completely or partially in fluid phase.
Islet Autoantibody Antigens
Over the years, numerous protein and non-protein targets in
pancreatic islets were reported as antigens of the autoantibody
response associated with diabetes. However, only a handful of
these proposed autoantigens survived the test of time and the
close scrutiny of standardization workshops. Currently, four
proteins are confirmed autoantigens of islet autoantibodies:
insulin [18], glutamic acid decarboxylase 2 (GAD65) [19],
the protein tyrosine phosphatase like protein IA-2 [20], and
the zinc transporter 8 (ZnT8) [21]. In addition, autoantibodies
can be found in T1D patients that recognize also two closely
homologous isoforms GAD67 [22] and IA-2β [23].
Very recently, Christie et al. [24] reported the identification
of the tetraspanin protein family member TSPAN7 as
glima38, a pancreatic islet glycosylated membrane-
associated protein of 38 kDa, previously reported to be the
target of autoantibodies in >30 % of T1D patients.
A common thread linking antigens recognized by islet auto-
antibodies is their involvement in the secretory machinery of
pancreatic islet beta cells. In fact, IA-2, ZnT8, and TSPAN7 are
found on the membrane of secretory granules (SG), in addition
to insulin contained within, while GAD65 is instead found on
the cytoplasmic site of synaptic-like micro-vesicles (SLMV).
GAD65 Autoantibodies
The Autoantigen of GADA
GAD65 is a key enzyme for the biosynthesis of gamma-
aminobutyric acid (GABA), a major inhibitory neurotransmitter
Curr Diab Rep (2016) 16: 53
in the central nervous system. The GAD65 structure includes a
NH2 terminal domain, a middle domain containing a pyridoxal
5′-phosphate (PLP) cofactor binding site, and a COOH terminal
catalytic domain [25, 26]. The GAD67 isoform is encoded by a
distinct gene and is overall 76 % homologous to GAD65. Both
isoforms are obligate functional dimers that can form both
homodimers and heterodimers, and GAD65 is generally found
in a multi-protein complex associated with the cytoplasmic side
membrane of SLMV, where GABA is stored upon synthesis.
GAD65 undergoes several post-translational modifications like
phosphorylation, palmitoylation, N-terminal cleavage, and re-
versible association with PLP [27] which are critical for its
association with SLMV and the control of enzymatic activity.
GAD65 Tissue Expression
GAD65 expression is not exclusive of pancreatic beta cells but
is present at the mRNA and protein levels in several tissues
where GABAergic systems exist [28]. In addition to neurons
in the central and peripheral nervous systems, GABA exert
signaling functions that only partially understood in several
peripheral tissues and cells of the intestine, stomach, pancreas,
fallopian tube, uterus, ovary, testis, kidney, urinary bladder,
lung, eye lens, and liver. However, while expression of
glutamic acid decarboxylase(s) is therefore expected to take
place in selected cells within those tissues, exhaustive
studies of GAD65 localization outside the brain are still
lacking in humans.
In the adult human pancreas, GAD65 is highly expressed
exclusively in pancreatic islets, where it is found mostly in β
cells and in a minority of α cells [29]. Expression of the
GAD67 isoform is instead absent at the protein level, suggest-
ing that the regulation of GABA production in pancreatic
islets must be at least partially different from the brain [30].
Finally, expression of GAD67, but not GAD65, has been
reported in human thymic medullary epithelial cells, that are
in charge of presenting self-antigen during thymic
education of immature lymphocytes [31], suggesting a
potential mechanism of escape from negative selection
of autoreactive lymphocytes recognizing GAD65.
GADA Epitopes
GAD65 autoantibodies (GADA) have been extensively
characterized in terms of their epitope recognition on the
antigen in T1D and other diseases. Three broad categories of
GADA reactivities against GAD65 can be identified:
GADA directed against epitopes in the NH2 terminal
domain; GADA to epitopes contained within the middle
PLP binding domain [32]; and GADA to epitopes in the
COOH terminal domain [33].
Curr Diab Rep (2016) 16: 53
GADA in Other Diseases
GADA have been described in several severe autoimmune
pathologies of the central nervous system like classical stiff
person syndrome [34], cerebellar ataxia, Batten disease, and
others [35, 36]. In addition, GADA are relatively common in
patients affected by autoimmune polyendocrinopathy (APS)
[37] or by the IPEX syndrome [38]. While development of
T1D is relatively frequent in these patients, GADA are often
present in the absence of diabetes. In particular, in APS,
presence of GADA has been associated more strongly with
gastrointestinal dysfunction than with diabetes [37]. Finally,
GADA are the hallmark of patients with latent autoimmune
diabetes of the adult (LADA) a slowly progressing form of
autoimmune diabetes [39].
IA-2 Autoantibodies
The Autoantigen of IA-2A
IA-2 is a receptor type protein tyrosine phosphatase (PTP) like
protein intrinsic to the membrane of SG [40]. IA-2 includes a
large ectodomain, localized in the SG lumen, followed by a
single transmembrane, a short cytoplasmic juxtamembrane
(JM), and PTP-like cytoplasmic domains. IA-2β is an
isoform, encoded by a distinct gene, that is 88 % homologous
at the level of the PTP-like domain. The biological role
of IA-2 is only partially known: no ligand has been identified
that binds the IA-2 ectodomain, and the IA-2 PTP-like domain
does not possess phosphatase activity. In pancreatic beta cells,
in addition to form homodimers and heterodimers with IA-2β,
IA-2 can establish interactions with a range of diverse proteins
[41] indirectly interacting with actin microfilaments. In
pancreatic islets, upon Ca++ stimulated insulin exocytosis,
the IA-2 cytosolic domain can be cleaved, promoting the mo-
bilization of additional SG towards the plasma membrane
[42]. Furthermore, the cleaved cytosolic fragment of IA-2
can translocate to the nucleus, where it promotes the up-
regulation of genes associated with SG function in beta cells,
including insulin and IA-2 themselves [43], and cell prolifer-
ation [44]. IA-2 can therefore influence insulin secretion, SG
biogenesis and homeostasis, and β-cell expansion.
IA-2 Tissue Expression
IA-2 expression is not exclusive of pancreatic beta cells
but is present at the mRNA and protein levels in several
neuroendocrine cells, i.e., enriched for dense core peptide
secretory vesicles. In rodents, it has been demonstrated in
the pituitary gland, amygdala, and hypothalamus in the brain,
the adrenal medulla, autonomic nerve fibers and ganglia,
thyroid C cells, lung Kulchitsky cells, stomach somatostatin-
Page 3 of 10 53
producing D cells, and selected cells in the basalis of the
gastric glands [40, 45]. Within the pancreas, IA-2 is absent
from the exocrine tissue and exclusively found in β, α, and δ
cells of pancreatic islets [40]. Additionally, IA-2 expression
has been also described, at both the mRNA and protein levels,
in a subset of splenocytes and in the thymus, where it is
thought to be implicated in the negative selection of self-
reactive immune cells [46].
IA-2A Epitopes
IA-2A at onset of disease can be divided in two major
categories: those directed against multiple short mostly, but
not exclusively, linear, epitopes within the cytoplasmic JM
domain [47, 48] and those against conformational epitopes
within the PTP-like domains [49, 50].
Recently, a close structural interaction between the two
domains and the modulation of proteolytic processing of both
IA-2 cytoplasmic domains by autoantibodies bound to JM
epitopes was described [51], suggesting a model mechanism
through which epitope spreading might occur.
IA-2A in Other Diseases
IA-2A have been reported in few patients with APS [37, 52],
Stiff-man syndrome, and other neurological diseases [36, 53].
However, unlike for GADA, presence of IA-2A in these
patients is in the majority of the cases a marker of actual or
future presence of T1D.
ZnT8 Autoantibodies
The Autoantigen of ZnT8A
ZnT8 is a multi-pass protein with six transmembrane domains
and a histidine-rich loop between transmembrane domains IV
and V, a classical feature of ZnT proteins, a family of trans-
membrane proteins involved in the regulation of cellular zinc
content [54]. ZnT8 forms homodimers that are inserted in the
membrane of SG, with NH2 and COOH terminal domains
facing the cytoplasm while the histidine-rich loop is directed
towards the lumen. Other ZnT transporters are expressed in β
cells, but ZnT8 appears to be critical for the accumulation of
zinc into SG and the maintenance of stored insulin as a tightly
packed hexamers [55].
Polymorphic variants of ZnT8 at the level of amino acid
325 in the cytoplasmic COOH terminal domain have been
linked to increased type 2 diabetes risk and impaired glucose
homeostasis [56, 57] and altered rate of proinsulin conversion
to mature hormone [58].
53 Page 4 of 10
ZnT8 Tissue Expression
Expression of ZnT8 in humans is largely restricted to pancreatic
islets at the mRNA level [55]. While few other tissue have been
reported to contain minimal amounts the ZnT8 gene mRNA,
like fetal retinal pigment epithelium, adipocytes, and a subset of
lymphocytes, so far the presence of ZnT8 at the protein level is
confirmed only in pancreatic islets and possibly in cells of the
retina [59, 60]. Within islets, ZnT8 is highly enriched in β cells
[59] but apparently present at lower levels in other cell types
like α cells [61].
ZnT8A Epitopes
ZnT8A epitopes are present in the NH2 or the COOH terminal
cytoplasmic domains [21]. Less than 10 % of ZnT8A-positive
patients have antibodies to the NH2 terminal domain, while
almost all recognize the COOH terminal domain.
Interestingly, ZnT8A have been identified that are specific
for either the arginine or tryptophan polymorphic variants at
amino acid residue 325 in the COOH cytoplasmic domain
[62]. These ZnT8A appear to be restricted by the presence
of the corresponding polymorphism in the genome of patients
[63]. However, additional reactivities against the COOH
domain exist that are not affected by the residue encoded at
amino acid 325 [64].
ZnT8A in Other Diseases
ZnT8A are rare in the absence of T1D and found only in a
minor fraction of APS patients, subjects with antibodies
to 21-hydroxylase without polyendocrinopathy, and in
few relatives of T1D patients with antibodies to tissue
transglutaminase [21].
Insulin Autoantibodies
Insulin Tissue Expression
Insulin in the human adult body is expressed almost
exclusively in pancreatic beta cells, and the claim of local
expression of insulin in extra-pancreatic tissues in humans
has raised controversy in the past [65, 66]. However, in
humans, extra-pancreatic expression of insulin has been clearly
demonstrated at both the mRNA and protein levels in thymic
medullary epithelial cells [67, 68]. Potentially relevant for the
negative selection of autoreactive lymphocytes is the observed
association between specific polymorphic variants the insulin
gene, known to confer different genetic risk for T1D, and a
diminished amount of insulin mRNA in the thymus.
Curr Diab Rep (2016) 16: 53
IAA Epitopes
IAA epitopes have been only indirectly characterized using
different recombinant insulins that form other species, ana-
logs, and proinsulin as competitors in binding experiment
[69]. IAA are directed against conformational epitopes and
do not react with denatured or reduced antigen. Key amino
acid residues for binding of high affinity IAA have been
mapped within residues 8 to 13 of insulin A chain, while most
low affinity IAA are dependent on residues 28–30 of the B
chain. However, in some subjects, IAA also exist which are
not influenced by either of these A or B chain residues.
Competition experiments suggest that only IAA depending
for binding on A chain residues 8–13, but not B chain residues
28–30, can bind also proinsulin.
IAA in Other Diseases
IAA have been described in other autoimmune diseases like
APS [37, 52] and Stiff-man syndrome [36]. Unlike for GADA,
and similar to IA-2A and ZnT8A, IAA in these patients are rare
and associated with co-occurrence or future development of
T1D. High-titer insulin antibodies can develop in patients after
insulin treatment but their binding characteristics are not those
of IAA [70]. Insulin autoantibodies with binding characteris-
tics different from IAA also develop in patients affected by
immune hypoglycemia syndrome (IAS) [71].
Natural History and Phenotype of Islet Autoantibodies
Several large prospective studies have focused on the devel-
opment of autoimmunity in subjects at risk of T1D, either first
degree relatives of patients with T1D or subjects with HLA
haplotypes predisposing to T1D. In all of these studies, islet
autoantibody appearance and phenotype were the key param-
eter used to track autoimmunity in sequential samples taken
from birth over a time span that, by now, for some individuals
covers more than two decades.
Ziegler et al. [72•] recently reported a combined analysis of
islet autoantibodies development in 13,377 at-risk children
enrolled in three prospective studies from Germany, Finland,
and the USA. During follow-up, 1059 children (7.9 %) con-
verted to islet autoantibody positive. Risk of developing T1D
was clearly associated with presence of islet autoantibodies; in
fact, 403 of 428 (94.1 %) children who developed diabetes
tested positive for islet autoantibodies before disease onset.
The Genetics of Islet Autoantibodies
A clear association between specific HLA haplotypes and
development of islet autoantibodies has been long known. In
particular, GADA are associated more strongly with the HLA
DR3-DQ2 haplotype, while IAA and IA-2A are more
Curr Diab Rep (2016) 16: 53
associated with the HLA DR4-DQ8 haplotype [73]. Islet
autoantibody development is further modulated by the
interaction of these HLA haplotypes with other polymor-
phisms outside the HLA, like those in the PTPN22, ERBB3,
SH2B3, and INS genes [74••].
Age at Seroconversion to Islet Autoantibody Positive
The age at which de novo seroconversion to islet autoanti-
bodies positive occurs has been carefully analyzed in children
at risk of T1D, taking into account the transplacental transmis-
sion in utero of islet autoantibodies, in children of mothers
with T1D, and the persistence of maternal autoantibodies after
birth for several months [75, 76].
Islet autoantibodies develop very rarely before the age of
6 months, and the peak of incidence of seroconversion takes
place between 9 and 24 months of age [73, 77]. In their paper,
Giannopoulu et al. [73] calculated the rate of seroconversion
at 9 and 24 months at 18.5 and 21 new cases per 1000 high-
risk children per year, respectively. Incidence then dropped to
9.1, 5.1, and 6.9 seroconverters per 1000 high-risk children-
years at 5, 11, and 14 years of age. In the same report, the
incidence of IAA at seroconversion peaked earlier and was
significantly higher than that of GADA, IA-2A, and ZnT8A.
Simultaneous development of multiple islet autoantibodies
is also highest at 9 and 24 months of age compared to later
time points, while seroconversion to single autoantibody re-
activities, other than IAA, is less pronounced at the same time
points and less variable over time [78]. In children with a
single autoantibody at seroconversion that during follow-up
will become multiple autoantibody positive, the development
of additional reactivities occurs within 2 years in around 70 %
of the cases [79]. Multiple islet autoantibodies confer in-
creased risk of developing T1D by age 15 years, with children
having two or more autoantibodies showing a diabetes risk of
61.6 and 79.1 % [72•], respectively. Seroconversion at a
young age is a further risk factor, and children who developed
multiple autoantibodies between 1 and 2 years of age
progress to diabetes in about 75 % of the cases within
6 years [72•, 80••]. Overall, seroconversion before age
of 3 is linked to an increase risk of developing T1D
risk within 10 years (74.9 vs 60.9 %) [72•].
Children instead who had antibodies to just one islet
antigen at seroconversion and remain single autoanti-
body positive during follow-up have a lower T1D risk
by age 15 of 12.7 % [72•]. These children are slightly
older at seroconversion compared to multiple autoanti-
body positive, but antibodies appear nonetheless in
75 % of the cases before 8 years of age. When occurs,
progression to T1D can be relatively rapid also in these
children, in the majority of the cases within 4 years
from seroconversion [72•].
Page 5 of 10 53
Islet Autoantibody Phenotype and T1D Risk
Several studies analyzed the phenotype of islet autoantibodies
at seroconversion measuring characteristics like combinations
of different autoantibodies, titer, isotype, IgG sub-class,
epitope specificity, and affinity [33, 73, 81–88].
Isolated IAA or GADA are the most frequent single
reactivities found in the first positive samples of children at
risk but multiple autoantibodies can be present already
at seroconversion in 60 % of samples children who will
remain persistently autoantibody positive during follow-
up [73, 77, 80••]. IAA and GADA are also the key
components of multiple autoantibodies at seroconversion, as
an example in the study of Streck et al. [80••] the autoantibody
combinations found were as follows: IAA-GADA-IA-2A in
32 %, IAA-GADA in 17 %, IAA-IA-2A in 7 %, and GADA-
IA-2A in 5 % of the cases. However, the contribution to T1D
risk of other islet autoantibodies like IA-2A, IA-2β
antibodies, and ZnT8A should not be discounted. In
fact, development of autoantibodies to these targets, that
all reside on SG in beta cells, was clearly associated in
other studies with a faster progression to T1D and a
risk of developing disease within 5 years from their
appearance greater than 45 % [63, 89, 90].
The titer of islet autoantibodies at seroconversion is also
significantly associated with T1D risk. Although islet autoan-
tibody titer can fluctuate during follow-up, children with IAA
or IA-2A, but not GADA, in the upper quartile of the range of
antibody titer, have a two to fourfold increase in risk of devel-
oping T1D within 10 years compared to children with titers of
the same antibodies in the lower quartile [85].
Islet autoantibodies show a predominance of immunoglob-
ulins mostly of the IgG1 class for GADA, IA-2A, and in
addition also of the IgG3 and IgG4 class for IAA, already at
seroconversion [69, 81]. This is unlike the sequential appear-
ance of different immunoglobulin classes from IgM to IgG of
a classical de novo immunization process.
Already at seroconversion, islet autoantibodies can be
directed against a variety of epitopes on their target antigens.
However, there is also clear evidence of epitope spreading
during follow-up [33, 91]. Within children who are single
GADA positive, the analysis of epitope reactivity suggests
that progression to T1D rarely, if ever, occurs when GADA
are directed exclusively to epitopes within the NH2 terminal
domain of GAD65 [92, 93]. Conversely, epitope spreading
that leads to the appearance of autoantibodies cross-reactive
between IA-2 and IA-2β or IA-2β specific is associated with a
more aggressive autoimmunity and faster progression to
clinical onset of T1D [89, 90].
Finally, the affinity of islet autoantibodies, i.e., the strength
with which they bind to their antigens, in at-risk children, can
be already high at seroconversion and generally show little
changes during follow-up in most cases [92–95]. Again, this
53 Page 6 of 10
is unlike the progressive increase over time, due to affinity
maturation, of a classical humoral response upon immuniza-
tion. The affinity of IAA and GADA at seroconversion is
particularly heterogeneous, with high affinity mostly limited
to subjects who are multiple autoantibody positive or will
develop multiple antibodies during follow-up, while low
affinity is usually associated with persistent positivity for a
single antibody [92, 94, 95]. The affinity of IA-2A instead,
while being particularly variable across IA-2A positive
children, is almost always confined to a high range [95]. All
these evidences indicate that high affinity is an additional
feature of islet autoantibodies which is associated with
increased risk of T1D.
After the analysis of islet autoantibody phenotypes at
seroconversion, some conclusions can be drawn [73]:
children positive for multiple autoantibodies, or for just
IAA with high affinity that later convert to multiple
antibody positive, are those at the greatest risk of
T1D; early development of autoimmunity, around or
within the first year of life, is clearly associated with
an antibody response to insulin; the GADA antibody
response occurs somewhat later in life, between 2 and
3 years of age, and can be directed against diverse
epitopes, only some of which are associated with T1D
risk.
Overall, the characteristics of islet autoantibodies found at
seroconversion appear to be those of a mature humoral
response with a particularly rapid development in at
least some at-risk children. These findings are consistent
with the emerging picture of the existence of T1D subtypes, in
some of which B cells likely play a major role in disease
pathogenesis [96••, 97, 98].
Islet Autoantibodies at and After T1D Onset
Frequency of autoantibody positivity at onset of disease range
in decreasing order from >80 % for GADA, ≥70 % for IA-2A
[99], ≥65 % for ZnT8A, and >50 % for IAA. Confirming the
prevalent concept that GAD65 and IA-2 are the primary
targets of autoimmunity in T1D [100], autoantibodies to the
GAD67 and IA-2β isoforms are less frequent and essentially
always coexist with antibodies to GAD65 or IA-2,
respectively.
Studies in long-standing patients with T1D from the
BGolden years^ and BJoslin medalist study^ cohorts demon-
strate that autoantibodies can persist more than 50 years after
onset of disease in up to 50 % of patients [101, 102]. Persisting
islet autoantibodies are in most cases GADA and less
frequently ZnT8A and IA-2A [102]. Overall, the number
and titer of islet autoantibodies decline after progression to
T1D, and the type of persistent antibodies can be influenced
by parameters like age at onset and disease duration [103].
Curr Diab Rep (2016) 16: 53
Islet Autoantibodies Upon Intervention Therapies
Allogeneic pancreas or pancreatic islet transplantation are
established therapies aimed at the restoration of insulin pro-
duction and secretion in T1D patients. Despite the invariable
administration of an immunosuppressive regimen, transplant
rejection is frequently preceded by recurrence of autoimmuni-
ty. In fact, in several studies that evaluated islet autoantibodies
before and after pancreas or islet transplantation, the reappear-
ance of islet autoantibodies, or an increase in their titers, pre-
ceded graft failure [104–106]. Usually, GADA are the earliest
autoantibodies to reappear in these patients but the recurrence,
or the increase in titers, of IA-2A and ZnT8A are the strongest
predictors of graft function loss [7]. However, in several pa-
tients in which graft function was maintained, islet autoanti-
bodies were already present at the time of transplant and
remained stable during follow-up. This observation suggests
that in transplanted patients with long-standing T1D, similarly
to new onset patients, islet autoantibodies are not mediators of
direct cytopathic islet or graft damage. Furthermore, B cell
depletion performed in T1D patients as a treatment for co-
occurring diseases does not invariably lead to a relevant
change in islet autoantibody titers, despite a clear positive
clinical response [107]. Similarly, GADA, IA-2A, and
ZnT8A autoantibody levels were unaffected by an experimen-
tal treatment of new onset T1D patients with an anti-CD3
monoclonal, known to be associated not only with T cell but
also partial B cell depletion [108]. Intriguingly, in patients that
had high IAA titers and greater beta cell function at T1D
onset, treatment with an anti-CD3 monoclonal resulted in bet-
ter preservation of beta cell function and a reduction of the
further rise of insulin antibodies that follows the start of insu-
lin therapy [108].These findings suggest that, both in new
onset and long-standing T1D patients, islet autoantibodies
might be produced either by long-lived plasmacells, usually
unaffected by common immunosuppressive therapies, or by
short-lived plasmablasts, following T cell-dependent B cell
activation and differentiation, or both.
Conclusions
The study of islet autoantibodies has provided fundament in-
sights on the natural history of T1D. Islet autoantibodies will
continue to be the main marker of T1D autoimmunity in the
near future both in diagnostics and in clinical research. Further
technical advancements are emerging aimed at improving islet
autoantibody assays and the accuracy of T1D develop-
ment predictions based on autoantibodies. Nevertheless,
fundamental questions about the basis for development
of islet autoantibodies in T1D and their role in disease
pathogenesis remain unanswered and will likely be the
focus of future research efforts.
Curr Diab Rep (2016) 16: 53
Compliance with Ethical Standards
Conflict of Interest Vito Lampasona and Daniela Liberati declare that
they have no conflict of interest.
Human and Animal Rights and Informed Consent This article does
not contain any studies with human or animal subjects performed by any
of the authors.
References
Papers of particular interest, published recently, have been
highlighted as:
• Of importance
•• Of major importance
1. Gepts W. Pathologic anatomy of the pancreas in juvenile diabetes
mellitus. Diabetes. 1965;14:619–33. doi:10.2337/diab.14.10.619.
2. Bottazzo GF, Florin-Christensen A, Doniach D. Islet-cell antibodies
in diabetes mellitus with autoimmune polyendocrine deficiencies.
Lancet. 1974;2:1279–83.
3. Eisenbarth GS. Type I diabetes mellitus. A chronic autoimmune
disease. N Engl J Med. 1986;314:1360–8. doi:10.1056/
NEJM198605223142106.
4. Winter WE, Pittman D. The clinical application of islet autoanti-
body testing for the diagnosis of autoimmune diabetes. MLO.
Med Lab Obs. 2013;45:16, 20, 22 passim.
5. Bonifacio E. Predicting type 1 diabetes using biomarkers.
Diabetes Care. 2015;38:989–96. doi:10.2337/dc15-0101.
6. Mujtaba MA, Fridell J, Book B, Faiz S, Sharfuddin A, Wiebke E,
et al. Re-exposure to beta cell autoantigens in pancreatic allograft
recipients with preexisting beta cell autoantibodies. Clin
Transplant. 2015;29:991–6. doi:10.1111/ctr.12619.
7. Piemonti L, Everly MJ, Maffi P, Scavini M, Poli F, Nano R, et al.
Alloantibody and autoantibody monitoring predicts islet
transplantation outcome in human type 1 diabetes. Diabetes.
2013;62:1656–64. doi:10.2337/db12-1258.
8. Knip M, Akerblom HK, Becker D, Dosch H-M, Dupre J, Fraser W,
et al. Hydrolyzed infant formula and early β-cell autoimmunity: a
randomized clinical trial. JAMA. 2014;311:2279–87. doi:10.1001/
jama.2014.5610.
9. Lernmark A, Molenaar JL, van Beers WA, Yamaguchi Y, Nagataki
S, Ludvigsson J, et al. The Fourth International Serum Exchange
Workshop to standardize cytoplasmic islet cell antibodies. The
Immunology and Diabetes Workshops and Participating
Laboratories. Diabetologia. 1991;34:534–5.
10. Bingley PJ, Bonifacio E, Mueller PW. Diabetes Antibody
Standardization Program: first assay proficiency evaluation.
Diabetes. 2003;52:1128–36.
11. Greenbaum C, Palmer J, Kuglin B, Kolb H. Insulin autoantibodies
measured by radioimmunoassay methodology are more related to
insulin-dependent diabetes mellitus than those measured by
enzyme-linked immunosorbent assay: results of the Fourth
International Workshop on the Standardization of Insulin
Autoantibody Measurement. J Clin Endocrinol Metab. 1992;74:
1040–4. doi:10.1210/jc.74.5.1040.
12. Schmidli RS, Colman PG, Bonifacio E, Bottazzo GF, Harrison
LC. High level of concordance between assays for glutamic acid
decarboxylase antibodies. The First International Glutamic Acid
Page 7 of 10 53
Decarboxylase Antibody Workshop. Diabetes. 1994;43:1005–9.
doi:10.2337/diabetes.43.8.1005.
13. Verge CF, Stenger D, Bonifacio E, Colman PG, Pilcher C, Bingley
PJ, et al. Combined use of autoantibodies (IA-2 autoantibody,
GAD autoantibody, insulin autoantibody, cytoplasmic islet cell
antibodies) in type 1 diabetes: Combinatorial Islet Autoantibody
Workshop. Diabetes. 1998;47:1857–66.
14. Liu E, Eisenbarth GS. Accepting clocks that tell time poorly: fluid-
phase versus standard ELISA autoantibody assays. Clin Immunol.
2007;125:120–6. doi:10.1016/j.clim.2007.08.005.
15. Lampasona V, Schlosser M, Mueller PW, Williams AJK, Wenzlau
JM, Hutton JC, et al. Diabetes antibody standardization program:
first proficiency evaluation of assays for autoantibodies to zinc
transporter 8. Clin Chem. 2011;57:1693–702. doi:10.1373/
clinchem.2011.170662.
16. Brooking H, Ananieva-Jordanova R, Arnold C, Amoroso M,
Powell M, Betterle C, et al. A sensitive non-isotopic assay for
GAD65 autoantibodies. Clin Chim Acta. 2003;331:55–9. doi:10.
1016/S0009-8981(03)00088-3.
17. Miao D, Guyer KM, Dong F, Jiang L, Steck AK, Rewers M, et al.
GAD65 autoantibodies detected by electrochemiluminescence
assay identify high risk for type 1 diabetes. Diabetes. 2013;62:
4174–8. doi:10.2337/db13-0534.
18. Palmer J, Asplin C, Clemons P, Lyen K, Tatpati O, Raghu P, et al.
Insulin antibodies in insulin-dependent diabetics before insulin
treatment. Science (80- ). 1983;222:1337–9. doi:10.1126/
science.6362005.
19. Baekkeskov S, Aanstoot HJ, Christgau S, Reetz A, Solimena M,
Cascalho M, et al. Identification of the 64K autoantigen in insulin-
dependent diabetes as the GABA-synthesizing enzyme glutamic
acid decarboxylase. Nature. 1990;347:151–6.
20. Rabin DU, Pleasic SM, Palmer-Crocker R, Shapiro JA. Cloning
and expression of IDDM-specific human autoantigens. Diabetes.
1992;41:183–6. doi:10.2337/diabetes.41.2.183.
21. Wenzlau JM, Juhl K, Yu L, Moua O, Sarkar SA, Gottlieb P, et al.
The cation efflux transporter ZnT8 (Slc30A8) is a major
autoantigen in human type 1 diabetes. Proc Natl Acad Sci U S A.
2007;104:17040–5. doi:10.1073/pnas.0705894104.
22. Kaufman DL, Erlander MG, Clare-Salzler M, Atkinson MA,
Maclaren NK, Tobin AJ. Autoimmunity to two forms of
glutamate decarboxylase in insulin-dependent diabetes mellitus.
J Clin Invest. 1992;89:283–92. doi:10.1172/JCI115573.
23. Wasmeier C, Hutton JC. Molecular cloning of phogrin, a protein-
tyrosine phosphatase homologue localized to insulin secretory
granule membranes. J Biol Chem. 1996;271:18161–70. doi:10.
1074/jbc.271.30.18161.
24. McLaughlin K, Richardson CC, Ravishankar A, Feltbower R,
Morgan D, Christie M. Identification of tetraspanin-7 as a target
of autoantibodies in type 1 diabetes. Diabetes. 2016. doi:10.2337/
db15-1058.
25. Fenalti G, Law RHP, Buckle AM, Langendorf C, Tuck K, Rosado
CJ, et al. GABA production by glutamic acid decarboxylase is
regulated by a dynamic catalytic loop. Nat Struct Mol Biol.
2007;14:280–6. doi:10.1038/nsmb1228.
26. Kass I, Hoke DE, Costa MGS, Reboul CF, Porebski BT,
Cowieson NP, et al. Cofactor-dependent conformational
heterogeneity of GAD65 and its role in autoimmunity and neuro-
transmitter homeostasis. Proc Natl Acad Sci. 2014;111:E2524–9.
doi:10.1073/pnas.1403182111.
27. Wei J, Jin Y, Wu H, Sha D, Wu J-Y. Identification and functional
analysis of truncated human glutamic acid decarboxylase
65. J Biomed Sci. 2003;10:617–24.
28. Erdö SL. GABA outside the CNS. Springer Berlin Heidelberg; 2012.
29. Mally MI, Cirulli V, Otonkoski T, Soto G, Hayek A. Ontogeny
and tissue distribution of human GAD expression. Diabetes.
1996;45:496–501. doi:10.2337/diabetes.45.4.496.
53 Page 8 of 10
30. Kanaani J, Cianciaruso C, Phelps EA, Pasquier M, Brioudes E,
Billestrup N, et al. Compartmentalization of GABA synthesis by
GAD67 differs between pancreatic beta cells and neurons. PLoS
One. 2015;10:e0117130. doi:10.1371/journal.pone.0117130.
31. Gotter J, Brors B, Hergenhahn M, Kyewski B. Medullary epithe-
lial cells of the human thymus express a highly diverse selection of
tissue-specific genes colocalized in chromosomal clusters. J Exp
Med. 2004;199:155–66. doi:10.1084/jem.20031677.
32. Piquer S, Belloni C, Lampasona V, Bazzigaluppi E, Vianello M,
Giometto B, et al. Humoral autoimmune responses to glutamic
acid decarboxylase have similar target epitopes and subclass that
show titer-dependent disease association. Clin Immunol.
2005;117:31–5. doi:10.1016/j.clim.2005.06.009.
33. Bonifacio E, Lampasona V, Bernasconi L, Ziegler AG. Maturation
of the humoral autoimmune response to epitopes of GAD in pre-
clinical childhood type 1 diabetes. Diabetes. 2000;49:202–8.
34. Solimena M, Folli F, Denis-Donini S, Comi GC, Pozza G, De
Camilli P, et al. Autoantibodies to glutamic acid decarboxylase
in a patient with stiff-man syndrome, epilepsy, and type I diabetes
mellitus. N Engl J Med. 1988;318:1012–20. doi:10.1056/
NEJM198804213181602.
35. Saiz A, Blanco Y, Sabater L, González F, Bataller L, Casamitjana
R, et al. Spectrum of neurological syndromes associated with
glutamic acid decarboxylase antibodies: diagnostic clues for this
association. Brain. 2008;131:2553–63. doi:10.1093/brain/
awn183.
36. McKeon A, Robinson MT, McEvoy KM, Matsumoto JY, Lennon
VA, Ahlskog JE, et al. Stiff-man syndrome and variants: clinical
course, treatments, and outcomes. Arch Neurol. 2012;69:230–8.
doi:10.1001/archneurol.2011.991.
37. Soderbergh A, Myhre AG, Ekwall O, Gebre-Medhin G,
Hedstrand H, Landgren E, et al. Prevalence and Clinical associa-
tions of 10 defined autoantibodies in autoimmune polyendocrine
syndrome type I. J Clin Endocrinol Metab. 2004;89:557–62. doi:
10.1210/jc.2003-030279.
38. Lampasona V, Passerini L, Barzaghi F, Lombardoni C,
Bazzigaluppi E, Brigatti C, et al. Autoantibodies to harmonin
and villin are diagnostic markers in children with IPEX syndrome.
PLoS One. 2013;8:e78664. doi:10.1371/journal.pone.0078664.
39. Buzzetti R, Di Pietro S, Giaccari A, Petrone A, Locatelli M, Suraci
C, et al. High titer of autoantibodies to GAD identifies a specific
phenotype of adult-onset autoimmune diabetes. Diabetes Care.
2007;30:932–8. doi:10.2337/dc06-1696.
40. Solimena M, Dirkx R, Hermel JM, Pleasic-Williams S, Shapiro
JA, Caron L, et al. ICA 512, an autoantigen of type I diabetes, is an
intrinsic membrane protein of neurosecretory granules. EMBO J.
1996;15:2102–14.
41. Hu YF, Zhang HL, Cai T, Harashima S, Notkins AL. The IA-2
interactome. Diabetologia. 2005;48:2576–81. doi:10.1007/
s00125-005-0037-y.
42. Trajkovski M, Mziaut H, Schubert S, Kalaidzidis Y, Altkrüger A,
Solimena M. Regulation of insulin granule turnover in pancreatic
beta-cells by cleaved ICA512. J Biol Chem. 2008;283:33719–29.
doi:10.1074/jbc.M804928200.
43. Trajkovski M, Mziaut H, Altkrüger A, Ouwendijk J, Knoch K-P,
Müller S, et al. Nuclear translocation of an ICA512 cytosolic
fragment couples granule exocytosis and insulin expression in
{beta}-cells. J Cell Biol. 2004;167:1063–74. doi:10.1083/jcb.
200408172.
44. Mziaut H, Kersting S, Knoch K-P, Fan W-H, Trajkovski M,
Erdmann K, et al. ICA512 signaling enhances pancreatic beta-
cell proliferation by regulating cyclins D through STATs. Proc
Natl Acad Sci U S A. 2008;105:674–9. doi:10.1073/pnas.
0710931105.
45. Takeyama N, Ano Y, Wu G, Kubota N, Saeki K, Sakudo A, et al.
Localization of insulinoma associated protein 2, IA-2 in mouse
Curr Diab Rep (2016) 16: 53
neuroendocrine tissues using two novel monoclonal antibodies.
Life Sci. 2009;84:678–87. doi:10.1016/j.lfs.2009.02.012.
46. Diez J, Park Y, Zeller M, Brown D, Garza D, Ricordi C, et al.
Differential splicing of the IA-2 mRNA in pancreas and lymphoid
organs as a permissive genetic mechanism for autoimmunity
against the IA-2 type 1 diabetes autoantigen. Diabetes.
2001;50:895–900.
47. Lampasona V, Bearzatto M, Genovese S, Bosi E, Ferrari M,
Bonifacio E. Autoantibodies in insulin-dependent diabetes
recognize distinct cytoplasmic domains of the protein tyro-
sine phosphatase-like IA-2 autoantigen. J Immunol.
1996;157:2707–11.
48. Bearzatto M, Naserke H, Piquer S, Koczwara K, Lampasona V,
Williams A, et al. Two distinctly HLA-associated contiguous
linear epitopes uniquely expressed within the islet antigen 2 mole-
cule are major autoantibody epitopes of the diabetes-specific
tyrosine phosphatase-like protein autoantigens. J Immunol.
2002;168:4202–8.
49. Bearzatto M, Lampasona V, Belloni C, Bonifacio E. Fine mapping
of diabetes-associated IA-2 specific autoantibodies. J Autoimmun.
2003;21:377–82.
50. Dromey JA, Weenink SM, Peters GH, Endl J, Tighe PJ, Todd I, et
al. Mapping of epitopes for autoantibodies to the type 1 diabetes
autoantigen IA-2 by peptide phage display and molecular model-
ing: overlap of antibody and T cell determinants. J Immunol.
2004;172:4084–90.
51. McLaughlin KA, Richardson CC, Williams S, Bonifacio E,
Morgan D, Feltbower RG, et al. Relationships between major
epitopes of the IA-2 autoantigen in type 1 diabetes: implications
for determinant spreading. Clin Immunol. 2015;160:226–36. doi:
10.1016/j.clim.2015.06.002.
52. Gylling M, Tuomi T, Björses P, Kontiainen S, Partanen J, Christie
MR, et al. beta-cell autoantibodies, human leukocyte antigen II
alleles, and type 1 diabetes in autoimmune polyendocrinopathy-
candidiasis-ectodermal dystrophy. J Clin Endocrinol Metab.
2000;85:4434–40. doi:10.1210/jcem.85.12.7120.
53. Martino G, Grimaldi LM, Bazzigaluppi E, Passini N, Sinigaglia F,
Rogge L. The insulin-dependent diabetes mellitus-associated ICA
105 autoantigen in stiff-man syndrome patients. J Neuroimmunol.
1996;69:129–34. doi:10.1016/0165-5728(96)00104-X.
54. Chimienti F, Devergnas S, Favier A, Seve M. Identification and
cloning of a beta-cell-specific zinc transporter, ZnT-8, localized
into insulin secretory granules. Diabetes. 2004;53:2330–7.
55. Lemaire K, Chimienti F, Schuit F. Zinc transporters and their role
in the pancreatic β-cell. J Diabetes Investig. 2012;3:202–11. doi:
10.1111/j.2040-1124.2012.00199.x.
56. Xu K, Zha M, Wu X, Yu Z, Yu R, Xu X, et al. Association
between rs13266634 C/T polymorphisms of solute carrier family
30 member 8 (SLC30A8) and type 2 diabetes, impaired glucose
tolerance, type 1 diabetes—a meta-analysis. Diabetes Res Clin
Pract. 2011;91:195–202. doi:10.1016/j.diabres.2010.11.012.
57. Shan Z, Bao W, Zhang Y, Rong Y, Wang X, Jin Y, et al.
Interactions between zinc transporter-8 gene (SLC30A8) and
plasma zinc concentrations for impaired glucose regulation
and type 2 diabetes. Diabetes. 2014;63:1796–803. doi:10.
2337/db13-0606.
58. Kirchhoff K, Machicao F, Haupt A, Schäfer SA, Tschritter O,
Staiger H, et al. Polymorphisms in the TCF7L2, CDKAL1 and
SLC30A8 genes are associated with impaired proinsulin
conversion. Diabetologia. 2008;51:597–601. doi:10.1007/
s00125-008-0926-y.
59. Chimienti F, Devergnas S, Pattou F, Schuit F, Garcia-Cuenca R,
Vandewalle B, et al. In vivo expression and functional character-
ization of the zinc transporter ZnT8 in glucose-induced insulin
secretion. J Cell Sci. 2006;119:4199–206. doi:10.1242/jcs.03164.
Curr Diab Rep (2016) 16: 53
60. Deniro M, Al-Mohanna FA. Zinc transporter 8 (ZnT8) expression
is reduced by ischemic insults: a potential therapeutic target to
prevent ischemic retinopathy. PLoS One. 2012;7:e50360.
doi:10.1371/journal.pone.0050360.
61. Mohanasundaram D, Drogemuller C, Brealey J, Jessup CF, Milner
C, Murgia C, et al. Ultrastructural analysis, zinc transporters, glu-
cose transporters and hormones expression in New world primate
(Callithrix jacchus) and human pancreatic islets. Gen Comp
Endocrinol. 2011;174:71–9. doi:10.1016/j.ygcen.2011.07.004.
62. Wenzlau JM, Liu Y, Yu L, Moua O, Fowler KT, Rangasamy S, et
al. A common nonsynonymous single nucleotide polymorphism in
the SLC30A8 gene determines ZnT8 autoantibody
specificity in type 1 diabetes. Diabetes. 2008;57:2693–7.
doi:10.2337/db08-0522.
63. Achenbach P, Lampasona V, Landherr U, Koczwara K, Krause S,
Grallert H, et al. Autoantibodies to zinc transporter 8 and
SLC30A8 genotype stratify type 1 diabetes risk. Diabetologia.
2009;52:1881–8. doi:10.1007/s00125-009-1438-0.
64. Andersson C, Larsson K, Vaziri-Sani F, Lynch K, Carlsson A,
Cedervall E, et al. The three ZNT8 autoantibody variants together
improve the diagnostic sensitivity of childhood and adolescent
type 1 diabetes. Autoimmunity. 2011;44:394–405. doi:10.3109/
08916934.2010.540604.
65. Rosenzweig JL, Havrankova J, Lesniak MA, Brownstein M, Roth J.
Insulin is ubiquitous in extrapancreatic tissues of rats and
humans. Proc Natl Acad Sci U S A. 1980;77:572–6. doi:
10.1073/pnas.77.1.572.
66. Eng J, Yalow RS. Evidence against extrapancreatic insulin
synthesis. Proc Natl Acad Sci U S A. 1981;78:4576–8. doi:10.
1073/pnas.78.7.4576.
67. Pugliese A, Zeller M, Fernandez A, Zalcberg LJ, Bartlett RJ,
Ricordi C, et al. The insulin gene is transcribed in the human
thymus and transcription levels correlated with allelic variation
at the INS VNTR-IDDM2 susceptibility locus for type 1 diabetes.
Nat Genet. 1997;15:293–7. doi:10.1038/ng0397-293.
68. Vafiadis P, Bennett ST, Todd JA, Nadeau J, Grabs R, Goodyer CG,
et al. Insulin expression in human thymus is modulated by INS
VNTR alleles at the IDDM2 locus. Nat Genet. 1997;15:289–92.
doi:10.1038/ng0397-289.
69. Achenbach P, Koczwara K, Knopff A, Naserke H, Ziegler A-G,
Bonifacio E. Mature high-affinity immune responses to
(pro)insulin anticipate the autoimmune cascade that leads to type
1 diabetes. J Clin Invest. 2004;114:589–97. doi:10.1172/
JCI21307.
70. Hall TR, Thomas JW, Padoa CJ, Torn C, Landin-Olsson M,
Ortqvist E, et al. Longitudinal epitope analysis of insulin-
binding antibodies in type 1 diabetes. Clin Exp Immunol.
2006;146:9–14. doi:10.1111/j.1365-2249.2006.03178.x.
71. Wong SL, Priestman A, Holmes DT. Recurrent hypoglycemia
from insulin autoimmune syndrome. J Gen Intern Med.
2014;29:250–4. doi:10.1007/s11606-013-2588-9.
72.• Ziegler AG, Rewers M, Simell O, Simell T, Lempainen J, Steck A,
et al. Seroconversion to multiple islet autoantibodies and risk of
progression to diabetes in children. JAMA. 2013;309:2473–9.
doi:10.1001/jama.2013.6285. This study presents a
combined analysis of islet autoimmunity markers and
disease risk in all of the major ongoing prospective
studies worldwide.
73. Giannopoulou EZ, Winkler C, Chmiel R, Matzke C, Scholz M,
Beyerlein A, et al. Islet autoantibody phenotypes and incidence in
children at increased risk for type 1 diabetes. Diabetologia.
2015;58:2317–23. doi:10.1007/s00125-015-3672-y.
74.•• Törn C, Hadley D, Lee H-S, Hagopian W, Lernmark Å, Simell O,
et al. Role of type 1 diabetes-associated SNPs on risk of autoanti-
body positivity in the TEDDY Study. Diabetes. 2015;64:1818–29.
doi:10.2337/db14-1497. This study presents a comprehensive
Page 9 of 10 53
analysis of genetic contribution to development of islet
autoantibodies.
75. Hämäläinen AM, Ronkainen MS, Akerblom HK, Knip M.
Postnatal elimination of transplacentally acquired disease-
associated antibodies in infants born to families with type 1
diabetes. The Finnish TRIGR Study Group. Trial to reduce
IDDM in the genetically at risk. J Clin Endocrinol Metab.
2000;85:4249–53. doi:10.1210/jcem.85.11.6987.
76. Naserke HE, Bonifacio E, Ziegler AG. Prevalence, characteristics
and diabetes risk associated with transient maternally acquired
islet antibodies and persistent islet antibodies in offspring of
parents with type 1 diabetes. J Clin Endocrinol Metab. 2001;86:
4826–33. doi:10.1210/jcem.86.10.7931.
77. Krischer JP, Lynch KF, Schatz DA, Ilonen J, Lernmark Å,
Hagopian WA, et al. The 6 year incidence of diabetes-associated
autoantibodies in genetically at-risk children: the TEDDY study.
Diabetologia. 2015;58:980–7. doi:10.1007/s00125-015-3514-y.
78. Ziegler A-G, Bonifacio E. Age-related islet autoantibody inci-
dence in offspring of patients with type 1 diabetes. Diabetologia.
2012;55:1937–43. doi:10.1007/s00125-012-2472-x.
79. Chmiel R, Giannopoulou EZ, Winkler C, Achenbach P,
Ziegler A-G, Bonifacio E. Progression from single to multiple islet
autoantibodies often occurs soon after seroconversion: implica-
tions for early screening. Diabetologia. 2015;58:411–3. doi:10.
1007/s00125-014-3443-1.
80.•• Steck AK, Vehik K, Bonifacio E, Lernmark A, Ziegler A-G,
Hagopian WA, et al. Predictors of progression from the appearance
of islet autoantibodies to early childhood diabetes: The
Environmental Determinants of Diabetes in the Young (TEDDY).
Diabetes Care. 2015;38:808–13. doi:10.2337/dc14-2426. This
study presents an updated analysis of the contribution of islet
autoantibody combinations and titers to T1D risk.
81. Bonifacio E, Scirpoli M, Kredel K, Füchtenbusch M, Ziegler AG.
Early autoantibody responses in prediabetes are IgG1 dom-
inated and suggest antigen-specific regulation. J Immunol.
1999;163:525–32.
82. Hawa MI, Fava D, Medici F, Deng YJ, Notkins AL, De Mattia G,
et al. Antibodies to IA-2 and GAD65 in type 1 and type 2 diabetes:
isotype restriction and polyclonality. Diabetes Care. 2000;23:228–
33. doi:10.1128/AAC.03728-14.
83. Seissler J, Eikamp K, Schott M, Scherbaum WA, DENIS Study
Group. IA-2 autoantibodies restricted to the IgG4 subclass are
associated with protection from type 1 diabetes. Horm Metab
Res. 2002;34:186–91. doi:10.1055/s-2002-26708.
84. Hoppu S, Ronkainen MS, Kimpimäki T, Simell S, Korhonen S,
Ilonen J, et al. Insulin autoantibody isotypes during the prediabetic
process in young children with increased genetic risk of type 1
diabetes. Pediatr Res. 2004;55:236–42. doi:10.1203/01.PDR.
0000100905.41131.3F.
85. Achenbach P, Warncke K, Reiter J, Naserke HE, Williams AJK,
Bingley PJ, et al. Stratification of type 1 diabetes risk on the basis
of islet autoantibody characteristics. Diabetes. 2004;53:384–92.
doi:10.2337/diabetes.53.2.384.
86. A.J.K. Williams, V. Lampasona, M. Schlosser, P.W. Mueller, D.
Pittman, W.E. Winter RW, B. Akolkar, P.J. Bingley1 PA.
N-terminally truncated GAD discriminates progression in
GAD antibody positive relatives of patients with type 1
diabetes. Diabetologia. 2013;65:56.
87. Williams AJK, Lampasona V, Wyatt R, Brigatti C, Gillespie KM,
Bingley PJ, et al. Reactivity to N-terminally truncated
GAD65(96-585) identifies GAD autoantibodies that are
more closely associated with diabetes progression in rela-
tives of patients with type 1 diabetes. Diabetes. 2015;64:
3247–52. doi:10.2337/db14-1694.
88. Williams AJK, Lampasona V, Schlosser M, Mueller PW, Pittman
DL, Winter WE, et al. Detection of antibodies directed to the N-
53 Page 10 of 10
terminal region of GAD is dependent on assay format and
contributes to differences in the specificity of GAD autoantibody
assays for type 1 diabetes. Diabetes. 2015;64:3239–46. doi:10.
2337/db14-1693.
89. Achenbach P, Bonifacio E, Williams AJK, Ziegler AG, Gale
EAM, Bingley PJ, et al. Autoantibodies to IA-2beta improve
diabetes risk assessment in high-risk relatives. Diabetologia.
2008;51:488–92. doi:10.1007/s00125-007-0912-9.
90. De Grijse J, Asanghanwa M, Nouthe B, Albrecher N, Goubert P,
Vermeulen I, et al. Predictive power of screening for antibodies
against insulinoma-associated protein 2 beta (IA-2beta) and zinc
transporter-8 to select first-degree relatives of type 1 diabetic pa-
tients with risk of rapid progression to clinical onset of the disease:
implications for prevention trials. Diabetologia. 2010;53:517–24.
doi:10.1007/s00125-009-1618-y.
91. Naserke HE, Ziegler AG, Lampasona V, Bonifacio E. Early
development and spreading of autoantibodies to epitopes
of IA-2 and their association with progression to type 1
diabetes. J Immunol. 1998;161:6963–9.
92. Mayr A, Schlosser M, Grober N, Kenk H, Ziegler AG, Bonifacio
E, et al. GAD autoantibody affinity and epitope specificity identify
distinct immunization profiles in children at risk for type 1
diabetes. Diabetes. 2007;56:1527–33. doi:10.2337/db06-1715.
93. Bender C, Schlosser M, Christen U, Ziegler AG, Achenbach P.
GAD autoantibody affinity in schoolchildren from the general
population. Diabetologia. 2014;57:1911–8. doi:10.1007/s00125-
014-3294-9.
94. Achenbach P, Schlosser M, Williams AJK, Yu L, Mueller PW,
Bingley PJ, et al. Combined testing of antibody titer and affinity
improves insulin autoantibody measurement: Diabetes Antibody
Standardization Program. Clin Immunol. 2007;122:85–90. doi:10.
1016/j.clim.2006.09.004.
95. Krause S, Chmiel R, Bonifacio E, Scholz M, Powell M,
Furmaniak J, et al. IA-2 autoantibody affinity in children at risk
for type 1 diabetes. Clin Immunol. 2012;145:224–9. doi:10.1016/
j.clim.2012.09.010.
96.•• Arif S, Leete P, Nguyen V, Marks K, Nor NM, Estorninho M, et al.
Blood and islet phenotypes indicate immunological heterogeneity
in type 1 diabetes. Diabetes. 2014;63:3835–45. doi:10.2337/db14-
0365. This paper present striking data regarding the existence
of diverse sub-types of islet autoimmunity in T1D.
97. Skyler JS. Characterizing subgroups of type 1 diabetes. Diabetes.
2014;63:3578–80. doi:10.2337/db14-1160.
98. Thompson WS, Pekalski ML, Simons HZ, Smyth DJ, Castro-
Dopico X, Guo H, et al. Multi-parametric flow cytometric and
genetic investigation of the peripheral B cell compartment in
Curr Diab Rep (2016) 16: 53
human type 1 diabetes. Clin Exp Immunol. 2014;177:571–85.
doi:10.1111/cei.12362.
99. Kawasaki E, Yu L, Rewers M, Hutton J, Eisenbarth G. Definition
of multiple ICA512/phogrin autoantibody epitopes and
detection of intramolecular epitope spreading in relatives
of patients with type 1 diabetes. Diabetes. 1998;47:733–42. doi:
10.2337/diabetes.47.5.733.
100. Jayakrishnan B, Hoke DE, Langendorf CG, Buckle AM, Rowley
MJ. An analysis of the cross-reactivity of autoantibodies to
GAD65 and GAD67 in diabetes. PLoS One. 2011;6:e18411.
doi:10.1371/journal.pone.0018411.
101. Keenan HA, Sun JK, Levine J, Doria A, Aiello LP, Eisenbarth G,
et al. Residual insulin production and pancreatic ß-cell turnover
after 50 years of diabetes: Joslin Medalist Study. Diabetes.
2010;59:2846–53. doi:10.2337/db10-0676.
102. Richardson CC, Dromey JA, McLaughlin KA, Morgan D,
Bodansky HJ, Feltbower RG, et al. High frequency of
autoantibodies in patients with long duration type 1 diabe-
tes. Diabetologia. 2013;14:2538–40. doi:10.1007/s00125-
013-3017-7.
103. Tridgell DM, Spiekerman C, Wang RS, Greenbaum CJ.
Interaction of onset and duration of diabetes on the percent of
GAD antibody and insulinoma-antigen 2 antibodies-
positive subjects in the Type 1 Diabetes Genetic
Consortium Database. Diabetes Care. 2011. doi:10.2337/
dc10-1903.
104. Roep BO, Stobbe I, Duinkerken G, van Rood JJ, Lernmark A,
Keymeulen B, et al. Auto- and alloimmune reactivity to human
islet allografts transplanted into type 1 diabetic patients. Diabetes.
1999;48:484–90.
105. Braghi S, Bonifacio E, Secchi A, Di Carlo V, Pozza G, Bosi E.
Modulation of humoral islet autoimmunity by pancreas allotrans-
plantation influences allograft outcome in patients with type 1
diabetes. Diabetes. 2000;49:218–24.
106. Bosi E, Braghi S, Maffi P, Scirpoli M, Bertuzzi F, Pozza G, et al.
Autoantibody response to islet transplantation in type 1 diabetes.
Diabetes. 2001;50:2464–71.
107. Madaschi S, Rossini A, Formenti I, Lampasona V, Marzoli SB,
Cammarata G, et al. Treatment of thyroid-associated orbitopathy
with rituximab—a novel therapy for an old disease: case report
and literature review. Endocr Pract. 2010;16:677–85. doi:10.4158/
EP09385.RA.
108. Demeester S, Keymeulen B, Kaufman L, Van Dalem A, Balti EV,
Van de Velde U, et al. Preexisting insulin autoantibodies predict
efficacy of otelixizumab in preserving residual β-cell function in
recent-onset type 1 diabetes. Diabetes Care. 2015;38:644–51.
doi:10.2337/dc14-1575.