The Journal of Immunology
Human Type 1 Diabetes Is Associated with T Cell
Autoimmunity to Zinc Transporter 8
MyLinh Dang,1Jennifer Rockell,1Rebecca Wagner,1Janet M. Wenzlau, Liping Yu,
John C. Hutton, Peter A. Gottlieb, and Howard W. Davidson
Recently we demonstrated that zinc transporter 8 (ZnT8) is a major target of autoantibodies in human type 1 diabetes (T1D).
Because the molecules recognized by T1D autoantibodies are typically also targets of autoreactive T cells, we reasoned that this
would likely be the case for ZnT8. To test this hypothesis, IFN-g–producing T cells specific for ZnT8 in the peripheral blood of 35
patients with T1D (,6 mo after onset at blood draw) and 41 age-matched controls were assayed by ELISPOT using a library of
23 overlapping dipeptide pools covering the entire 369 aa primary sequence. Consistent with our hypothesis, patients showed
significantly higher T cell reactivity than the matched controls, manifest in terms of the breadth of the overall response and the
magnitude of responses to individual pools. Therefore, the median number of pools giving positive responses (stimulation index ‡ 3)
in the control group was 1.0 (range, 0–7) compared with 6.0 (range, 1–20; p , 0.0001) for the patients. Similarly, the median
stimulation index of positive responses in controls was 3.1 versus 5.0 in the patients (p , 0.0001). Individually, 7 of 23 pools showed
significant disease association (p , 0.001), with several of the component peptides binding the disease associated HLA-DR3 (0301)
and -DR4 (0401) molecules in vitro. We conclude that ZnT8 is also a major target of disease-associated autoreactive T cells in
human T1D, and we suggest that reagents that target ZnT8-specific T cells could have therapeutic potential in preventing or
arresting the progression of this disease.The Journal of Immunology, 2011, 186: 6056–6063.
principally the result of the activation and expansion of autore-
active T cells specific for b-cell Ags (1). Consequently, consid-
erable efforts have been made over the past 25 y to identify the
molecular targets of potentially diabetogenic T cells and the
immunodominant epitopes within them (2). Such information has
contributed significantly to our understanding of the pathophy-
siology of T1D, for example by revealing possible molecular
mimicry between viral and islet Ags (3). It also underpins the
development of peptide-based, Ag-specific therapeutic strategies,
and it is responsible for the identification of novel biomarkers of
preclinical disease (4).
Zinc transporter 8 (ZnT8) is primarily restricted to the islets of
Langerhans, with the highest expression being in pancreatic b cells
lthough autoantibodies are currently the best biomar-
kers of type 1 diabetes (T1D) in humans, it is generally
accepted that the destruction of pancreatic b-cells is
(5). Recently we demonstrated that, depending on the age of onset,
autoantibodies to ZnT8 (ZnT8A) are present in 60–80% of newly
diabetic subjects attending the Barbara Davis Center clinic (6)
(J.M. Wenzlau, H.W. Davidson, and J.C. Hutton, unpublished ob-
servations). This result has since been replicated by ourselves
and others in different populations (7, 8); it is now evident that
ZnT8A occur with a similar prevalence to those against the “gold
standard” T1D autoantigens proinsulin, the 65-kDa form of glu-
tamic acid decarboxylase (GAD65), and insulinoma Ag 2 (IA-2).
In addition, ZnT8A overlap with, but are independent of, these
other biomarkers. Consequently, protocols that measure ZnT8A in
addition to Abs against the other three major targets have a sig-
nificantly enhanced detection rate of diabetes-related autoimmu-
nity (6, 7, 9, 10).
are believed to be activated independently (11), previous studies
have shown that T cells recognizing epitopes from proinsulin,
GAD65, and IA-2 can be detected in the peripheral blood of
patients atthe onsetofclinical disease(2).Given thisprecedent,we
reasoned that ZnT8 is also likely to be a target of autoreactive
T cells in human T1D. To investigate this hypothesis, we analyzed
the frequency of proinflammatory IFN-g secreting cells in PBMCs
from 35 patients with recently diagnosed diabetes (,6 mo after
diagnosis) and 41 age-matched controls, using a library of over-
lapping peptides spanning the entire 369 aa primary sequence of
ZnT8. The results of this initial study suggest that ZnT8 is indeed
a significant T cell target in T1D, and identify 6 regions of the
molecule that likely contain key disease associated epitopes.
Materials and Methods
Participants of either gender aged 6–30y (Table I) were recruited from
patients, relatives, and volunteers attending the Barbara Davis Center for
Childhood Diabetes in accordance with protocols approved by the Colo-
rado Multiple Institute Review Board. Subjects with clinical disease of .6
mo duration at the time of blood draw were excluded from the study, as
Barbara Davis Center for Childhood Diabetes, University of Colorado Denver, Au-
rora, CO 80045
1M.D., J.R., and R.W. contributed equally to this work.
Received for publication November 19, 2010. Accepted for publication March 13,
This work was supported by Juvenile Diabetes Research Foundation Autoimmunity
Prevention Consortium Center Grant 4-2007-1056, National Institutes of Health Grant
R01 DK052068 (to J.C.H.), National Institutes of Health Grant R56 DK052068 (to
J.C.H. and H.W.D.), National Institutes of Health Grant P30 DK57516 “University of
Colorado Health Sciences Center Diabetes and Endocrinology Research Center”, and
the Children’s Diabetes Foundation of Denver.
Address correspondence and reprint requests to Dr. Howard Davidson, Barbara Davis
Center for Childhood Diabetes, Mailstop B140, 1775 Aurora Court, Aurora, CO
80045. E-mail address: firstname.lastname@example.org
The online version of this article contains supplemental material.
Abbreviations used in this article: GAD65, glutamic acid decarboxylase; GADA,
autoantibodies to GAD65; IA-2, insulinoma Ag 2; IAA, insulin autoantibodies;
ICA512, autoantibodies to IA-2; SI, stimulation index; T1D, type 1 diabetes; ZnT8,
zinc transporter 8; ZnT8A, autoantibodies to ZnT8.
were control subjects who tested positive for one or more diabetes auto-
antibodies (insulin autoantibody [IAA], autoantibody to GAD65 [GADA],
ICA512, or ZnT8A). PBMCs were prepared from heparinized blood using
Ficoll-paque Plus (GE Healthcare, Piscataway, NJ), and aliquots were used
immediately for either ELISPOT analysis or DNA preparation.
Control peptides P1 (Proinsulin C19-A3; GSLQPLALEGSLQKRGIV),
Insulin B9-23 (SHLVEALYLVCGERG), GAD3 (GAD65 335-352;
TAGTTVYGAFDPLLAVAD), GAD4 (GAD65 554-575; VNFFRMVIS-
NPAATHQDIDFLI), R2 (IA-2 853-872; SFYLKNVQTQETRTLTQFHF),
and R5 (IA-2 709-736; LAKEWQALCAYQAEPNTCATAQGEGNIK) (12,
13) were synthesized at .95% purity (University of Colorado Cancer
Center Proteomics Core) and added from stocks of 10 mM in DMSO. A
PEPscreen library of overlapping 20mers spanning the entire 369 residue
primary sequence of ZnT8 was obtained from Sigma Genosys (St. Louis,
MO). The library was designed to contain 51 20mers, with consecutive
peptides overlapping by 13 residues. However, 5 failed synthesis, pro-
viding an actual library of 46 peptides. Individual peptides were resus-
pended in DMSO to a final concentration of 50mM. (Fig. 1A)
Indirect ELISPOT analyses were conducted essentially as described else-
where (13) using the human IFN-g ELISPOT kit (U-CyTech Biosciences,
Utrecht, The Netherlands). To optimize the use of the available blood draw
(typically 30–70 ml) while minimizing potential competition between
peptides, the ZnT8 library was divided into 23 pools, each containing two
sequential peptides (each at 10 mM; Supplemental Table II). Freshly iso-
lated PBMCs (1 3 106) were cultured in 250 ml RPMI 1640 containing
10% heat-inactivated human Ab serum (PAA Laboratories, Dartmouth,
MA) and 10 mM control peptide or di-peptide pool. An additional 250 ml
medium was added after 24 h, and the cells were harvested 24 h later. After
washing, the cells were resuspended in 300 ml medium and transferred as
three 100-ml aliquots to 96-well clear polystyrene culture plates previously
coated with the anti–IFN-g capture monoclonal and subsequently treated
with 13 blocking solution (U-CyTech). Seventeen hours later, the cells
were removed by decanting, and the wells were washed extensively (23
PBS, and 53 PBS containing 0.05% Tween-20). Spots were then formed
by sequential incubations with the biotinylated second site anti–IFN-g,
gold-labeled goat anti-biotin, and a precipitating silver substrate, and
enumerated with a Bioreader 4000 Pro X (BIOSYS, Karben, Germany).
Results are expressed either as the total number of specific spots or as
stimulation indices (SIs). The former is calculated by adding the number of
spots formed in the three wells derived from incubations in the presence of
peptides and subtracting the total number of spots detected in the three
wells containing cells incubated in the presence of vehicle (DMSO) only to
define the specific signal for each peptide or peptide pool. Negative values
are set to zero. Total (spots 2 background) is the sum of the specific
signals for each individual. SIs are calculated by adding the total number
of spots formed in the three wells derived from incubations in the presence
of peptides and dividing either by the total number of spots detected in
the three wells containing cells incubated in the presence of DMSO
alone, or by 1 if no spots were detected in these wells. Based on the be-
havior of ELISPOT assays for other diabetes autoantigens (14), an SI $ 3
was selected as the cut-off for positivity. Positive control samples
comprising incubations with Pentacel (Sanofi Pasteur, Swiftwater, PA:
a mixture of diphtheria and tetanus toxoids, acellular pertussis, adsorbed
and inactivated poliovirus, and Haemophilus influenzae type b capsular
polysaccharide conjugated to tetanus toxoid) were also included in each
using established assays (15–17). ZnT8Awere either determined using the
“standard” RIA (6), or a modified procedure using a trimeric probe con-
taining sequentially the R, Q, and W variants of the ZnT8 C-terminal
domain. The detailed design of the trimeric probe will be reported else-
where (J.M. Wenzlau, H.W. Davidson, and J.C. Hutton, manuscript in prep-
HLA genotyping was performed by the UCD DERC clinical core. In-
dividual DRB1 and DQB1 alleles were identified by reverse hybridization
of PCR amplicons (18) to either sequence specific oligonucleotide bead
arrays (DRB1; Luminex xMAP; One L, Canoga Park, CA), or linear arrays
(DQB1; Roche Molecular Systems, Alameda, CA), respectively.
In vitro binding assay
Binding of ZnT8 peptides to recombinant HLA-DR3 (0301) and -DR4
(0401) was performed by Proimmune (Oxford, UK) using their Class II
REVEAL binding assay.
Statistical analyses were conducted using Prism 5 software (GraphPad
Software, La Jolla, CA). Group comparisons used the Mann–Whitney U
test, and categorical variables used Fischer’s exact test. In each case, p ,
0.05 was considered significant.
Overall responses to ZnT8 in patients and controls
ZnT8 is a recently described humoral autoantigen in T1D (6).
However, at present little is known regarding its relevance as a
target of potentially diabetogenic T cells. The goal of this study
was to determine whether ZnT8 is a significant target of proin-
flammatory T cells in patients with recently diagnosed T1D. In
contrast to proinsulin, GAD65, and IA-2, ZnT8 is a polytopic
integral membrane protein with six predicted transmembrane he-
lices (5), and we are currently unable to express the intact re-
combinant molecule in a form suitable for conducting T cell
assays. Thus, as a surrogate for the intact protein, we used a li-
brary of overlapping peptides encompassing the entire 369 aa
primary sequence (Fig. 1A). Given the relatively low frequency of
islet Ag-specific autoreactive T cells in the peripheral blood of
most patients with T1D, and the complexity of the human MHC,
which exhibits codominant expression of alleles thereby creating
the possibility for both cis and trans-pairing of the polymorphic
HLA-DP and -DQ a and b-chains (for example, Ref. 19) and
potential expression of up to 12 distinct class II molecules by
a single individual, each with its own unique binding motif, it is
unsurprising that previous studies have suggested that T1D asso-
ciation is most evident when multiple epitopes are considered
together, rather than on the basis of responses to a single antigenic
peptide (12, 14, 20, 21). Accordingly, we first examined the total
number of ZnT8-specific T cells detected in the samples from
each donor (Fig. 1B). A highly significant disease association was
observed. Although there was no statistically significant difference
between the background signals in the two groups (median 0 spots
[range, 0–16] for controls and 1 spot [range, 0–24] for patients;
p = 0.11), the median number of spots above background in the
summed incubations from the control group was 13 (range, 0–
300) compared with 81 (range, 18–689; p , 0.0001) in PBMCs
from the subjects with a recent diagnosis of diabetes. Using a cut-
off defined as the upper 99% confidence limit of the control
subjects (50.2 spots) 24 in 35 (68.6%) of the patients but only 3 in
41 (7.3%) of controls, showed significant ZnT8-specific responses.
To estimate the breadth of the autoresponse in each individual,
we next calculated the total number of peptide pools giving
a positive response (SI $ 3) (14) in the two groups (Fig. 1C).
Again, a highly significant expansion in the patient group was
observed. In the control group the median number of positive
peptide pools was 1.0 (range, 0–7), compared with 6.0 (range, 1–
20; p , 0.0001) in PBMCs from the patients. By this criterion all
of the subjects with T1D, but only 29/41 (70.7%) of the control
subjects, responded to at least one ZnT8 peptide pool, although all
control subjects responded to the positive control that contained
a mixture of pediatric recall Ags (data not shown). The significant
association between this measure of ZnT8 autoreactivity and T1D
was unchanged if either less stringent (SI $ 2.1) or more stringent
(SI $ 5) cut-offs were applied (1.0 versus 9.0 pools, p , 0.0001;
and 0 versus 3.0 pools, p , 0.0001, respectively; Fig. 1D, 1E).
However, using the more stringent cut-off, only 29 in 35 (82.9%)
The Journal of Immunology6057
of the subjects with T1D, and 10 in 41 (24.4%) of the control
subjects, responded to any ZnT8 peptide pool.
Our use of a library of overlapping peptides was essential to
ensure that as many specificities as possible were detected, but it
measured by the sum of positive pools. We therefore repeated our
analysis disregarding the second of sequential positive responses in
which any duplication could occur (i.e., if positive responses were
observed in pools 1–4, only the nonoverlapping pools 1 and 3 were
counted; Fig. 1F). As expected, this correction selectively reduced
the value for the median number of positive pools (SI $ 3) in the
patients (controls, 1.0 versus 1.0; patients, 5.0 versus 6.0), but did
not alter the level of statistically significant difference between
There was also a significant difference in the magnitude of the
positive responses between the two groups. Overall the median SI
in control individuals who responded (SI $ 3) to at least one
peptide pool was 3.1 (29 individuals, 84 positive responses, range
3.0–30) compared with 5.0 in the patient group (35 individuals;
258 positive responses; range, 3.0–222; p , 0.0001). Similarly, on
an individual basis the median SIs of responders were also sig-
nificantly different in the two groups (controls [29 individuals,
median SI, 3.2; range 3.0–9.0] versus patients [35 individuals,
median SI, 4.5; range, 3.2–18.0; p , 0.0001]; Fig. 1G). However
there was no correlation between the number of peptide pools to
which an individual responded and the median SI of the positive
responses in that individual (controls, p = 0.73; patients, p = 0.63;
We also analyzed responses in our subjects to a series of six
previously validated control peptides from proinsulin, IA-2, and
GAD65 (12, 13). Of these only GAD65335–352showed statistically
significant disease association when considered alone (p = 0.0047;
Supplemental Fig. 1A). In contrast, as has been reported pre-
viously (14), when the combined responses to the control peptides
were examined a highly significant association was revealed
(Supplemental Fig. 1B).
HLA-association of ZnT8 T cell autoreactivity
T1D shows a strong association with the expression of particular
MHC class II alleles, with the HLA-DRB1, -DQA1, and -DQB1
loci on chromosome 6p21 responsible for ∼50% of familial ag-
gregation of the disease (22). In particular there is a strong posi-
tive association with the DRB1*0301-DQA1*0501-DQB1*0201,
DRB1*0401-DQA1*0301-DQB1*0302, and DRB1*0405-DQA1*
0301-DQB1*0302 (DR3/DQ2 and DR4/DQ8) haplotypes. Con-
sistent with this association, 30 in 35 of the patient group, but only
28 in 41 of the control group, expressed at least one HLA-
DQB1*0201 or -DQB1*0302 allele (Table I, Supplemental Table
I). Preliminary bioinformatics analyses predicted that both low-
and high-risk molecules would likely bind similar numbers of
Schematic representation of the ZnT8 protein and peptide library. The
transmembrane helices (solid bars), peptide pools (alternating pairs of
faint and bold lines), and polymorphic residue 325 are indicated. B–G,
PBMCs from 35 subjects with a recent diagnosis of diabetes and 41 age-
matched controls were analyzed by IFN-g ELISPOT as described in
Materials and Methods using 23 ZnT8 dipeptide pools. B, Total (spots 2
background) values of summed ZnT8 incubations from each subject are
shown. C, The number of ZnT8 peptide pools giving positive responses
(SI $ 3) in each subject is shown. D, The number of ZnT8 peptide pools
giving positive responses (SI $ 2.1) in each subject is shown. E, The
number of ZnT8 peptide pools giving positive responses (SI $ 5) in each
subject is shown. F, The number of positive ZnT8 peptide pools (SI $ 3)
in each subject is shown after correction for potentially nonredundant
responses. G, The median SI of positive responses to ZnT8 (SI $ 3) in
each subject is shown. In B–E, the median and interquartile ranges of each
group are indicated.
T cell responses to ZnT8 in newly diabetic individuals. A,
Table I.Characteristics of study participants
Number of subjects
Age at blood draw (y; median, range)
aZnT8A positivity was an exclusion criterion for the control group.
bZnT8A data were unavailable for three subjects in the T1D group.
cNumbers in parentheses report the high risk DR4/DQ8 haplotype.
6058AUTOREACTIVE T CELLS TO ZnT8
peptides from ZnT8. Consequently, there was no a priori reason to
expect that the expression of high-risk alleles alone would result in
increased autoreactivity to ZnT8. However, to exclude the possi-
bility that the higher proportion of individuals in our patient group
who expressed high-risk haplotypes was significantly influencing
our analysis, we examined the degree of disease association after
stratification on the basis of the disease associated DQB1 alleles.
When only HLA-DQ2 and -DQ8–positive individuals were con-
sidered, the median number of spots above background in the
patient samples was 94.0 (range, 20–689) compared with 13.0
(range, 0–140; p , 0.0001) in the samples from the control sub-
jects (Fig. 2A). Similarly, the increased breadth of the response
was maintained (controls median = 1.0 pools, patients median =
6.0 pools; p , 0.0001; Table II). Moreover, disease associated
expanded responses were not restricted to either the HLA-DR3/
DQ2 or HLA-DR4/DQ8 haplotypes, being evident in both DQ2+
individuals who do not express DQ8 and DQ8+individuals who do
not express DQ2 (Table II). However, the greatest breadth of re-
activity in the patient group was seen in individuals expressing
HLA-DR1. Thus, the median response in DR1+patients (n = 6)
was 11.0 pools (range, 4–20) versus 5.0 pools (range, 1–20) in
DR12subjects (n = 29; p = 0.035). Consistent with this obser-
vation, DRB1*0101-containing molecules are predicted to bind
peptides from 22 of the 23 ZnT8 peptide pools (data not shown),
although no significant difference in reactivity associated with
DR1 expression was observed in control subjects (DR1+[n = 10]
median 1.5 pools, DR12[n = 31] median 1.0 pools; p = 0.852).
Unlike HLA-DRB1*0301, *0401, and *0405, alleles such as
HLA-DRB1*0403 and HLA-DQB1*0602 show a negative associ-
ation with T1D, and are therefore considered protective (22). At
present the mechanism responsible for this genetic protection from
T1D remains a matter of debate, but has variously been proposed
to be due to a failure of the protective molecules to productively
present key diabetogenic peptides (23) or an enhanced ability to
bind key tolerogenic peptides (24). Consequently, we were also
concerned by the fact that 13 in 41 controls but 0 in 35 patients
expressed one or more of the molecules encoded by these pro-
tective alleles. Accordingly, we repeated our analysis stratifying
the control group on the basis of the expression of either HLA-
DRB1*0403 or HLA-DQB1*0602 (protected), or the absence of
these protective alleles (unprotected). No difference between the
two control groups was observed. Thus, the median number of
spots above background in the samples from the protected subjects
was 13.0 (n = 13; range, 3–124) compared with 13.5 (n = 28;
range, 0–300; p = 0.5277) in the samples from the unprotected
subjects (Fig. 2B). Similarly, the breadth of the responses in the
two groups was equivalent (unprotected median = 1.0 pool; range,
0–7; genetically protected median = 2.0 pools; range 0–7; p =
0.336). Unsurprisingly, comparison of each group with the patient
cohort again revealed a highly statistically significant disease as-
sociation in each case (p , 0.0001; Fig. 2C, 2D).
These data indicate that individuals with clinical T1D show
a significantly greater number of circulating proinflammatory
T cells reactive with ZnT8 than age and HLA-matched controls,
and that this auto-response is present in individuals with both the
DR3/DQ2 and DR4/DQ8 haplotypes. The data also suggest that
HLA molecules present in individuals with both genetically pro-
tective and susceptible haplotypes are equally able to present ZnT8
peptides to proinflammatory T cells, and that the increased re-
sponse in the patient group is directly related to disease, rather than
simplyaconsequence ofthe particular MHCclass IImolecules that
Association of ZnT8 T cell immunity with gender or ZnT8A
Unlike many autoimmune conditions T1D does not show any
significant gender association (25). Consequently, gender was con-
sidered irrelevant in the recruitment of our study subjects. How-
ever, despite the fact that ∼50% of individuals attending the
Barbara Davis Center clinic are female (P.A. Gottlieb, unpub-
lished observations) there was a distinct male bias in the T1D
group, with females comprising only 12 in 35 (34.3%) of the total
(Table I). In contrast, only 15 in 41 (36.6%) of the control group
was male (Table I). Thus, although we considered it highly un-
reactivity to ZnT8. PBMCs from 35 subjects with a recent diagnosis of
diabetes and 41 age-matched controls were analyzed by IFN-g ELISPOT
as described in Materials and Methods using 23 ZnT8 di-peptide pools.
Individuals were stratified on the basis of their expression of selected HLA
molecules, and the total responses (spots 2 background) in each included
subject is shown. A, Subjects expressing HLA-DR3/DQ2 and/or HLADR4/
DQ8. B, Controls were stratified on the basis of HLA-DQB1*0602 or HLA-
DRB1*0403 expression. C, The control group was restricted to subjects
who did not express either HLA-DQB1*0602 or HLA-DRB1*0403. D,
The control group was restricted to subjects who expressed either HLA-
DQB1*0602 and/or HLA-DRB1*0403. The median and interquartile ranges
of each group are indicated.
Effect of the HLA genotype on proinflammatory T cell
Table II.Summary of overall responses to ZnT8 assessed by the total number of positive pools (SI $ 3.0)
n Positive Pools (range)nPositive Pools (range)p
HLA-DR3 and/or DR4(8)
HLA-DR3 / non DR4(8)
HLA-DR4(8) / non -DR3
Data are median and range. Statistical comparisons were conducted with the Mann–Whitney U test.
The Journal of Immunology6059
likely, we examined whether this gender discrepancy contributed
to the significant differences in ZnT8 T cell responses that we
observed. As expected, no significant correlation with gender was
observed in either the patient or control groups (Supplemental Fig.
The generation of high-affinity class-switched Abs, includ-
ing diabetes autoantibodies, is a T-dependent process. Neverthe-
less, previous studies that investigated potential relationships be-
tween humoral and cellular autoimmunity to proinsulin, IA-2, and
GAD65 did not reveal any significant association (11, 26), likely
because of the dependence of B cell help and tissue inflammation
on separate effector T cell populations (27). Consistent with the
results obtained for the other diabetes autoantigens, no correlation
between the ZnT8A index and the magnitude of the T cell re-
sponse was observed (Supplemental Fig. 1E). Likewise, there was
also no association between T cell reactivity to ZnT8 and humoral
autoimmunity to insulin, GAD65, or IA-2 (data not shown).
Multiple peptide pools from ZnT8 bind to HLA-DR4 in vitro
Patients with recently diagnosed diabetes who express at least one
copy of the risk-conferring HLA-DR4/DQ8 and/or -DR3/DQ2
haplotypes exhibit a significantly higher frequency of proin-
flammatory ZnT8-specific T cells in their peripheral blood than do
age- and HLA-matched control subjects (Fig. 2A). Preliminary
bioinformatics analyses using several public domain MHC class II
prediction servers suggested that multiple peptides from ZnT8
would be expected to bind to the HLA molecules expressed by
these subjects (data not shown). However, there was considerable
divergence between the various algorithms, precluding a definitive
conclusion regarding the behavior of individual peptides from
being made. To begin to address this issue empirically, and to
corroborate our ex vivo data, we used a commercial in vitro
binding assay (Reveal and Prove; ProImmune) to directly examine
binding of a library of 52 15mer peptides spanning the entire
primary sequence of ZnT8 to recombinant HLA-DR3 (0301) and
-DR4 (0401). As shown in Fig. 3B, 21/52 (40.4%) peptides
showed positive binding to HLA-DR4 in vitro, of which eight had
assay values more than twice the threshold for positivity. Com-
parison between the ex vivo and in vitro libraries indicated that 16
of the 23 pools used for the T cell assays contained at least one
peptide capable of binding to HLA-DR4 (0401), including 5 of 6
pools that showed statistically significant disease association in
subjects expressing HLA-DR4 but not -DR3 (Fig. 3B). In contrast,
only 4 in 52 (7.7%) peptides showed significant binding to re-
combinant HLA-DR3 (0301), with only one giving a value more
than 200% of the threshold (Fig. 3A). Nevertheless, HLA-DR3
binding peptides were present in 2 of 4 pools that showed statis-
tically significant disease association in subjects expressing HLA-
DR3 but not -DR4.
A limited number of individual peptide pools from ZnT8 show
The magnitude and diversity of the overall response to ZnT8 (Fig.
1) suggests that T cells recognizing multiple epitopes within this
autoantigen are expanded in the peripheral blood of subjects with
recent onset T1D. Although the data shown in Fig. 3 indicate that
multiple peptides from ZnT8 can bind both HLA-DR3 and -DR4,
they do not determine which are naturally processed and pre-
sented. For the other major autoantigens, CD4+T cell responses
restricted to a particular MHC molecule are typically focused on
a limited subset of immunodominant peptides (28). We therefore
analyzed the frequency of positive responses to individual ZnT8
peptide pools in the diabetic and control groups (Table III). Per-
haps surprisingly, all the peptide pools elicited a response in at
least 4 in 35 (11%) of the newly diabetic subjects, with T cells
reactive to four pools (7, 9, 17, 21) being present in .40% of the
members of this group. In contrast, only 13 in 23 pools elicited
a positive response in more than 2 in 41 (4.9%) of the control
subjects, with reactivity to two pools (3, 18) being undetectable in
these individuals. Pair-wise analysis (Fischer’s exact test) revealed
that 13 in 23 pools showed a statistically significant disease as-
-DR4 in vitro. A library of 52 15mers encompassing the entire 369 aa
sequence of ZnT8 was tested for binding to recombinant HLA-DR3(0301)
(A) and HLA-DR4(0401) (B), using the Class II REVEAL binding assay
(Proimmune). The boxes above each profile indicate the peptide pools used
in the ex vivo assays and are filled to indicate statistical association with
T1D in relevant groups. *p , 0.05, **p , 0.01 (Supplemental Table III).
The solid lines show the cut-off for positivity of the assay.
Binding of ZnT8 peptides to recombinant HLA-DR3 and
pools (all subjects)
Frequency of positive responses to individual ZnT8 peptide
PoolControl (%) T1D (%)p
Data are the percentages of the control group (n = 41) and T1D group (n = 35)
who responded (SI $ 3) to a particular ZnT8 peptide pool. Statistical comparisons
used Fischer’s exact test.
*p , 0.05, **p , 0.01, ***p , 0.001.
NS, not significant.
6060AUTOREACTIVE T CELLS TO ZnT8
sociation at the 95% confidence level, with seven (pools 1, 5, 9,
17, 18, 19, and 21) also being significant at the 99.9% confidence
interval (Table III). The same group of pools also showed the
highest statistical significance when the data were stratified to
include only those individuals who expressed at least one HLA-
DRB1*03 or *04 allele (Supplemental Table III).
Pairwise analysis of the responses to individual peptide pools in
PBMCs from subjects expressing HLA-DR4 but not HLA-DR3
revealed that 6 in 23 showed T1D association, with pools 1 and
17 having the greatest statistical significance (Supplemental Table
III). Comparison with the results of the in vitro binding assay
indicated that the positive responses to pools 1 and 17, which were
each detectable in 9 of 19 (47%) subjects with T1D, might be
explained by DR4-binding peptides (Fig. 3B). Consistent with the
lower level of in vitro binding to HLA-DR3 (Fig. 3A), analysis of
the responses to individual peptide pools in PBMCs from subjects
expressing HLA-DR3 but not HLA-DR4 revealed only 4 of 23
that showed significant disease association (Supplemental Table
III). The most significant of these (pool 21) gave positive re-
sponses in 4 of 6 (66.7%) subjects with T1D, but failed to elicit
a positive response in all 12 control subjects (p = 0.0049). Con-
sistent with the involvement of this molecule, pool 21 contains
a peptide that showed modest in vitro binding to HLA-DR3 (Fig.
In addition to the disease association in individuals who express
HLA-DR4 but not HLA-DR3, pool 17 also showed significant
disease association in subjects expressing HLA-DR3 but not HLA-
of T1D subjects with 95.1% specificity (Table III). However, the
most effective assay was obtained when analysis was restricted to
the six nonoverlapping pools that showed the most significant
disease association (pools 1, 5, 9, 17, 19, and 21; Fig. 4). In the
control group, the median number of positive pools was 0 (range,
0–2), compared with 2.0 (range, 0–6; p , 0.0001) in PBMCs from
the patients. Using a randomly selected cut-off predefined as the
mean + 3 SD of the control group (1.9 pools), the assay showed
74.3% sensitivity at 97.5% specificity (Fig. 4A). A similar result
was obtained when only those individuals expressing one or more
HLA-DQ2 or -DQ8 molecule were included (Fig. 4B), although in
this case the calculated cut-off was marginally higher (2.03),
giving an assay with 46.7% sensitivity at 100% specificity.
The results of this initial study of cellular autoimmunity to ZnT8
clearly suggest that, like the other gold-standard humoral targets,
ZnT8 is a significant target of autoimmune T cells in human T1D.
Based on overall activity, .68% of patients but ,8% of controls
showed significantly expanded numbers of ZnT8-specific proin-
flammatory T cells in their peripheral blood. The presence of
positive responses in some controls may appear surprising, but has
often been reported in studies of human T cell responses to other
diabetes autoantigens (reviewed in Ref. 1), and may be indicative
of the inherent immunogenicity of the Ag. In addition, it should be
noted that some of the control subjects are autoantibody negative,
first-degree relatives of diabetic subjects, who might be more
prone to islet autoimmunity than those with more protective
genotypes. Using an established cut-off, all the newly diabetic
subjects tested responded to at least one peptide from ZnT8, with
the majority exhibiting elevated numbers of proinflammatory
T cells specific to multiple noncontiguous regions of the protein,
likely indicative of epitope-spreading. Moreover, the magnitude of
the signal elicited by individual pools among responders was also
considerably greater in the patients than controls. The time course
of the ELISPOT assay is too short to allow significant maturation
of naive cells to occur (29). Consequently the responses we ob-
served presumably reflect the presence of elevated pools of ZnT8-
specific effector or memory T cells in the peripheral blood of the
diabetic subjects, with the most likely explanation for this being
that it is indicative of a role of ZnT8-specific T cells in diabeto-
genesis. However, as is also true for all other human T cell
responses to diabetes autoantigens, the relationship between pe-
ripheral T cells and those in the target organ remains a subject of
debate (30, 31). Nevertheless, consistent with an Ag-driven pro-
cess, the peripheral frequency of ZnT8-specific, IFN-g producing
T cells after onset appeared greatest within 6 mo of clinical di-
agnosis, with individuals retested at later ages typically showing
a much reduced response (data not shown). Given that cross-
presentation of free 20mer peptides is typically inefficient (32),
we assume that the responses we observed were predominantly, if
not exclusively, derived from CD4+T cells. However, because the
library we used may contain some truncated peptides, we cannot
entirely exclude the possibility that CD8+T cells may also have
contributed, although our recent studies using highly purified pep-
tides suggest that this is unlikely to have significantly impacted
the results we obtained.
When considered individually, 13 of the 23 dipeptide pools
showed statistically significant disease association at the 95%
confidence level. Although at first glance this may seem un-
expectedly high, the human MHC is highly complex, with any in-
dividual expressing up to 12 different class II molecules, most if
not all of which have the potential to bind multiple peptides from
ZnT8. The existence of ZnT8-specific T cells restricted to many
different class II molecules, both within an individual, and in the
control and patient populations as a whole, likely explains both the
breadth of the autoresponse we observed, and the fact that all of
the pools gave positive responses in at least 11% of the patients
tested. The degree of complexity of the response only increased
when the components of the most reactive pools were tested in-
dividually in a separate cohort of patients (data not shown). To date
we have only conducted direct binding studies with two of the
ZnT8. PBMCs from 35 subjects with a recent diagnosis of diabetes and 41
age-matched controls were analyzed by IFN-g ELISPOT as described in
Materials and Methods using 23 ZnT8 di-peptide pools, and stratified on
the basis of the six nonredundant pools giving the most significant disease
association (Table III). A, The number of selected peptide pools giving
positive responses (SI $ 3) in each subject is shown. B, The number of
selected peptide pools giving positive responses in subjects who expressed
at least 1 HLA-DR3/DQ2 or HLA-DR4/DQ8 haplotype is shown. The
median and interquartile ranges of each group are indicated. The dotted
line shows a cut-off for positivity based on the mean + 3 SD of the re-
spective control group.
T1D association of proinflammatory T cell reactivity to
The Journal of Immunology 6061
HLA-DR molecules expressed by the subjects in our study.
However, the level of T cell reactivity we observed appears con-
sistent with in silico predictions that suggest that most of the HLA-
DR molecules expressed by our subjects, including some of those
deemed protective, have the capacity to bind peptides from mul-
tiple pools of the library. The bioinformatics-based predictions
suggest that ZnT8 is inherently immunogenic, and the magnitude
of the response we observe implies that its potential as a target of
cellular immunity is realized in many patients, perhaps because
ZnT8-specific T cells are subject to minimal negative selection.
For tissue-specific Ags such as proinsulin, central tolerance is be-
lieved to depend on thymic and extrathymic expression driven by
transcriptional activators such as AIRE (33, 34) and Deaf1 (35).
Interestingly, ZnT8 (slc30a8) was not identified among the genes
selectively expressed by mouse thymic medullary epithelial cells
(36), and we are unaware of any evidence of thymic expression
in humans. Therefore, it is tempting to speculate that tolerance to
ZnT8 is largely the result of immunologic ignorance, and once
this is overcome a robust response may ensue.
It is possible that the actual diversity of the auto-response might
be slightlyexaggeratedbyour methodology,withthe overlapofthe
constituent peptides in consecutive pools meaning that some of the
disease-associated responses we observed might not be redundant.
For example, part or all of the reactivity to pool 18 could be due to
epitopes also in pools 17 and 19. Nonetheless, our data strongly
suggest the presence of a minimum of nine disease-associated
T cell epitopes in human ZnT8. Coincidentally, this value is
similar to those obtained in studies using overlapping peptide li-
braries encompassing GAD65 (37) and the cytoplasmic domain of
IA-2 (38). Because the HLA haplotypes exhibited by the two
groups were not identical, we cannot discount the possibility that
variations in detection limits within the two groups might have
influenced the magnitude of the differences in overall reactivity
that we observed. However, its contribution is clearly insufficient
to account for the results we obtained, and a highly statistically
significant disease association was also evident when our analysis
was limited to nonoverlapping peptide pools (Figs. 1F, 4), when
this concern no longer applies.
Our decision to use a library of overlapping peptides rather than
focusing solely on those predicted to bind to high risk alleles was
intended to ensure an unbiased analysis of the overall proin-
flammatory CD4+response in patients and controls. However, the
fine mapping of epitopes is highly problematic using this experi-
mental approach, and currently we cannot be certain of the total
number of immunodominant ZnT8 epitopes or of their restric-
tion to any particular class II molecule. Nevertheless, our results
clearly indicate that like ZnT8A, ZnT8-directed T cell autoim-
munity is associated with, but not restricted to, both the DR4/DQ8
and DR3/DQ2 high-risk haplotypes. In particular the HLA-DR4
(0401) molecule, which was expressed by 19 in 35 (54.3%) of the
diabetic group, appears to be a major element in ZnT8 autoim-
munity, with several peptides that bind this molecule including
ZnT88–22and ZnT815–29(pool 1), ZnT8120–134and ZnT8134–148
(pool 9), ZnT8260–274(pool 17), ZnT8267–281(pools 17 and 18),
and ZnT8295–309(pool 19) possibly containing disease-related
epitopes. Similarly, ZnT8155-169(pool 10) and ZnT8323-337(pool
21) may contain HLA-DR3 restricted epitopes.
In a previous study of autoreactivity to IA-2 and proinsulin, Arif
et al. (14) reported a reciprocal polarization toward IL-10 or IFN-g
production for peripheral T cells from patients and controls spe-
cific for the same epitope. In the current study, we were unable to
measure both IFN-g and IL-10 responses to the entire peptide
library in the majority of our study subjects because of the limited
amount of blood available, although preliminary results from the
subset tested for both cytokines revealed essentially equivalent
ZnT8-specific IL-10 responses in both patients and controls (data
not shown). Our identification of seven key disease-associated
ZnT8 peptide pools will enable us to address this and other im-
portant questions relating to the role of ZnT8 in T1D pathogen-
esis, such as the temporal appearance and disappearance of ZnT8-
specific T cells during disease progression, and whether ZnT8 is
also a significant target of natural regulatory T cells. Our identi-
fication of seven key disease-associated ZnT8 peptide pools also
provides the basis for the development of novel reagents that ei-
ther alone or in combination could be used for Ag-specific ther-
apeutic intervention to arrest the progression of the disease.
We thank the patients, relatives, and volunteers attending the Barbara
Davis Center for Childhood Diabetes Clinic who donated blood for this
study; Whitney Kastelic, Jesse Temple-Trujillo, and Rachael Jenison for
coordinating sample collection; Danny Zipris and George Eisenbarth for
helpful discussions; Sunanda Babu, Taylor Armstrong, Lisa Fitzgerald-
Miller, and Lisa Frisch for technical assistance; and Kim McFann for sta-
H.W.D., J.M.W., and J.C.H. are coinventors of U.S. patent No. 12/521,022
based on PCT/US2007/089125.
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