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Journal of Autoimmunity xxx (2013) 1e6
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Journal of Autoimmunity
journal homepage: www.elsevier.com/locate/jautimm
Short communication
CD8 T-cell reactivity to islet antigens is unique to type 1 while CD4
T-cell reactivity exists in both type 1 and type 2 diabetes
Ghanashyam Sarikonda a,1, Jeremy Pettus a, b,1, Sonal Phatak b,
Sowbarnika Sachithanantham a, Jacqueline F. Miller a, Johnna D. Wesley c, Eithon Cadag d,
Ji Chae b, Lakshmi Ganesan b, Ronna Mallios a, Steve Edelman b, Bjoern Peters a,
Matthias von Herrath a, c, *
a
La Jolla Institute for Allergy and Immunology, La Jolla, CA, USA
University of California San Diego, San Diego, CA, USA
c
Novo Nordisk Type 1 Diabetes R & D Center, Seattle, WA, USA
d
Ayasdi, Inc., Palo Alto, CA, USA
b
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 12 August 2013
Received in revised form
6 December 2013
Accepted 8 December 2013
Previous cross-sectional analyses demonstrated that CD8þ and CD4þ T-cell reactivity to islet-specific
antigens was more prevalent in T1D subjects than in healthy donors (HD). Here, we examined T1Dassociated epitope-specific CD4þ T-cell cytokine production and autoreactive CD8þ T-cell frequency on
a monthly basis for one year in 10 HD, 33 subjects with T1D, and 15 subjects with T2D. Autoreactive CD4þ
T-cells from both T1D and T2D subjects produced more IFN-g when stimulated than cells from HD. In
contrast, higher frequencies of islet antigen-specific CD8þ T-cells were detected only in T1D. These observations support the hypothesis that general beta-cell stress drives autoreactive CD4þ T-cell activity
while islet over-expression of MHC class I commonly seen in T1D mediates amplification of CD8þ T-cells
and more rapid beta-cell loss. In conclusion, CD4þ T-cell autoreactivity appears to be present in both T1D
and T2D while autoreactive CD8þ T-cells are unique to T1D. Thus, autoreactive CD8þ cells may serve as a
more T1D-specific biomarker.
! 2013 Elsevier Ltd. All rights reserved.
Keywords:
Type 1 diabetes
Antigen specific T-cells
CD8þ T-cells
HLA-Qdot-multimer analysis
ELISpot
1. Introduction
Epidemiological studies indicate that the incidence of type 1
(T1D) and type 2 diabetes (T2D) is increasing [1,2]. While both
conditions share the end result of hyperglycemia, they are classically defined as distinct clinical entities. Generally, T1D is considered an autoimmune disease [3] while T2D is thought to be a
metabolic disorder driven by insulin resistance. However, recent
postmortem studies suggest that b-cell destruction occurs even in
T2D subjects [4,5]. Additional studies have found evidence of islet
autoimmunity in patients with T2D [6,7]. Collectively, these findings support the hypothesis that islet-directed autoimmunity could
play a role in both T1D and T2D. This is evident clinically with the
existence of overlapping syndromes such as latent autoimmune
* Corresponding author. La Jolla Institute for Allergy and Immunology, La Jolla,
CA, USA. Tel.: þ1 858 752 6817; fax: þ1 858 752 6993.
E-mail addresses: [email protected], [email protected] (M. von Herrath).
1
These authors contributed equally.
diabetes of adulthood (LADA), lean type 2 diabetics, and ketosisprone T2D. Our ability to detect autoimmunity in diabetes is
currently limited to determining the presence and titer of classic
T1D autoantibodies. While these antibodies have proven invaluable
in predicting disease onset, their ability to predict disease course
post-onset is severely limited (discussed in Ref. [8]). Therefore, a
need exists to develop a biomarker that will indicate underlying
autoimmunity, changes as the disease progresses, and can be followed serially for evidence of therapeutic efficacy.
In T1D subjects, frequencies of islet antigen reactive CD4þ, and
CD8þ T-cells [9e12] are higher compared to healthy donors (HD),
and thus, have the potential to function as such a biomarker (discussed in Ref. [13]). However, how these parameters differ between
patients with T1D and T2D is largely unknown. Further, previous
cross-sectional studies have not taken into account the inherent
biological variation within a given subject that could lead to
misinterpretation of results due to stochastic fluctuations (discussed in Ref. [14]). In the current report, we followed subjects with
T1D or T2D and HDs over a one-year period with monthly blood
draws. At each visit, we determined IFN-g and IL-10 cytokine
0896-8411/$ e see front matter ! 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.jaut.2013.12.003
Please cite this article in press as: Sarikonda G, et al., CD8 T-cell reactivity to islet antigens is unique to type 1 while CD4 T-cell reactivity exists in
both type 1 and type 2 diabetes, Journal of Autoimmunity (2013), http://dx.doi.org/10.1016/j.jaut.2013.12.003
2
G. Sarikonda et al. / Journal of Autoimmunity xxx (2013) 1e6
Table 1
Participant information is shown including male to female ratio, age, and disease
duration.
Number of males (%)
Age (Yrs)
Disease duration (Yrs)
BMI
Insulin dose (units)
TDD insulin (U/kg)
HbA1c (%)
DRB1*04 e number (%)
DRB1*03 e number (%)
HD
T1D
T2D
1 (10)
31.3 $ 5.27
N/A
N/A
N/A
N/A
N/A
0 (0)
1 (14)
18 (55)
46.06 $
18.55 $
25.39 $
40.48 $
0.525 $
7.039 $
19 (58)
11 (36)
10 (67)
60.47 $ 8.48
14.57 $ 1.98
35.43 $ 7.00
90.09 $ 75.11
0.75 $ 0.49
7.45 $ 0.26
4 (27)
1 (7)
15.91
14.93
3.04
16.6
0.22
1.22
Mean $ SD values are shown. N/A represents not available (BMI and HbA1c for HD)
or not applicable (Duration and Insulin usage for HD).
production by CD4þ T-cells in response to diabetes-associated antigens, and frequencies of autoreactive CD8þ T-cells against HLAA2-restricted antigenic epitopes. To improve the robustness of the
data and reduce the influence of inherent subject-specific variation,
data from all available visits for each subject were averaged. Our
intent in this study was to determine features of autoimmunity that
are potentially shared between both disease states and those that
are unique to T1D. If a unique feature of T1D could be identified, this
would help to define a true biomarker for T1D. We found that CD4þ
T-cell reactivity to islet antigens was shared between T1D and T2D
while increased CD8þ T-cell autoreactivity was unique to subjects
with T1D.
the University of California-San Diego and LIAI Institutional Review
Boards. Informed consent, study identification numbers, clinical
case histories, and other information were collected and recorded
by clinical investigators. HDs were recruited from a normal blood
donor program at LIAI and did not require separate consent forms.
To maintain the anonymity of HDs, only the age and sex of the
donors were recorded. Use of glucocorticoids at the time of study
served as an exclusion criterion. HD and T1D cohorts in this study
have also been described in a related paper (Sarikonda et al., submitted to PLoS ONE). Each subject had w40 mL non-fasting blood
drawn once every month for 12 months.
2.2. PBMC isolation
Isolation of PBMCs using FicollePaque (Sigma) density gradient
centrifugation was performed as described [15]. PBMCs were
adjusted to a concentration of 8 # 106/mL in AIM-V medium
(Invitrogen Life Sciences, San Diego, CA) and used fresh for ELISpot
or cryopreserved in AIM-V medium containing 40% FCS and 10%
DMSO and stored in liquid nitrogen for up to 18-months prior to
analysis.
2.3. HLA-typing
Donors were first screened using an anti-HLA-A2 antibody
(eBioscience, San Diego, CA) and flow cytometry. Those who were
positive were further typed using genomic DNA. All enrolled donors had HLA-DR typing performed. HLA-A2 and HLA-DR typing
from genomic DNA was performed as described [16].
2. Materials and methods
2.4. ELISpot
2.1. Subject recruitment, study design, and scheduled blood draws
We enrolled subjects clinically defined as having T1D (n ¼ 33) or
T2D (n ¼ 15), along with HD (n ¼ 10) as controls. Patients were
classified as T1D or T2D by their referring practitioner using
commonly accepted clinical criteria including age at diagnosis,
initial presentation, autoantibody status, family history, dependence on exogenous insulin, and BMI. Diabetic donors were
recruited at the Veterans Affairs (VA) hospital in San Diego, La Jolla
Institute for Allergy and Immunology (LIAI), and at Taking Control of
Your Diabetes educational conferences. Protocols were approved by
The ELISpot assay was performed according to the original
standardized reports [9] with minor modifications. High purity
(>95%, Genscript) DR4-restricted epitopes of IA-2 (752e775) and
proinsulin (C19eA3), as well as the DR3-restricted GAD65 epitope
(335e352), were used to stimulate PBMCs [15]. A pool of immunogenic viral peptides (CEF) was used as a positive control (Mabtech Inc., Mariemont, OH) and cells incubated only in AIM-V
medium served as a negative controls. Development of IFN-g and
IL-10 spots was performed using U-Cytech antibodies and reagents
as described [15]. Spots above 65-mM in size were counted as
Fig. 1. CD4þ T-cell autoreactivity exists in both T1D and T2D subjects. A shows response among all donors irrespective of HLA-DR4 or -DR3 positivity while B shows data only from
DR4þ donors. A: Net IFN-g and IL-10 production (spots with antigen e spots in media) against DR4-restricted epitopes of IA-2 (752e775), proinsulin (C19eA3) and a DR3-restricted
epitope of GAD65 (335e352), were pooled at each visit and averaged over all visits. Statistical significance between the groups was determined using ManneWhitney test. B: IFN-g
(top panels) or IL-10 (bottom panels) production against IA-2752e775 (left half) or PIC19eA3 (right half) was assessed as net spot numbers (spots with antigen e spots in media), or as
stimulation index, SI (spots with antigen O spots in media). Mean net spot numbers.
Please cite this article in press as: Sarikonda G, et al., CD8 T-cell reactivity to islet antigens is unique to type 1 while CD4 T-cell reactivity exists in
both type 1 and type 2 diabetes, Journal of Autoimmunity (2013), http://dx.doi.org/10.1016/j.jaut.2013.12.003
G. Sarikonda et al. / Journal of Autoimmunity xxx (2013) 1e6
cytokine spots using the KS-ELISPOT reader [16] (Zeiss, Munich,
Germany). All epitopes were run in triplicate and averaged.
2.5. Qdot-HLA-A2-multimer assay
HLA-A2-restricted peptides (>95% purity, Genscript) from islet
antigens and their labeling with quantum dots (Qdots) have been
described previously [12]: insulin (B-chain, aa 10e18), IA-2 (aa
797e805), IGRP (aa 265e273), PPI (aa 15e23), GAD65 (aa 114e
123), and ppIAPP (aa 5e13). Peptide-loaded HLA-A*0201 monomers were produced by the NIH Tetramer Core Facility (Emory
University, Atlanta, GA), according to standard protocols, http://
tetramer.yerkes.emory.edu/client/protocols.
Streptavidinconjugated Qdots (Qdot-585, -605, -655, -705, and -800; Invitrogen, San Diego, CA) were added to achieve a 1:20 streptavidinQdot:biotinylated-pHLA ratio to generate multimeric HLA-A2peptide complexes. Each HLA-A2-monomer was labeled with two
different Qdots and each peptide-multimer had its own unique
Qdot combination [12].
For each HLA-A2þ subject, samples from all available visits were
labeled, acquired and analyzed simultaneously, as described [12].
Briefly, cryopreserved PBMC were thawed and 2 # 106 cells were
immediately stained first with all Qdot-labeled multimers (0.1 mg
each multimer) followed by Alexa Flour-700-CD8 along with FITClabeled anti-CD4, -CD14, -CD20, -CD40, and -CD16 antibodies
(eBiosciences, San Diego, CA) and resuspended in PBS supplemented with 0.5% BSA and containing 7-AAD (eBioscience, San
Diego, CA, USA). Sample data were acquired using an LSRII (BD
Biosciences, San Diego, CA) with the following filter settings; for the
488-nm laser: Qdot 655, 635LP, and 655/20. For the 405-nm laser:
QD800, 770LP, 800/30; QD705, 685LP, 710/40; QD605, 595LP, 605/
20 and QD585, blank, and 585/22.
3
determined IFN-g and IL-10 cytokine production from CD4þ T-cells
after stimulation with two DR4-restricted peptides (PIC19eA3 & IA2752e775) and one DR3-restricted epitope (GAD65335e352). As previous studies have shown that these peptides are also reactive in
some DR4/DR3-negative donors, we included data from all donors
regardless of their DR3/4 status in this first analysis. Net spot
numbers (spots with antigen e spots in media) against all three
epitopes were pooled at each visit and averaged over all visits.
Surprisingly, T2D subjects had significantly more IFN-g producing
CD4þ T-cells than controls (Fig. 1A, left panel). In contrast, T1D
subjects (but not T2D subjects) had more IL-10 producing CD4þ
T-cells than control subjects (Fig. 1A, right panel). Importantly,
while cytokine production differed between control subjects and
those with diabetes, there was no statistically significant difference
between the T1D and T2D cohorts in either IFN-y or IL-10 production. Further, in T2D subjects, higher IFN-g production by CD4þ
T-cells suggests the existence of either general inflammation or a
degree of underlying autoimmunity. In support of the latter, a
2.6. Statistical analysis
All data were analyzed and statistical analyses performed using
GraphPad Prism 5 software (GraphPad Software, Inc.). Additional
topological-based data analyses were conducted using the Ayasdi
IRIS software (Ayasdi, Inc.). Statistical significance was determined
using Spearman’s Rho test, ManneWhitney test, unpaired t-test,
and ANOVA.
3. Results and discussion
Herein we described CD4þ and CD8þ T-cell islet antigen reactivity in a cohort of patients with T1D compared to T2D and HDs.
Our study design provided a robust amount of data in that each
patient was followed for one year with monthly blood draws, and to
our knowledge, is the first study comparing these immunological
parameters between disease states longitudinally. We found that
only subjects with T1D consistently had increased numbers of
circulating, autoreactive CD8þ T-cells yet both T1D and T2D subjects had similar autoreactive CD4þ T-cell-derived cytokine
production.
We recruited 33 subjects with T1D, 15 with T2D, and 10 HD as
controls. A summary of participants’ baseline characteristics is
shown in Table 1. Detailed subject profiles with age, duration of
disease, HLA phenotypes, and autoantibody status are listed in
Table S1. A higher proportion of T1D donors were HLA-DR4þ and
HLA-A2þ (58% and 49%) compared to T2D donors (27% each) or HDs
(14% and 40%); this was expected given the known association
between HLA and T1D [17e19].
Extensive validation of the indirect ELISpot assay [9] showed
low intra- and inter-assay co-efficient of variation in assay performance (7.3% and 13%, respectively, data not shown). We then
Fig. 2. Ag-specific CD8þ T-cell numbers tend to be higher in HLA-A2þ T1D donors than
control or T2D donors. A: Mean frequencies of each antigen-specific CD8þ T-cells
averaged over all visits among HD, T1D, and T2D cohorts are shown. B: At each visit,
CD8þ T-cell frequencies against each antigenic epitope were counted as a positive
event when they were >0.01% of total CD8þ T-cells. Number of antigen-specific events
detected over all visits was pooled for each antigen. C: Antigen-specific CD8þ T-cell
positive events against all six antigenic-epitopes, detected over all visits were pooled.
D: At each visit, CD8þ T-cell frequencies were counted as a positive event when they
were >0.02% of CD8þ T-cells. Cumulative number of antigen-specific CD8þ T-cell
positive events against all six antigenic-epitopes, detected over all visits is shown.
Statistical values shown in C and D are from unpaired Student’s t-test.
Please cite this article in press as: Sarikonda G, et al., CD8 T-cell reactivity to islet antigens is unique to type 1 while CD4 T-cell reactivity exists in
both type 1 and type 2 diabetes, Journal of Autoimmunity (2013), http://dx.doi.org/10.1016/j.jaut.2013.12.003
4
G. Sarikonda et al. / Journal of Autoimmunity xxx (2013) 1e6
Fig. 3. Distinct populations comprise the T1D and T2D cohorts and highlight the existence of unique immune profiles. A: Distinct sub-groups within each disease type were
identified when the data (ELISpot þ Clinical Parameters þ Additional Immune Response Data) were used to construct the topological network. The similarity metric and mathematical
functions used to generate the topological network were normalized cosine (metric) and principal and secondary principal components (embedded). B: The subgroups identified in
A differed from each other in the mean total spot count, regardless of stimulating antigen.
Please cite this article in press as: Sarikonda G, et al., CD8 T-cell reactivity to islet antigens is unique to type 1 while CD4 T-cell reactivity exists in
both type 1 and type 2 diabetes, Journal of Autoimmunity (2013), http://dx.doi.org/10.1016/j.jaut.2013.12.003
G. Sarikonda et al. / Journal of Autoimmunity xxx (2013) 1e6
significant proportion of T2D patients exhibited T-cell proliferation
when exposed to human islet preparations [20].
To analyze our data more stringently, we assessed cytokine
production against the DR4-restricted epitopes (PIC19eA3 & IA-2752e
þ
775) in only DR4 subjects (T1D n ¼ 19, T2D n ¼ 4). As with the
analysis of entire cohort above, there were no significant differences in cytokine production between T1D and T2D cohorts in this
subgroup whether we assessed the average of net spots or Stimulation Index (SI) values over all visits (Fig. 1B). Thus, CD4þ T-cells
from HLA-DR4þ T1D or T2D subjects appear to elicit similar cytokine production in response to islet antigens. This finding argues for
a potential shared component of immune dysregulation between
the disease states.
Next, to determine antigen-specific CD8þ T-cell frequencies, we
employed HLA-A2-Qdot-multimer assay [12]. Mean frequencies of
each of the six epitope-specific CD8þ T-cells averaged over all visits
were higher in the T1D cohort compared to HD or T2D cohorts
(Fig. 2A and data not shown). Using a positivity cutoff of 0.01% of
CD8þ T cells [12], the T1D cohort had greater number of visits
(events) positive, in all but one of the antigenic epitopes, compared
to HD and T2D cohorts (Fig. 2B and data not shown). Further, when
all antigen specificities were pooled, the distribution and mean
number of positive events were higher in T1D subjects (Fig. 2C);
however, these differences were not statistically significant. Upon
setting a higher positivity threshold (>0.02% of CD8þ T-cells), the
increase in CD8þ T-cell frequencies in T1D donors became apparent
as they had significantly higher number of positive events
compared to T2D and control donors. Taken together, this data
suggests the increase in frequency of islet antigen-specific CD8þ Tcells is specific to T1D subjects, compared to HDs or T2D subjects.
While both T1D and T2D result in relative insulin deficiency, the
underlying b-cell destruction in T1D is much more rapid and severe
(reviewed in Ref. [21]). Additional analyses using a topological
approach for detecting nonlinear patterns in high-dimensional data
in an unsupervised fashion [22,23] revealed the possibility of
distinct subpopulation clusters within the T1D and T2D cohorts,
but not the HD group (Fig. 3A). These unique clusters were characterized by cytokine response, hinting at underlying differences in
immune function within both T1D and T2D populations that may
impact disease progression (Fig. 3B).
We hypothesize that CD4þ T-cell activation is common to both
forms of diabetes resulting in a mild degree of b-cell loss and/or
dysfunction. There is a range of inflammation that was easily seen
in the topological analyses but has not been appreciated previously.
In contrast, CD8þ T-cell activation is a feature unique to T1D and
leads to the massive b-cell destruction characteristic of this disease.
We propose a simplified model (Fig. S1) in which a primary insult to
the islet results in release of autoantigens. This initial insult may be
a metabolic derangement such as obesity-induced lipotoxicity and/
or dysregulated Kþ ATP channel [24e26] for T2D; whereas in T1D it
may be environmental or viral infections [27,28]. The exact mechanisms for b-cell injury are multifactorial and complex but,
regardless of the insult, the released autoantigens are taken up by
antigen presenting cells (APCs) that migrate to pancreatic lymph
nodes and activate T-cells. CD4þ T-cells are activated in both T1D
and T2D and result in inflammation that promotes functional b-cell
decline. The initiation of CD8þ T-cell activation, in contrast, is
specific to the T1D disease process. It has been shown that the
majority of patients with T1D have CD8þ T-cell infiltrates in the
islets. This is most pronounced in patients close to diagnosis but
also in a portion of patients with longstanding disease [29]. Similarly, cadaveric pancreatic samples from patients with T1D display
upregulation of MHC class I up to 8-years post-diagnosis [29]. We
propose that a possible secondary insult in individuals genetically
predisposed to develop T1D leads to upregulation of MHC I on the
5
islets and directed CD8þ T-cell killing. The resulting release of b-cell
antigens perpetuates the attack by recruiting more CD8þ T-cells
with different antigen specificities. This process could happen
slowly over time in response to repeated insults as has recently
been proposed [30]. While a proportion of T1D subjects did not
have significant, detectable circulating autoreactive CD8þ T-cells in
our study, the presence of such cells was almost exclusively found
in T1D. In those T1D subjects without detectable autoreactive CD8þ
T-cells, the frequency of these T-cells may wane as more islets are
destroyed, reducing the antigenic load. This would explain previously published observation that some, but not all, pancreatic
samples of T1D patients had insulitis [29].
The amount of remaining b-cells clearly differs between type 1
and type 2 diabetics, although recent investigations have shown
that some b-cells regularly survive even in type 1 patients over long
periods of time [31]. Induction of autoreactive T cells and their
maintenance clearly has to occur after exposure to islet antigens.
Two aspects of our study are, in this respect, novel and remarkable:
First, CD8þ T-cells reacting with islet antigens are only found in
type 1 patients, indicating over-expression of MHC class I on b-cells
in T1D [29] is instrumental for their sensitization. Second, CD4
autoreactivity is present in both types of diabetes, suggesting that
mere b-cell stress can lead to presentation of autoantigens via the
MHC class II pathway.
The current report indicates that CD4þ T-cell autoreactivity may
be common to both T1D and T2D, and could represent a shared
mechanism of b-cell dysfunction. Conversely, autoreactive CD8þ Tcells appear to be unique to T1D and may be responsible for the
accelerated loss of b-cell mass. This evidence gives credence to the
concept that the different forms of diabetes represent a continuum
from autoimmunity to metabolic disturbances with some overlapping and some unique features for each disease. Thus,
measuring CD8þ autoreactivity in T1D subjects could serve as a
more specific immune biomarker to study disease progression and,
possibly, response to therapeutics in future clinical trials.
In conclusion, the data presented here demonstrates that there
is a shared autoimmune component between T1D and T2D. The
complexity of both diseases is further highlighted by topological
analyses; the emergence of distinct subgroups underscores the
need for better understanding of the impact of the diabetogenic
immune response on disease progression and therapeutic outcomes. Identifying and understanding immune-based differences
within the spectrum of diabetesdbeyond T1D and T2Ddwill ultimately facilitate the development and use of more targeted,
personalized therapies.
Funding
GS was supported by a postdoctoral fellowship from the T1D
center of San Diego. This work was supported by a subaward
number 3002074563 from University of Michigan, Ann Arbor
(UMich), to MvH, issued under NIH award number 3UL1RR02498605S1 to UMich under the direction of Dr. Thomas P. Shanley, P.I.
Conflict of interest
The authors declare that there are no conflicts of interest pertaining to this manuscript.
Acknowledgments
We thank Ken Coppieters, (NNRC, Seattle, WA) and Bart Roep
(LUMC, Leiden, Netherlands) for many helpful discussions and
advice in manuscript preparation; Joana RF Abreu and Bart Roep
(LUMC, Leiden, Netherlands) for assistance with the Qdot-HLA-
Please cite this article in press as: Sarikonda G, et al., CD8 T-cell reactivity to islet antigens is unique to type 1 while CD4 T-cell reactivity exists in
both type 1 and type 2 diabetes, Journal of Autoimmunity (2013), http://dx.doi.org/10.1016/j.jaut.2013.12.003
6
G. Sarikonda et al. / Journal of Autoimmunity xxx (2013) 1e6
multimer assay; Tobias Boettler, Darius Schneider, Renee Parker
and Mark Bouchard (LIAI) and the nurses at UCSD VA hospital for
their assistance in obtaining blood samples for this study; Denise
Baker and Duy Le (LIAI) for their assistance with HLA typing. Finally,
we would like to thank the NIH tetramer core facility at Emory
University for their help in providing HLA-A2-peptide monomers.
GS, JP, SS, SP JFM, and JC participated in obtaining samples and
performing assays. GS, JP, RM, BP, JDW, EC, and MvH performed the
data analysis. GS, JP, BP and MvH wrote the manuscript. GS, SE and
MvH conceptualized and designed the project. GS, JP and MvH are
the guarantors of this work and, as such, had full access to all the
data in the study and take responsibility for the integrity of the data
and accuracy of data analysis.
Parts of the data shown in this manuscript were presented at the
Immunology of Diabetes Society (IDS) conference 2012, held in
Victoria, British Columbia, Canada.
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.jaut.2013.12.003
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Please cite this article in press as: Sarikonda G, et al., CD8 T-cell reactivity to islet antigens is unique to type 1 while CD4 T-cell reactivity exists in
both type 1 and type 2 diabetes, Journal of Autoimmunity (2013), http://dx.doi.org/10.1016/j.jaut.2013.12.003