Islet-Specific Glucose-6-Phosphatase
Catalytic Subunit-Related Protein-Reactive
CD4+ T Cells in Human Subjects
This information is current as
of June 17, 2017.
Junbao Yang, Nancy A. Danke, DeAnna Berger, Sandra
Reichstetter, Helena Reijonen, Carla Greenbaum, Catherine
Pihoker, Eddie A. James and William W. Kwok
J Immunol 2006; 176:2781-2789; ;
doi: 10.4049/jimmunol.176.5.2781
http://www.jimmunol.org/content/176/5/2781
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References
The Journal of Immunology
Islet-Specific Glucose-6-Phosphatase Catalytic Subunit-Related
Protein-Reactive CD4ⴙ T Cells in Human Subjects1
Junbao Yang,* Nancy A. Danke,* DeAnna Berger,* Sandra Reichstetter,* Helena Reijonen,*
Carla Greenbaum,* Catherine Pihoker,† Eddie A. James,* and William W. Kwok2*‡
T
ype 1 diabetes (T1D)3 is an autoimmune disease in which
T cell-mediated autoimmune responses eventually lead to
the destruction of insulin-producing ␤ cells in the pancreatic islets (1). Susceptibility to T1D in humans is strongly associated with the HLA class II loci. The DRB1*0401 (DR0401)DQB1*0302 and DRB1*0301(DR0301)-DQB1*0201 haplotypes
are disease susceptible, whereas the DRB1*1501-DQB1*0602
haplotype offers disease protection (2). The NOD mouse (NOD)
develops autoimmune diabetes spontaneously, and has been well
established as a model for studying diabetes (3). Studies in NOD
mice have suggested that both CD4⫹ and CD8⫹ T cells are involved in the pathogenesis of autoimmune diabetes (4). A high
percentage of islet-infiltrating CD8⫹ T cells in NOD mice are specific for a peptide mimotope NRP (5). In subsequent reports, the
NRP mimotope was revealed to be an epitope derived from isletspecific glucose-6-phosphatase catalytic subunit-related protein
(IGRP) (6). Progression of autoimmune diabetes in NOD mice is
also correlated with an increase in the avidity of NRP/IGRP-reactive
T cells, and administration of NRP/IGRP peptides has an antidiabetogenic effect (7, 8). These results suggest that IGRP is a major
autoantigen and plays a role in islet destruction in the NOD model.
More recently, CD4⫹ T cells specific for IGRP were also identified in
*Benaroya Research Institute at Virginia Mason, Seattle, WA 98101; †Children’s
Hospital and Regional Medical Center, Seattle, WA 98105; and ‡Department of Immunology, University of Washington, Seattle, WA 98195
Received for publication September 15, 2005. Accepted for publication December
30, 2005.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
This work was supported by Juvenile Diabetes Foundation International and the
Immune Tolerance Network.
2
Address correspondence and reprint requests to Dr. William W. Kwok, Benaroya
Research Institute at Virginia Mason, 1201 9th Avenue, Seattle, WA 98101. E-mail
address: [email protected]
3
Abbreviations used in this paper: T1D, type 1 diabetes; IGRP, islet-specific glucose6-phosphatase catalytic subunit-related protein; Treg, regulatory T cells; G6Pase, glucose-6-phosphatase; UGRP, ubiquitously expressed glucose-6-phosphatase catalytic
subunit-related protein; TGEM, tetramer-guided epitope mapping.
Copyright © 2006 by The American Association of Immunologists, Inc.
NOD mice (9). In adoptive T cell transfer experiments, IGRP-specific
CD4⫹ T cells delayed the onset of diabetes, implying that IGRPspecific CD4⫹ T cells acted as regulatory T cells (Treg).
IGRP is a member of the glucose-6-phosphatase (G6Pase) family of proteins. Other members of this family include G6Pase-␣
and ubiquitously expressed G6Pase catalytic subunit-related protein (UGRP or G6Pase-␤). Expression of IGRP is restricted to the
islet. G6Pase-␣ is found in all gluconeogenic tissues, including the
liver, kidney, and intestine, whereas UGRP is widely expressed
(10, 11). IGRP shows 50% sequence homology to G6Pase-␣ and
35% sequence homology to UGRP. The G6Pase catalytic subunit
couples with glucose-6-phosphate transporter to form a G6Pase
complex that hydrolyzes glucose-6-phosphate to glucose. This
complex is involved in both the glycogenolysis and gluconeogenesis pathways and plays a major role in the control of blood glucose
homeostasis (12). Recently, the enzymatic function of the IGRP has
also been demonstrated. Membrane fractions from cells that overexpressed IGRP converted glucose-6-phosphate to glucose (13). It is
speculated that IGRP is the major enzyme that is responsible for
G6Pase activity in the islet. Like other G6Pases, IGRP is a glycoprotein anchored in the endoplasmic reticulum by multiple transmembrane helices. The major portion of the protein is embedded
in the membrane, with short lumens and cytoplasmic loops (14).
The precise role of IGRP in human T1D is unclear. The T1D
susceptibility locus IDDM7 was mapped within the IGRP locus (11,
15), suggesting the possibility that IGRP is a disease susceptibility
gene. Studies in human subjects have identified GAD65, insulin, and
IA-2 as the major T1D autoantigens, and CD4⫹ T cells that are
directed against each of these proteins have been described (16 –21).
However, the presence of IGRP-specific CD4⫹ T or CD8⫹ T cells in
human subjects has not yet been reported. In this study, we examined
CD4⫹ T cell responses to IGRP in both healthy and T1D subjects
using MHC class II tetramers. The tetramer-guided epitope mapping
(TGEM) approach (22) was used to identify human IGRP epitopes.
The prevalence of these IGRP epitopes in healthy and T1D subjects
was also examined.
0022-1767/06/$02.00
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Islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP) is recognized as a major autoantigen for autoimmune
type 1 diabetes (T1D) in the NOD mouse model. This study was undertaken to examine CD4ⴙ T cell responses toward IGRP in human
subjects. The tetramer-guided epitope mapping approach was used to identify IGRP-specific CD4ⴙ T cell epitopes. IGRP23–35 and
IGRP247–259 were identified as DRA1*0101/DRB1*0401-restricted epitopes. IGRP13–25 and IGRP226 –238 were identified as DRA1*0101/
DRB1*0301-restricted epitopes. IGRP-specific tetramers were used to evaluate the prevalence of IGRP-reactive T cells in healthy and
T1D subjects. More than 80% of subjects with either DRB1*0401 or DRB1*0301 haplotype have IGRP-specific CD4ⴙ T cell responses
for at least one IGRP epitope. IGRP-specific T cells from both healthy and T1D groups produce both ␥-IFN and IL-10. DRA1*0101/
DRB1*0401 IGRP247–259-restricted T cells also show cross-reactivity to an epitope derived from liver/kidney glucose-6-phosphatase. The
detection of IGRP-reactive T cells in both type 1 diabetic subjects and healthy subjects and recent reports of other autoreactive T cells
detected in healthy subjects underscore the prevalence of potentially autoreactive T cells in the peripheral immune system of the general
population. The Journal of Immunology, 2006, 176: 2781–2789.
IGRP CD4⫹ T CELLS IN HUMAN T1D
2782
␮g/ml streptomycin (T cell medium). Each well of cells was stimulated
with a different pooled peptide (a total of nine different wells for the nine
different IGRP-pooled peptides). After 7 days, the cells were split into two
wells with addition of fresh medium containing 5% of natural human IL-2
(Hemagen). The culture was maintained by changing half volume of culture supernatant every 3 days with fresh medium and IL-2. On day 14, the
cultured cells were stained with pooled tetramers. Cells that gave positive
staining with pooled tetramers were identified and subsequently stained
with a corresponding set of individual peptide tetramers.
For other experiments in detecting IGRP-reactive T cells, 2 ⫻ 106 unfractionated CD4⫹ T cells or 3 ⫻ 106 PBMC were plated out per well onto
a 48-well plate. Cells were stimulated with either one or two IGRP peptides
per well (10 ␮g/ml per peptide). Cells were cultured as indicated above.
Tetramer staining was conducted with 2 ⫻ 105 cells and 10 ␮g/ml
tetramers in 100 ␮l of T cell medium at 37°C for 2 h followed by anti-CD4
staining at 4°C for 15 min. Cells were analyzed on a FACSCalibur (BD
Biosciences). In most experiments, the background tetramer staining was
⬍0.2%, and a staining of 0.4% or higher was considered to be positive. In
some experiments, cells that were positive for a particular tetramer were
single-cell sorted by using a BD Biosciences FACSVantage. Sorted cells
were expanded with 1.5 ⫻ 105 unmatched, irradiated PBMC per well as
feeders with 2.5 ␮g/ml PHA and IL-2.
Materials and Methods
Healthy and T1D subjects
DR0301 and/or DR0401 T1D subjects ⬍3.5 years after diagnosis were
recruited at the Diabetes Clinical Research Unit at Benaroya Research
Institute (Seattle, WA). Healthy DR0301 and/or DR0401 donors were recruited from normal volunteers with consent. A total of 10 T1D subjects
and 10 healthy subjects were recruited for this study. The HLA status of
these groups is shown in Table I. The mean age of the patient group was
22 ⫾ 9 years, whereas the mean age of the healthy group was 30 ⫾ 10 years.
Peptides and tetramers
A panel of 43 overlapping IGRP peptides (p1 to p43) were synthesized on
polyethylene pins with 9-fluorenylmethoxycarbonyl chemistry by MIMOTOPES. These peptides, each 20-aa in length with a 12-aa overlap between
adjacent peptides covered the entire IGRP protein. Peptides were dissolved
in DMSO, and peptide pools were generated by mixing five consecutive peptides. There were a total of nine IGRP peptide pools. The individual peptides—IGRP13–25 QHLQKDYRAYYTF, IGRP23–35 YTFLNFMSNVGDP,
IGRP226 –238 RVLNIDLLWSVPI, IGRP247–259 DWIHIDTTPFAGL, G6Pase␣228 –240 KGLGVDLLWTLEK, G6Pase-␣249 –261 EWVHIDTTPFASL,
UGRP218 –230 FTLGLDLSWSISL, and UGRP239 –251 EWIHVDSRPFASL—
were synthesized with an Applied Biosystems 433A Peptide Synthesizer.
Soluble DR0401 molecules were produced as described previously (23).
Soluble DR0301 molecules were produced from Schneider cells using a
similar approach. Empty biotinylated HLA-DR0401 or DR0301 monomer
was loaded with 0.2 mg/ml of either pooled IGRP peptides or individual
IGRP peptides. Loadings were conducted at 37°C for 72 h in the presence
of 2.5 mg/ml (0.25%) n-octyl-␤-D-glucopyranoside and 1 mM Pefabloc SC
(Sigma-Aldrich). Peptide-loaded HLA-DR monomers were tetramerized
with PE- or allophycocyanin-conjugated streptavidin (BioSource International) at a molar ratio of 8:1, respectively.
DR0301/Flu MP169 –181 and DR0401/Flu HA306 –318 were used as control tetramers in staining. The sequence of Flu MP169 –181 is PLIRHENRMVLAS and Flu HA306 –318 is PRYVKQNTLKLAT.
Cytokine assay
Validation of tetramer reagents
T cell proliferation assay
Each individual preparation of class II protein was loaded with a reference
peptide (Flu MP169 –181 for DR0301 or Flu HA306 –318 for DR0401),
assembled as a tetramer, and used to stain a defined reference T cell
clone (DR0301-restricted MP169 –181-specific clone or DR0401-restricted HA306 –318-specific clone). Only preparations that give acceptable staining of the reference clone and low background staining when
loaded with an irrelevant peptide were used for experiments.
T cells were plated in 96-well plates at a concentration of 104/well. Irradiated DR0301 or DR0401-expressing BLS-1 lines were used as APCs and
plated at 2.5 ⫻ 104/well (25). Peptides were added at various concentrations. Assays were performed in triplicate wells in 150 ␮l of T cell medium. After a 3-day incubation at 37°C, 1 ␮Ci [3H]thymidine/well was
added. Cells were harvested, and tritium uptake was measured after 15 h of
further incubation.
TGEM and tetramer staining
Results
The TGEM approach for epitope identification has been described in detail
previously (22). Briefly, CD4⫹ T cells were isolated from donor PBMCs
by no-touch CD4⫹ isolation kit (Miltenyi Biotec). To augment T cell responses, CD4⫹CD25⫹ T cells were removed from total CD4⫹ T cells by
FACS sorting (24). Two million purified CD4⫹CD25⫺ T cells were seeded
into wells of a 48-well plate, which had been coated with adherent cells (to
be used as APCs) from the same donor. Cells were stimulated with 10
␮g/ml of pooled peptides in RPMI 1640 supplemented with 10% pooled
human AB serum, 1 mM sodium pyruvate, 50 U/ml penicillin, and 50
Detection of IGRP-specific CD4 T cells and identification of
DR0301- and DR0401-restricted IGRP T cell epitopes
The TGEM approach was used to detect IGRP-specific T cells for
the identification of DR0301 and DR0401-restricted IGRP T cell
epitopes. A set of 43 overlapping peptides spanning the entire
IGRP protein, each 20 residues in length with an offset of 8 residues, was synthesized. The peptides were divided up into 9 pools.
Table I. DR0301 and DR0401 donors
T1D
Subject
no.
Healthy
HLA-DR genotype
Duration of disease in
months
Subject
no.
HLA-DR genotype
1
2
3
4
5
0301/0404
0301/0401
0301/0404
0301/1301
0301/0101
31
35
28
13
15
11
12
13
14
15
0301/0701
0301/1201
0301/0101
0301/1301
0301/1101
6
7
8
9
10
0401/0101
0401/0901
0401/1301
0401/04- 0401/0301
5
27
39
1
7
16
17
18
19
20
0301/0401
0401/1501
0401/04- 0401/0701
0401/1401
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CD4⫹ T cells were stimulated with IGRP peptides as described above and
assayed on day 14. For the cytokine assay, 1 ⫻ 106 T cells were incubated
with 10 ␮g/ml tetramers, 10 U/ml IL-2, and 10 ␮g/ml anti-CD28. After 3 h of
incubation, IFN-␥ and IL-10 secretions were determined by use of a cytokine
secretion capture assay (Miltenyi Biotec). Briefly, cells were washed twice in
PBS and incubated in 100 ␮l of medium on ice for 5 min with an Ab-Ab
conjugate directed against both CD45 and the cytokine (i.e., IFN-␥ or IL-10).
Prewarmed medium was added to a final volume of 2 ml, and cells were
incubated at 37°C for 45 min under gentle rotation to allow cell surface capture
of secreted cytokines. Cells were washed once in PBS and then stained for 15
min on ice with allophycocyanin-conjugated Ab directed against the cytokine
of interest and PerCP-conjugated anti-CD4 Ab.
The Journal of Immunology
2783
FIGURE 1. Mapping of IGRP T cell epitopes. A,
CD4⫹CD25⫺ T cells were isolated from PBMC of a
healthy DR0301 subject and were stimulated with
IGRP-pooled peptides. Fourteen days poststimulation,
cells were stained with PE-conjugated DR0301/pooled
IGRP peptide tetramers, and anti-CD4 and anti-CD3
mAbs. Cells were gated on light scattering and antiCD3 positivity. Fifty thousand events were analyzed. B,
CD4⫹CD25⫺ T cells were isolated from PBMC of a
healthy DR0401 subject and were stimulated with
IGRP-pooled peptides. Fourteen days poststimulation,
cells were stained with PE-conjugated DR0401/pooled
IGRP peptide tetramers, and anti-CD4 and anti-CD3
mAbs. Cells were gated on scattering and anti-CD3
positivity.
positive tetramer staining was observed with DR0301 pool 1 and
pool 6 tetramers (Fig. 1A). For the DR0401 subject, positive staining
was observed with DR0401 pool 1 and pool 7 tetramers (Fig. 1B).
Single peptide tetramers were generated with individual peptides from
the positive pools, and the staining was repeated. For the DR0301
subject, tetramers with peptide p2 from pool 1 and peptide p29 from
pool 6 gave positive staining (Fig. 2A) (peptide p30 gave a staining
above background; however, the result was irreproducible), and for
the DR0401 subject, tetramers with peptide p3 from pool 1 and peptide p31 from pool 7 gave positive staining (Fig. 2B). These experiments were repeated with two additional DR0301 T1D subjects (samples were obtained within 16 mo after disease onset) and one DR0401
FIGURE 2. Fine mapping of DR0301 and DR0401restricted IGRP epitopes. A, The positive wells shown
in Fig. 1A were stained with DR0301 tetramers generated with individual peptides p1, p2, p3, p4, and p5
from pool 1 and peptides p26, p27, p28, p29, and p30
from pool 6. B, The positive wells shown in Fig. 1B
were stained with DR0401 tetramers generated with individual peptides p1, p2, p3, p4, and p5 from pool 1 and
peptides p31, p32, p33, p34, and p35 from pool 7.
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Pools 1– 8 consisted of 5 peptides each, whereas pool 9 consisted
of 3 peptides. These pooled peptides were used to stimulate
CD4⫹CD25⫺ T cells from a DR0301 healthy subject and a
DR0401 healthy subject. As described in Materials and Methods,
CD4⫹CD25⫺ T cells were used as responder cells. CD4⫹CD25⫹
T cells were depleted because CD4⫹ T cells are more responsive
to antigenic challenge when these regulatory cells are absent (24).
Fourteen to 16 days after the peptide stimulation, cells were examined by staining with DR0301 or DR0401-pooled IGRP peptide
tetramers. Results of tetramer staining of cells from the DR0301
subject are shown in Fig. 1A, and results of staining cells from the
DR0401 subject are shown in Fig. 1B. For the DR0301 subject,
IGRP CD4⫹ T CELLS IN HUMAN T1D
2784
Table II. DR0301 and DR0401-restricted IGRP epitopes
Minimal Epitopec
Peptide Name
Epitope
Sequence
p2a
p29
p3
p31
IGRP8 –27
IGRP225–244
IGRP17–36
IGRP241–260
GVLIIQHLQKDYRAYYTFLNb
LRVLNIDLLWSVPIAKKWCA
KDYRAYYTFLNFMSNVGDPR
KWCANPDWIHIDTTPFAGLV
IGRP13–25
IGRP226 –238
IGRP23–35
IGRP247–259
QHLQKDYRAYYTF
RVLNIDLLWSVPI
YTFLNFMSNVGDP
DWIHIDTTPFAGL
a
Peptides p2 (IGRP8 –27) and p29 (IGRP225–244) have a DR0301 binding motif, and peptides p3 (IGRP17–36) and p31
(IGRP241–260) have a DR0401 binding motif.
b
The proposed p1, p4, and p9 anchor residues are underlined.
c
Truncated peptides that were 13 aa in length containing the respective binding motifs from p2, p29, p3, and p31 were
shown. These peptides were IGRP13–25, IGRP226 –238, IGRP23–35, and IGRP247–259, respectively.
majority of DR0401 T1D subjects have T cell responses toward at
least one IGRP epitope. For the healthy group, most of the T responses are directed toward the IGRP247–259 epitope. One subject
in the T1D group and one subject in the healthy group did not
respond to either epitope. Although the sample set was too small
to evaluate whether there might be a significant difference in the
magnitude of responses and the extent of intramolecular determinant spreading between the healthy and T1D groups, the data demonstrated convincingly that IGRP-specific T cells were present in
both healthy and T1D subject subjects. An unexpected finding of
these experiments was that IGRP T cell responses could be detected in the majority of the healthy subjects, even without the
depletion of CD4⫹CD25⫹ Treg.
Prevalence of IGRP CD4⫹ T cells in healthy and T1D subjects
The prevalence of IGRP13–25 and IGRP226 –238-specific T cells in
DR0301 healthy and T1D subjects was examined by stimulating
PBMC or total CD4⫹ T cells from DR0301 healthy and T1D subjects with IGRP13–25 and IGRP226 –238 peptides. The presence of
IGRP13–25 and IGRP226 –238 T cells were evaluated by IGRP peptide-specific DR0301 tetramers 14 days after in vitro stimulation.
The results from five T1D and five healthy DR0301 subjects are
shown in Fig. 4, A and B, respectively. All five DR0301 T1D
subjects have significant T cell responses toward both the
IGRP13–25 and IGRP226 –238 epitopes (Fig. 4A). For the DR0301
healthy subjects, robust IGRP-reactive T cells toward both
epitopes were also detected in some subjects. There was also one
subject in which the responses toward both epitopes were minimal
(subject number 15). Similarly, the prevalence of IGRP247–259 and
IGRP23–35-specific T cells in DR0401 subjects was evaluated with
IGRP peptide-specific DR0401 tetramers. The results from five
T1D and five healthy DR0401 subjects are shown in Fig. 5. The
FIGURE 3. Fine mapping of DR0301 and DR0401-restricted IGRP
epitopes. A, Staining of DR0301-restricted IGRP p2 clone with DR0301/p2
and DR0301/IGRP13–25 tetramers. B, Staining of DR0301-restricted IGRP
p29 clone with DR0301/p29 and DR0301/IGRP226 –238 tetramers. C, Staining of DR0401-restricted IGRP p31 clone with DR0401/p31 and DR0401/
IGRP247–259 tetramers. D, CD4⫹ T cells from a DR0401 subjects were
stimulated with peptide p3, the cells were then stained with DR0401/p3 and
DR0401/IGRP23–35 tetramers. A DR0301/Flu MP169 –181 tetramer was used
as an irrelevant control tetramer in A and B. A DR0401/Flu HA306 –318
tetramer was used as the negative control in C and D.
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T1D subject (sample was obtained within 7 mo after disease onset).
Although not all of the epitopes were detected in the repeated experiments, no new additional IGRP epitopes were identified (data not
shown). These experiments indicated IGRP p2 and p29 as the major
DR0301-restricted epitopes and IGRP p3 and p31 as the major
DR0401-restricted epitopes. DR0301-restricted T cells specific for p2
and p29, respectively, were single-cell cloned by sorting for tetramerpositive T cells. Similarly, DR0401-restricted T cells that were specific for p31 were also cloned. Attempts to clone DR0401/p3-specific
T cells failed.
The IGRP residues corresponding to p2, p29, p3, and p31 peptides are shown in Table II. Peptides p2 (IGRP8 –27) and p29
(IGRP225–244) have a DR0301 binding motif (26) as underlined,
and peptides p3 (IGRP17–36) and p31 (IGRP241–260) have a
DR0401 binding motif (27) as underlined. Peptides that were 13 aas
in length containing the putative binding motifs from p2, p29, p3, and
p31 were then synthesized. These peptides were IGRP13–25,
IGRP226 –238, IGRP23–35, and IGRP247–259, respectively (Table II).
DR0301/IGRP13–25, DR0301/IGRP226 –238, DR0401/IGRP23–35,
and DR0401/IGRP247–259 tetramers were also assembled. IGRPspecific T cell clones isolated as indicated above were stained with
the respective tetramers generated with the long and short peptides
(Fig. 3). Staining of T cell clones with tetramers generated from
the shorter peptides confirmed that peptides IGRP13–25 and
IGRP226 –238 encompassed the DR0301-restricted p2 and p29
epitopes, respectively, and peptide IGRP247–259 encompassed the
DR0401-restricted p31 epitopes. Proliferation assays also confirmed the specificities of these T cell clones (data not shown). T
cells that were specific for p3 were not isolated, but a p3-stimulated T cell line could be easily stained with IGRP23–35-loaded
tetramers, confirming that peptide IGRP23–35 encompassed the p3
epitopes (Fig. 3D).
The Journal of Immunology
2785
Cytokine profiles of IGRP-reactive T cells in healthy and T1D
subjects
Because frequencies of autoreactive T cells are usually very low,
we have not attempted to assay for the cytokine profiles of CD4⫹
IGRP-reactive T cells directly ex vivo. We did compare the cyto-
FIGURE 5. Detection of IGRP autoreactive T cells in DR0401 T1D and
healthy subjects. A, PBMC or CD4⫹ T
cells from DR0401 T1D subjects were
stimulated with peptides IGRP247–259
and IGRP23–35. Cells were analyzed by
flow cytometry 14 days poststimulation
with anti-CD3 and anti-CD4 mAbs,
DR0401/IGRP247–259, and DR0401/
IGRP23–35 tetramers. Results from
five T1D subjects (numbers 6 –10) are
shown. B, PBMC or CD4⫹ T cells
from DR0401 healthy subjects were
stimulated with peptides IGRP247–259
and IGRP23–35. Cells were analyzed
by flow cytometry 14 days poststimulation with anti-CD3 and anti-CD4
mAbs, DR0401/IGRP247–259, and
DR0401/IGRP23–35 tetramers. Results
from five healthy subjects (numbers
16 –20) are shown. DR0401/Flu
HA306 –318 was used as irrelevant control tetramers in both A and B.
kine profiles of DR0401/IGRP247–259-reactive T cells after in vitro
expansion in both healthy and T1D subjects. IGRP-reactive T cells
from both groups produced both ␥-IFN and IL-10. Examples of
these assays are shown in Fig. 6. For T1D subjects, the percentage
of ␥-IFN-positive cells within the tetramer-positive population
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FIGURE 4. Detection of IGRP autoreactive T cells in DR0301 T1D and healthy subjects. A, PBMC or CD4⫹ T cells from DR0301 T1D subjects were
stimulated with peptides IGRP13–25 and IGRP226 –238. Cells were analyzed by flow cytometry 14 days poststimulation with anti-CD3 and anti-CD4 mAbs,
DR0301/IGRP13–25, and DR0301/IGRP226 –238 tetramers. Results from five T1D subjects (numbers 1–5) are shown. B, PBMC or CD4⫹ T cells from
DR0301 healthy subjects were stimulated with peptides IGRP13–25 and IGRP226 –238. Cells were analyzed by flow cytometry 14 days poststimulation with
anti-CD3 and anti-CD4 mAbs, DR0301/IGRP13–25, and DR0301/IGRP226 –238 tetramers. Results from five healthy subjects (numbers 11–15) are shown.
DR0301/Flu MP169 –181 tetramers were used as irrelevant control tetramers in both A and B.
IGRP CD4⫹ T CELLS IN HUMAN T1D
2786
FIGURE 6. Cytokine profiles of IGRP-reactive T cells in healthy and T1D subjects. CD4⫹ T cells were isolated from PBMC of DR0401 subjects and
stimulated with 10 ␮g/ml IGRP247–259 peptide. Fourteen days poststimulation, cells were incubated with 10 ␮g/ml DR0401/IGRP247–259 tetramers for 3 h.
Cells were then assayed for cytokine secretion with the Miltenyi Biotec cytokine capturing reagents. For analyses, cells were gated on light scattering and
CD4⫹. The figure shows the profiles of two T1D subjects, and two healthy subjects. For the two T1D subjects as shown, the percentages of ␥-IFN cells
within the tetramer-positive populations were 40 and 38%, the percentages of IL-10 cells were 30 and 14%. For the two healthy subjects as shown, the
percentages of ␥-IFN cells within the tetramer-positive populations were 11 and 41%, the percentages of IL-10 cells were 16 and 14%.
Cross-reactivity between IGRP epitopes and G6Pase epitopes
IGRP is one member of the G6Pase family. The other members of
this family of proteins are G6Pase-␣ and UGRP. IGRP protein
shows roughly 50% aa sequence homology to other G6Pases.
Amino acid alignment indicated that the homology between
IGRP23–35 and the corresponding region in G6Pase-␣ and UGRP
is minimal. In contrast, amino acid alignment indicated IGRP247–
259 sequence has extensive homology to the corresponding sequences with the other G6Pases. The IGRP247–259 sequence is
DWIHIDTTPFAGL, whereas the corresponding sequence for
G6Pase-␣249 –261 and UGRP239 –251 are EWVHIDTTPFASL and
EWIHVDSRPFASL, respectively (Table III). IGRP247–259 and
G6Pase-␣249 –261 bound to DR0401 with an IC50 of 0.1 ␮M
and 0.3 ␮M, respectively, in an in vitro peptide binding assay (data
not shown). Because UGRP239 –251 did not bind to DR0401 in the
same assay, this peptide was not investigated further. To examine
whether T cells that responded to IGRP247–259 could cross-react
with G6Pase-␣249 –261, IGRP247–259 clones were stimulated with
either IGRP247–259 or G6Pase␣249 –261 peptide using BLS-DR0401
transfectants as APC. Strong proliferation was observed in all of
the 10 clones examined. Representative results from three different
clones are shown in Fig. 7A. In addition, DR0401-restricted CD4⫹
T cells stimulated with IGRP247–259 could be stained with both
DR0401/IGRP247–259 and DR0401/G6Pase-␣249 –261 tetramers
(Fig. 7B).
Similarly, we examined whether DR0301-restricted T cells that
responded to IGRP epitopes could cross-react with homologous
G6Pase epitopes. No homology occurs between IGRP13–25 and the
corresponding regions of both G6Pase-␣ and UGRP, but limited
homology was observed between IGRP226 –238 and the corresponding regions in G6Pase-␣ and UGRP. The peptide sequence for
IGRP226 –238 is RVLNIDLLWSVPI. The corresponding sequences
in G6Pase-␣228 –240 and UGRP218 –230 are KGLGVDLLWTLEK
and FTLGLDLSWSISL, respectively (see Table III). Five different
DR0301-restricted IGRP226 –228-specific T cells were tested for
their reactivity toward IGRP226 –238, G6Pase-␣228 –240, and
UGRP218 –230 peptides in proliferation assays using BLS-DR0301
as APC. Although all clones proliferated upon stimulation with the
cognate IGRP peptide, no cross-reactivity was observed with the
other two peptides. Representative results from three of these
clones are shown in Fig. 7C. In another series of experiments,
CD4⫹ T cells from a DR0301 subject were stimulated with
IGRP226 –238. Fourteen days poststimulation, these cells were
stained with DR0301/IGRP226 –238, DR0301/G6Pase-␣228 –240,
and DR0301/UGRP218 –230 tetramers. A strong staining signal was
observed with the DR0301/IGRP226 –228 tetramers; however, no
staining signal was observed with DR0301/G6Pase-␣228 –240 tetramers and DR0301/UGRP218 –230 tetramers (Fig. 7D).
In summary, cross-reactivity was observed between IGRP247–
259 and G6Pase-␣249 –261 for T cells from DR0401 subjects, but no
homology or cross-reactivity was observed between other corresponding epitopes within IGRP and UGRP. Limited homology
was observed between IGRP226 –238, G6Pase-␣228 –240, and
UGRP218 –230. However, cross-reactivity was not observed with
either of these homologous epitopes for T cells from DR0301
subjects.
Table III. Homology between IGRP, G6Pase-␣, and UGRP
Peptide
Sequence
IGRP247–259
G6Pase-␣249 –261
UGRP239 –251
DWIHIDTTPFAGLa
EWVHIDTTPFASLb
EWIHVDSRPFASL
IGRP226 –238
G6Pase-␣228 –240
UGRP218 –230
RVLNIDLLWSVPI
KGLGVDLLWTLEK
FTLGLDLSWSISL
a
The proposed p1, p4, and p9 anchor residues for IGRP are underlined.
Residues of G6Pase-␣ and UGRP sequences that are identical to the corresponding sequence in IGRP are in bold italics.
b
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ranged from 33 to 40%, with an average of 36 ⫾ 4%, and the
percentage of IL-10-positive cells within the tetramer-positive
population ranged from 14 to 33%, with an average of 26 ⫾ 8% for
four subjects. For healthy subjects, the percentage of ␥-IFN-positive cells ranged from 11 to 41%, with an average of 25 ⫾ 14%,
and the percentage of IL-10-positive cells ranged from 5 to 16%,
with an average of 11 ⫾ 5% for three subjects. Although the sample size was too small to conclude any significant difference in
cytokine profiles between the two groups, it is notable that the
tetramer-positive T cells from healthy subjects and T1D produce
both ␥-IFN (suggesting an effector phenotype) and IL-10 (suggesting a regulatory phenotype).
The Journal of Immunology
2787
Discussion
In previous studies, TGEM was used successfully to identify
CD4⫹ T cell epitopes within viral Ags (22). In this context, TGEM
provided a rapid and consistent approach for MHC-specific
epitope identification and the isolation of MHC-specific T cell
clones. However, epitope mapping using tetramers relies on the
binding of tetramers to high-avidity T cells. It has been previously
demonstrated that many high-avidity self-reactive T cells are deleted within the thymus, and that self-reactive T cells that are
found in the periphery tend to bind weakly to tetramers (28, 29).
Therefore, it was unclear whether the TGEM approach could be
used to identify T cell epitopes for autoantigens. In the current
study, the TGEM approach was successfully used to identify a set
of CD4⫹ T cell epitopes for an islet-specific autoantigen IGRP for
both DR0301 and DR0401 individuals. IGRP13–25 and IGRP226 –238
were identified as DR0301-restricted epitopes, whereas IGRP23–35
and IGRP247–259 were identified as DR0401-restricted epitopes.
Because tetramer-based assays rely on high-avidity interactions, it
is possible that additional low-avidity epitopes also exist. There is
also the possibility that some of the epitopes identified are not
naturally processed epitopes. Nevertheless, these results indicate
that the TGEM approach is valid for the discovery of autoimmune
epitopes.
This study demonstrated the prevalence of CD4⫹ IGRP-specific
T cells not only in T1D subjects, but also in healthy individuals
that carry the DR0301 or DR0401 haplotypes. DR0301-restricted
IGRP-specific T cells were detected in 100% of the healthy subjects and T1D subjects that were examined. DR0401-restricted
IGRP-specific T cells were detected in ⬃80% of the subjects that
were examined. In contrast to our earlier report on GAD65-reactive T cells, which demonstrated that the depletion of
CD4⫹CD25⫹ Treg significantly enhanced responses to a GAD65
peptide in healthy subjects (24), responses to IGRP peptides could
be detected in most healthy and T1D subjects even without
CD4⫹CD25⫹ Treg depletion. Based on these results, the specific
role of IGRP-responsive CD4⫹ T cells in autoimmune patients and
healthy subjects is unclear. Differences between patients and
healthy subjects may be subtle, or it is possible that certain aspects
of the tetramer assay mask these differences. In any case, the detection of IGRP-restricted T cells within DR0301 and DR0401
patients and normal volunteers confirm that high-avidity cells with
autoimmune potential exist and can escape into the periphery.
Therefore, the presence of high-avidity autoreactive T cells alone
must be insufficient to induce autoimmunity.
The current study also investigated the cytokine profile of
IGRP-restricted T cells isolated from T1D subjects and healthy
controls. Secretion of ␥-IFN and IL-10 by IGRP-stimulated CD4
cells was measured using a Miltenyi capture assay. Tetramer-positive T cells isolated from patients and controls after in vitro expansion secreted both ␥-IFN and IL-10. Whether IGRP-reactive T
cells produce both cytokines in vivo has not been determined. The
detection of ␥-IFN (an inflammatory cytokine) and IL-10 (a suppressive cytokine) secreting cells as observed in these experiments
suggests that IGRP-specific CD4 cells can play multiple roles. We
speculate that naive IGRP-specific T cells in the peripheral immune system have the plasticity to become either proinflammatory
pathogenic T cells or Treg. In a proinflammatory environment,
naive IGRP CD4⫹ T cells differentiate toward a Th1 pathway and
perpetuate the inflammatory condition. Because CD4⫹ T cells are
required for both the priming and maintenance of CD8⫹ T cells,
IGRP-specific CD4⫹ T cells could then provide help for CD8⫹
IGRP-specific T cells that have cytotoxic function. Although a
definitive report of the presence of IGRP-specific CD8⫹ T cells in
human subjects has not yet been published, it is likely that these T
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FIGURE 7. Cross-reactivity of IGRPreactive T cells. A, Proliferation assays
of DR0401-restricted IGRP clones with
either IGRP247–259 peptide or G6Pase␣249 –261 peptide, with BLS-DR0401 as
APC. Assays for three different clones
are shown. B, CD4⫹ T cells from a
DR0401 T1D subject were stimulated
with 10 ␮g/ml IGRP247–259 peptide.
Fourteen days poststimulation, cells
were stained with DR0401/IGRP247–259APC and DR0401/G6Pase-␣249 –261-PE.
C, Proliferation assays of DR0301-restricted IGRP clones with either
IGRP226 –238 peptide, G6Pase-␣228 –240
peptide, or UGPR218 –230 peptide, using
BLS-DR0301 as APC. Assays for three
different clones are shown. D, CD4⫹ T
cells from a DR0301 T1D subject were
stimulated with 10 ␮g/ml IGRP226 –238
peptide. Fourteen days poststimulation,
cells were stained with DR0301/
IGRP226 –238, DR0301/G6Pase-␣228 –240,
and DR0301/UGRP218 –230 tetramers.
IGRP CD4⫹ T CELLS IN HUMAN T1D
2788
transient autoimmune response could actually be beneficial to the
host. They also suggested that such a “benign” autoimmune response may be an essential step for the induction of adaptive Treg
to control autoimmunity. The current findings demonstrating the
presence of IGRP-autoreactive T cells in healthy subjects appear to
support this benign autoimmunity hypothesis. Clearly, more investigation is needed to determine whether IGRP-specific CD4⫹ T
cells are primarily effector cells that accelerate or trigger disease,
or regulatory cells that down-regulate the inflammatory process
associated with disease progression.
In summary, we reported in this study the identification and
isolation of tetramer-positive IGRP-specific CD4⫹ T cells from
human subjects for the first time. IGRP epitopes could be readily
mapped using the TGEM approach, even without the removal of
CD25⫹ Tregs. Cross-reactivity was observed between IGRP and
G6Pase-␣ epitopes for DR0401-restricted T cells. The equal prevalence of autoreactive T cells in healthy subjects and T1D subjects
and their production of both IL-10 and ␥-IFN support the hypothesis that autoreactive T cells with different functional roles coexist.
Additional studies aimed at understanding the significance of Agspecific effector and Treg and the differences between transient
benign autoimmunity and prolonged autoimmunity will be crucial.
Acknowledgments
We thank Aru Arumuganathan and Kelly Geubtner for technical assistance
and Matt Warren for secretarial assistance.
Disclosures
The authors have no financial conflict of interest.
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