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,
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Program in Immunology, Departments of
Microbiology and Immunology,
Pediatrics, and
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Medicine, Stanford University School of Medicine, Stanford, CA 94305;
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Children’s Hospital Oakland Research Institute, Oakland, CA 94609; and
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Department of Human Genetics, Roche Molecular Systems, Alameda, CA 94501
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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- and ß-chains form a
peptide binding groove that presents peptides to
CD4+ T cells (4). Analysis of the
gene sequences encoding these chains in diabetic individuals revealed
that the strongest association with disease lies in the presence or the
absence of an aspartic acid residue at position 57 of the HLA-DQ
ß-chain (ß57),3
(5). Alleles encoding a noncharged serine, valine, or
alanine residue at this position, such as the HLA-DQ2 (DQ2) and HLA-DQ8
(DQ8) alleles, confer susceptibility to type I diabetes, while closely
related alleles, such as HLA-DQ7 (DQ7) and HLA-DQ9 (DQ9) encoding ß57
aspartic acid, are not associated with susceptibility. Exceptions to
this have been noted, and other residues in HLA-DR as well as HLA-DQ
molecules have been shown to play a critical role in susceptibility,
for example in other ethnic groups (1, 2).
The observation that specific allelic polymorphisms in an HLA molecule
lead to disease susceptibility suggests that the role of these
molecules in presenting unique sets of peptides to T cells could be the
critical factor for determining susceptibility. Class II/self peptide
complexes are involved in both positive and negative selection events
in the thymus, where particular allele differences could lead, for
example, to a lack of negative selection of autospecific
CD4+ T cells (6). Alternately,
diabetes-associated HLA class II molecules might present distinct
disease-promoting self peptides to CD4+ T cells
in the periphery (7). The P1 through P9 pockets in an HLA
class II molecule correspond to the anchoring residues of the typically
9-aa core peptide required for binding to the molecule
(8). By homology to the known crystal structure of
HLA-DR1, an allele without an aspartic acid residue at ß57 is unable
to form a salt bridge with arginine at position 76 of the DR -chain
(9). This salt bridge contributes to the shape of the P9
pocket. The lack of this salt bridge has been postulated to affect
disease susceptibility by selecting for negatively charged residues in
the peptide at P9 (10, 11). Such residues in the peptide
might substitute for the lack of aspartic acid at ß57 by replacing
this salt bridge and conferring increased molecular stability.
Although peptide motifs for the DQ8 molecule have been defined by elution studies (12, 13), specific autoantigenic peptides recognized by human T cells in the context of the DQ8 molecule are poorly defined. Due to the difficulty of obtaining DQ8-restricted human T cell clones, no clones specific for autoantigens such as glutamic acid decarboxylase 65 (GAD65) are available (14). GAD65 is a protein expressed in neural and pancreatic tissue and is a target of the autoimmune process in type I diabetes (15). Several groups have been able to show that bulk cultures of human PBMC from diabetic individuals respond to GAD65 (16). The importance of GAD65 in the disease process is evidenced by the presence of GAD65 autoantibodies in >70% of patients (17, 18, 19). Autoantibodies to GAD65 in combination with anti-IA-2 and anti-insulin Abs in individuals with a susceptible HLA haplotype have a high predictive value for subsequent development of diabetes.
To analyze peptides from human GAD65 presented by the DQ8 molecule, we
have studied the T cell response to this protein in a DQ8 transgenic
(Tg) mouse model. In this system, the Tg DQ8 molecule is the only class
II restriction element due to targeted mutation of the mouse class II
I-Aß locus (I-Aß0/0), and a natural deletion
in the I-E gene promoter region. HLA Tg mice have been used
successfully in other systems to analyze responses restricted by human
class II molecules in lieu of studying responses directly in humans
(20). DQ8 Tg mice have been used as a model for
collagen-induced arthritis and to generate GAD65-specific mouse T cell
clones (21, 22). An initial characterization of regions of
GAD65 presented by the DQ8 molecule in Tg mice was published during
preparation of this manuscript (23). We independently
generated a similar model to determine GAD65-derived peptides that are
processed and presented by the DQ8 molecule and are immunogenic to T
cells. Furthermore, we have performed careful analyses of the GAD65
epitopes determined in DQ8 Tg mice, leading to the identification of an
unexpected peptide motif without a negatively charged residue at P9 of
the peptide. Additionally, we observed special features for these
peptides, including a 10 mer minimal core epitope. To validate that
these studies identify epitopes relevant to human type I diabetes, we
showed that human PBMC from DQ8+ diabetic and
nondiabetic individuals proliferate to the human GAD65 epitopes defined
in our DQ8 Tg mouse system.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A DNA construct for Tg expression of DQ8 was generated using
genomic sequences for the DQA1*0301 (provided by Dr. Jack Strominger)
and DQB1*0302 genes, each under the control of the I-E promoter in
the vector pSV5neo (provided by Drs. Diane Mathis and
Christophe Benoist, Strasbourg, France). The I-E promoter, in which
SalI and XhoI sites were destroyed, was
engineered upstream of the genomic sequence of DQA1*0301 and DQB1*0302.
These two fragments were then subcloned into pSV5neo using
engineered 5' XhoI and 3' SalI sites (see Fig. 1
A). The XhoI
fragment containing the promoters and transgene sequences was
microinjected into (FVB x nonobese diabetic
(NOD))F1 mouse embryos according to standard Tg
mice production protocols (24). Eight separate founder Tg
mice expressing DQ8 on the surface of PBL were identified by isolating
mouse PBMC on a Lympholyte-M gradient (Cedarlane, Ontario, Canada),
staining with a pan-anti-HLA-DQ Ab (Leu 10, Becton Dickinson, San
Jose, CA), and analyzing cells by flow cytometry. Cells from spleen,
lymph node, and thymus were also analyzed by flow cytometry (FACScan,
Becton Dickinson, San Jose, CA). Mice were sacrificed, and organs were
removed and prepared as single-cell suspensions. The cells were
preblocked with FcBlock (PharMingen, San Diego, CA); stained with
anti-HLA-DQ and/or Abs specific for I-Ag7
(OX6), B220 (RA3-6B2), CD4 (GK1.5), or CD3 (145-2C11; PharMingen, San
Diego, CA); and analyzed as described above. Tg mice (at N5 backcross
to the NOD background) were crossed to I-Aß0/0
mice on the B6 background (provided by Drs. Diane Mathis and Christophe
Benoist), and intercrossed to obtain mice expressing human Tg DQ8 as
the only class II molecule. Mice were screened for this phenotype by
flow cytometric analysis of PBMC, stained with anti-HLA-DQ or Abs
specific for I-Ag7 and mouse CD4, as described
above.
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Recombinant human GAD65 was produced using a baculovirus expression system (Invitrogen, San Diego, CA) as previously described (25). Histidine 6-tagged human GAD65 was purified using NTA-Ni2+ agarose (Qiagen, Valencia, CA). For immunization, the protein was purified further by gel electrophoresis and electroelution of the 65-kDa species. Purified protein was dialyzed into PBS for injection and/or hybridoma screening.
Generation and screening of GAD-specific T cell hybridomas
(NOD x B6)F2, homozygous DQ8 Tg, I-Aß0/0 mice were immunized in each hind footpad and at the base of the tail with 50 µg of purified recombinant human GAD65 emulsified in IFA (Difco, Detroit, MI). T cell hybridomas were generated from the draining lymph nodes, as previously described (25, 26), and propagated in complete medium: RPMI 1640, 10% FCS, 10 mM HEPES, 2 mM L-glutamine, 100 U/ml each of penicillin and streptomycin, and 50 mM 2-ME, with 1x hypoxanthine/aminopterin/thymidine (Life Technologies-BRL, Gaithersburg, MD). Cells were first screened for specificity to whole human GAD65 by culturing T cell hybridomas and APC with 20 µg/ml human GAD65 or medium only, as previously described (25). Human EBV-transformed B cells of the Priess line, homozygous for DQ8, were used for APC. Supernatants were tested by immunoassay for IL-2 production, using a europium-based detection system (Wallac, Gaithersburg, MD), (25). Cells specific for whole human GAD65 were then tested for peptide specificity in the same assay, using peptide in pools of 12 or singly at 6.7 µg/ml instead of whole Ag. Ab blocking studies were performed by adding 50 µg/ml IVD12 (American Type Culture Collection, Manassas, VA), specific for DQ8, to the cultures of hybridomas, APC, and specific peptide described above and testing for IL-2 production.
Peptides
Peptides were synthesized using the PIN method to generate 117 15 mers overlapping by 10 aa, spanning the entire 585-aa sequence of human GAD65 (Chiron Technologies, San Diego, CA), as previously described (25). Truncation peptide sets of 11 mers shifting the register by 1 aa were produced in the same way.
Determination of core minimal epitope for hybridoma stimulation
Truncation sets of 11 mer peptides were included in cultures of T cell hybridomas and irradiated human DQ8+ Priess APC as described above. IL-2 production was assayed using eight concentrations of peptide at 4-fold dilutions ranging from 0–100 µM, in duplicate wells for each experiment.
Analysis of responses from human peripheral blood
Blood was isolated from recent-onset diabetic patients at the Stanford Pediatric Endocrinology Clinic headed by Dr. Darrell Wilson at their first clinical visit after diagnosis or from healthy control volunteers. PBMC were isolated by density centrifugation. Blood was layered 1/1 on a Histopaque (Sigma, St. Louis, MO) gradient and centrifuged at 2200 rpm for 20 min at room temperature. Cells were harvested and washed in complete medium (as described for hybridomas, except without FCS). Cells were then incubated with autologous serum at 1 x 106 cells/ml in 1.0-ml cultures in the presence or the absence of human GAD65 peptides at 20 µg/ml or with 20 µg/ml tetanus Ag as a positive control. After 5 days, wells were split into five wells of 250 µl, and 1 µCi/well of [3H]thymidine was added to the cultures for 8–12 h. Cells were harvested, and counts were determined on a Tomtec harvester (Orange, CT). Data were calculated as the mean ± SD of five replicate cultures. HLA typing was performed using a PCR method, as previously described (27).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Transgenic mice were produced that express the diabetes-associated
DQ8 molecule. The genomic sequences containing the DQA1*0301 and
DQB1*0302 genes were each engineered to be under the control of the
mouse I-E promoter (Fig. 1
A). This approach was taken
after generation of DQ Tg mice using human genomic promoters led to
inappropriate expression of human class II molecules (data not shown).
Spleen lymphocytes from Tg mice established using the I-E
promoter-DQ8 construct were analyzed by flow cytometry for HLA-DQ and
mouse class II I-A expression (Fig. 1B). DQ8 Tg mice, but
not nontransgenic (non-Tg) littermates, express HLA-DQ on the same
cells that express endogenous mouse class II molecules. We tested
spleen, thymus, lymph node, and peripheral blood and found the DQ8
product expressed on the same cells as mouse class II I-A (Fig. 1B; data not shown).
DQ8 Tg mice at the N5 backcross to the NOD strain were crossed to B6
strain I-Aß0/0 mice. Heterozygotes were
intercrossed to produce (NOD x B6)F2
I-Aß0/0 pups with homozygous Tg DQ8 as the only
class II molecule. Flow cytometric analysis of PBMC from a
representative mouse confirms the DQ8 Tg phenotype (Fig. 1C). PBMC from the same mouse were analyzed for mouse class
II expression (I-A) and mouse CD4, showing that the human DQ8 Tg is
able to select mouse CD4+ T cells in the absence
of mouse class II (Fig. 1D). The addition of a human CD4
transgene to the DQ8 Tg mice described above did not enhance the number
of mouse CD4+ T cells in peripheral blood (data
not shown; human CD4 Tg mice provided by Dr. Dan Littman). In the
(NOD x B6)F2 background,
CD4+ T cells make up 20–30% of PBL in
homozygous Tg DQ8, I-Aß0/0 animals (Fig. 1D).
Determination of human GAD65 epitopes in Tg mice
We have used the Tg mice described above as a means to identify
the molecular nature of the DQ8-restricted T cell response to human
GAD65, an important diabetes autoantigen expressed in the pancreas. DQ8
Tg I-Aß0/0 mice (see Fig. 1, C and
D) were immunized with recombinant human GAD65 protein.
Responding T cells were used to generate T cell hybridomas, which were
screened for reactivity to whole human GAD65 protein. Different
hybridoma clones responded to whole GAD65 with IL-2 production ranging
from 1.5- to 9-fold over medium alone (data not shown). Specific
epitopes were determined by testing for IL-2 production by the
GAD-specific hybridomas in response to 15 mer peptides from a peptide
set spanning the whole protein. The set consisted of 15 mer peptides
overlapping by 10 residues to represent every possible 10 mer epitope
in the protein.
A total of 35 DQ8-restricted hybridomas was obtained from two
independent fusion events. The panel of hybridomas represents
reactivity to at least nine epitopes within human GAD65 (Fig. 2). Most clones respond to one of three
epitopes, in regions 51–120, 111–180, or 521–585. T cell
responsiveness was identified in multiple regions of the protein. Six
epitopes have been characterized to the level of a 15 mer peptide
(Figs. 2 and 3; data not shown). Six
hybridomas responded to 101–115, six hybridomas responded to 126–140,
one hybridoma responded to 206–220, and nine hybridomas responded to
536–550. Two hybridomas each responded to 431–445 and 461–475. Fig. 3A shows an example of the IL-2 response of one hybridoma to
whole human GAD65 and a series of 10 pools of peptides using a
DQ8+ human EBV-transformed B cell line as APC.
Hybridoma 20D4.1 recognizes whole human GAD65, specifically an epitope
in the C-terminal region. The response to this region can be mapped to
the peptide 536–550 (Fig. 3B). This response is DQ8
restricted, since the human APC and Tg mice have only DQ8 in common.
Additionally, an Ab specific for DQ8 (IVD12) included in the cultures
with APC and specific peptide could completely inhibit the production
of IL-2 from all hybridomas tested, specific for five of the six
epitopes identified in this study (data not shown).
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To examine the interaction of peptide, DQ8, and TCR, we determined
the core minimal sequence required for stimulation of the T cell
hybridomas by truncation analysis. Fig. 4
shows the IL-2 responses of four hybridomas to their specific 15 mer
peptides and truncations for the following epitopes of human GAD65:
101–115 (Fig. 4A), 126–140 (Fig. 4B), 206–220
(Fig. 4C), and 536–550 (Fig. 4D). Hybridoma 15H2
responds to the 101–115 epitope of human GAD65 in a dose-dependent
fashion as well as responds to some truncations of that sequence
spanning amino acids 102–112, 103–113, and 104–114 (Fig. 4A). Fig. 4B shows the same type of analysis for
hybridoma 17G12.7, specific for an epitope from 126–140. The
dose-dependent IL-2 response of hybridoma 8G5.4 to the 206–220 epitope
is confirmed in Fig. 4C. Finally, the epitope found at
536–550 elicits a response from representative hybridoma 20D4.1 (Fig. 4D).
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replots the responses of
the hybridomas to the maximal peptide dose of 100 µM from Fig. 4, with the peptide sequences shown at the left. Truncating the
glutamic acid at the N-terminal position 104 completely abrogates IL-2
production from clone 15H2 (Fig. 5A). C-terminal truncation
of the glutamine at position 112 also abrogates IL-2 production,
indicating a 9-aa core from glutamic acid 104 to glutamine at position
112. The aspartic acid at position 113 is preferred, but not required,
for T cell stimulation. Hybridoma 17G12.7 responds to a 10-aa-long core
sequence, from the aspartic acid residue at position 127 to the
histidine at position 136, as evidenced by the lack of response to
121–135 (Fig. 5B). The 8G5.4 hybridoma also sees a 10 mer
core peptide, from a glutamic acid at 208 to a second glutamic acid
residue at position 217 (Fig. 5C). Responsiveness is also
lost when residues are present at 218 and 219, but returns with
206–217, suggesting that 208–217 is the core. Truncation analysis of
the 536–550 epitope demonstrates that clone 20D4.1 recognizes a 10-aa
core that extends from a glutamic acid at position 539 to the glutamine
residue at 548 (Fig. 5D).
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reveals a
striking pattern for peptides derived from human GAD65 that bind to DQ8
and are immunogenic to T cells (Table I).
Alignment of other known DQ8 binding peptides, a 
repressor peptide
and a herpes virus VP-16 peptide, confirms agreement of these sequences
with a motif determined from the human GAD65 epitopes
(10). Table I also shows a putative alignment for two
epitopes found in this study from 431–445 and 461–475 that were not
analyzed by truncation. At the amino terminus P1, we found a negatively
charged acidic residue. There was a small polar or proline residue at
P4. P6 and P7 were predominantly hydrophobic residues. No definitive
pattern emerged at P9, which presumably would be the position affected
by the aspartic acid/noncharged residue polymorphism at DQ8 ß57.
Interestingly, we did see a trend indicating a negatively charged
residue at P10, which is required for T cell stimulation of most clones
(Table I and Fig. 5).
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To ask whether the epitopes determined in DQ8 Tg mice are relevant
in the human disease process, we stimulated PBMC from diabetic and
nondiabetic control individuals with the human GAD65 peptides and
tested for proliferation. Individuals with positive responses to GAD65
peptides and >3-fold increased response to tetanus are presented
(Table II). Not all individuals responded
to GAD65 peptides (data not shown). Overall, 11 of 16 recent-onset
diabetic individuals and 9 of 16 control individuals show proliferative
responses to at least one GAD65 peptide. All six epitopes from this
study generate a proliferative response by PBMC from at least one
diabetic individual (Table II). PBMC from control individuals also
proliferate in response to the peptides. Some of these responses are
not restricted to DQ8, because not all responders were
DQ8+. Fifteen of 16 diabetic and 4 of 16 control
individuals were DQ8+ (Table II and data not
shown). Responses demonstrated in diabetic and control individuals
could be restricted by DQ2 or DQA1*0101/DQB1*0501, both
diabetes-associated HLA molecules lacking aspartic acid at ß57
(2).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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D). We were able to generate GAD65-specific hybridomas from
DQ8 Tg mice, defining the presence of at least nine epitopes in human
GAD65 presented by the DQ8 molecule (Fig. 2). Although human GAD65 is
not a self Ag in these mice, mouse and human GAD65 are 96% identical
at the amino acid level, and all core peptides determined in this study
are identical between the mouse and human sequences (data not shown).
We were able to specifically identify 101–115, 126–140, 206–220,
431–445, 461–475, and 536–550 as epitopes. Interestingly, the
206–220 epitope is the immunodominant epitope presented by the
diabetes-associated I-Ag7 class II molecule in
NOD mice (26, 28). Although the structure of the two
molecules may be very similar (29), the DQ8-restricted
206–220 specific hybridoma does not recognize this epitope in the
context of I-Ag7 (data not shown).
Human GAD65 regions presented by the DQ8 molecule were determined in a
similar DQ8 Tg mouse system in a study published during the preparation
of this manuscript (23). This study also identified
121–140 and 201–220 as well as 231–250 and 471–490 as regions
eliciting proliferative responses from bulk cultures of splenocytes
from human GAD65-immunized mice. The identification by both studies of
126–140 and 206–220 provides independent confirmation that these
regions are presented by the DQ8 molecule. Our study also detected
reactivity in the regions spanning 231–250 and 471–490, although
those hybridomas became nonresponsive before a specific epitope could
be mapped (see Fig. 2). The hybridoma methodology may have allowed us
to identify the additional epitopes from 101–115, 431–445, 461–475,
and 536–550 due to greater ability to detect specific T cells that are
not detectable in bulk culture. The differences between the two studies
could be due to the inclusion of a human CD4 transgene in the DQ8 Tg
mice studied by Liu et al. (23), to the use of bacterial
vs baculovirus-derived GAD65, or to the differences in immunization and
detection of the T cell response between the two studies. Preserving
the T cell specificities as hybridomas allowed us to prove that the six
epitopes identified in this study can be processed by human APC. The
IL-2 responses demonstrated to the whole human GAD65 protein are
stimulated by human EBV-transformed DQ8+ B cells
presenting Ag (Figs. 2 and 3; data not shown). This underscores the
usefulness of the mouse model, because it appears that the murine
processing machinery is similar enough to generate many epitopes also
generated by the human processing machinery.
Analyses of the responses by hybridomas to their specific peptides and
truncations shows several interesting characteristics for peptides
presented by the DQ8 diabetes-associated molecule. One observation
common to all clones is that they produce significant amounts of IL-2
with a minimum requirement of 6.25 µM of their specific peptide (Fig. 4). A similar high concentration of peptide is required to stimulate
GAD65-specific hybridomas restricted to the diabetes-associated
I-Ag7 molecule (C.-C. Chao, personal
communication). In contrast, hybridomas restricted to HLA-DR class II
alleles can respond to peptide at concentrations of <0.3 µM
(25) (A. Cope, unpublished observation). This observation
could reflect the low affinity binding of these peptides to the DQ8
molecule, as has been documented for epitopes from autoantigens
important in other autoimmune diseases (30). Alternately,
the high concentration of peptide required could reflect a relatively
low avidity TCR interacting with the DQ8/peptide complex.
Another feature is that a majority of the hybridomas require a 10-aa
core peptide for stimulation, in contrast to a 9 mer core required by
most class II-restricted T cell responses (Fig. 5) (8).
Our data do not distinguish between the 10th residue being required for
binding to the DQ8 molecule or for recognition by the TCR. The crystal
structure of mouse I-Ak/hen egg lysozyme peptide,
the closest HLA-DQ homologue determined, suggests involvement of
peptide-flanking residues outside P1–P9 in TCR engagement
(31). In this structure, residues that flank the core nine
residues of the peptide on either end point up and angle toward the
TCR. Positions P10 and P11 at the C terminus of the hen egg lysozyme
peptide have been shown to be critical for TCR recognition of several
clones (32). Additionally, naturally processed sets of
peptides with differences in the number of peptide-flanking residues
have been shown to act as antagonists for T cells dependent on these
residues for recognition (33). A similar recognition
pattern by GAD65-specific DQ8-restricted clones as a general feature
could be biologically relevant and potentially important for
Ag-specific disease therapy. Clone 8G5.4, specific for the core epitope
208–217, appears to be sensitive to the presence of additional
flanking residues beyond P10 (Fig. 5C). It is unclear why
the addition of residues 218 or 218 and 219 produces this effect, while
the addition of residues 218–220 does not.
The pattern discerned from the alignment of the core epitopes
determined in this study in Table I is similar in part to the pattern
that emerges from DQ8 peptide elution studies or binding studies, but
differs at the C terminus (12, 13). DQ8 peptide elution
studies found predominantly a negatively charged residue at P1,
confirming our result and the alignment of the P1 position
(13). Unlike our study, the elution studies found
negatively charged residues at P9. They demonstrated that peptides with
negatively charged P9 residues bound with higher affinity than peptides
without such residues. However, elution studies have the disadvantage
of detecting predominantly epitopes of high affinity or high
availability and do not take into account the T cell response to the
peptides studied. By determining immunogenic T cell epitopes, we find
many epitopes from an Ag important in the disease process that do not
contain a negatively charged residue at P9. This may again indicate
that these GAD65 epitopes have low affinity interactions with the DQ8
molecule. This result is consistent with the idea that MHC/peptide
complexes of high affinity, and therefore high availability, would
delete specific T cells during negative selection in the thymus
(34).
Kwok et al. (10) found using the HSV-2 VP-16 peptide that
the DQ8 molecule prefers negatively charged residues at the putative P9
residue, while the reverse is true for diabetes-nonassociated DQ9,
which differs only at ß57. Applying the DQ8 motif in Table I to this
peptide puts the aspartic acid residue 433 in the P1 position, placing
the aspartic acid residue 442 critical for the binding difference
between DQ8 and DQ9 into the P10 position (see Table I). Using this
motif with the repressor 12–24 peptide also puts the glutamic acid
23 residue, critical for DQ8/DQ9 binding differences (35),
in P10. Our results indicate that P10 is absolutely required for T cell
stimulation for most epitopes, and P10 is commonly a negatively charged
residue (Fig. 5 and Table I). Taken together, these studies suggest the
possibility that a negatively charged residue at P10 is important in
binding differences caused by a nonaspartic acid residue at ß57.
The ultimate test of the mouse model system was to determine whether
human PBMC from diabetic individuals could respond to the GAD65
epitopes defined in DQ8 Tg mice. This is indeed the case, although
responses are not restricted to diabetic individuals (Table II). Prior
studies have shown that both individuals with autoimmune disease and
healthy subjects have T cells that respond to self peptides (36, 37). Although responses are generated in many nondiabetic
individuals, pathogenic responses could be qualitatively or
quantitatively different. Alternately, the difference may be a lack of
ability to regulate a pathogenic response in a diabetic individual.
Another notable finding is that the response to these GAD65 peptides is not necessarily restricted to the DQ8 molecule, since in a number of cases DQ8-negative individuals can respond to these peptides. The products of DQ2 and other alleles may be able to present some of the same peptides from human GAD65 as the DQ8 molecule. In a Sardinian population, DQ2 homozygous diabetic individuals also respond to the 206–220 peptide, indicating that the DQ2 molecule can present this peptide (M. Congia, unpublished observation). Recent evidence from this laboratory using HLA-DQ6 (DQA1*0103/DQB1*0601) Tg mice shows that this molecule can present at least one peptide from human GAD65 presented by the DQ8 molecule (G. Hwang, personal communication).
Our data suggest that the Tg mouse model is a useful system for identifying epitopes from human autoantigens that are recognized by human T cells. Epitopes that are naturally processed from the whole protein can be identified (38). The model allows us to address questions that cannot be addressed in the human system due to the difficulty of human T cell cloning or the large volumes of patient blood needed for proliferative studies. In addition to examining peptide presentation, the HLA Tg mouse model provides an opportunity to study how T cell responses restricted to disease-associated class II molecules may differ from those restricted to class II molecules not associated with type I diabetes.
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Acknowledgments |
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promoter, and the pSV5neo vector; Dr. Jack Strominger for a
construct containing the DQA1*0301 sequence; Dr. Dan Littman for human
CD4 transgenic mice; May Koo, Peggy Sullivan, Mary Vadeboncoeur, Sharon
Phillips, Jon Toma, and Robert Pesich for excellent technical
assistance; Lou Hildalgo and Ricardo Salazar for animal care; and Dr.
Mark M. Davis, Igor Brodsky, Dr. Iris Ferber, Marcos
García-Ojeda, Dr. Frances Hall, Dr. Sybil Munson, Dr. Joshua
Rabinowitz, and Kristin Tarbell for critical reading of the
manuscript. |
Footnotes |
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2 Address correspondence and reprint requests to Dr. Hugh McDevitt, D345 Fairchild Building, Stanford University, Stanford, CA 94305-5124. E-mail address:
3 Abbreviations used in this paper: ß57, HLA-DQ ß-chain position 57; DQ2, HLA-DQ2 (DQA1*0501/DQB1*0201); DQ7, HLA-DQ7 (DQA1*0301/DQB1*0301); DQ8, HLA-DQ8 (DQA1*0301/DQB1*0302); DQ9, HLA-DQ9 (DQA1*0301/DQB1*0303); GAD65, glutamic acid decarboxylase 65; I-Aß0/0, homozygous targeted disruption of the I-Aß locus; NOD, nonobese diabetic; non-Tg, nontransgenic; Tg, transgenic.
Received for publication June 23, 1999. Accepted for publication September 14, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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1*0101,ß1*0401) transgenic mice. Proc. Natl. Acad. Sci. USA 94:8082.This article has been cited by other articles:
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  K. Y. Chang and E. R. Unanue Prediction of HLA-DQ8 {beta} cell peptidome using a computational program and its relationship to autoreactive T cells Int. Immunol., June 1, 2009; 21(6): 705 - 713. [Abstract] [Full Text] [PDF]
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 A. Toma, T. Laika, S. Haddouk, S. Luce, J.-P. Briand, L. Camoin, F. Connan, M. Lambert, S. Caillat-Zucman, J.-C. Carel, et al. Recognition of Human Proinsulin Leader Sequence by Class I-Restricted T-Cells in HLA-A*0201 Transgenic Mice and in Human Type 1 Diabetes Diabetes, February 1, 2009; 58(2): 394 - 402. [Abstract] [Full Text] [PDF]
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X. Ge, J. D. Piganelli, H. M. Tse, S. Bertera, C. E. Mathews, M. Trucco, L. Wen, and W. A. Rudert Modulatory Role of DR4- to DQ8-restricted CD4 T-Cell Responses and Type 1 Diabetes Susceptibility Diabetes, December 1, 2006; 55(12): 3455 - 3462. [Abstract] [Full Text] [PDF]
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B. Faideau, E. Larger, F. Lepault, J. C. Carel, and C. Boitard Role of {beta}-Cells in Type 1 Diabetes Pathogenesis Diabetes, December 1, 2005; 54(suppl_2): S87 - S96. [Abstract] [Full Text] [PDF]
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 A. Toma, S. Haddouk, J.-P. Briand, L. Camoin, H. Gahery, F. Connan, D. Dubois-Laforgue, S. Caillat-Zucman, J.-G. Guillet, J.-C. Carel, et al. Recognition of a subregion of human proinsulin by class I-restricted T cells in type 1 diabetic patients PNAS, July 26, 2005; 102(30): 10581 - 10586. [Abstract] [Full Text] [PDF]
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 E. Havari, A. M. Lennon-Dumenil, L. Klein, D. Neely, J. A. Taylor, M. F. McInerney, K. W. Wucherpfennig, and M. A. Lipes Expression of the B7.1 Costimulatory Molecule on Pancreatic {beta} Cells Abrogates the Requirement for CD4 T Cells in the Development of Type 1 Diabetes J. Immunol., July 15, 2004; 173(2): 787 - 796. [Abstract] [Full Text] [PDF]
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K. Kelemen, P. A. Gottlieb, A. L. Putnam, H. W. Davidson, D. R. Wegmann, and J. C. Hutton HLA-DQ8-Associated T Cell Responses to the Diabetes Autoantigen Phogrin (IA-2{beta}) in Human Prediabetes J. Immunol., March 15, 2004; 172(6): 3955 - 3962. [Abstract] [Full Text] [PDF]
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 S. E. Starwalt, E. L. Masteller, J. A. Bluestone, and D. M. Kranz Directed evolution of a single-chain class II MHC product by yeast display Protein Eng. Des. Sel., February 1, 2003; 16(2): 147 - 156. [Abstract] [Full Text] [PDF]
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J. Sidney, M.-F. del Guercio, S. Southwood, and A. Sette The HLA Molecules DQA1*0501/B1*0201 and DQA1*0301/B1*0302 Share an Extensive Overlap in Peptide Binding Specificity J. Immunol., November 1, 2002; 169(9): 5098 - 5108. [Abstract] [Full Text] [PDF]
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P. Halbout, J.-P. Briand, C. Becourt, S. Muller, and C. Boitard T Cell Response to Preproinsulin I and II in the Nonobese Diabetic Mouse J. Immunol., September 1, 2002; 169(5): 2436 - 2443. [Abstract] [Full Text] [PDF]
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P. Y. Arnold, N. L. La Gruta, T. Miller, K. M. Vignali, P. S. Adams, D. L. Woodland, and D. A. A. Vignali The Majority of Immunogenic Epitopes Generate CD4+ T Cells That Are Dependent on MHC Class II-Bound Peptide-Flanking Residues J. Immunol., July 15, 2002; 169(2): 739 - 749. [Abstract] [Full Text] [PDF]
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R. S. Abraham, L. Wen, E. V. Marietta, and C. S. David Type 1 Diabetes-Predisposing MHC Alleles Influence the Selection of Glutamic Acid Decarboxylase (GAD) 65-Specific T Cells in a Transgenic Model J. Immunol., January 15, 2001; 166(2): 1370 - 1379. [Abstract] [Full Text] [PDF]
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S. Trembleau, G. Penna, S. Gregori, G. Magistrelli, A. Isacchi, and L. Adorini Early Th1 Response in Unprimed Nonobese Diabetic Mice to the Tyrosine Phosphatase-Like Insulinoma-Associated Protein 2, an Autoantigen in Type 1 Diabetes J. Immunol., December 15, 2000; 165(12): 6748 - 6755. [Abstract] [Full Text] [PDF]
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