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yvind Molberg*
*
Institute of Immunology and
Section of Gastroenterology, Medical Department, Rikshospitalet, University of Oslo, Oslo, Norway
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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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MHC tetramers have predominantly been applied in studies of CD8+ T lymphocytes (reviewed in Refs. 2 and 3). This class I tetramer technology has had a profound impact on our understanding of the biology of the CD8+ T cell immune response to viruses and tumors. In contrast, only a few studies with murine MHC class II tetramers have been reported (4, 5, 6, 7, 8, 9) and only recently have data from human class II tetramers been published (10, 11, 12, 13, 14, 15).
HLA class II molecules are implicated in susceptibility (and
resistance) to many severe autoimmune diseases such as type I diabetes,
rheumatoid arthritis, multiple sclerosis, and systemic lupus
erythematosus (16). The generation of appropriate class II
tetramers could provide valuable new insights into the role
CD4+ T cells play in these chronic diseases.
However, the development of such technology is hindered by the lack of
knowledge about which Ags and T cell epitopes play a role in human
autoimmune diseases. One exception is celiac disease
(CD),3 an
HLA-associated disease where an abnormal immune response to wheat
gluten drives pathology in the small intestine (17).
Gluten peptides have been identified that are recognized by
CD4+ T cells isolated from the intestines of CD
patients (18, 19, 20, 21). The relevance of these T cells to
disease is clear, because the majority are restricted by HLA-DQ2 or
-DQ8 (22, 23), the same two molecules that have been
identified as conferring susceptibility to CD using immunogenetic
approaches (17). Identification of the epitopes recognized
by the intestinal T cells in CD is complicated by the complexity of the
gluten Ag, which is made up of alcohol-soluble 
-, 
-, and
-gliadins and alcohol-insoluble glutenins. Three DQ2-restricted
gliadin epitopes have been characterized to date, and for each of them
conversion of specific glutamine residues improves their binding to DQ2
and is critical for their recognition by intestinal T cells (18, 21). Interestingly, this modification is thought to be mediated
in vivo by the enzyme tissue transglutaminase (24), an
enzyme which is also the major target of autoantibodies in CD
(25). Two of the gliadin epitopes stem from -gliadin
(-I and -II), and these appear to be major epitopes, as all patients
tested so far responded to at least one of them (21). The
third gliadin epitope is derived from -gliadin (-I) and is more
infrequently recognized by patients (17, 18). Because it
is clear that these well-characterized peptides play a role in the T
cell response to gluten, and because they are all restricted by the
same HLA molecule, this system lends itself well to testing the class
II tetramer technology in a human disease setting.
In this study we produced soluble DQ2 molecules in the baculovirus
expression system for the purpose of peptide-DQ2 tetramer generation.
The DQ2 and DQ2
chains were engineered to ensure stable formation
of soluble DQ2 molecules with covalently linked peptide. We succeeded
in expressing soluble peptide-DQ2 complexes that, when coated onto
plastic, efficiently stimulated T cell clones (TCCs). Moreover, by
incubating these enzymatically biotinylated peptide-DQ2s with
streptavidin-R-PE (SA-R-PE) we were able to generate tetrameric and
multimeric complexes that we refer to in this study as peptide-DQ2
multimers. Using a panel of peptide-specific TCCs and lines derived
from CD patients, we demonstrated that the peptide-DQ2 multimers
stained T cells in a peptide-specific manner. However, we observed a
dramatic loss of multimer binding to Ag-specific T cells after
antigenic challenge, due to an extensive down-modulation of the TCRs.
This phenomenon must be taken into consideration when using peptide-DQ2
multimers to stain T cells of active celiac lesion, and has serious
implications for the use of class II multimer reagents in general.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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cDNA of DQA1*0501 and DQB1*0201
(generously provided by A.-K. Jonsson, Swedish University of
Agricultural Science, Uppsala, Sweden, and B. Mach, University of
Geneva Medical School, Geneva, Switzerland, respectively) were
truncated by PCR and added to fos and jun
encoding sequences in a TA vector (generously provided by L.
Fugger, Aarhus University Hospital, Aarhus, Denmark). These
constructs, which were originally used for the expression of empty
recombinant DQ2 molecules (G. Paulsen, L. Fugger, and L. M. Sollid,
unpublished observations), were further modified for our
purpose. A factor Xa site was introduced between the DQ and the
fos segment by megaprimer PCR using the
DQA1*0501-fos construct as template.
BglII sites were introduced on both sides of the complete
DQA gene to be able to introduce the gene into a
BglII restriction site of the baculovirus expression vector.
The amplified and modified DQA gene was cloned into pMOSBlue
vector and sequenced. A synthetic gene encoding the native DQB leader
peptide, the sequence encoding a flexible linked peptide, the 3' DQB
extracellular region with a factor Xa site, the jun motif,
and a birA site (26) were purchased from Operon
Technologies (Alameda, CA) (Fig. 1
). The
15-aa-long linker (GAGSLVPRGSGGGGS) included a thrombin site. The
synthetic gene was flanked by BamHI sites which were
exploited to clone into the BamHI sites in a modified
pGEM-112f+ plasmid DNA vector in which the
AvaI site had been removed. The major part of DQB was
excised and purified from the TA vector by
AvaI/BstX digestion. This fragment was then
inserted into AvaI/BstX sites included in the
synthetic gene as shown in Fig. 1.
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Production and purification of recombinant peptide-DQ2 complexes
The DQA- and DQB-carrying pAcAB3 plasmid was cotransfected with linearized baculovirus DNA (BD PharMingen, BaculoGold kit 21100K) into Sf9 insect cells according to the manufacturer’s recommendation. One or two rounds of plaque purification generated clonal virus isolates. High-titer virus stocks were produced in Sf9 cells and used to infect the serum-free, suspension expresSF+ cell line (Protein Science, Meriden, CT). Spinner cultures of 1.5–2.0 liters in total were infected at a concentration of 2.5 x 106 cells/ml with a multiplicity of infection of 3–5. Supernatants harvested at day 4 or 5 were affinity-purified using the mAb 2.12.E11 (27), coupled, and cross-linked to protein A-Sepharose beads. Soluble peptide-DQ2 molecules were eluted at pH 11.5 with 50 mM of diethylamine containing 0.15 M of NaCl and immediately neutralized using 2 M of Tris (pH 6.2). Buffer exchange (PBS 0.02% NaN3) and concentration of eluates were performed in Vivaspin centrifugal concentrators (cutoff 10,000; Vivascience, Binbrook Lincoln, U.K.). The protein content was determined by the bicinchoninic acid method (Pierce, Oud-Beijerland, The Netherlands) and purity was confirmed by SDS-PAGE under reducing conditions.
Generation of gliadin-specific T cells
Briefly, cells were isolated from small intestinal biopsies from DQ2+ CD patients that had been challenged overnight with pepsin/trypsin-digested gliadin (T cell line (TCL) CD 429, 430, 431), chymotrypsin-digested gliadin (TCL CD 433), or pepsin/trypsin-digested gluten (TCC CD 380). The TCL CD 444 was generated from biopsies that were not challenged ex vivo with gluten or gliadin. Isolated cells were cultured with 1 x 106 autologous, irradiated PBMC and 5–10 IU/ml recombinant human IL-2. Expansion of TCL was done with 1 µg/ml PHA (Murex Diagnostics, Dartford, U.K.), 5 IU/ml IL-2, and allogeneic, irradiated PBMC. TCCs were established from peptide-specific TCL by seeding 0.3–1.0 T cells/well in 20-µl wells in the presence of 2 x 104 allogeneic, irradiated PBMC, 1 µg/ml PHA, and 5 IU/ml IL-2. The TCCs and TCLs tested were isolated from different CD patients. The TCLs analyzed were selected toward being reactive to all three gliadin epitopes. The generation of TCC 4.66, a sister clone of TCC 4.32, and the TCC CD 380 is reported elsewhere (21, 22). We generated a negative control TCL (TCL CTRL) by challenging intestinal biopsies from a DQ2+ individual not having CD with human astrovirus serotype 1, and the cells were expanded following the same protocol as described above.
T cell assay
Soluble DQ2 molecules in PBS 0.02% NaN3
were adsorbed to flat-bottom 96-well plates (Costar, Corning,
NY) at the given concentration in triplicates by overnight
incubation at 4°C (100 µl/well). The wells were washed three times
in medium before 5 x 104 TCCs were added in
200 µl of culture medium (RPMI 1640 (Life Technologies, Paisley,
Scotland) and 15% pooled, inactivated human serum). In the cases of
peptide stimulation of TCLs, 3–5 x 104 T
cells were added to 5 x 104 irradiated (80
Gy) APC (DQ2+ B-lymphoblastoid cell line) which
had been incubated overnight with different peptide dilutions in
triplicates, at a total volume of 150 µl of culture medium. T cell
proliferation was measured as [3H]thymidine
incorporation 72 h after stimulation. All assays were performed
twice with similar results. For the experiments shown in Fig. 6, 5
x 104 TCC 4.66 were mixed with 1 x
105 irradiated APC (DQ2+
B-lymphoblastoid cell line) that had been incubated overnight with
peptide in a 96-well plate. Wells were harvested in triplicates at days
1 (16 h after stimulation), 2, 3, 5, and 8 for analysis by flow
cytometry.
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Peptide-DQ2 molecules were biotinylated with BirA enzyme (Avidity, Denver, CO) in 10 mM of Tris-HCl (pH 8). The peptide-DQ2 molecules at concentration of 1–2 mg/ml were incubated with BirA (Avidity) at a concentration of 10 µg/ml in room temperature overnight in a biotin-containing buffer provided by the manufacturer. Unbound biotin was removed and buffer was exchanged with PBS containing 0.02% NaN3 by centrifugal concentrators (Vivascience; cutoff 10,000). The biotinylation efficiency was estimated by ELISA quantification of DQ2 molecules before and after removal of biotinylated protein with streptavidin-agarose (Pierce). Typically >95% of the DQ2 molecules were biotinylated. Biotinylated peptide-DQ2s were incubated with 70% of SA-R-PE conjugate (Molecular Probes, Eugene, OR), giving a final 6:1 molar ratio (DQ2: SA-R-PE) at room temperature for 2 h in the dark. Thereafter, the rest of the SA-R-PE conjugate was added and incubated for another 2 h before storage at 4°C. The peptide-DQ2 staining reagent gave stable staining for months.
Gel filtration assay
Soluble peptide-DQ2 molecules, SA-R-PE conjugate, or a mix of these two reagents (prepared as when generating peptide-DQ2 multimers) was analyzed by size exclusion fast protein liquid chromatography on a Superdex 200 (Amersham Pharmacia Biotech, Uppsala, Sweden) column.
Staining of gliadin-specific T cells
Pellets of 1–5 x 105 T cells were
incubated in 30 µl of DQ2 multimers at a concentration of 30 µg/ml
(RPMI 1640, 5% FCS, 0.1% NaN3) for 30 min at
37°C. Prior to staining of the cells with other surface
markers (FITC anti-CD4 (clone SK3), PE anti-CD4 (clone
SK3), FITC anti-TCR (clone WT31); BD Biosciences, San Jose,
CA), they were chilled for 5 min, supplemented with staining reagents
for 30 min, and subsequently washed twice before analysis by flow
cytometry. The TCCs in Fig. 4 were stained directly after thawing,
whereas the TCLs in Fig. 5
were stained either directly from culture or
after thawing. The analyses of multimer staining were performed on
FACSort or FACSCaliber instruments (BD Biosciences).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Soluble DQ2 molecules were expressed in the baculovirus expression
system with the transmembrane DQ and DQ regions replaced by
fos and jun leucine zipper dimerization motifs,
respectively (28). To further facilitate assembly of DQ2
dimers, and to ensure complete loading of desired Ag in the binding
site, the soluble DQ2 molecules were produced with peptides covalently
linked to the DQ2 chain (29, 30). Using this approach
we made soluble DQ2 molecules with four different peptides (Table I). Three of the peptides were antigenic
gliadin T cell epitopes (-gliadin I, -gliadin I, and -gliadin
II) while the fourth was an endogenous self-peptide eluted from DQ2
which derives from the HLA class I H chain and is known to bind to DQ2
with a high affinity (control-DQ2) (31, 32).
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-I-DQ2, -II-DQ2, and
-I-DQ2 was typically around 0.25–0.4 mg/L, while the control-DQ2
expression was only 0.1–0.2 mg/L of cell culture. Stimulation of TCCs by immobilized gliadin-DQ2 complexes
Affinity-purified DQ2 molecules with covalently attached peptides
were coated onto plastic and tested to see whether they could
specifically stimulate TCCs derived from the intestines of CD patients.
Two unrelated TCCs were tested for each of the -I-DQ2 and -II-DQ2
molecules, whereas only one TCC was available for testing the soluble
-I-DQ2 preparation. All TCCs responded to the cognate immobilized
peptide-DQ2 complex (stimulation of one TCC of each specificity is
shown in Fig. 2). The stimulatory
capacity of the soluble gliadin peptide-DQ2 complexes compared well
with the stimulation seen when peptide-pulsed APCs were used (data not
shown).
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Peptide-DQ2 molecules were biotinylated at a single site in the
C-terminal tail of the DQ chain and then used to generate PE-labeled
tetrameric complexes on a streptavidin backbone. Optimal staining with
tetramer staining reagent is obtained when all streptavidin molecules
are complexed with four peptide-MHC molecules (6). To
achieve this, we used a MHC:streptavidin ratio of 6:1. In addition,
70% of total streptavidin used was preincubated with the peptide-DQ2
molecules before the rest of the streptavidin was added and the
incubation was extended. The step-wise incubation was performed to
optimize the excess of DQ2 molecules able to bind streptavidin.
We used SA-R-PE conjugate from Molecular Probes for generation of the
peptide-DQ2 staining reagents. The main fraction of the SA-R-PE eluted
as conjugates of molecular mass lower than 440 kDa when
subjected to Superdex 200 gel filtration (Fig. 3). This indicates that most SA-R-PE
conjugates consist of only one PE molecule of 240 kDa and that no
significant amounts of free streptavidin molecules were present. Gel
filtration of the peptide-DQ2 staining reagent demonstrated that a
fraction eluted as complexes of between 440 and 660 kDa and, in
addition, parts of the reagent eluted in the void volume as aggregates
(Fig. 3). When aggregates and complexes were separated, both stained T
cells specifically and in a similar manner to the staining given by
unseparated reagent (data not shown). Therefore, the reagents were used
in an unseparated form in all additional experiments and are referred
to as peptide-DQ2 multimers.
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-I-specific TCC 4.66 was
stained with -I- and control-DQ2 multimers for 5, 15, 30, and 60
min. Optimal staining was observed with incubation between 15 and 30
min (Fig. 4A). All further
staining experiments were incubated for 30 min. The specificity of the
peptide-DQ2 multimers was analyzed by staining a panel of
well-characterized gluten peptide-specific TCCs. Peptide-DQ2 multimers
stained TCCs in accordance with their known peptide specificity (Fig. 4B). The TCCs displayed no cross-reactivity when noncognate
peptide was presented by APC (data not shown), and likewise no
cross-reactive staining was observed with multimers of other gluten
specificities. Several additional TCCs were also stained by the
peptide-DQ2 multimers in a specific manner. Interestingly, while the
intensity of staining remained the same for the same T cell stained on
different occasions, it varied among TCCs of the same specificity. This
was not due to different cell-surface expression levels of the TCR, as
staining these T cells with a TCR-specific Ab gave almost identical
results (data not shown). All the T cells used were expanded by cyclic
weekly mitogenic restimulation, and they were always applied in assays
in a nonproliferating state 8 or 9 days after the last stimulation.
Thus, the differences in staining intensities are not likely to be
caused by dissimilarities in activation status of the T cells. The
differential staining may thus reflect differences in affinity of the
TCRs from the different T cells for the same peptide-DQ2 complexes. We
also stained several TCLs derived from the intestines of CD patients to
assess the ability of the gliadin-DQ2 multimers to stain distinct
populations in a more heterogeneous cell population (Fig. 5 and Table II). We demonstrated that the number of
positive cells of heterogeneous TCLs with particular gliadin-DQ2
multimers largely corresponded to the responses elicited by the
peptides in T cell proliferation assays (Table II). In the case of TCL
CD 429, this TCL was highly responsive to -I peptide-pulsed APC,
whereas the staining with the corresponding multimers gave few cells
with high staining intensities. The control peptide (HLA class I) is a
dominant natural ligand of DQ2 and hence already expressed by the APC
used in our experiments (32). No external peptide was thus
added to the culture where T cell reactivity to this peptide was
tested. An astrovirus-specific TCL from a DQ2+
individual not having CD (TCL CTRL) was used as a control cell line and
did not stain with any of the peptide-DQ2 multimers (Fig. 5).
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The level of TCRs expressed in the cell surface of T cells is
influenced by activation stimulus, and we wondered whether an
Ag-induced receptor down-modulation could influence the binding of
multimers. To examine this we activated TCC 4.66 with different
concentrations of the -I peptide presented by DQ2-expressing APCs
and used an irrelevant peptide (native sequence of the -I peptide)
as a control. The staining by multimers on day 1 was dramatically
reduced after challenge by antigenic peptide but not by the irrelevant
peptide (Fig. 6A). The
reduction in staining intensity obtained with the multimers reagent was
equivalent to the reduced staining with the TCR-specific Ab. The higher
the concentration of the antigenic peptide used in the assay the more
extensive the down-modulation of the TCR and, consequently, the poorer
the multimers stained the activated cells. Furthermore, we analyzed the
dynamics of the receptor up-regulation and the concurrent improvement
of multimer staining by repeating the analysis on days 2, 3, 5, and 8
(Fig. 6B). Throughout the experiment, the reactivity of the
multimers strongly related to the level of TCR cell-surface expression.
We observed that the highest antigenic peptide concentration induced a
faster (and probably greater) down-regulation than the lower
concentrations of peptide. On the contrary, low Ag concentration
resulted in a more rapid reappearance of the TCRs than when exposed to
the highest Ag dose. In our experiments, at the highest peptide
concentration the multimers staining intensity did not return to the
preactivation level even after several days.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-heterodimer was
facilitated by leucine zipper motifs and covalent linkage of a peptide
to the -chain. The gliadin-DQ2 multimers specifically stained clones
and lines derived from CD patients. Following activation of a
CD4+ gliadin-specific TCC by APCs presenting the
antigenic gliadin peptide we observed that staining was dramatically
reduced. We found that the intensity of the multimer staining was
critically dependent on the TCR surface expression level.
Biochemical studies have indicated that DQ2 is highly susceptible for
decomposition into - and -chains when exposed to SDS (33, 34). This is in contrast to the behavior of most DR molecules
and some DQ molecules, suggesting that DQ2 is fairly unstable. We
substituted the transmembrane region of the DQ and DQ chains by
fos and jun leucine zipper domains, respectively,
to enhance the stability of DQ2. The acidic and basic zippers
specifically adhere and facilitate the formation of dimers
(28). In addition, we took advantage of another strategy
and genetically engineered a sequence encoding a flexible thrombin
linker and a peptide into the construct (29, 30). The
peptide coupled to the DQ chain, in addition to having a stabilizing
effect, ensured proper loading of the DQ2 molecules.
The yields of the soluble peptide-DQ2 molecules were relatively low. This was a general finding independent of which cell line (Sf9, Hi-Five, or expresSF+) we used and independent of which viral recombinants derived from the same constructs we used. ExpresSF+ cells were chosen because they were easy to grow in large cultures under serum-free conditions which avoided contamination of the DQ2 preparations by serum proteins. Properties of DQ2 itself could be responsible for the low yield of the soluble complexes, but the sequence of the peptides clearly influenced the production level.
The immobilized peptide-DQ2 molecules specifically activated all the TCCs tested in the proliferation assay. This indicates that the molecules expressed are correctly folded and, importantly, that the linker for the peptides does not impose a major hindrance for TCR binding. The two crystal structures of TCR-peptide-MHC class II complexes reported so far found that both the TCRs span nine peptide residues from P-1 to P-8 (35, 36). If this is common to all class II-restricted TCRs, this may indicate that the linker extending the peptide to the C terminus would not be expected to interfere with the TCR binding.
Our kinetic data on multimer staining demonstrated that at 37°C an incubation time of 30 min gave optimal staining. A similar incubation time was also used in a previous study with MHC class II multimers (9), but in most studies longer incubation times (1.5–5 h) have been used (4, 5, 7, 8, 10, 11, 12, 13, 14, 15). However, few of these papers have documented the gain of lengthy incubations.
The staining intensities for the different TCCs specific for the same peptide-MHC complex varied despite having almost identical levels of TCR expression (data not shown). This is unlikely to be attributable to variation in the multimer reagents themselves, because the intensity of staining for individual clones remained consistent when stained on different occasions. The activation state of the cells is also unlikely to be responsible for this phenomenon, as all the T cells used in this experiment were taken at least 8 days after restimulation and when in a "resting" state. Thus, this phenomenon is likely to reflect differences in affinity of the TCR for the peptide-DQ2 complexes as was recently shown by Crawford et al. (4) using T cell hybridomas and by Reichstetter et al. (14), who observed the same phenomenon for two TCCs which were specific for the same HSV peptide-DQ6 complex.
Evidence is accumulating that there exists an array of distinct gluten peptides which are recognized by DQ2-restricted T cells of the celiac lesion. Exactly how many epitopes exist is currently unclear, but there may be more than 10 (Ref. 37 and H. Arentz-Hansen, unpublished observations). Some of these epitopes have been identified in young children, and a study of adult Norwegian patients has indicated that the number of peptides associated with responses in these patients may be relatively limited (21). Analysis with multimers should allow a more detailed understanding of the hierarchy of epitopes in different patients and patient groups. As a first approach to this we have tested polyclonal gut-derived TCLs. We found that the staining of the TCLs mainly correlated with the proliferation data obtained by peptide-pulsed APC in T cell assays. The correlation between stimulation and staining was poor for the TCL CD 429. However, the few multimer-positive cells stained with high intensity, and these might have had a particular high-proliferative capacity. Notably, the spectrum of staining intensities often varied more for the lines than for the clones, which could reflect variation in TCR affinities between different peptide-specific T cells within the TCLs.
The TCLs tested in this study were selected for their
reactivity to the -I epitope so that we were able to demonstrate
staining of all three gluten-peptide multimers in the same samples.
However, the percentage of T cells that were stained with the -I-DQ2
and -II-DQ2 multimers was higher than that obtained with the
-I-DQ2 multimer. This further supports the notion that the two
-gliadin epitopes are important among the epitopes, which are
recognized by adult CD patients (17).
We found that Ag-induced TCR down-modulation led to poor multimers
staining and this reduction in staining was dependent on the Ag
concentration used to activate the T cells. These data highlight some
important limitations for the application of MHC multimers that should
be taken into account when using them in monitoring immune responses.
In studying recently activated T cells, there seems to be a period of
days where MHC-multimers appear to have reduced potential to stain all
Ag-specific cells due to reduced levels of TCRs in the cell surface.
Similar findings have very recently been reported for polyoma-specific
CD8+ mouse T cells and MHC class I tetramers
(38). They found an unexpected discordance between
peptide-stimulated T cells producing IFN- and staining by tetramers
detecting T cells of the same peptide specificity during viral
infection. Lack of staining of peptide-specific T cells dependent on
the activation state has also been observed for a
CD4+ TCL and HLA class II multimers
(15). The authors claimed that the effect was independent
of TCR down-modulation, but direct evidence demonstrating this was not
presented.
Initial data from limiting dilution experiments (. Molberg and
S. N. McAdam, unpublished observations) suggest that the
frequency of gluten peptide-specific T cells in the intestinal lesions
of CD patients may be very high and thus suited for direct detection
using the DQ2 multimers. However, the staining of biopsy-derived cells
is technically more demanding than the staining of TCCs and TCLs
because the analysis is performed on heterogeneous cell populations
prepared from solid tissue. The future development of robust protocols
to purify CD4+ T cells from intestinal biopsies
is thus needed. Moreover, in the active lesions present in celiacs on a
gluten-containing diet, the peptide-specific T cells are most likely
regularly stimulated, so it is conceivable that at the time the biopsy
is taken there is extensive down-modulation of the TCRs on the
gluten-specific T cells. To be able to determine the precursor
frequencies in active lesions these problems have to be solved.
In summary, we have generated DQ2 multimers carrying gliadin epitopes that specifically stain T cells recognizing the corresponding gliadin epitopes. The gliadin-DQ2 multimers do not exhibit any cross-reactive staining of DQ2-restricted T cells specific for different peptides, and specific staining for T cells was obtained for polyclonal TCLs derived from gut biopsies of CD patients. The staining of the polyclonal lines mainly correlated with the corresponding proliferative data when stimulated with peptide-pulsed APC. This further supports the concept that there is a hierarchy among the gluten T cell epitopes and that some appear to be more important than others. We also demonstrate that Ag stimulation led to an extensive down-modulation of the TCR cell-surface expression, resulting in impaired multimer staining. This observation has relevance for studies using multimers to characterize gluten-specific T cells that likely are involved in the development of CD.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Hanne Quarsten, Institute of Immunology, Rikshospitalet, 0027 Oslo, Norway. E-mail address: hanne.quarsten{at}labmed.uio.no
3 Abbreviations used in this paper: CD, celiac disease; TCC, T cell clone; TCL, T cell line; SA-R-PE, streptavidin-R-PE; TCL CTRL, negative control TCL.
Received for publication April 10, 2001. Accepted for publication August 22, 2001.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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. Molberg, R. Körner, S. N. McAdam, D. Anthonsen, H. Quarsten, O. Norèn, P. Roepstorff, E. Thorsby, L. M. Sollid. 1998. Identification of a gliadin T-cell epitope in coeliac disease: general importance of gliadin deamidation for intestinal T-cell recognition. Scand. J. Immunol. 48:111.[Medline]
. Molberg, K. E. A. Lundin, L. M. Sollid, T. Mutis, W. E. Benckhuijsen, et al 1998. Small intestinal T cells of celiac disease patients recognize a natural pepsin fragment of gliadin. Proc. Natl. Acad. Sci. USA 95:10050.. Molberg, H. Quarsten, W. Vader, Y. M. Kooy, K. E. A. Lundin, F. Koning, P. Roepstorff, L. M. Sollid, S. N. McAdam. 2000. The intestinal T cell response to -gliadin in adult celiac disease is focused on a single deamidated glutamine targeted by tissue transglutaminase. J. Exp. Med. 191:603.1*0501,1*0201) restricted T cells isolated from the small intestinal mucosa of celiac disease patients. J. Exp. Med. 178:187.., S. N. McAdam, R. Körner, H. Quarsten, C. Kristiansen, L. Madsen, L. Fugger, H. Scott, O. Noren, P. Roepstorff, et al 1998. Tissue transglutaminase selectively modifies gliadin peptides that are recognized by gut-derived T cells in celiac disease. Nat. Med. 4:713.[Medline]
1*0202. Hum. Immunol. 42:319.[Medline]
heterodimers. J. Biol. Chem. 271:20156.1*0501, 1*0201) molecule. Immunogenetics 44:246.[Medline]
1*0501, 1*0201) molecule. Eur. J. Immunol. 26:2764.[Medline]
protein dimer, the class II MHC molecule associated with protection from insulin-dependent diabetes mellitus. J. Immunol. 161:6439. T-cell receptor, influenza HA peptide and MHC class II molecule, HLA-DR1. EMBO J. 19:5611.[Medline]
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 T. Kinnunen, W. W. Kwok, A. Narvanen, M. Rytkonen-Nissinen, A. Immonen, S. Saarelainen, A. Taivainen, and T. Virtanen Immunomodulatory potential of heteroclitic analogs of the dominant T-cell epitope of lipocalin allergen Bos d 2 on specific T cells Int. Immunol., December 1, 2005; 17(12): 1573 - 1581. [Abstract] [Full Text] [PDF]
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 R P Anderson, D A van Heel, J A Tye-Din, M Barnardo, M Salio, D P Jewell, and A V S Hill T cells in peripheral blood after gluten challenge in coeliac disease Gut, September 1, 2005; 54(9): 1217 - 1223. [Abstract] [Full Text] [PDF]
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 K. A. Latham, K. B. Whittington, R. Zhou, Z. Qian, and E. F. Rosloniec Ex Vivo Characterization of the Autoimmune T Cell Response in the HLA-DR1 Mouse Model of Collagen-Induced Arthritis Reveals Long-Term Activation of Type II Collagen-Specific Cells and Their Presence in Arthritic Joints J. Immunol., April 1, 2005; 174(7): 3978 - 3985. [Abstract] [Full Text] [PDF]
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 R. Mallone, S. A. Kochik, E. M. Laughlin, V. H. Gersuk, H. Reijonen, W. W. Kwok, and G. T. Nepom Differential Recognition and Activation Thresholds in Human Autoreactive GAD-Specific T-Cells Diabetes, April 1, 2004; 53(4): 971 - 977. [Abstract] [Full Text] [PDF]
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C.-Y. Kim, H. Quarsten, E. Bergseng, C. Khosla, and L. M. Sollid Structural basis for HLA-DQ2-mediated presentation of gluten epitopes in celiac disease PNAS, March 23, 2004; 101(12): 4175 - 4179. [Abstract] [Full Text] [PDF]
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