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Original Studies |
Thyroid Study Unit, Department of Medicine, University of Chicago, Chicago, Illinois 60637
Address all correspondence and requests for reprints to: Dr. Leslie J. DeGroot, Thyroid Study Unit, Department of Medicine, University of Chicago, 5841 South Maryland Avenue, Chicago, Illinois 60637.
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Abstract |
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There are many reports that Graves’ disease (GD) is associated with certain human leukocyte antigen (HLA) molecules, in particular DR3. Here we examined the characteristics of binding of human TSH receptor (TSHR) peptides to this disease-associated HLA class II molecule. DR3 molecules bind TSHR immuonodominant peptide epitopes with intermediate affinity. On the contrary, DR3 binds nonimmunogenic peptides either with poor affinity or not at all, with one exceptional peptide that has extremely high affinity. These results suggest that susceptibility to GD associated with inheritance of a specific HLA class II gene is due to the influence of the HLA molecule-TSHR peptide complex on the T cell repertoire.
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Introduction |
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We previously investigated T cell responses to a battery of synthetic peptides spanning the human TSHR extracellular domain. The results in patients with GD suggested that peptides containing amino acid residues 158–176, 207–222, 237–252/248–263, and 343–362/357–376 of TSHR are important or possibly immunodominant T cell epitopes (5, 6). These sequences most commonly stimulated peripheral blood mononuclear cells or T cell lines and T cell clones of patients with GD and had the least stimulatory effect on peripheral blood mononuclear cells or T cell lines from normal subjects.
In this study we use the TSHR peptides to examine their binding characteristics to a GD-predisposing HLA class II molecule. The immunogenic peptides bind to the HLA molecule, whereas nonimmunogenic peptides do not, with one exceptional peptide that has extremely high affinity.
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Materials and Methods |
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Monclonal antibody L243 (anti-DR) was purchased from American Type Culture Collection (Manassas, VA). An Epstein-Barr virus-transformed B lymphocyte cell line, QBL, homozygous for DR3 (DRB1*0301) was a gift from Dr. Gerald T. Nepom (Virginia Mason Research Center, Seattle, WA). QBL cells were cultured in RPMI 1640, which was supplemented with 2 mmol/L L-glutamine, 100 U/100 µg/mL penicillin/streptomycin, and 10% heat-inactivated FCS (Life Technologies, Inc., Grand Island, NY).
Purification of DR
A total of 108 cells were harvested and washed three times with phosphate-buffered saline (PBS). Cells were lysed in 20 mL of 10 mmol/L PBS (pH 8.0), 0.1% Triton X-100, 1 mmol/L phenylmethylsulfonylfluoride, and 1 mmol/L ethylenediamine tetraacetate. The lysates were cleared by centrifugation at 100,000 x g for 60 min at 4 C. Lysates were then passed through a protein A column followed by passage through an L243 affinity column. The L243 affinity column was washed extensively with 10 mmol/L PBS (pH 8.0) and 0.1% Triton X-100. The DR molecules were then eluted with 50 mmol/L glycine-NaOH (pH 11.5), and 0.1% Triton X-100, and the eluates were neutralized with 2 mol/L glycine-HCl (pH 2.0) and then dialyzed in 10 mmol/L PBS (pH 7.4) and 0.1% Triton X-100.
DR molecules purified from QBL consist of DR3 (DRB1*0301) and DR52b (DRB3*0202). DR52b was not removed, but a DR3-specific indicator peptide [which binds to DR3 and not DR52b, Mycobacterium tuberculosis 65-kDa heat shock protein 3–13 (hsp-(3–13); see Peptide synthesis] (7, 8) allowed us to examine DR3-specific binding.
Peptide synthesis
Peptides corresponding to the sequence of TSHR extracellular domain were synthesized by a solid phase peptide synthesis method (9). The peptides overlapped each other by five or six residues to minimize the risk of missing epitopes split between peptides. N-Terminal biotinylated Mycobacterium tuberculosis 65-kDa hsp-(3–13) (KTIAYDEEARR) was used as a DR3-specific indicator peptide. This peptide is known to bind to HLA DR3 specifically and with high affinity. In the system defined below, using nonlabeled hsp-(3–13) to compete with labeled hsp-(3–13), we found its IC50 to be between 100–500 nmol/L.
Peptide binding assays
Ten microliters of N-terminal biotinylated indicator peptide in PBS [hsp-(3–13) at final concentration of 100 nmol/L] were dispensed into 96-well polypropylene plates (binding plates). Ten microliters of serially diluted TSHR peptides in PBS-1.5% dimethylsulfoxide were added to each well of the binding plates. Thirty microliters of affinity-purified HLA class II molecules (100 ng protein) in a 5-µl volume and 25 µL binding buffer [50/100 mmol/L citrate-phosphate (pH 4.5), 0.15 mol/L NaCl, 0.2% Triton X-100, 2 mmol/L phenylmethylsulfonylfluoride, 2 mmol/L ethylenediamine tetraacetate, 0.15 µmol/L aprotinin, 2 µmol/L leupeptin, and 1.5 µmol/L pepstatin A] were added to each well of the binding plates and incubated for 21 h at 37 C.
The enzyme-linked immunosorbent assay (ELISA) plates were prepared as follows. Fifty microliters per well of 10 µg/mL anti-DR L243 (American Type Culture Collection HB 55) monoclonal antibodies were used to coat 96-well polystyrene plates overnight at 4 C. The wells were washed 3 times with DW, blocked with 200 µL blocking buffer (PBS, 1% BSA fraction, and 0.1% Tween-20) for 30 min at 37 C, and washed 3 times with DW. Twenty-five to 450 mmol/L Tris-HCl (pH 7.5), 3% BSA, 0.1% Triton X-100, and 0.3% Tween-20 were dispensed to each well of the ELISA plates. Subsequently, 50 µL of the mixture from each well of the binding plates was transferred to the corresponding wells of the ELISA plates and incubated for 7 h at room temperature. After washing wells 3 times with PBS-0.05% Tween-20 and 3 times with DW, 50 µL 2 µg/mL streptavidin-alkaline-phosphatase (Pierce Chemical Co., Rockford, IL) in PBS, 1% BSA fraction, and 0.1% Tween-20 were added to each well and incubated for 60 min at room temperature. The plates were then washed 5 times with PBS-0.05% Tween-20 and 5 times with DW. Then, 200 µL/well of 1 mg/mL p-nitrophenyl phosphate (Sigma, St. Louis, MO) were added and incubated for 12 h at room temperature. The amount of substrate hydrolyzed was assessed with a spectrophotometer at 405 nm.
The binding of indicator peptides without DR molecules was measured as nonspecific binding, and it was always the same as background.
The percent inhibition was calculated as follows: % inhibition = [100 - (sample binding - background binding) x 100]/(maximum binding - background binding).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We previously identified immunodominant T cell epitopes in human TSHR extracellular domain in GD [TSHR-(158–176), -(207–222), -(237–252/248–263), and –(343–362/357–376)]. These peptides caused significantly greater stimulation of T cells from patients with GD than of cells from normal control subjects (5, 6).
In this study the immunogenic peptides exhibited intermediate affinity
for a GD-predisposing DR3 molecules, with the exception of
TSHR-(248–263), which exhibited high affinity. Most peptides had an
IC50 of 10–50 µmol/L. TSHR-(248–263) had a
higher IC50 of 1–5 µmol/L (Fig. 1A
).
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). TSHR-(183–198) seemed to be a
nonbinder to DR3. However to determine whether TSHR-(183–198) is a
nonbinder or a very poor binder, a higher concentration of
TSHR-(183–198) needs to be tested for inhibition. The percent inhibitions by immunodominant TSHR peptides are significantly higher than those by nonimmunogenic TSHR peptides at 50 µmol/L (P < 0.05). Among nonimmunogenic TSHR peptides, TSHR-(109–124) is exceptional and appears to have a different way of affecting susceptibility to GD, as discussed below. Statistical difference is much greater when TSHR-(109–124) is excluded (P < 0.001).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We found that a GD-predisposing DR3 molecule binds multiple
immunoreactive TSHR peptides with intermediate affinity, whereas DR3
binds nonimmunogenic TSHR peptides with low affinity or does not bind
the peptides at all (one exceptional peptide showed extremely high
affinity; Fig. 1 and Table 1). Our result
is consistent with several studies indicating that most autoantigenic
peptides show intermediate or low binding affinity to the
disease-predisposing major histocompatibility complex (MHC) class II
molecules (10, 11, 12). These findings suggest that the relatively low
affinity of autoantigenic peptides for MHC molecules may allow
autoreactive T cells to escape from thymic deletion.
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What factors choose immunodominant peptides from among all intermediate to poor affinity peptides One possible idea relates to their higher affinity. For example, Kozhich et al. reported that enhanced affinity of an autoimmune peptide for MHC molecules increases both immunogenicity and immunopathogenicity (16). Other than the binding affinity of peptides for MHC molecules, several other factors may contribute to the immunodominance of peptides. These include the variations in antigen proteolysis in antigen-presenting cells, composition of the T cell repertoire, and molecular mimicry.
Geluk et al. (17) proposed a DR3-specific peptide binding motif composed of two different submotifs: n = I, L,V, M, Y, F; n + 3 = D (submotif 1); or n = I, L,V, M,Y, F, A; n + 3 = D, N,Q, E, S, T; n + 5 = K, R, H (submotif 2). Mycobacterium tuberculosis 65-kDa hsp-(3–13) (KTIAYDEEARR), which was used as an indicator peptide in this study, includes the submotif 1. It is noteworthy that TSHR-(109–124), which has the submotif 1, showed extremely high affinity almost equal to that of the hsp-(3–13) peptide.
However, there is no clear relation between the motifs and
immunogenicity of the TSHR peptides and binding affinity of the TSHR
peptides for DR3 molecule (Tables 1 and 2), suggesting that surrounding amino
acids or other factors may alter binding. If peptides with high
affinity cannot be immunodominant, as we described above, the lack of
relation may be logical.
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Our finding that immunogenic TSHR peptides bind to a GD-predisposing DR3 molecule with intermediate affinity provides additional information on the mechanism of HLA-associated susceptibility to autoimmune diseases and suggests that these interactions may shape the T cell repertoire. These observations may be applied to therapy by use of competing peptides with altered affinity for HLA molecule.
Received April 2, 1999.
Revised September 17, 1999.
Accepted October 20, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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