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The Journal of Clinical Endocrinology & Metabolism Vol. 89, No. 9 4734-4745
Copyright © 2004 by The Endocrine Society

Affinity-Enrichment of Thyrotropin Receptor Autoantibodies from Graves’ Patients and Normal Individuals Provides Insight into Their Properties and Possible Origin from Natural Antibodies

Francesco Latrofa, Gregorio D. Chazenbalk, Pavel Pichurin, Chun-Rong Chen, Sandra M. McLachlan and Basil Rapoport

Autoimmune Disease Unit, Cedars-Sinai Research Institute and School of Medicine, University of California, Los Angeles, Los Angeles, California 90048

Address all correspondence and requests for reprints to: Basil Rapoport, Cedars-Sinai Medical Center, 8700 Beverly Boulevard, Suite B-131, Los Angeles, California 90048. E-mail: rapoportb{at}cshs.org.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Note Added in Proof
 References
 
We used purified recombinant TSH receptor (TSHR) antigen prepared in mammalian cells to affinity-enrich TSHR autoantibodies from Graves’ patients’ IgG. Autoantibody enrichment, assayed by TSH binding inhibitory activity, was 20- to 1000-fold. Thyroid-stimulating antibody activity enrichment, although more difficult to quantitate, was comparable. TSHR-autoantibody approximate affinities for the holoreceptor assessed indirectly by TSH binding inhibition were 4–27 x 10–9 M, an underestimate because 100% TSHR autoantibody purity was not attained. Consistent with previous data for serum, highly enriched TSHR autoantibodies in three of four patients showed {lambda} light chain bias. However, in contrast to expectations, antigen-enriched IgG was skewed primarily toward IgG2 and IgG3, subclasses associated with polysaccharides and microorganisms, respectively. Subclass depletion studies on antigen-enriched IgG indicated that TSHR autoantibodies were predominantly IgG1 and, surprisingly, IgG4. As controls, we affinity-enriched pooled IgG from normal individuals on TSHR antigen. This enriched IgG had detectable TSH binding inhibitory activity, although with lower specific activity than, and lacking the thyroid stimulatory activity of, Graves’ IgG. Moreover, these natural IgG class autoantibodies largely recognized the same conformational variation in the TSHR N-terminal region as disease-associated TSHR autoantibodies. These studies suggest that TSHR autoantibodies may arise from natural autoantibodies, possibly by class switching from cross-reacting antibodies to microorganisms.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Note Added in Proof
 References
 
IgG CLASS AUTOANTIBODIES to the TSH receptor (TSHR) are the direct cause of hyperthyroidism in Graves’ disease (reviewed in Ref. 1). Consequently, there has been much interest in the properties of these antibodies. Thyroid-stimulating antibodies (TSAbs) have a very narrow isoelectic point, unlike other thyroid-specific autoantibodies (2). In the majority of Graves’ patients, TSAbs have restricted light chain usage (frequently lambda) (3, 4, 5). In addition, TSHR autoantibodies in thyrotoxic Graves’ disease are reported to be limited to the IgG1 subclass (6). This restricted heterogeneity of TSAbs has been suggested to reflect IgG oligoclonality, at least in some patients (4). Unlike many autoantibodies, TSHR autoantibodies are extremely difficult to detect in patients’ sera by direct binding assays. One explanation for this phenomenon is the very low TSHR autoantibody concentration in serum (7, 8, 9), orders of magnitude lower than thyroid peroxidase autoantibodies. Steric hindrance for autoantibody binding to the TSH holoreceptor on the cell surface may also be a contributing factor (10). It is also generally recognized that the TSHR is a sticky antigen, producing a high background when tested with serum or IgG from normal individuals. Speculation that the failure over many years to obtain stimulating human monoclonal autoantibodies could be related to their low affinity or the need for multiple antibodies to simultaneously engage the same receptor has very recently been settled. Sanders et al. (11) attained this long-standing quest and isolated one human monoclonal TSAb whose affinity was found to be very high. Whether the properties of this antibody are representative of the spectrum of polyclonal autoantibodies in multiple patients remains to be determined.

Analysis of polyclonal TSHR autoantibodies affinity- enriched from patients’ sera has also been a long-standing but difficult goal attempted by numerous investigators over the past three decades. Earlier studies were hampered by lack of purified TSHR antigen, necessitating the use of thyroid (12) or adipocyte (13) plasma membranes containing a multitude of antigens. Even after the molecular cloning of the TSHR (reviewed in Ref. 1), the task has remained difficult. TSHR synthetic peptides have been used for autoantibody affinity purification with limited success (14). Moreover, the discontinuous (15) and highly conformational nature of TSHR autoantibody epitopes has made mammalian cells the optimal expression system for antigen expression (reviewed in Ref. 1).

In recent years a number of groups have succeeded in generating and purifying substantial quantities of soluble TSHR ectodomain preparations that are well recognized by human autoantibodies. The approach we used was to truncate the TSHR ectodomain at its C terminus in the approximate region of intramolecular cleavage into subunits (9). The product, secreted by transfected mammalian cells, corresponds essentially to the thyroid stimulating autoantibody binding A subunit. Other groups have generated the entire TSHR ectodomain by a variety of approaches including use of vaccinia virus vectors (16, 17) and release of the ectodomain from the holoreceptor at an inserted thrombin cleavage site (18) or from a glycosyl phosphatidylinositol anchor using phospholipase C (19).

In the present report, using purified, recombinant TSHR A subunits, we highly enriched polyclonal TSHR autoantibodies from the sera of Graves’ patients. These IgG autoantibodies, of high affinity, yielded unexpected findings regarding their subclass distribution. Furthermore, in contrast to the only previous study of this nature (13), we were able to affinity-enrich IgG class TSHR antibodies, although of low specific activity, from the sera of normal individuals. Remarkably, these natural TSHR autoantibodies recognized the conformational epitopic determinant at the N terminus of the TSHR characteristic of disease-associated autoantibodies.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Note Added in Proof
 References
 
IgG purification

Feasibility of this project required the processing of large volumes of serum or plasma from patients with high titers of TSHR autoantibodies as detected by a commercial TSH binding inhibition (TBI) assay, described below. The majority of the studies in this report were on plasma from three Graves’ patients (A, B, and C) who underwent plasmapharesis for treatment of extrathyroidal manifestations of Graves’ disease (kindly provided by Dr. Yuji Nagayama, University of Nagasaki, Nagasaki, Japan, and Dr. Leslie DeGroot, University of Chicago, Chicago, IL). For each patient, 300–500 ml plasma were available. Small volumes of serum (5 ml) from two other patients (D and E) with severe Graves’ disease and ophthalmopathy were obtained from our clinics with institutional review board approval. All sera/plasma had TBI values greater than 75% except for patient D, who had a TBI of 40%. The TBI on patient C plasma was 80–90%, even at a 1:10 dilution. As controls, we pooled serum from 10 normal individuals (10–15 ml serum each). None of these individuals had a history of autoimmune thyroid disease, none had thyroid peroxidase or thyroglobulin autoantibodies detectable by ELISA (data not shown), and serum TBI values ranged from 3.2 to 4%, consistent with the absence of TSHR autoantibodies. Eight of these normal individuals had blood samples taken 15 months later to provide a second pool.

IgG was purified from plasma or serum using protein A (5 ml Hi Trap rProtein A FF column, Amersham Bioscience Corp., Piscataway, NJ; ~100 mg IgG capacity) according to the protocol of the manufacturer. In brief, plasma or serum (3–20 ml) was diluted 1:1 with 20 mM Na phosphate buffer (pH 7.0) and particles removed by centrifugation (3000 x g) and filtration (0.45 µm) and applied twice to the column. After rinsing the column with buffer, IgG was eluted with 0.1 M Na citrate (pH 3.0), neutralized with 1 M Tris HCl (pH 9.0), and dialyzed against 10 mM Tris and 50 mM NaCl (pH 7.4). IgG was concentrated using a Centriprep YM-50 (Millipore, Bedford, MA) to approximately 5–10 mg/ml. In preliminary experiments at higher concentrations, IgG was quite soluble at 50 mg/ml (Fig. 1DGo). Recovered IgG was quantitated by ELISA (human IgG-Fc ELISA quantitation kit, Bethyl Laboratories, Montgomery TX).



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  		FIG. 1. Physical stability of TSHR autoantibodies and antigen for recovery of the former from autoantibody-antigen complexes. A, Stability of TBI activity in acid. Aliquots of protein A-purified Graves’ IgG concentrated to approximately 2 mg in 20 µl were acidified with 0.2 M glycine (pH 2.3) (160 µl). After incubation at room temperature for the indicated periods of time, samples were neutralized with 40 µl of 2 M Tris (pH 8.0) and dialyzed against TBI assay buffer [50 mM NaCl, 10 mM Tris (pH 7.4)]. To compensate for volume changes occurring on dialysis, assay buffer was added as needed to equalize sample volumes before TBI assay. B, Temperature stability of Graves’ IgG TBI activity and IgG-A subunit complexes. Graves’ IgG (approximately 1 mg in 20 µl) was incubated alone or, after precomplexing with purified TSHR A subunits (~1.5 µg in 1.5 µl for 45 min at ambient temperature), for the indicated periods of time at 50 C. Aliquots were then assayed for TBI activity. C, Combined acid and temperature stability. Graves’ IgG alone or Graves’ IgG precomplexed with A subunits were subjected to both acid (pH 2.3) and high temperature (50 C) as described above. Note the rapid loss of TBI activity in the Graves’ IgG, compared with the transient restoration of TBI activity in the IgG-A subunit complex after 5 min of incubation. In different experiments, the time course of TBI restoration was too variable for practical use. D, Recovery of IgG with full TBI activity from antibody-TSHR antigen complexes using a size-exclusion centrifugal filter. Aliquots of Graves’ IgG (~1 mg in 20 µl) were incubated alone or after precomplexing with excess purified TSHR A subunits sufficient to neutralize TBI activity. One aliquot of complexed material was left untreated. To dissociate antibody-antigen complexes, a second aliquot was acidified with 0.2 M glycine (pH 2.3) (160 µl) and applied to a 100-kDa size exclusion centrifugal filter. After concentration while still under acid conditions to maintain antibody-antigen dissociation, the material was then brought to neutral pH and reequilibrated in TBI binding buffer using the centrifugal filter (see Materials and Methods). Volumes of all aliquots were equalized before TBI assay. Data shown are mean + SD of duplicate determinations. Similar observations were made in two experiments.

 
TSHR antigen purification

Purification of milligram quantities of recombinant TSHR-289 (comprising the first 289 amino acids including the signal peptide) of the TSHR ectodomain has been described previously (9, 20, 21). This protein exists in two conformational forms that are reciprocally recognized by Graves’ TSHR autoantibodies (active form) and a mouse monoclonal antibody (mAb) (inactive form) (20, 21). Differential affinity chromatography using this mouse mAb and another mAb ({alpha}-5H mAb; Qiagen, Valencia, CA), each coupled to Sepharose 4B (Amersham Biosciences), permits the separate purification of the active and inactive forms of the protein. The latter mAb recognizes a six-histidine epitope at the C terminus of TSHR-289. Because TSHR-289 essentially comprises the A subunit to which thyroid-stimulating autoantibodies bind (reviewed in Ref. 1), it is subsequently referred to as the recombinant A subunit.

Enrichment of TSHR autoantibodies by affinity capture with purified TSHR A subunits

Purified IgG from the five Graves‘ patients and the two normal pools were incubated with active TSHR A subunits (90 min at room temperature). The amount of antigen used for each preparation was based on the amount of IgG (Table 1Go) as well as preliminary, smaller-scale titering experiments involving neutralization of TBI activity (see below). For example (in one of the three enrichments performed on IgG from patient C), 29 µg purified A subunit (160 µl) was added to 38 mg IgG (6.4 ml). After incubation for 90 min at room temperature with slow mixing to permit TSHR antibody-A subunit complex formation, samples were diluted 1:1 in 10 mM Tris, 50 mM NaCl (pH 7.4) (binding buffer) and passed twice over a 1-ml affinity column of the {alpha}-5H mAb (see above). TBI activity was not present in the flow-through, consistent with absorption of TBI activity by the A subunit in the material applied to the column. After extensive washing with binding buffer, adherent proteins were eluted with 0.2 M glycine (pH 2.3). In preliminary experiments (see Results), the eluted material was immediately neutralized with 2 M Tris-HCl (pH 8) and dialyzed against binding buffer. In subsequent experiments, the eluted material was not neutralized, and BSA was added (0.05% final concentration). The sample was concentrated using a Biomax-100 (Millipore), rediluted in glycine buffer, concentrated once again, and then reequilibrated in binding buffer using the same procedure.


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  		TABLE 1. TBI activity in IgG before and after enrichment by TSHR antigen capture

 
TBI assays

TSHR autoantibody inhibition of TSH binding to the TSH holoreceptor was measured using a commercial kit (Kronus, Boise, ID). In brief, different amounts of Graves’ IgG (as described in text) added to 25 µl normal serum as carrier (50 µl final volume) was incubated for 20 min with detergent-solubilized TSHR. After addition of 125I-TSH (2 h at room temperature, final volume of 200 µl) TSHRs were precipitated with polyethylene glycol. TBI values were calculated as follows:

For neutralization (or reversal) of autoantibody TBI activity by TSHR A subunits, the indicated amounts of IgG were first diluted in 25 µl normal serum and mixed with different amounts of A subunits in binding buffer (50 µl final volume). After preincubation for 45 min at room temperature, solubilized TSHRs were added and the TBI assay performed as described above.

A different form of TBI assay was used for saturation analysis of autoantibody binding to the TSH holoreceptor. Unlike the previous assay, autoantibodies do not interact with TSH holoreceptors in solution but with TSH holoreceptors immobilized in tubes (Dynotest TRAK, ALPCO, Windham, NH). Radiolabeled TSH was diluted with the indicated concentrations of IgG (200 µl final volume) and added to the tubes. After 2.5 h at room temperature, the tubes were extensively washed [10 mM Tris HCl (pH 7.4), 50 mM NaCl, 0.1% BSA], and the radioactivity was counted.

TSAb assay

TSAb was detected by its ability to increase intracellular cAMP levels in monolayers of TSHR-expressing Chinese hamster ovary (CHO) cells as described previously, with minor modifications (22). In brief, monolayers of TSHR-expressing CHO cells in 96-well plates were incubated with different amounts of IgG in Ham’s F12 medium with 10% fetal bovine serum and 1 mM isobutylmethylxanthine (final volume 0.1 ml/well). After 3 h at 37 C, intracellular cAMP was extracted with 95% ethanol, evaporated to dryness, redissolved in 50 mM sodium acetate buffer (pH 6.2), acetylated, and measured by RIA. Values (triplicate wells measured in duplicate) were expressed as a percent of cAMP values obtained with IgG from normal individuals.

Detection of TSHR autoantibodies by flow cytometry on TSHR-expressing cells

The following cell types were used: 1) untransfected CHO (CHO-K1) cells; 2) CHO cells stably expressing the wild-type TSH holoreceptor (WT-TSHR); this cell line with an amplified transgenome has previously been referred to as TSHR-10,000 cells (23); 3) CHO cells expressing the TSHR ectodomain tethered to the plasma membrane by a glycosylphosphatidylinositol (ECD-GPI) anchor, kindly provided by Dr. Alan Johnstone (St. George’s Hospital Medical School, London, UK) (24). Cells were propagated in Ham’s F12 medium supplemented with 10% fetal bovine serum, penicillin (100 U/ml), gentamicin (50 µg/ml), and amphotericin B (2.5 µg/ml). Flow cytometric analysis was performed as described previously (10). In brief, cells were harvested from 10-cm-diameter dishes using 1 mM EDTA and 1 mM EGTA in Dulbecco’s PBS (pH 7.4). After incubation (30 min at room temperature) with the indicated concentrations of human IgG, cells were rinsed twice and then incubated for 30 min at 4 C with fluorescein isothiocyanate-conjugated goat antihuman IgG (Caltag, Burlingame, CA; 1:100; or BD Biosciences PharMingen, San Diego, CA; 1:10). After rinsing, cells were analyzed using a Beckman FACScan flow cytometer (Beckton Dickinson Immunocytometry Systems, San Jose, CA). Cells stained with propidium iodide (1 µg/ml) were excluded from analysis. The results were expressed as geometric mean fluorescence intensity values.

ELISA for total IgG, IgG light chain, and IgG subclass concentrations

Assays to determine concentrations of total IgG and {kappa} and {lambda} light chains were from Bethyl Laboratories, according to the protocol of the manufacturer, except for the use of o-phenylene-diamine + H2O2 as substrate, and the OD values were read at 490 nm. IgG subclass concentrations were assayed using a kit from Zymed Laboratories (San Francisco, CA), exactly according to the recommended protocol. For all assays, sample concentrations were calculated from standard curves generated using known amounts of IgG.

IgG subclass depletion

For determination of subclass distribution of TBI activity, enriched IgG samples, titrated to provide midrange TBI values of 50–60% (0.1–3 µg in 3–5 µl), were incubated for 1 h at room temperature with 20 µl biotin-conjugated mouse {alpha}hIgM, {alpha}hIgG, {alpha}hIgG1, {alpha}hIgG2, {alpha}hIgG4 (0.5 mg/ml; all from BD PharMingen) and 25 µl {alpha}hIgG3 (0.4 mg/ml; Caltag Laboratories). Subsequently, 20 µl rinsed Immunopure Immobilized Streptavidin (Pierce Biotechnology, Rockford, IL) were added to the samples. After incubation for 30 min at room temperature, the samples were centrifuged and the supernatants evaluated for TBI activity as described above.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Note Added in Proof
 References
 
Recovery of TSHR autoantibodies by capture with TSHR antigen

Autoantibodies complexed to the TSHR A subunit can be captured via the C-terminal 6 His residue tag on the antigen. In principle, acid elution should dissociate the complexes, allowing autoantibody recovery. However, after sample neutralization and buffer exchange, we could not detect TSHR autoantibodies in the TBI assay. Two possible explanations for this finding were antibody denaturation by acid or reformation of autoantibody-A subunit complexes during buffer exchange. TBI activity was stable in acid [0.2 M glycine (pH 2.3)] for up to 30 min (Fig. 1AGo), suggesting reassociation of antibody and antigen. The temperature lability of active TSHR A subunits (21) suggested that heating the autoantibody-A subunit complex could inactivate the antigen without damaging the autoantibody. However, although TBI activity was relatively stable for up to 3 h at 50 C, similar heating of the autoantibody-A subunit complex did not restore TBI activity (Fig. 1BGo). Therefore, unlike in the free state, the A subunit maintains its antigenic stability when complexed with autoantibody. Therefore, to recover affinity- enriched autoantibodies, TSHR antigen required to be removed (physically or by inactivation) while dissociated from the autoantibodies under acid conditions. For this purpose we tried a number of approaches. Heating (50 C) autoantibody-A subunit complexes in acid did expose some TBI activity (Fig. 1CGo). However, this activity was short lived and variable (2–5 min). Under the same harsh conditions, TSHR antibodies alone or complexed with A subunits rapidly lost TBI activity (Fig. 1CGo). Neither cationic nor anionic chromatography under acid conditions led to functional TSHR autoantibody recovery (data not shown). Finally, we attempted to separate antibody and antigen using a size exclusion (100 kDa) membrane under acid conditions before neutralization and buffer exchange. In principle, only IgG and not 60-kDa A subunits would be retained on the membrane. Indeed, by this approach we fully recovered IgG and TBI activity from autoantibodies whose TBI activity had been neutralized by addition of excess TSHR antigen (Fig. 1DGo).

Affinity enrichment of TSHR autoantibodies from Graves’ serum

TSHR autoantibodies complexed with purified TSHR A subunits were recovered by affinity capture of the epitope-tagged antigen (see Materials and Methods). The extent of specific TSHR autoantibody enrichment relative to the starting material was determined by titering TBI activity after removal of antigen (see Fig. 1DGo). For this comparison we used an arbitrary midrange TBI value of 40%. In the example shown (Fig. 2AGo), the amount of IgG required to obtain comparable TBI values was approximately 280-fold lower in the affinity-enriched IgG than in the starting IgG. In 13 TBI enrichments performed on IgG from five Graves’ patients, the extent of enrichment varied widely (Table 1Go). As expected, greater enrichment was attained with IgG containing less TBI activity. For example, with IgG from patient A (for whom we had a large volume of serum), enrichment reached 1000-fold in one preparation (mean of 500-fold in five preparations). However, with little TBI activity in the starting material (patient D), TBI recovery was too low to permit calculation of enrichment, and this IgG was not studied further. IgG from patient C is noteworthy in having the most potent TBI activity both before and after enrichment. Indeed, after enrichment, as little as 50 ng IgG (mean of three preparations) was sufficient to inhibit TSH binding by 40%. Finally, we processed IgG from pools of normal individuals, the data for which are addressed below.



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  		FIG. 2. Affinity enrichment of TSHR autoantibodies from Graves’ serum. A, Example of one of 13 separate antigen enrichment studies on IgG from five Graves’ patients. TSHR A subunits (His-tagged; 20 µg) were added to 80 mg IgG (patient A) in a volume of 10 ml. After 90 min at ambient temperature, the sample was diluted 1:1 in TBI assay binding buffer, applied twice to the anti-His mAb affinity column, and eluted with glycine (see Materials and Methods). The extent of specific TSHR autoantibody enrichment relative to the starting IgG material was determined by titering TBI activity after enrichment. Bars, Mean + SD of duplicate determinations. B, TSAb in antigen-enriched IgG from patient C. CHO cell monolayers expressing the wild-type TSHR were incubated with the indicated concentrations of IgG before (starting IgG) or after affinity enrichment using TSHR A subunits. The cAMP generated in response to Graves’ IgG is expressed as a percentage of cAMP levels generated in the presence of 25 µg/well IgG purified from a normal individual (dashed line). Each bar represents the mean + SE of cAMP values in triplicate wells, each well measured in duplicate. In another IgG enrichment from the same patient, we obtained approximately 250-fold enrichment in TSAb activity. C, Flow cytometry with TSHR autoantibodies affinity-enriched from IgG from Graves’ patients. Upper panel, CHO cell lines incubated with IgG before affinity enrichment (1 µg in 0.1 ml/tube). CHO-K1, untransfected cells; WT-TSHR, wild-type TSH holoreceptor; ECD-GPI, the TSHR ectodomain anchored to the membrane by a GPI tail (24 ). Upper panel, The same concentration of TSHR antigen-enriched IgG was incubated with the indicated CHO cell lines. Bars, Mean + SE fluorescence values for IgG from four patients. Differences between binding to CHO-K1 cells vs. WT-TSHR or ECD-GPI cells were analyzed by paired t tests.

 
In addition to TBI activity, TSHR antigen-captured Graves’ IgG was also enriched for TSAb bioactivity as assessed by stimulation of cAMP generation by cultured CHO cells expressing the recombinant TSHR (22). Enrichments determined in IgG from patients A, B, and C ranged from 50- to 250-fold (e.g. Fig. 2BGo).

A characteristic of stimulatory TSHR autoantibodies in Graves’ patients’ sera is their poor recognition of the TSH holoreceptor on the cell surface in contrast to their better recognition of the TSHR ectodomain anchored to the membrane by a glycosyl-phosphatidylinositol (GPI) tail (10). Before antigen enrichment, IgG (1 µg per tube) from four Graves’ patients provided no specific signals on flow cytometry with cells expressing either form of the TSHR. In contrast, the same quantity of TSHR antigen-enriched IgG provided a clear signal on TSHR ectodomain-GPI expressing CHO cells relative to untransfected CHO cells (Fig. 2CGo). Signals of this magnitude were rarely attained with whole Graves’ serum (1:50 dilution) containing 20- to 30-fold more IgG, as reported previously (10). Consistent with the properties of serum TSHR autoantibodies, antigen-enriched IgG recognized more weakly a CHO cell line overexpressing the TSH holoreceptor.

Affinity of TSHR autoantibody interaction with the TSHR

Availability of substantially enriched TSHR autoantibodies and purified TSHR antigen permitted estimation of TSHR autoantibody affinity for its cognate receptor. The technical difficulties for this determination should be appreciated. The yield of small amounts of enriched IgG could be accomplished only by using albumin as carrier, precluding radiolabeling of the IgG. Also, the specific activity of TSHR autoantibody in our enriched IgG was unknown. Nevertheless, we used two approaches to estimate the affinities of TSHR autoantibodies in IgG enriched from four Graves’ patients. First, we assessed autoantibody interaction with the free TSHR A subunit in solution. The principle of the assay is that autoantibodies complexed with A subunits cannot bind to the TSH holoreceptor. TSH holoreceptors would then remain accessible to radiolabeled TSH binding, with neutralization (or reversal) of autoantibody-mediated TBI activity (schematically depicted in Fig. 3AGo). This assay does not require autoantibody purity. However, it was first necessary to titer the enriched IgG preparations to determine the lowest concentrations that can provide a practical TSHR autoantibody assay signal (50–60% TBI). Subsequently, we determined the A subunit concentrations required for half-maximal neutralization of TBI activity (Fig. 3AGo). The EC50 for the four IgG preparations ranged from 4 to 9 x 10–9 M. Because of antibody divalency, autoantibody affinities for the TSHR A subunit can be estimated to be in the range of 2–5 x 10–9 M.



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  		FIG. 3. Affinity of TSHR autoantibody interaction with the TSHR. A, Autoantibody interaction with the free TSHR A subunit in solution. As depicted in the cartoon, TSHR autoantibodies in affinity-enriched Graves’ IgG bind to the TSH holoreceptor and inhibit TSH binding. Preadsorption of autoantibodies with soluble A subunits, to which TSH does not bind, restores TSH binding to the holoreceptor. Affinity-enriched IgG preparations from patients A, B, C, and E were titered to provide TBI values of 50–60%. These quantities of IgG (nanogram amounts shown in parentheses) were preincubated with increasing concentrations of TSHR A subunits. Complete neutralization of TBI activity by added A subunits is designated as 100%, and TBI activity in the absence of added A subunits is depicted as 0% reversal. The EC50 producing 50% reversal of TBI are indicated. Each point represents the mean of duplicate determinations. B, Saturation analysis of Graves’ autoantibody binding to the TSH holoreceptor. TSH holoreceptors were preincubated with increasing concentrations of affinity-enriched IgG preparations from patients A, B, C, and E. The extent of IgG binding was assessed by inhibition of radiolabeled TSH binding to the holoreceptor (see cartoon). Binding affinity estimates were based on the IgG concentrations required to saturate half the TSHR. Each point represents the mean of duplicate determinations. Similar data were obtained in a repeated experiment. Note that these values are overestimates (affinities underestimated) because they assume purity of the IgG ligand.

 
In the second approach, we estimated autoantibody affinity by saturation analysis. Autoantibody binding to the TSHR was assessed by inhibition of TSH binding (schematically shown in Fig. 3BGo). Unlike the preceding assay, this assay examines autoantibody interaction with the TSH holoreceptor rather than with the free A subunit. Apparent autoantibody affinities were slightly lower than observed with the A subunit competition assay, with EC50 values of 4 x 10–9 to 3 x 10–8 M (Fig. 3BGo). However, these calculations for the holoreceptor (unlike for the A subunit in the previous assay) assume autoantibody purity and are, therefore, underestimates.

Enrichment of TSHR autoantibodies from serum of normal individuals

As a control, we subjected IgG from normal individuals to the TSHR antigen enrichment procedure described above. IgG pooled from 10 individuals (500 µg/tube, equivalent to 2.5 mg/ml) was devoid of TSH binding inhibitory activity (Fig. 4AGo). Surprisingly, however, after affinity enrichment, TBI activity was clearly detectable, albeit at a low level. Unlike for Graves’ IgG, the degree of enrichment could not be determined because of the absence of TBI activity in the starting material as well as the low level of TBI activity in the enriched IgG (compare Fig. 4AGo with Fig. 2AGo). On flow cytometry, 4 µg of affinity-enriched normal IgG provided a greater fluorescent signal than did 500 µg of starting IgG (Fig. 4BGo). However, this signal was enhanced on both untransfected CHO-K1 cells and the same cell line expressing the GPI-anchored TSHR ectodomain (ECD-GPI) (Fig. 4BGo). These data suggested cross-reactivity between IgG in normal sera for both the TSHR and other unidentified antigens on the CHO cell surface.



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  		FIG. 4. Enrichment of TSHR autoantibodies from normal serum. A, Pooled IgG from 10 normal individuals was affinity enriched using TSHR A subunits (see Materials and Methods). Starting IgG and affinity-enriched IgG was tested for TBI activity at the indicated concentrations. Each bar represents the mean + SD of duplicate determinations. Similar data were obtained using IgG affinity enriched from eight of these individuals 15 months later. B, Flow cytometry of the same IgG as in A using untransfected CHO-K1 cells and the same cells expressing the TSHR ECD-GPI. C, Differential reversal of TBI activity using active and inactive purified A subunits. Active, but not inactive, A subunits are specifically recognized by TSHR autoantibodies in the sera of Graves’ patients (20 21 ). Each of these conformational A subunit forms was purified (see Materials and Methods) and tested individually for their ability to neutralize TBI activity affinity enriched from Graves’ patient B (see Table 1Go) (upper panel) and pooled IgG from eight normal individuals (middle panel). The concentrations of A subunits and IgG used in these assays are indicated. The lower panel indicates positive and negative internal controls (serum from a Graves’ patient and a normal individual, respectively) included in the same assay. The negative control is also present as carrier for the small amounts of enriched IgG (top and middle panels). Each bar represents the mean + SD of duplicate determinations. *, Values significantly different from the negative control; ANOVA on ranks, P < 0.05.

 
To assess the specificity of enriched, normal IgG for the TSHR we used two conformational forms of recombinant A subunits. Only the active, not the inactive, form neutralizes human autoantibodies (20, 21). Both forms are in the native state and differ only in the conformation of critical epitope at the N terminus of the ectodomain. As anticipated, active but not inactive A subunits completely neutralized TBI activity in affinity-enriched Graves’ IgG (Fig. 4CGo, upper panel). Surprisingly, for the affinity enriched normal IgG, active A subunits were also more effective than inactive A subunits in reversing TBI activity (Fig. 4CGo, middle panel). Internal assay controls with serum from a normal individual and a Graves’ patient are included in Fig. 4CGo (lower panel). These data indicate that a substantial portion of the affinity enriched IgG molecules from normal serum recognize the TSHR antigen in the same manner as Graves’ autoantibodies. We should note that in the experiment shown in Fig. 4CGo, 8 µg enriched normal IgG per tube (the maximum within the volume limitation of the TBI assay) used a total of 48 µg IgG. This experiment was repeated, with similar results, but insufficient material was available for dose-response experiments. IgG enriched from normal individuals had no TSAb activity at the highest concentration that could be tested (4 µg/0.1 ml; data not shown).

Light chains and subclasses of TSHR affinity-enriched IgG

We analyzed the proportions of {kappa} and {lambda} light chains in the TSHR antigen affinity-enriched IgG preparations. In support of previous light chain observations, IgG from three of four Graves’ patients (A, C, and E) showed significant enrichment for {lambda} light chains (Fig. 5Go; paired t test, P = 0.004). None were enriched for {kappa} light chains. One of the Graves’ IgG (patient B) and the IgG pool from normal individuals showed no light chain bias after enrichment on TSHR antigen. Turning to the IgG subclass distribution, all four Graves’ IgG preparations, as well as the normal IgG pool, showed a decrease in the proportion of IgG1 after affinity enrichment on TSHR antigen vs. the starting material that was associated with reciprocal increases in IgG2 (Fig. 6Go; paired t test, P = 0.002). The low concentration of IgG3 before enrichment is attributable to the low affinity of this subclass for protein A that was used for initial IgG purification from serum. Nevertheless, IgG3 was also increased in all four enriched Graves’ IgG preparations, as was IgG4 but only in two of four Graves’ patients.



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  		FIG. 5. Light chain type in IgG affinity-enriched using TSHR antigen. IgG from four Graves’ patients (A, B, C, and E) and IgG pooled from 10 normal individuals was affinity enriched for TSHR antibodies using epitope-tagged purified TSHR A subunits (see Materials and Methods). {kappa} and {lambda} light chain concentrations in the starting IgG (S) and after affinity enrichment (E) were determined by quantitative ELISA. Each bar represents the mean + SD of duplicate determinations. The proportion of {kappa} and {lambda} light chains in an individual IgG sample is shown above each bar as a percent of the total light chain concentration.

 


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  		FIG. 6. Subclass distribution of IgG affinity-enriched on TSHR antigen. IgG from four Graves’ patients (A, B, C, and E) and IgG pooled from 10 normal individuals were affinity enriched for TSHR antibodies using epitope-tagged purified TSHR A subunits (see Materials and Methods). IgG subclass concentrations in the IgG before (starting) and after affinity enrichment were determined by quantitative ELISA. Each bar represents the mean + SD of duplicate determinations. The proportions of the four IgG subclasses in an individual IgG sample are shown above each bar as a percent of the total IgG concentration.

 
At face value, the foregoing data suggest that TSHR autoantibodies are skewed away from IgG1 toward the other IgG subclasses, particularly IgG2 and IgG3. However, because this deduction diametrically opposes prior evidence of TSHR autoantibody IgG1 restriction (4, 6), we performed IgG subclass depletion studies on TSHR antigen-enriched IgG from the three patients for whom sufficient plasma was available (plasmapharesis). After incubation of enriched IgG with excess biotinylated, subclass-specific murine monoclonal antibodies, the latter were removed using streptavidin beads and residual TBI activity was measured in the supernatant. As expected, pretreatment with anti-IgG, but not anti-IgM, removed virtually all TBI activity. Unexpectedly, however, although TSHR adsorption enriched for IgG2 and IgG3, the TBI activity in these enriched preparations resided primarily in the IgG1 and IgG4 components in patients A and C, respectively (Fig. 7Go). In patient B, TBI activity was more widely distributed; primarily in IgG1, IgG4, and, to a lesser degree, in IgG2.



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  		FIG. 7. TBI activity in antigen-enriched IgG preparations after IgG subclass depletion. TSHR antigen- enriched IgG from the three patients for whom sufficient plasma was available (plasmapharesis) were titrated to provide TBI values of 50–60%. After incubation of excess biotinylated, subclass-specific murine monoclonal antibodies, the latter were removed using streptavidin beads, and residual TBI activity was measured in the supernatant. As negative and positive controls, we used antihuman IgM and anti-IgG, respectively. Each bar represents the mean + SD of duplicate determinations. Arrows indicate the most marked subclass depletions. The data shown are representative of three separate experiments.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Note Added in Proof
 References
 
Only recently have ideal reagents become available to enable the long-standing goal of affinity-enriching IgG class TSHR autoantibodies from the serum of patients with Graves’ disease. Previous studies adsorbing Graves’ IgG with very impure antigen, thyroid (12) or adipocyte (13) plasma membranes, were suboptimal. We report the use of purified, soluble recombinant TSHR A subunits produced in mammalian cells to enrich for highly potent, polyclonal TSHR autoantibodies. The A subunit can adsorb all TBI (9, 20) and TSAb (25) activity in the sera of the great majority of patients. We attained up to 1000-fold enrichment, as judged by the specific activity of autoantibody binding to the TSH holoreceptor. At the completion of our study, Na et al. (26) reported a similar degree of TSHR autoantibody enrichment using immobilized TSHR ectodomains; however, they used their material for a purpose different from ours, namely identification of peptide mimotopes. It must be emphasized that our study, of necessity, involved the investigation of IgG from only a few patients with high TSHR autoantibody levels and for whom large volumes of serum (or plasma) were available and should not be regarded as a survey of the general Graves’ population.

For reasons mentioned above, the affinity of TSHR autoantibodies for their cognate antigen has been of long-standing interest. Very recently Sanders et al. (11) isolated a human monoclonal thyroid-stimulating autoantibody and found its affinity to be very high: 2 x 10–11 M as determined by direct radiolabeled antibody binding. The question remained, however, whether the properties of this single monoclonal autoantibody were representative of the spectrum of polyclonal autoantibodies in multiple patients. Although unable to directly label our affinity-enriched polyclonal TSHR autoantibodies (necessary addition of albumin as carrier), our indirect estimate using absolute IgG concentrations provided values in the nanomolar range. Given that our affinity enrichment did not attain complete purity, the average affinities of the polyclonal TSHR autoantibodies must be higher, perhaps approaching that of the human monoclonal TSHR autoantibody. Also of note, thyroid-stimulating mAbs have recently been obtained by immunization of mice (27, 28) and hamsters (29). However, the affinities of these induced antibodies are reported to be lower than that of the human mAb (30).

Previous studies on TSHR autoantibody light chain usage and IgG subclass distribution have been indirect, involving autoantibody assay after depletion of different IgG components from unenriched serum IgG using light chain- or subclass-specific antibodies. The TSHR autoantibody enrichment that we attained permitted, for the first time, direct assessment of autoantibody light chain and IgG subclasses. Our data support previous evidence (3, 4, 5) for restricted TSHR autoantibody {lambda} light chain usage in the majority of Graves’ patients. Also consistent with these previous data, TSHR autoantibodies in one patient (B) did not show any light chain bias. None of our patients had {kappa} light chain autoantibody skewing. Overall, our study confirms that TSHR autoantibody light chain bias does occur. However, our sample size is too small to assess the frequency or direction of this bias in the general Graves’ population.

In contrast to the light chain data, direct analysis of the IgG subclass distribution in affinity-enriched TSHR autoantibodies conflicted with previous reports of restriction to the IgG1 component (6). Indeed, we observed a reduced proportion of IgG1 content in all four patients studied. Reciprocally, the proportions of IgG2 and IgG3 subclasses were clearly increased. These data were especially puzzling because the earlier IgG subclass depletion studies were expertly performed and well controlled (6). A possible reason for this difference was that we measured TSHR autoantibodies by TBI assay vs. bioassay (6). However, this explanation is unlikely because our TSHR autoantibodies were enriched for both TBI and bioactivity. We chose the TBI assay because it is more quantitative and reproducible than the bioassay. IgG subclass depletion studies on microgram quantities of antigen-enriched TSHR IgG (more straightforward than on milligram quantities of total IgG in serum) suggested an explanation for the discrepant data. Surprisingly, the proportionate increases of IgG2 and IgG3 in the affinity-enriched IgG did not correlate with TSHR autoantibody activity. Rather, in three Graves’ patients (for whom sufficient plasma was available after plasmapharesis), TBI activity was predominantly in the IgG1 component in one patient and the IgG4 component in another. In the third patient, TBI activity was primarily in IgG1 and IgG4, with a lesser contribution from IgG2. These data clearly indicate that, contrary to expectations, TSHR autoantibodies in thyrotoxic Graves’ disease are not necessarily restricted to the IgG1 subclass. An important corollary of our data (discussed below) is that TSHR antigen adsorbed two distinct types of antibodies, namely IgG2 and IgG3, devoid of TBI activity, as well as IgG1 and IgG4 with TBI activity. Adsorption of IgG2 and IgG3 by purified TSHR A subunits explains our inability to enhance TBI enrichment by subjecting TSHR-enriched Graves’ IgG to a second enrichment cycle (data not shown).

Numerous reports over the past two decades have described the apparent presence of TSHR antibodies in serum of normal individuals. Interpretation of such data has been fraught with difficulty. Studies with unfractionated serum are subject to pitfalls. In animals, serum proteins that inhibit TSH action (31) or, conversely, provide a false-positive signal in a TSAb assay (32) are present in non-IgG as well as IgG fractions. More recent studies involving the recombinant TSHR report IgG binding as detected by ELISA (33) or flow cytometry (34) in 55 and 12% of normal individuals, respectively. Again, although these studies were carefully performed, the results are not unequivocal. In particular, ELISA was performed with sera at very low titer (1:20 dilution), and the signal with normal IgG was not very different from that of Graves’ IgG. These data contrast with our present study in which we were unable to detect TSHR autoantibodies in normal IgG before enrichment. As reported by Metcalfe et al. (34), and consistent with our experience, flow cytometry signals with normal IgG are very weak.

How, then, can the existence of natural IgG autoantibodies to the TSHR be definitively established? Affinity enrichment of TSHR-specific antibody-specific activity from the serum of normal individuals would provide strong supportive evidence. However, previous studies have mitigated against the concept of natural TSHR autoantibodies. IgG with TSH binding inhibitory activity could be enriched by adsorption to thyroid plasma membranes but also to kidney and liver membranes, indicating lack of organ specificity (35). Ingbar and coworkers (13) provided even stronger evidence, failing to detect any TSHR autoantibody activity in IgG enriched from normal individuals as opposed to potent activity obtained from Graves’ IgG. It was for this reason that we were surprised by our present observations. Using purified TSHR antigen, we did not expect to enrich TSHR autoantibodies from normal IgG and we performed the enrichment process with this material as a control. Surprisingly, we obtained IgG with weak but clearly detectable TBI activity (but lacking stimulatory bioactivity). The final validation of our finding was that the trace amounts of IgG class TSHR autoantibodies in normal individuals recognized the same active conformational epitope at the TSHR N terminus as do TSHR autoantibodies from Graves’ patients (discussed below). Could the TSHR antibody activity enriched from normal IgG contribute to that enriched from Graves’ sera? We consider this possibility unlikely because of the low specific activity of the former. For example, 4–8 µg enriched IgG from normal individuals produced a TBI value of only 20–25%. In contrast, from Graves’ patients, 0.33 µg of our weakest IgG yielded a robust 40% TBI value.

Enrichment of the natural IgG class TSHR autoantibodies raises the question of their pathophysiological importance. As with TSHR-enriched Graves’ IgG, the enriched normal IgG also had proportionately more IgG2 and IgG3. The potential significance of this bias is the preferential use of IgG2 and IgG3 by antibodies to polysaccharides and other microorganisms, respectively. The low TBI-specific activity in the enriched IgG from normal individuals precluded reasonably accurate subclass depletion studies, as performed for Graves’ IgG. However, an important question that we could answer was whether the TSHR was simply a sticky antigen or there was epitopic specificity to the natural TSHR autoantibodies. Two conformational forms of recombinant TSHR subunits (TSHR-289) are secreted by CHO cells and can be separately purified (21). TSHR autoantibodies recognize only the active and not the inactive form. The conformational difference between these two forms lies in an epitope containing the cysteine-rich extreme N terminus of the A subunit (20, 21, 22). Remarkably, a major component of the natural TSHR autoantibodies showed specificity for the active form of the TSHR A subunit. Our data for TSHR autoantibodies are noteworthy because the same phenomenon does not apply in the case of thyroglobulin epitopic determinants, which differ between patients and healthy subjects (i.e. see Ref. 36).

Sharing some, but not all, of the fine specificity of Graves’ TSHR autoantibodies (TBI, but not thyroid-stimulatory activity) raises the possibility that natural TSHR autoantibodies may be precursors of the more potent, disease-causing autoantibodies in Graves’ disease. It is possible that an antigen-driven process in genetically susceptible individuals leads to affinity maturation and IgG class switching of natural IgG class TSHR autoantibodies. Also of interest is enrichment of IgG2 and IgG3 class antibodies by purified TSHR A subunits. Enrichment of these subclasses may relate to xenogeneic forms of carbohydrate on hamster cell-derived proteins. However, preferential recognition by natural TSHR autoantibodies of the active subunit (dependent on polypeptide, not carbohydrate, structure) speaks against this likelihood. Cross-reactivity with microorganism antigens also can not be excluded. There is epidemiological evidence of a role for prior infections in the incidence of Graves’ disease (reviewed in Ref. 37). TSH is also reported to bind specifically to microorganisms such as Yersinia enterocolitica (38, 39, 40, 41). However, the pathophysiological significance of such findings remains unclear.

In summary, using purified recombinant antigen, we have succeeded in highly enriching polyclonal, IgG class TSHR autoantibodies from Graves’ serum. We determined that these autoantibodies are of high affinity. Contrary to expectations, direct analysis of IgG subclass distribution in highly enriched TSHR autoantibody preparations indicated skewing toward IgG2 and IgG3. However, most TSHR autoantibodies were not in these subclasses but primarily in IgG1 and, surprisingly, in IgG4. Finally, we provide the first unequivocal evidence for the presence in normal individuals of natural, IgG class TSHR autoantibodies with some of the characteristics of disease-associated autoantibodies. These observations raise the possibility that some TSHR autoantibodies may arise from natural autoantibodies, possibly by class switching from cross-reacting antibodies to microorganisms.


   Note Added in Proof
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Note Added in Proof
References
 
Since submission of our paper, Morgenthaler et al. (42) have reported affinity enrichment of TSHR autoantibodies from Graves’ patients’ serum using a different approach.


   Acknowledgments
 
This work was supported by National Institutes of Health Grant DK 19289.

The generous contribution of Dr. Boris Catz (Los Angeles, CA) is gratefully acknowledged. We thank BRAHMS (Berlin, Germany) for kindly providing some of the radiolabeled TSH used in this study.


   Footnotes
 
Abbreviations: CHO, Chinese hamster ovary; ECD-GPI, ectodomain tethered to the plasma membrane by a GPI; GPI, glycosylphosphatidylinositol; mAb, monoclonal antibody; TBI, TSH binding inhibition; TSAb, thyroid-stimulating antibody; TSHR, TSH receptor.

Received December 2, 2003.

Accepted May 19, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Note Added in Proof
 References
 

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