help button home button Endocrine Society Endocrinology
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Endocrinology, doi:10.1210/en.2006-1096
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
148/2/752    most recent
Author Manuscript (PDF)
Right arrow Purchase Article
Right arrow View Shopping Cart
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Martinez, O.
Right arrow Articles by Prabhakar, B. S.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Martinez, O.
Right arrow Articles by Prabhakar, B. S.
Endocrinology Vol. 148, No. 2 752-761
Copyright © 2007 by The Endocrine Society

Diversity in the Complementarity-Determining Region 3 (CDR3) of Antibodies from Mice with Evolving Anti-Thyroid-Stimulating Hormone Receptor Antibody Responses

Osvaldo Martinez, Eryn Gangi, David Mordi, Sonal Gupta, Samuel Dorevitch, Marie-Paule Lefranc and Bellur S. Prabhakar

Department of Microbiology and Immunology (E.G., D.M., S.G., B.S.P.), School of Public Health, Epidemiology, and Biostatistics (S.D.), University of Illinois at Chicago, Chicago, Illinois 60612; Laboratoire d’ImmunoGénétique Moléculaire (M.-P.L.), Université Montpellier II, Unité Propre de Recherche, Centre National de la Recherche Scientifique 1142, Institut de Génétique Humaine, 34396 Montpellier, France; and Department of Microbiology (O.M.), Mount Sinai School of Medicine, New York, New York 10029

Address all correspondence and requests for reprints to: Bellur S. Prabhakar, Department of Microbiology and Immunology (M/C 790), Room E-709, Building 935, 835 South Wolcott Avenue, Chicago, Illinois 60612. E-mail: bprabhak{at}uic.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
In a mouse model of autoimmune Graves’ disease, stimulatory anti-TSH receptor (TSHR) antibodies (TSAbs) slowly evolve upon repeated immunization with TSHR and lead to hyperthyroidism. Although all immunized mice developed high levels of TSH-binding inhibitory Ig (TBII), only a subset of these mice become hyperthyroid, suggesting that the generation of pathogenic antibodies (Abs) may require affinity maturation. We analyzed the complementarity-determining region 3 (CDR3) of IGHV1 and IGHV5 heavy chains from mice at different stages of disease development. Subcloned CDR3 PCR products were amplified from RNA isolated from enriched splenic B/plasma cells of a control mouse, and mice with low TBII and normal T4 levels (LTNT4), high TBII and normal T4 levels (HTNT4), and high TBII and high T4 levels (HTHT4). Using statistical analyses, we correlated usage of D and J genes and the amino acid composition and length of and mutations within the CDR3 with different outcomes after TSHR immunization. CDR3 sequences from TSHR-immunized mice contained a higher frequency of D gene SP2.9 relative to control, whereas sequences from HTHT4 contained a higher frequency of D gene Q52 compared with sequences from LTNT4. Furthermore, HTHT4 sequences also contained higher CDR3 replacement mutations, relative to LTNT4 and HTNT4 mice, that are indicative of somatic hypermutation. Collectively, our results suggest that higher somatic mutations within the CDR3 may correlate with pathogenic antibodies against the TSHR.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE EUTHYROID STATE is maintained through a hypothalamo-pituitary-thyroid feedback loop (1) that is regulated by the levels of TSH and thyroid hormones. Binding of TSH or thyroid-stimulating antibodies (TSAbs) to the TSH receptor (TSHR) results in thyroid hormone secretion (2). Several monoclonal TSAbs have been described (3, 4, 5, 6), and most of them bind to TSHR with high affinity (Kd = 1 x 10–9 to 7.1 x 10–11) and block TSH binding, suggesting that TSH and TSAbs may have a common binding specificity (7).

In patients with Graves’ disease (GD), the TSAbs are restricted to IgG isotypes (8, 9) and show evidence of affinity maturation (10, 11). High-affinity antibodies (Abs) are produced through a regulated process in which somatic mutations are randomly introduced into the Ig genes. After mutating, B cells that lose their affinity for the cognate antigen are eliminated, whereas B cells that competitively bind the remaining antigen survive and may increase their affinity for the antigen by undergoing additional somatic mutations. The complementarity-determining regions (CDR) and the framework regions (FR) of the Ab are primarily important for antigen binding and maintaining the Ab structure, respectively (12, 13). The Ig gene heavy chain CDR3 that results from a recombination of variable-diversity-joining (V-D-J) genes is translated into a protein loop (14) that comes in direct contact with the antigen and thus plays a critical role in determining the specificity and affinity of Abs. Therefore, antigen-driven selection that results in affinity maturation is a consequence of higher frequencies of mutations in the CDR relative to those in FR of Ig genes.

It is likely that pathogenic Abs to TSHR (15) appear slowly over a period of time in patients with GD. Whether this slow appearance of pathogenic Abs is dependent upon repeated exposure to the target antigen (i.e. TSHR) and emergence of high-affinity Abs through somatic hypermutation, or repertoire spreading, is not known. We have developed a mouse model of experimental autoimmune GD (EAGD) in which the disease is induced through repeated immunization of BALB/c mice with isogenic cells expressing high levels of TSHR (mM12 cells) (16, 17). Anti-TSHR Abs that can be readily detected in an ELISA are induced shortly after the second immunization. However, the TSH-binding inhibitory Ig (TBII) activity emerges after three to four immunizations, but the animals remain euthyroid. The TBII activity steadily increases with each immunization, and TSAbs appear in a subset of these mice leading to hyperthyroidism. Therefore, the mere presence of high levels of TBII activity does not correlate with hyperthyroidism (18, 19, 20, 21). Little is known about the evolution of somatic hypermutation and its relation to pathogenic anti-TSHR Abs in both humans and mice (15). We hypothesized that the pathogenic anti-TSHR Ab production is related, at least in part, to affinity maturation as indicated by increased somatic mutations in the CDR3. To begin to understand the molecular evolution of TSAbs, we analyzed the CDR3 sequences from mice with different stages of disease for Ig heavy chain (IGH) V-D-J gene usage and somatic mutations.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cells and antibodies
The M12 cells (BALB/c isogenic B cell line) or M12 cells stably transfected with a TSHR cDNA (mM12 cells) that allows cell surface expression of the protein (17) were used for immunization. The HEK293 cells expressing chimeric protein consisting of the extracellular (or ectodomain) domain of the TSHR (ECD-TSHR), intervening thrombin site, and a C-terminal CD8 transmembrane domain (17, 20) (i.e. TTCD8 cells) were used as a source of soluble TSHR ectodomain, sufficient for anti-TSHR antibody induction (22). The antimouse CD4, antimouse CD8, antimouse B220, antimouse IgG, antimouse CD19, antimouse IgM, and antimouse CD11b antibodies were purchased from Becton Dickinson (PharMingen, San Diego, CA).

Induction of GD
Six- to eight-week-old BALB/c mice were purchased from Jackson Laboratories (Bar Harbor, ME). Mice were immunized seven times with mitomycin C-treated M12 or mM12 cells 2 wk apart. Each mouse was immunized with approximately 107 cells. Harvested cells were washed and resuspended in 50 µg/ml mitomycin C and incubated for 30 min in the dark at 37 C followed by three washings in PBS. Each mouse was immunized ip with mitomycin C-treated cells and 5 µg cholera toxin B as described before (17). All animal studies were carried out according to the procedure approved by the Animal Care and Use Committee of the University of Illinois at Chicago.

TBII and T4 assays
Sera were obtained from blood samples that were collected periodically from mouse tail vein. Fifty microliters sera were tested for TBII activity using a commercial radioreceptor (Kronus, Dana Point, CA) assay following the manufacturer’s protocol. Mouse serum (25 µl) was used to determine the blood levels of total T4, using a commercial kit, Coat-a-Count (Diagnostic Products, Los Angeles, CA), following the manufacturer’s protocol.

Purification of the ECD-TSHR protein
TTCD8 cells were grown to confluence in T-150 flasks and washed once gently, and serum-free medium containing 100 U/ml {alpha}-thrombin was added. Flasks were incubated overnight at 37 C in a humidified incubator containing 5% CO2. Supernatants were collected, and histidine-tagged TSHR was purified from the spent medium using a nickel column (Pharmacia Amersham, Piscataway, NJ), following the manufacturer’s protocol. Purity of the protein was confirmed using SDS-PAGE followed by Western blotting for TSHR.

Expansion of anti-TSHR Ab-producing cells
Mice previously immunized with mM12 cells were rechallenged iv with either 100 µg purified ECD-TSHR or 100 µg ovalbumin (OVA). Three days later, mice were killed and splenocytes harvested. Red blood cells were removed by resuspending splenocytes in 0.83% ammonium chloride for 5 min at room temperature followed by washing with PBS. To enrich for plasma cells, IgM+, CD4+, CD8+, and CD11b+ cells were depleted by staining splenocytes with anti-CD4, anti-CD8, anti-IgM, and anti-CDllb phycoerythrin- or fluorescein-isothiocyanate-conjugated antibodies at 5 µg/ml for 30 min in PBS plus 2% fetal calf serum at 4 C. Stained cells were washed with PBS and then incubated with anti-phycoerythrin and anti-fluorescein-isothiocyanate MACS beads (Milteny Biotech, Auburn, CA) for 10 min. The beads were washed, and bound cells were removed with a MACS sorter using the deplete program. The depleted cells from different mice were tested for viability (~75%) and B220+ staining (55–60%) and were found to be comparable (not shown). Depleted and nondepleted cells were washed and resuspended in 5% RPMI 1640 medium and plated into 96-well plates at 1 x 105 cells per well. Six days later, culture supernatants were tested for the presence of spontaneously secreted anti-BSA, anti-OVA, and anti-TSHR IgG antibodies by ELISA.

ELISA
For ELISA, 96-well flat-bottomed Maxi-Sorp (Nunc, Rochester, NY) plates were coated with 200 ng/well of OVA or ECD-TSHR protein in buffer overnight at 4 C. Plates were then washed three times with PBS containing 0.05% Tween 20 and blocked with PBS plus 10% FCS at room temperature (RT) for 1 h. Plates were washed three times with PBS/0.05% Tween 20, and culture supernatants were added to appropriate wells and incubated for 2 h at RT followed by five washes. Antimouse IgG conjugated to horseradish peroxidase (Caltag, Burlingame, CA) was added at a 1/2000 dilution to all wells and incubated at RT for 1 h followed by seven washes. 3,3',5,5'-Tetramethylbenzidine substrate was added to each well and incubated for 15 min in the dark until the horseradish peroxidase reaction was stopped with 1/4 vol of 1 N HCl. Plates were read using an ELISA plate reader (Bio-Rad, Hercules, CA) at 450 nm wavelength.

RNA purification and cDNA synthesis
Total RNA was harvested from splenocytes using TRIzol reagent following the manufacturer’s protocol (Invitrogen, Carlsbad, CA). The RNA was resuspended in water containing 1 µl RNAGuard (Pharmacia-Amersham, Piscataway, NJ) at 5 x 106 cellular equivalents/50 µl. The first-strand cDNA was synthesized using Superscript II (Invitrogen) following the manufacturer’s protocol with a minor modification. Total RNA (11 µl) was added to 1 µl oligo-dT (Fermentas, Hanover, MD) and heated to 60 C for 10 min. Two microliters of of 10x Superscript buffer was added along with 1 µl of 10 mM dNTP mix and 1 µl of 0.1 M dithiothreitol. The rest of the protocol was identical to that suggested by the manufacturer.

Subcloning of CDR3
PCR samples were prepared in UV-irradiated sterile hoods with all components on ice before placing them into an iCycler PCR machine (Bio-Rad, Hercules, CA). CDR3 was amplified using 0.4 µM of IGHV subgroup-specific primers and 0.2 µM of each of the four IGHJ primers (see Fig. 2Go) in 1x PCR buffer (Roche Molecular Biochemicals, Indianapolis, IN) in a final volume of 50 µl. The reaction mix was supplemented with 0.2 mM dNTP mix, 3 U high-fidelity polymerase mix (Roche Molecular Biochemicals, Mannheim, Germany), and 1 µl sample cDNA. The PCR was performed with a 2-min hot start at 94 C and 30 cycles at 91 C, 72 C, and 60 C, each for a period of 30 sec ending with a 5-min extension at 72 C. The PCR product was then separated on a 1.5% agarose gel to confirm size and purity of the product. Each PCR product was concentrated and purified using the PCR purification and concentration kit (QIAGEN, Valencia, CA) and eluted in a 10-µl vol of water, and 1.5 µl of the products was then used in a ligation reaction consisting of 1x ligation buffer, 0.5 µl T4 ligase, and 0.5 µl pGEM vector (Fisher, Pittsburgh, PA) and incubated overnight at 16 C. Then, 2 µl was used to transform MAX efficiency DH5{alpha}-competent cells (Invitrogen). Ampicillin-resistant colonies were harvested and grown overnight in LB broth, and their DNA was prepared using QIAGEN mini-prep kit. The presence of insert was confirmed using SacI and SacII digests, and positive clones were sequenced using universal T7 short primers.


Figure 2
View larger version (10K):
[in this window]
[in a new window]

  		FIG. 2. Frequency of IGHV subgroup usage in representative mice. Total RNA from depleted splenocytes was used to generate oligo-dT-primed cDNAs, which were used to amplify each IGHV separately using an IGHV subgroup-specific primer and a combination of all four IGHJ primers. A standard curve was used to determine IGHV subgroup usage as described in Materials and Methods. The SD are shown for the real-time PCR performed on three separate occasions. A, Each IGHV gene subgroup usage shown as a percentage of all IGHV subgroups amplified; B, each IGHV subgroup shown as a proportion of their genomic distribution.

 
Real-time PCR
One microliter of cDNA was used for each real-time PCR using the iCycler PCR system (Bio-Rad). In the final PCR cocktail, a single IGHV primer (0.4 µM) and a combination of four IGHJ primers (0.2 µM each) that can prime all four IGHJ genes in mouse were used to produce an approximately 165-bp PCR product. PCR was carried out for 50 cycles at 91 C, 72 C, and 60 C, each for a period of 30 sec. The cocktail of primers and cDNA was diluted with an equal volume of 2x Sybr Green qPCR mix (Invitrogen). For each mouse, two to three PCR using all the subgroup-specific primers were performed. To estimate the copy numbers, a standard curve, using cloned VH1 cDNA was used in each reaction. This plasmid was diluted 10-fold over a previously determined range of 10–35 cycle number thresholds. Cycle number thresholds for the IGHV PCR were typically in the 20s except for IGHV1 (18, 19, 20). Copy numbers calculated from the standard curve using regression equations were added together to represent 100% of all IGHV expressed per mouse. The copy number per mouse per individual IGHV reaction was then divided by the total per mouse and multiplied by 100 to determine the percent usage of that particular IGHV subgroup.

CDR3-IMGT analysis
Sequences were aligned and analyzed using the Seqlab program from the University of Illinois’ online GCG Wisconsin package software. The pileup program was used to determine the nucleotide and amino acid similarities of all sequences and their translated products. Sequences were then exported in FASTA format. The IMGT/V-QUEST tool (23) from IMGT, the international ImMunoGeneTics information system http://imgt.cines.fr (initiator and coordinator Marie-Paule Lefranc, Montpellier, France) (24) was used to determine IGHV gene and IGHJ gene usage for each sequence, which was then used in the IMGT/JunctionAnalysis tool (25) to determine IGHD gene usage, V-D-J JUNCTION diversity, and JUNCTION mutation analysis. IMGT/JunctionAnalysis explores the JUNCTION that is delimited in 5' by the V-REGION 2nd-CYS 104 and in 3' by the J-REGION J-TRP 118 (for the IGHJ). The JUNCTION includes 2nd-CYS 104 and J-TRP 118, whereas the CDR3-IMGT does not include these positions and extends from position 105–117. Possible permutations of exact positions of mutations within the JUNCTION were determined using a newly improved IMGT/JunctionAnalysis program. For all sequences, the permutation with the highest levels of mutations was chosen. Finding two replacement mutations within one codon was counted as a single-replacement mutation. Sequences used for the analysis have been deposited into GenBank and are identified with accession numbers AY877924–AY877941 and AY877943–AY878052.

Statistical analysis
Associations were analyzed between D gene usage, J gene usage, and CDR3 amino acid usage on the one hand and TSHR-immunization status of mice (immunized vs. control) and the thyroid status [hyperthyroid with high TBII and high T4 levels (HTHT4) vs. euthyroid with normal TBII and normal T4 levels and high TBII and normal T4 levels (HTNT4)] on the other. The {chi}2 or odds ratio with confidence intervals (95%) were calculated using SAS, version 8.12 (SAS Institute, Cary, NC). Because frequencies of mutations among gene sequences were not normally distributed among groups, Wilcoxon rank-sum test was performed to determine whether statistically significant differences existed in the number of CDR3 R and S mutations, FWR3 R and S mutations and CDR3 lengths for sequences from TSHR-immunized mice vs. control mouse and high-T4 mouse vs. other TSHR-immunized mice with normal T4 mice existed. A P value < 0.05 was deemed to be significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
We monitored the production of anti-TSHR antibodies by testing serum TBII activity in mice immunized with mM12 cells. Mice had developed either high (>40%) (HTNT4) or low (≤20%) TBII activity, and some mice with high TBII activity had also developed higher than normal T4 levels (Table 1Go). We chose to study the IGH CDR3 derived from mice highlighted in Table 1Go (in bold) that featured either low TBII activity (20%) with normal T4 (4 µg/dl) (LTNT4), high TBII activity (50%) with little T4 perturbation (5 µg/dl) (HTNT4), or high TBII values (49%) with high (8.5 µg/dl) T4 levels (HTNT4), compared with control (12% TBII, 3.9 µg/dl T4).


View this table:
[in this window]
[in a new window]

  		TABLE 1. Serum TBII and T4 hormone levels in TSHR-immunized mice

 
Before isolating IGH CDR3 sequences, we tested a plasma cell enrichment protocol (26) modified to deplete IgM+ cells. IgM+ cells were depleted because the TSAb activity is found in serum IgG in GD patients (8, 9, 27) and hyperthyroid mouse (not shown). Table 2Go demonstrates an example of the results of plasma cell enrichment. In this experiment, mice previously immunized with mM12 cells were immunized with either 100 µg OVA or 100 µg purified recombinant ECD-TSHR protein while the other served as a control. Three days after immunization, plasma cells were enriched from harvested splenocytes and tested for viability (~75%) and B220+ staining (55–60%) and found to be similar in all depleted populations (not shown). MACS negative depletion prevents nonspecific activation of target cells and allowed us to test for spontaneous antibody production. To test for the presence of Ab-producing cells, spent supernatants of the plasma-cell-enriched cultures were tested for the presence of anti-BSA (irrelevant antigen), anti-OVA, and anti-TSHR IgG Abs by ELISA. Table 2Go shows that supernatants from both control and OVA-immunized mice were negative for anti-TSHR, anti-BSA, or anti-OVA IgG Abs. Splenocytes from mM12 and OVA-immunized mice failed to produce anti-OVA Abs, likely because these mice were primed once with OVA 3 d before being killed and the assay detected only IgGs. In contrast, splenocytes from mM12 and ECD-TSHR-immunized mice readily produced anti-TSHR IgGs. The number of anti-TSHR IgG Abs secreted per splenocyte per 6 d was estimated to be approximately 158,000 for the depleted population (Table 2Go). There was approximately a 3-fold increase in the levels of anti-TSHR Abs in depleted, relative to the nondepleted, populations (Table 2Go).


View this table:
[in this window]
[in a new window]

  		TABLE 2. Spontaneous secretion of anti-TSHR Ab by splenocytes from TSHR-immunized mice

 
Mice primed with TSHR (Table 1Go), but not the control, were immunized iv with 100 µg purified ECD-TSHR protein and killed 3 d later when plasma cell populations peak (d 3–5) in secondary lymphoid organs (28, 29, 30, 31). After enrichment, depleted cells were tested for B220 expression levels and were found to be similar in all mice (55–65% B220+ cells, not shown). To take advantage of the increase in steady-state Ig RNA levels in plasma cells (32, 33) compared with resting B cells (~10- to 60-fold), total RNA was extracted from enriched B/plasma cells and cDNA synthesized using oligo-dT primers. To amplify the heavy chain CDR3, previously published IGHV subgroup-specific and IGHJ primers were used (34, 35) (Fig. 1Go). These primers have been used to distinguish CDR3 sequences corresponding to the antihapten Ab response from those derived from total spleen by CDR3 length spectratyping (i.e. searching for overrepresented CDR3 molecular characteristic). Using these IGHV subgroup-specific primers (36), we obtained approximately 165 nucleotides products consisting of approximately 129 nucleotides of FR3 and approximately 36 nucleotides of the CDR3. PCR conditions consistently produced expected size products without spurious bands as demonstrated by single-peak melt curves and single-sized products when separated in 1.5% agarose gels (not shown).


Figure 1
View larger version (24K):
[in this window]
[in a new window]

  		FIG. 1. Primers used to amplify CDR3 of murine IGHV-D-J sequences. The end result of heavy-chain V-D-J recombination is the CDR3 (boxed). IGHV subgroup-specific primers prime within the FR3 and were used with a cocktail of four IGHJ antisense primers, representing all four IGHJ genes in mouse. In the final PCR cocktail, a single IGHV primer (0.4 µM) and a combination of all four IGHJ primers (0.2 µM each) that are able to prime all four IGHJ genes in mouse were used to produce an approximately 165-bp PCR product. On average, the length of the CDR3 was 36 nucleotides long, and the upstream FR3 sequence amplified was 129 nucleotides long.

 
Before B cells enter the periphery during their development, Ab diversity is generated through V-D-J recombination (13). V-D-J recombination is a random process that results in the formation of the CDR3 (Fig. 1Go). This involves the 3' end of a V gene recombining with a D gene that has already recombined with the 5' end of a J gene. By using real-time PCR and subgroup-specific primers, we determined the frequency of usage of IGHV subgroups (Fig. 2AGo). For comparison, we have shown the IGHV subgroup usage as a proportion of their genomic distribution (Fig. 2BGo). The repertoire in each mouse was similar but showed distinct differences between each other. For example, the HTHT4 mouse showed increased usage of IGHV5 in the IGHV-D-J repertoire. Transcripts in all mice used mostly IGHV1 and IGHV5 subgroup genes.

Therefore, IGHV1 and IGHV5 CDR3 were PCR amplified and cloned into the pGEM cloning vector, and a representative number (~35–50 per mouse) of PCR products were sequenced. Of the more than 200 IGHV1 and IGHV5 rearranged sequences analyzed in this study, most translated into proteins without stop codons (>70%), none was a pseudogene, and subgroup-specific IGHV primers amplified genes from only their corresponding subgroup (not shown). There was an average 4-fold increase in IGHV1 transcripts among immunized mice relative to the naive control, but there was no statistical difference between immunized mice (not shown).

CDR3 from all mice was analyzed both at the nucleotide and amino acid level. No identical or similar (defined as >98% identical at nucleotide level) sequences were identified when comparing sequences from one mouse to any other mouse. However, similar CDR3 sequences were found when sequences were compared within each mouse, except for the control mouse (Table 3Go). For example, one sequence from the LTNT4 mouse was repeated twice, whereas another one was repeated five times (see Table 3Go). Repeated sequences from the HTNT4 and HTHT4 mice were not always 100% identical, and interestingly, some sequences displayed a few nucleotide substitutions, which could represent clonal sets.


View this table:
[in this window]
[in a new window]

  		TABLE 3. Repeated sequences from control and immunized mice

 
V-D-J recombination results in the formation of the CDR3 (Fig. 1Go). We determined the IGHD and IGHJ genes used by the IGHV-D-J transcripts analyzed. IGHD gene usage in all CDR3 (Fig. 3AGo) and in repeated sequences (Fig. 3BGo) relative to the control is shown. The IGHD genes used by the control demonstrated a bias toward the IGHD-FL16.1*01 gene, whereas other IGHD genes were used at a lower frequency. The IGHD genes used by the repeated sequences from the LTNT4 mouse were IGHD-FL16.1 (~30%) and IGHD-SP2.9 (~70%). The HTNT4 mouse preferentially used IGHD-FL16.1*01 (~55%) and several others to a much lower extent including IGHD-SP2.9 (11%). In contrast, the HTHT4 mouse used mostly IGHD-Q52*01 (33%), IGHD-FL16.1*01 (22%), IGHD-SP2.9 (22%), and IGHD-FL16.2 (22%) (Fig. 3BGo). The IGHD-SP2.9 gene was used exclusively by transcripts from all immunized mice tested and not the control.


Figure 3
View larger version (26K):
[in this window]
[in a new window]

  		FIG. 3. CDR3 IGHD gene usage. All CDR3 (A) or repeated CDR3 (B) from immunized mice and control CDR3 were analyzed for the relative frequency of IGHD gene usage. Note that only those IGHD genes that were used at least once are shown in the histogram.

 
The IGHJ gene usage in all CDR3 (Fig. 4AGo) and in repeated sequences (Fig. 4BGo) showed that the control sequences mostly used IGHJ4 followed by IGHJ2 and IGHJ3 (Fig. 4Go, A and B). Repeated sequences from the LTNT4 mouse used exclusively IGHJ3, whereas repeated sequences from the HTNT4 mouse used all four IGHJ genes. In contrast, repeated sequences from the HTHT4 mouse used IGHJ1 and IGHJ4 (Fig. 4BGo).


Figure 4
View larger version (8K):
[in this window]
[in a new window]

  		FIG. 4. CDR3 IGHJ gene usage. All CDR3 (left) or repeated CDR3 (right) from immunized mice and all control CDR3 were analyzed to determine the relative frequency of IGHJ gene usage.

 
V-D-J recombination also generates diversity in the CDR3 region through nuclease-mediated shortening of the recombining V, D, and J genes and in the random N and P additions between the recombining V-D-J genes (13). All sequences were analyzed by using IMGT/JunctionAnalysis (http://imgt.cines.fr) (20) to delineate the V, D, and J genes in the rearrangement and to determine the junctional diversity of each sequence. In Fig. 5Go, the junctional diversity of the repeated CDR3 nucleotide sequences from TSHR-immunized mice (Table 3Go) is shown. This includes the N and P nucleotide additions and nuclease-mediated shortening of the IGHV, IGHD, and IGHJ genes represented by dots (Fig. 5Go). Boxed groups in Fig. 5Go represent sequences that contain less than 2% difference at the nucleotide level.


Figure 5
View larger version (41K):
[in this window]
[in a new window]

  		FIG. 5. CDR3 diversity of similar sequences. CDR3 of repeated sequences is shown. IMGT/V-QUEST (23 ) and IMGT/JunctionAnalysis (25 ) tools from IMGT (http://imgt.cines.fr) (24 ) were used to determine the V-D-J diversity of each sequence with respect to nucleotide (N) additions (TdT enzyme mediated), palindrome (P) additions and V, D, and J gene end-shortening (shown as dots). The number of repeated sequences that each CDR3 represents is shown at the end of CDR3. CDR3 that are boxed form part of a set of repeated similar sequences.

 
Mature B cells may further diversify their antibodies through random somatic mutations in the variable regions (including FR3 and CDR3) of their Igs. Therefore, we determined the number of mutations within the 3' part of the FR3 (codons at positions 82–104) by comparing with the germline IGHV genes identified by IMGT/V-QUEST. Similarly, the number of mutations in the CDR3 (codons at positions 105–117) was determined by comparing with the nucleotides encoded by the germline 3'V-REGION, D-REGION, and 5'-J-REGION and identified by IMGT/JunctionAnalysis. The analysis was performed for all sequences and repeated sequences compared with control sequences (Table 4Go). The data are presented as the average number of mutations per sequence. Table 4Go shows that there were 0.46, 0.07, 0.4, and 0.93 replacement (R) mutations per sequence in the CDR3 and 0.96, 0.57, 0.96, and 1 R mutation per sequence in the 3' part of the FR3 for the control, LTNT4, HTNT4, and HTHT4 mice, respectively.


View this table:
[in this window]
[in a new window]

  		TABLE 4. Mutations in the CDR3 and FR3

 
The R and silent (S) mutations denote nucleotide changes that lead to and do not lead to amino acid changes, respectively. Therefore, a higher R/S ratio would indicate accumulation of mutations that are more relevant for affinity maturation. Importantly, the R/S values in these groups were 6.6, 1, 2.5, and 9.3 for the CDR3 and 1.9, 0.6, 1.1, and 1.8 for the 3' part of the FR3, respectively. The trends for the mutations from all sequences compared with repeated sequences (Table 4Go) did not change significantly. Within CDR3, the LTNT4 sequences contained fewer R mutations (approximately 10-fold less than HTHT4), whereas control and HTNT4 sequences contained a similar number of mutations. However, HTHT4 sequences showed twice as many R mutations as those of either control or HTNT4 sequences. Moreover, the CDR3 R/S values were higher in the HTHT4 sequences than in any of the other mouse sequences. In contrast, the R mutations and R/S values in the 3' part of the FR3 for HTHT4 sequences were not high. Although the R/S value in the CDR3 of the HTHT4 sequences was a minimum of 9, R/S values in the 3' part of the FR3 of all or repeated sequences were never higher than 1.9.

To determine whether associations between molecular characteristics of sequences and TSHR immunization status or T4 hormone levels could be explained by chance alone, statistical analyses were performed (Table 5Go). Specifically, we tested to see whether there were any associations between the V, D, or J gene usage (Figs. 2–4GoGoGo), CDR3 amino acid usage (i.e. basic, acid, aliphatic, aromatic, S-containing, proline, alcohol, or amides) (not shown), mean CDR3 length (not shown) and mean R and S mutations in CDR3 and FR3 (Table 4Go), with TSHR immunization status, and TBII and T4 levels. In Table 5Go, we show that the IGHD-SP2.9 gene was found at a higher frequency in sequences from TSHR-immunized mice relative to control (P = 0.04), but the sequences from HTHT4 used IGHD-Q52 at a higher frequency than sequences from the other TSHR-immunized mice (P = 0.05). IGHJ1 was found at a higher frequency in sequences from TSHR-immunized mice compared with control (P = 0.005), and the reverse was found with IGHJ4 (P = 0.0001). IGHJ3 was found in a higher frequency (P = 0.004) of TSHR-immunized mice sequences compared with the HTHT4 sequences.


View this table:
[in this window]
[in a new window]

  		TABLE 5. Associations between molecular characteristics of sequences and TSHR immunization status and T4 hormone levels

 
There were fewer sulfur-containing amino acids in sequences from TSHR-immunized mice, particularly in CDR3 of HTHT4, compared with control (P = 0.0002). There were no statistically significant associations with CDR3 length, which averaged 12 amino acids (Table 5Go). There were significantly fewer FR3 S mutations in the sequences from TSHR-immunized mice (P = 0.048). There were no significant differences in the R mutations or S mutations of CDR3 between TSHR-immunized mice compared with control. However, there was a significant (P = 0.002) difference in the mean CDR3 R mutations when CDR3 from the HTHT4 was compared with the other TSHR-immunized mice with normal T4 levels.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In our EAGD model, animals are repeatedly immunized with TSHR, and anti-TSHR antibodies slowly evolve into TSAbs resulting in hyperthyroidism. TSHR-immunized mice contained an average 4-fold increase in IGHV1 CDR3 transcript levels compared with naive control; however, there was no significant difference between different immunized mice (not shown). The percentage of B220+ cells after depletion was similar for all mice (not shown) and indicated that the increase in Ig transcripts was probably not due to increased cell numbers and was a consequence of TSHR immunization. This is consistent with the robust spontaneous secretion of anti-TSHR IgG Abs from splenocytes isolated from mice that were immunized with TSHR (Table 2Go).

The IGHV subgroup usage in control mice mirrored the percentages of genes reported for each subgroup (Fig. 2Go). This suggested that the IgG repertoire is chosen, at least in part (37, 38), stochastically (39). A comparison of data from all mice showed that the IGHV response was similar (Fig. 2AGo) and the observed moderate increase in IGHV5 frequency in the HTHT4 mouse might indicate a more focused response. It is interesting to note that the percentage of IGHV1 gene subgroup usage in our control mouse is approximately 15% higher than that reported in other studies (38, 40). The reason for this discrepancy is unclear, although it could be due to different assays used to determine IGHV usage (hybridizations as opposed to PCR) and/or due to the depletion of IgM+ B cells.

We sequenced a representative number of the two most abundant (34) IGHV subgroups (i.e. IGHV1 and IGHV5) that account for 55–80% of the mouse Ab repertoire (Fig. 2Go), including the 3' part of the FR3 and CDR3 of the rearranged transcripts (Fig. 1Go). The translated CDR3 showed no identity with each other when sequences from different mice were compared, indicating the diversity of responses in these mice. There were, however, CDR3 amino acid sequences that were repeated (Table 3Go) within the same immunized mouse but not in the naive mouse. Because immunized mice had higher levels of CDR3 transcripts, it is likely that the repeated products in these mice are not a consequence of limited initial transcripts but dominant CDR3 usage in what is most likely an oligoclonal B cell response. It is interesting to note that the groups of similar sequences or clonal groups (Fig. 3Go) in the high-TBII mice (HTNT4 and HTHT4) contained small nucleotide differences that translate into different amino acids (R mutations) in their CDR3 that are indicative of an evolving Ab response.

Somatic hypermutation of Igs allows further diversification and refinement of Ab-antigen binding including increased affinity. The highest R mutation value in the CDR3 (>0.93) was found in the diseased mouse (HTHT4) (Table 4Go). The frequency of R mutations in HTHT4 was 0.93, which is higher than the frequencies of 0.46, 0.07, and 0.4 from control, LTNT4, and HTNT4 mice, respectively. The R mutation value in the CDR3 for repeated sequences (Table 4Go) from HTHT4 (1.13) was also higher than the frequency of R mutations in LTNT4 (0), HTNT4 (0.56), and control (0.46) sequences. On average, CDR3 sequences from the HTHT4 mouse had accumulated more amino acid changes than any other mouse. Statistical analysis of HTHT4 sequences demonstrated no significant difference in the mean CDR3 S and FR3 R and S mutations relative to sequences from other TSHR-immunized mice. However, a statistically higher mean value of CDR3 R mutations was noted in HTHT4 sequences relative to sequences from other TSHR-immunized mice. These results indicate that Igs from HTHT4 mice contained a higher frequency of somatic mutations that alter the protein sequence.

Several sets of murine anti-TSHR monoclonal Abs (mAbs) with TSAb activity have been generated. Analyses of two of these murine mAbs showed that one set of 2 Abs used IGHJ2 and IGHD-ST4 or -Q52 (41) whereas the other set of 3 mAbs used IGHJ4 and IGHD-Q52 (5). Although repeated sequences from the hyperthyroid mouse demonstrate a preferential use of IGHJ1 and IGHJ4 and IGHD-Q52 (Fig. 5Go), additional studies are required to draw a firm conclusion regarding an association with hyperthyroidism. More interestingly, a set of murine monoclonal TSAbs used IGHD-Q52 and IGHJ4 (5) and contained average R/S values of 1.2, 0.63, and 1.0 in the FR and 6, 6, and 4 in the CDR of the Abs. The R/S values of the HTNT4 sequences (Table 4Go) were (<2.5) less than the R/S values (4–6) of the monoclonal TSAbs. In contrast, the sequences from the HTHT4 mouse showed R/S values of more than 9. Based on these results, we speculate that in an evolving anti-TSHR Ab response, there is an increased probability that a pathogenic autoantibody will emerge as mutations accumulate within the CDR3 of that autoantibody (i.e. increasing CDR3 R/S values).

Tolerance to TSHR is incomplete (42, 43, 44) and thus allows the possibility that stimulation of relevant B cells may lead to anti-TSHR Ab production. An earlier study demonstrated that concentrated TSHR-specific autoantibodies from healthy individuals not only contain TBII activity but also show the same TSHR N-terminal binding specificity as that of pathogenic autoantibodies from GD patients (45). This raises the possibility that diversification of these Abs through somatic hypermutation may increase their affinity and lead to the production of pathogenic TSAbs.

Our CDR3 analysis focused on the antibody response generated in the spleen after immunization with purified TSHR protein. Unlike generating monoclonal TSAbs to TSHR or separating out TSHR-specific B cells, potential anti-TSHR CD3 sequences were identified by certain molecular characteristics that were overrepresented (34). Despite this, we were able to identify certain molecular characteristics associated with the TSAb activity. However, our ability to draw a more definitive conclusion is tempered by the paucity of data on monoclonal TSAb sequences. It should also be noted that although a large number of studies have reported generation of monoclonal antibodies to TSHR, there is very limited information available on molecular characteristics of TSAbs. As more monoclonal TSAb sequences become available, we will have a greater ability to identify common pathogenic antibody molecular characteristics. Similarly, development of new techniques to isolate TSHR-specific B cells should allow a clearer characterization of the repertoire of activated TSHR-specific B cells. This later approach will not only help detect the emergence of dominant specific B cell clones but also provide the context from which this population emerges.


   Acknowledgments
 
We are very grateful to Véronique Giudicelli for her help in the sequence analysis with the newly improved IMGT/JunctionAnalysis program. We are also grateful to David Ucker and Prasad Kanteti for helpful commentary.


   Footnotes
 
This work was supported by National Institutes of Health Grant RO1 DK047417.

Disclosure: The authors have nothing to disclose.

First Published Online October 26, 2006

Abbreviations: Ab, Antibody; CDR, complementarity-determining region; EAGD, experimental autoimmune GD; ECD, extracellular domain; FR, framework region; GD, Graves’ disease; HTHT4, high TSH-binding inhibitory Ig and high T4 levels; HTNT4, high TSH-binding inhibitory Ig and normal T4 levels; IGH, Ig heavy chain; LTNT4, low TSH-binding inhibitory Ig and normal T4 levels; mAb, monoclonal Ab; OVA, ovalbumin; R, replacement; RT, room temperature; S, silent; TBII, TSH-binding inhibitory Ig; TsAb, thyroid-stimulating Ab; TSHR, TSH receptor; V-D-J, variable-diversity-joining.

Received August 11, 2006.

Accepted for publication October 17, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Prabhakar BS, Fan JL, Seetharamaiah GS 1997 Thyrotropin-receptor-mediated diseases: a paradigm for receptor autoimmunity. Immunol Today 18:437–442[CrossRef][Medline]
  2. Kim WB, Chung HK, Park YJ, Park DJ, Lee HK, Cho BY 2001 Clinical significance of classification of Graves’ disease according to the characteristics of TSH receptor antibodies. Korean J Intern Med 16:187–200
  3. Ando T, Latif R, Pritsker A, Moran T, Nagayama Y, Davies TF 2002 A monoclonal thyroid-stimulating antibody. J Clin Invest 110:1667–1674[CrossRef][Medline]
  4. Costagliola S, Franssen JD, Bonomi M, Urizar E, Willnich M, Bergmann A, Vassart G 2002 Generation of a mouse monoclonal TSH receptor antibody with stimulating activity. Biochem Biophys Res Commun 299:891–896[CrossRef][Medline]
  5. Sanders J, Jeffreys J, Depraetere H, Richards T, Evans M, Kiddie A, Brereton K, Groenen M, Oda Y, Furmaniak J, Rees Smith B 2002 Thyroid-stimulating monoclonal antibodies. Thyroid 12:1043–1050[CrossRef][Medline]
  6. Nordyke RA, Gilbert Jr FI, Miyamoto LA, Fleury KA 1993 The superiority of antimicrosomal over antithyroglobulin antibodies for detecting Hashimoto’s thyroiditis. Arch Intern Med 153:862–865[CrossRef][Medline]
  7. Costagliola S, Vassart G 2002 Monoclonal antibodies with thyroid stimulating activity, at last. Thyroid 12:1039–1041[CrossRef][Medline]
  8. Weetman AP, Yateman ME, Ealey PA, Black CM, Reimer CB, Williams Jr RC, Shine B, Marshall NJ 1990 Thyroid-stimulating antibody activity between different immunoglobulin G subclasses. J Clin Invest 86:723–727[Medline]
  9. MacNeil S, Munro DS, Metcalfe R, Cotterell S, Ruban L, Davies R, Weetman AP 1994 An investigation of the ability of TSH and Graves’ immunoglobulin G to increase intracellular calcium in human thyroid cells, rat FRTL-5 thyroid cells and eukaryotic cells transfected with the human TSH receptor. J Endocrinol 143:527–540[Abstract/Free Full Text]
  10. McLachlan SM, Rapoport B 2004 Thyroid stimulating monoclonal antibodies: overcoming the road blocks and the way forward. Clin Endocrinol (Oxf) 61:10–18[CrossRef][Medline]
  11. Sanders J, Evans M, Premawardhana LD, Depraetere H, Jeffreys J, Richards T, Furmaniak J, Rees Smith B 2003 Human monoclonal thyroid stimulating autoantibody. Lancet 362:126–128[CrossRef][Medline]
  12. Kabat EA, Wu TT, Reid-Miller M, Perry HM, Gottesman KS 1987 Sequences of proteins of immunological interest. Bethesda, MD: National Institutes of Health
  13. Lefranc MP, Lefranc G 2001 The immunoglobulin factsbook. London: Academic Press
  14. Ruiz M, Lefranc MP 2002 IMGT gene identification and Colliers de Perles of human immunoglobulins with known 3D structures. Immunogenetics 53:857–883[CrossRef][Medline]
  15. McIntosh R, Watson P, Weetman A 1998 Somatic hypermutation in autoimmune thyroid disease. Immunol Rev 162:219–231[CrossRef][Medline]
  16. Dogan RN, Vasu C, Holterman MJ, Prabhakar BS 2003 Absence of IL-4, and not suppression of the Th2 response, prevents development of experimental autoimmune Graves’ disease. J Immunol 170:2195–2204[Abstract/Free Full Text]
  17. Kaithamana S, Fan J, Osuga Y, Liang SG, Prabhakar BS 1999 Induction of experimental autoimmune Graves’ disease in BALB/c mice. J Immunol 163:5157–5164[Abstract/Free Full Text]
  18. Shimojo N, Kohno Y, Yamaguchi K, Kikuoka S, Hoshioka A, Niimi H, Hirai A, Tamura Y, Saito Y, Kohn LD, Tahara K 1996 Induction of Graves-like disease in mice by immunization with fibroblasts transfected with the thyrotropin receptor and a class II molecule. Proc Natl Acad Sci USA 93:11074–11079[Abstract/Free Full Text]
  19. Valente WA, Vitti P, Yavin Z, Yavin E, Rotella CM, Grollman EF, Toccafondi RS, Kohn LD 1982 Monoclonal antibodies to the thyrotropin receptor: stimulating and blocking antibodies derived from the lymphocytes of patients with Graves disease. Proc Natl Acad Sci USA 79:6680–6684[Abstract/Free Full Text]
  20. Kohn LD, Suzuki K, Hoffman WH, Tombaccini D, Marcocci C, Shimojo N, Watanabe Y, Amino N, Cho BY, Kohno Y, Hirai A, Tahara K 1997 Characterization of monoclonal thyroid-stimulating and thyrotropin binding-inhibiting autoantibodies from a Hashimoto’s patient whose children had intrauterine and neonatal thyroid disease. J Clin Endocrinol Metab 82:3998–4009[Abstract/Free Full Text]
  21. Shimojo N, Arima T, Yamaguchi K, Kikuoka S, Kohn LD, Kohno Y 2000 A novel mouse model of Graves’ disease: implications for a role of aberrant MHC class II expression in its pathogenesis. Int Rev Immunol 19:619–631[Medline]
  22. Kikuoka S, Shimojo N, Yamaguchi KI, Watanabe Y, Hoshioka A, Hirai A, Saito Y, Tahara K, Kohn LD, Maruyama N, Kohno Y, Niimi H 1998 The formation of thyrotropin receptor (TSHR) antibodies in a Graves’ animal model requires the N-terminal segment of the TSHR extracellular domain. Endocrinology 139:1891–1898[Abstract/Free Full Text]
  23. Giudicelli V, Chaume D, Lefranc MP 2004 IMGT/V-QUEST, an integrated software program for immunoglobulin and T cell receptor V-J and V-D-J rearrangement analysis. Nucleic Acids Res 32:W435–W440
  24. Lefranc MP, Giudicelli V, Kaas Q, Duprat E, Jabado-Michaloud J, Scaviner D, Ginestoux C, Clement O, Chaume D, Lefranc G 2005 IMGT, the international ImMunoGeneTics information system. Nucleic Acids Res 33:D593–D597
  25. Monod MY, Giudicelli V, Chaume D, Lefranc MP 2004 IMGT/JunctionAnalysis: the first tool for the analysis of the immunoglobulin and T cell receptor complex V-J and V-D-J JUNCTIONs. Bioinformatics 20(Suppl 1):I379–I385
  26. Underhill GH, Kolli KP, Kansas GS 2003 Complexity within the plasma cell compartment of mice deficient in both E- and P-selectin: implications for plasma cell differentiation. Blood 102:4076–4083[Abstract/Free Full Text]
  27. Waldmann TA, Strober W 1969 Metabolism of immunoglobulins. Prog Allergy 13:1–110[Medline]
  28. Slifka MK, Matloubian M, Ahmed R 1995 Bone marrow is a major site of long-term antibody production after acute viral infection. J Virol 69:1895–1902[Abstract]
  29. Manz RA, Lohning M, Cassese G, Thiel A, Radbruch A 1998 Survival of long-lived plasma cells is independent of antigen. Int Immunol 10:1703–1711[Abstract/Free Full Text]
  30. McHeyzer-Williams MG 1997 Immune response decisions at the single cell level. Semin Immunol 9:219–227[CrossRef][Medline]
  31. Sze DM, Toellner KM, Garcia de Vinuesa C, Taylor DR, MacLennan IC 2000 Intrinsic constraint on plasmablast growth and extrinsic limits of plasma cell survival. J Exp Med 192:813–821[Abstract/Free Full Text]
  32. Jack HM, Wabl M 1988 Immunoglobulin mRNA stability varies during B lymphocyte differentiation. EMBO J 7:1041–1046[Medline]
  33. Chen-Bettecken U, Wecker E, Schimpl A 1987 Transcriptional control of µ- and {kappa}-gene expression in resting and bacterial lipopolysaccharide-activated normal B cells. Immunobiology 174:162–176[Medline]
  34. Delassus S, Gey A, Darche S, Cumano A, Roth C, Kourilsky P 1995 PCR-based analysis of the murine immunoglobulin heavy-chain repertoire. J Immunol Methods 184:219–229[CrossRef][Medline]
  35. Dattamajumdar AK, Jacobson DP, Hood LE, Osman GE 1996 Rapid cloning of any rearranged mouse immunoglobulin variable genes. Immunogenetics 43:141–151[Medline]
  36. Lefranc MP, Pommie C, Ruiz M, Giudicelli V, Foulquier E, Truong L, Thouvenin-Contet V, Lefranc G 2003 IMGT unique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains. Dev Comp Immunol 27:55–77[CrossRef][Medline]
  37. Gu H, Tarlinton D, Muller W, Rajewsky K, Forster I 1991 Most peripheral B cells in mice are ligand selected. J Exp Med 173:1357–1371[Abstract/Free Full Text]
  38. Haines BB, Angeles CV, Parmelee AP, McLean PA, Brodeur PH 2001 Germline diversity of the expressed BALB/c VhJ558 gene family. Mol Immunol 38:9–18[CrossRef][Medline]
  39. Dildrop R, Krawinkel U, Winter E, Rajewsky K 1985 VH-gene expression in murine lipopolysaccharide blasts distributes over the nine known VH-gene groups and may be random. Eur J Immunol 15:1154–1156[Medline]
  40. Freitas AA, Andrade L, Lembezat MP, Coutinho A 1990 Selection of VH gene repertoires: differentiating B cells of adult bone marrow mimic fetal development. Int Immunol 2:15–23[Abstract/Free Full Text]
  41. Costagliola S, Bonomi M, Morgenthaler NG, Van Durme J, Panneels V, Refetoff S, Vassart G 2004 Delineation of the discontinuous-conformational epitope of a monoclonal antibody displaying full in vitro and in vivo thyrotropin activity. Mol Endocrinol 18:3020–3034[Abstract/Free Full Text]
  42. Akkaraju S, Canaan K, Goodnow CC 1997 Self-reactive B cells are not eliminated or inactivated by autoantigen expressed on thyroid epithelial cells. J Exp Med 186:2005–2012[Abstract/Free Full Text]
  43. Akkaraju S, Ho WY, Leong D, Canaan K, Davis MM, Goodnow CC 1997 A range of CD4 T cell tolerance: partial inactivation to organ-specific antigen allows nondestructive thyroiditis or insulitis. Immunity 7:255–271[CrossRef][Medline]
  44. Pichurin PN, Pichurina O, Marians RC, Chen CR, Davies TF, Rapoport B, McLachlan SM 2004 Thyrotropin receptor knockout mice: studies on immunological tolerance to a major thyroid autoantigen. Endocrinology 145:1294–1301[Abstract/Free Full Text]
  45. Chazenbalk GD, Pichurin P, Chen CR, Latrofa F, Johnstone AP, McLachlan SM, Rapoport B 2002 Thyroid-stimulating autoantibodies in Graves disease preferentially recognize the free A subunit, not the thyrotropin holoreceptor. J Clin Invest 110:209–217[CrossRef][Medline]




This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
148/2/752    most recent
Author Manuscript (PDF)
Right arrow Purchase Article
Right arrow View Shopping Cart
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Martinez, O.
Right arrow Articles by Prabhakar, B. S.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Martinez, O.
Right arrow Articles by Prabhakar, B. S.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Endocrinology Endocrine Reviews J. Clin. End. & Metab.
Molecular Endocrinology Recent Prog. Horm. Res. All Endocrine Journals