Relationship between Thyroid Peroxidase T Cell Epitope Restriction and Antibody Recognition of the Autoantibody Immunodominant Region in Hum
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《内分泌学杂志》
Autoimmune Disease Unit (J.G., S.M.M., P.N.P., C.-R.C., N.P., H.A.A., B.R.), Cedars-Sinai Research Institute and University of California, Los Angeles School of Medicine, Los Angeles, California 90048
Department of Immunology (C.S.D.), Mayo Clinic, Rochester, Minnesota 55905
Abstract
We investigated the relationship between thyroid peroxidase (TPO) antibody and T lymphocyte epitopes in TPO-adenovirus (TPO-Ad) immunized BALB/c mice and mice transgenic for the human class II molecule DR3 associated with human thyroid autoimmunity. TPO autoantibodies are largely restricted to an immunodominant region (IDR). BALB/c mice immunized with fewer (107 vs. 109) TPO-Ad particles developed TPO antibodies with lower titers that displayed greater restriction to the IDR. However, as with higher-dose TPO-Ad immunization, T cell epitopes (assessed by splenocyte interferon- response to TPO in vitro) were highly diverse and variable in different animals. In contrast, DR3 mice immunized the higher TPO-Ad dose (109 particles) had high TPO antibody levels that showed relative focus on the IDR. Moreover, T cell epitopes recognized by splenocytes from DR3 mice showed greater restriction than BALB/c mice. Antibody affinities for TPO were higher in DR3 than in BALB/c mice. The present study indicates that weak TPO-Ad immunization of BALB/c mice (with consequent low TPO antibody titers) is required for enhanced IDR focus yet is not associated with T cell epitopic restriction. Humanized DR3 transgenic mice, despite stronger TPO-Ad immunization, develop higher titer TPO antibodies that do focus on the autoantibody IDR with T cells that recognize a more limited range of TPO peptides. These data suggest a relationship between major histocompatibility complex class II molecules and the development of antibodies to the IDR, a feature of human thyroid autoimmunity.
Introduction
AUTOIMMUNE (HASHIMOTO’S) THYROIDITIS, the most common organ-specific autoimmune disease affecting humans, involves loss of tolerance to thyroid peroxidase (TPO), a thyroid cell surface antigen. The immune response to TPO is both humoral and cell-mediated. TPO autoantibodies can activate the complement cascade (1); are mainly associated with complement-fixing IgG1, not IgG4 (2); and can damage thyroid cells in vitro by an antibody-dependent cell cytotoxic mechanism involving NK cells (3, 4, 5, 6). Nevertheless, whether TPO autoantibodies directly cause thyrocyte damage has not been definitively established. In contrast, recent evidence in a humanized T cell transgenic model establishes that T cells alone are sufficient to induce thyroiditis (7).
Even if not directly involved in thyrocyte damage, TPO autoantibodies have two important roles in studying Hashimoto’s disease. First, TPO autoantibodies in patients’ sera are excellent clinical markers of disease, perhaps unrivaled among organ-specific autoimmune diseases. Second, autoantibodies on the B cell surface function as antigen receptors that (unlike macrophages and dendritic cells) capture specific antigen for processing and presentation to T cells. Antibodies (both soluble and membrane associated) can profoundly effect antigen processing within the antigen-presenting cell, introducing bias in antigenic determinants (peptides) presented to T cells (reviewed in Ref.8). In particular, human TPO autoantibodies with different epitopes can influence which TPO peptides are presented by antigen-presenting cell to human T cell clones (9, 10). A remarkable feature of polyclonal human TPO autoantibodies is their relative restriction to epitopes within an immunodominant region (IDR) on the surface of the native antigen (11, 12). Understanding the factors contributing to this epitopic focus of TPO autoantibodies is clearly of value in understanding the pathogenesis of autoimmune thyroiditis with, ultimately, the potential of immunotherapy for this disease.
Animal models are important tools in studying the pathogenesis of autoimmunity. Conventional immunization of mice with soluble TPO protein in adjuvant effectively induces antibodies, but their epitopes, unlike human autoantibodies, are not largely to restricted to the IDR (11, 13). Antibodies with the epitopic profiles of human autoantibodies have been produced by nonconventional immunization using fibroblasts coexpressing TPO and major histocompatibility complex (MHC) class II molecules (13) as well as by TPO-DNA vaccination (14, 15). However, these two approaches have not permitted identification of the TPO T cell epitopes associated with IDR-focused TPO antibodies. The TPO-MHC class II expressing fibroblasts induce very high background responses by splenocytes in vitro, most likely because of high costimulatory molecule (B7) expression (16). On the other hand, TPO plasmid DNA vaccination led to splenocyte responses to a highly diverse array of synthetic TPO peptides (both intra- and interanimal) (17). More recently TPO-adenovirus (TPO-Ad) immunization induced high-titer TPO antibodies in BALB/c mice but without the IDR restriction typical of human autoantibodies (14).
In the present study, we tried two new approaches to facilitate investigation of the relationship between antibody (B cell) and T cell epitopes on TPO. First, we tested the hypothesis that immunizing BALB/c mice with fewer TPO-Ad particles would generate TPO antibodies with greater IDR restriction than observed with high-dose TPO-Ad immunization and whose T cell epitopes would also be more restricted. Second, because human leukocyte antigen (HLA)-DRB1*0301 (DR3) is a class II molecule associated with human thyroid autoimmunity (although more strongly in Graves’ disease than Hashimoto’s thyroiditis; reviewed in Ref.18), we examined whether TPO-Ad immunization of DR3 transgenic mice (19) would present T cell epitopes that could be related to TPO antibodies with greater focus on the IDR.
Materials and Methods
Immunization protocols
Adenovirus expressing human TPO, kindly provided by Dr. Yuji Nagayama (Nagasaki University School of Biomedical Sciences, Nagasaki, Japan), was propagated in 293 HEK cells and purified through two rounds of CsCl density gradient centrifugation (20). The viral particle concentration was determined by measuring the absorbance at 260 nm as described; an absorbance of 1 corresponds to 1.1 x 10 (12) particles per milliliter (21).
Female BALB/c mice (6–7 wk old; Jackson Laboratories, Bar Harbor, ME) were injected in the thigh muscle with 109, 107, or 105 TPO-Ad viral particles in 50 μl PBS per injection (five animals in each group). Mice received three injections at 3-wk intervals. A control group (five mice) was immunized similarly with 109 particles of -galactose-expressing adenovirus. Mice transgenic for DR3 were bred on to mice lacking murine MHC class II genes (IAb knockouts) (22, 23). The background non-MHC genes of these mice, previously used to study TSH receptor-DNA vaccination (24), is 50% C57BL/10 and a 50% contribution from CBA, C57BL6, and 129. Mice were bred in a pathogen-free facility (Cedars-Sinai) and expression of HLA-DR3 was tested by flow cytometry using antibody L227 (American Type Culture Collection, Manassas, VA) and/or PCR. We immunized 10 DR3 animals with 109 TPO-Ad particles and five DR3 animals with 109 control-Ad per injection according to the protocol described above. One of the 10 DR3 mice died before completion of the protocol, leaving nine animals for analysis. Four weeks after the third injection, BALB/c or DR3 mice were euthanized to obtain blood, spleens, and thyroids. Thyroids were fixed in paraformaldehyde and serial sections prepared from paraffin blocks were stained with hematoxylin and eosin. All animal studies were approved by the local Institutional Animal Care and Use Committees and performed with the highest standards of care in pathogen-free facilities.
ELISA for TPO antibodies
TPO produced by Chinese hamster ovary cells was affinity purified from culture supernatants (25). ELISA wells were coated with TPO (1 mg/ml) and incubated with test sera (duplicate aliquots, diluted 1:100). Antibody binding was detected with horseradish peroxidase-conjugated goat antimouse IgG (Sigma Chemical Co., St. Louis, MO), the signal developed with o-phenylenediamine, and the OD read at 490 nm.
Antibody binding of 125I-TPO and affinity for TPO
Purified TPO protein was labeled using Iodogen to a specific activity of approximately 50 mCi/mg) as previously described (26). Duplicate serum aliquots (diluted 1:100) were incubated 1 h at room temperature with 125I-TPO (15,000 cpm). For determination of antibody affinity, purified unlabeled TPO at concentrations indicated in the text was included in the incubation mixture. Antigen-antibody complexes were precipitated with antimouse IgG coupled to a solid phase (Sac Cel; IDS, Boldon, Tyne and Wear, UK) and radiolabeled TPO remaining in the pellets was counted. Nonspecific 125I-TPO binding by normal mouse serum (5% of total cpm) was subtracted in calculating the percent 125I-TPO bound by antibodies in sera from immunized mice.
Recognition of the TPO autoantibody immunodominant region
125I-TPO binding by mouse antibodies was examined in the absence and presence of a pool of four human TPO-specific autoantibody Fab (SP1.4, WR1.7, TR1.8, and TR1.9) (12) that define the immunodominant region recognized by patients’ TPO autoantibodies (reviewed in Ref.27). As previously reported (26), duplicate aliquots of serum were incubated with 125I-TPO (20,000 cpm) alone or with the pool of four Fab (each Fab at 10–8 M final concentration; prepared as previously described (28). After incubation (1 h, room temperature), complexes were precipitated using Sac Cel and radiolabeled TPO remaining in the pellets counted. The human Fab is not precipitated by Sac Cel. Preliminary experiments were performed to determine the murine serum antibody dilutions required to provide binding values of 10–15% in the absence of TPO Fab. Such dilution is necessary to attain maximal inhibition of TPO binding by the addition of an excess concentration of Fab. Nonspecific 125I-TPO binding (3% of total counts per minute) was subtracted to calculate the percent inhibition by the TPO-specific Fab. This value is referred to in the text as the proportion (percent) of serum antibodies in a mouse directed to the human autoantibody IDR.
Spleen cell cultures for T cell responses to TPO protein and TPO synthetic peptides
Spleen cells (duplicate 200-ml aliquots of 4 x 105 cells) were incubated in round-bottomed 96-well plates in the presence and absence of soluble TPO (10 μg/ml) or synthetic TPO peptides. The amino acid composition of these peptides has been reported previously (17). In brief, the panel comprised 55 peptides each 20 amino acids in length with a five-residue overlap. Peptides were numbered sequentially (1–55) and spanned amino acid residues 27–853, corresponding to TPO extracellular region without the 26-residue signal peptide (29). Peptides were synthesized, HPLC purified, and their structures confirmed by mass spectrometry by methodology previously reported (30). The peptides were resuspended in sterile distilled water and used at a final concentration of 10 μg/ml. Concanavalin A (5 μg/ml) (Sigma) was used to determine the maximum responses. Culture medium was RPMI 1640, 10% heat-inactivated fetal bovine serum, 2 mM glutamine, 1 mM sodium pyruvate, 50 μg/gentamicin, 50 μM -mercaptoethanol, and 100 U/ml penicillin. The cells were cultured for 6 d and, after centrifugation, supernatants (100 μl in duplicate) were tested for interferon (IFN)- by ELISA using capture and biotinylated detection antibodies (PharMingen, San Diego, CA). IFN values are reported as picograms per milliliter extrapolated from recombinant IFN standards.
Statistical analysis
Differences between the properties of antibodies in mice immunized by different protocols were tested by ANOVA and Student’s t test, where indicated (SigmaStat; Jandel Scientific Software, San Rafael, CA).
Results
TPO antibodies induced by TPO-Ad immunization
Previously BALB/c mice were immunized with 1011 TPO-Ad particles per injection according to the immunization protocol initially used for TSH receptor adenovirus induction of Graves’ hyperthyroidism in mice (31). These animals developed TPO antibodies but with epitopes largely outside the IDR when compared with human autoantibodies and antibodies in mice injected with TPO-expressing fibroblasts or after TPO-DNA vaccination (14). Because injection of fewer TSH receptor adenovirus particles increased the pathogenicity of induced TSH receptor antibodies (32), in the present study, we injected female BALB/c mice with fewer (109, 107, and 105) TPO-Ad particles. As a control, we injected mice with 109 -galactose-expressing adenovirus. HLA-DR3 transgenic mice (that lack endogenous MHC) were injected with 109 TPO-Ad particles because it has been reported that such mice have a reduced number of CD4+ cells (33).
TPO antibodies were determined in sera from BALB/c and DR3 mice 4 wk after three injections at three weekly intervals. Injection of 109 TPO-Ad particles led to strong TPO antibody responses as measured by ELISA (Fig. 1A) and 125I-TPO immunoprecipitation (Fig. 1B). Injection of fewer (107) TPO-Ad particles generated a much weaker TPO antibody response, although still detectable relative to control adenovirus. The lowest dose of TPO-Ad (105 particles) did not induce detectable TPO antibodies in either assay (data not shown). DR3 mice injected with 109 TPO-Ad particles also developed TPO antibodies detectable at the same serum dilution (1:100) by ELISA (Fig. 1A) and 125I-TPO immunoprecipitation (Fig. 1B). However, TPO reactivity was lower in the DR3 mice than in BALB/c mice injected with a similar TPO-Ad dose (109 particles).
The antigen binding affinity of TPO antibodies in TPO-Ad immunized animals was tested by competition inhibition for binding to radiolabeled TPO. DR3 mice injected with 109 TPO-Ad particles developed TPO antibodies with the highest affinity, affinity constant of approximately 1.2 x 10–9 M (Fig. 2). In contrast, BALB/c mice injected with the same dose of TPO-Ad developed antibodies with a lower affinity (affinity constant 4.5 x 10–9 M) than the DR3 mice. This lower affinity was associated with the higher TPO antibody titer attained in BALB/c than DR3 mice (Fig. 1, A and B).
Antibody recognition of the human immunodominant region in TPO-Ad immunized mice
Four monoclonal human TPO autoantibodies define the IDR on the surface of native TPO (12). Competition by a pool of these four autoantibodies (expressed as Fab) for TPO antibody binding in an individual mouse serum determines the proportion of these polyclonal antibodies directed to the IDR. Previously, injecting BALB/c mice with a high dose of TPO-Ad particles (1011 per injection) induced antibodies whose epitopes lay largely outside the IDR (14). In the present study, BALB/c mice injected with 100-fold fewer (109) TPO-Ad particles also developed antibodies that poorly recognized the autoantibody IDR (Fig. 3, left panel). Indeed, sera from only two of five of these mice exceeded 20% recognition of the IDR. Injection of BALB/c mice with even fewer TPO-Ad particles (107) (Fig. 3, left panel) increased the average IDR recognition from 11 ± 15 to 41 ± 21% (mean ± SD; P = 0.034, Student’s t test).
TPO antibodies in DR3 mice injected with 109 TPO-Ad particles (Fig. 3, right panel) were directed to the IDR to a significantly greater extent than BALB/c mice receiving the same TPO-Ad dose (Fig. 3, left panel), namely 44 ± 21 vs. 11 ± 15%; mean ± SD, P = 0.009). This difference is associated with the lower TPO antibody titer in the DR3 mice (Fig. 1, A and B). Indeed, analysis of the data for all BALB/c and DR3 mice revealed a significant inverse correlation (r = –0.538; P = 0.017) between TPO antibody titer and the proportion of TPO antibodies within an individual serum directed to the IDR (Fig. 4).
Splenocyte responses to challenge with TPO antigen in vitro
Previously after TPO-plasmid DNA vaccination, memory T cell responses were elicited by culturing splenocytes with TPO antigen and measuring secretion of IFN (not IL-4) (17). In the present study, regardless of the TPO-Ad particle dose injected (105, 107, 109), splenocytes from all BALB/c and DR3 mice responded to TPO challenge, unlike splenocytes from control adenovirus-injected mice (Fig. 5). Of note is the IFN response, even in BALB/c mice injected with 105 TPO-Ad, a dose insufficient to induce TPO antibodies. However, consistent with the low antibody levels induced by TPO-Ad in DR3 mice, their splenocytes had a significantly lower IFN response to TPO challenge than BALB/c mice injected with the same number of TPO-Ad particles (109) (Fig. 5).
Next we challenged splenocytes with individual synthetic TPO peptides encompassing the intact antigen used in the foregoing experiments. Peptides were 20-mers overlapping by five amino acid residues, spanning amino acid residues 27–853 and numbered sequentially as 1–55. A positive IFN response to an individual peptide was defined as being greater than 2 SD above the mean of the responses to the entire panel of 55 peptides.
For TPO-Ad injected BALB/c mice, the range of peptides inducing a splenocyte IFN response (between one and three peptides in each animal) was diverse, showing no common pattern among different animals (Fig. 6). Surprisingly, unlike the greater IDR focus of antibodies in animals injected with fewer (107 vs. 109) viral particles (Fig. 3), there was no corresponding restriction in TPO peptides recognized (Fig. 6). As for IFN responses to the intact antigen, splenocytes from 105 TPO-Ad immunized BALB/c mice (with no detectable TPO antibodies) nevertheless responded to TPO peptides. Splenocytes from DR3 transgenic mice immunized with 109 TPO-Ad particles responded to a more restricted set of TPO peptides, namely peptides 5, 7, 30, and a cluster between 40 and 48 (Fig. 6). Moreover, there was minimal overlap in the peptides eliciting IFN responses between BALB/c and DR3 mice.
Discussion
The epitopic specificity of both B cell antigen receptors and soluble antibodies can influence the epitopes (peptides) presented to T cells (8), a phenomenon also demonstrated in thyroid autoimmunity (9, 10). The important question then arises as to why in Hashimoto’s thyroiditis the epitopes of TPO autoantibodies are largely restricted to an IDR and whether there is a relationship between autoantibody epitopic specificity and presentation of particular, potentially pathogenic TPO T cell epitopes. Previous studies in mice on the immune response to TPO, elicited by various immunization approaches, have not permitted study of this relationship. TPO antibodies from conventionally immunized animals (13) and animals injected with adenovirus expressing TPO in vivo (14) do not demonstrate relative restriction to the IDR. In other models involving in vivo TPO expression, splenocytes have not been amenable to study (13), have not been investigated (15), or respond to an excessively broad and random array of TPO peptides (17).
In the present study, we hypothesized that less vigorous TPO-Ad immunization, involving a lower level of in vivo TPO expression and consequently less antibody epitopic spreading, would generate TPO antibodies with greater restriction to the IDR. Support for this hypothesis was that injection of fewer TSH receptor adenoviral particles altered the epitopic balance of TSH receptor antibodies (32). Consistent with this hypothesis, reducing the dose of TPO-Ad injected into BALB/c mice increased the proportion of TPO antibodies directed to the IDR. In addition, we found that TPO-Ad immunization of mice transgenic for DR3 developed TPO antibodies with relative IDR restriction. It should be pointed out that DR3 is more strongly associated with Graves’ disease than with Hashimoto’s thyroiditis (18).
Turning to T cell epitopes, recognition of diverse, random TPO peptides, observed previously after TPO-DNA vaccination (17), was not altered by progressively reducing the number of TPO-Ad particles injected into BALB/c mice. This finding occurred even when immunization with 105 adenovirus particles was insufficient to elicit a detectable TPO antibody response. Remarkably, even when injected with the highest dose (109 TPO-Ad particles), the T cell epitopic response was more restricted in humanized DR3 mice. TPO peptides recognized by DR3 mice were predominantly near the C terminus of the myeloperoxidase-like domain. Perhaps coincidentally, because T cell and B cell epitopes do not necessarily overlap, this C-terminal region of the TPO extracellular region is also an autoantibody epitopic hot spot (34, 35).
Taken together, these data raise the interesting question of whether the greater propensity of DR3 mice than BALB/c mice to develop TPO antibodies to the IDR relates to TPO antibody titer or to different TPO peptides restricted by different MHC class II molecules. On the one hand, our data indicate that focus on the autoantibody IDR is greater at lower TPO antibody titers. This conclusion is supported by our previous findings using other TPO immunization protocols of inverse proportionality between antibody titer and IDR restriction (13, 14). On the other hand, the present data also suggest a relationship between IDR recognition and MHC-dependent TPO peptide restriction. DR3 mice injected with 109 TPO-Ad particles develop higher TPO antibody titers than BALB/c mice receiving only 107 TPO-Ad particles (Fig. 1) without a reduction in IDR recognition (Fig. 3). Therefore, independent of antibody titer, TPO antibodies in DR3 mice are biased toward the IDR. Our data for DR3 mice are consistent with the findings of Flynn et al. (15) that the majority of TPO antibodies induced by TPO-DNA vaccination were to the IDR in HLA-DR3 transgenic mice on the nonobese mouse model of autoimmune diabetes (NOD) background. However, the DR3 mice used in the present and a previous study (24) have mixed background genes (50% C57BL/10 and a 50% contribution from CBA, C57BL6, and 129). For this reason, background genes are unlikely to contribute to antibody IDR recognition.
Support for the notion that DR3 class II restriction of T cell epitopes is related to TPO antibody IDR recognition is the marked difference in TPO peptide responses between antibody IDR resistant BALB/c mice and greater IDR recognition in DR3 mice. The tendency of BALB/c mice to have diverse and variable TPO peptide responses after TPO-DNA vaccination (17) persisted even when TPO-Ad vaccination was so weak (105 particles per injection) that no TPO antibody response could be detected. On the other hand, in DR3 mice the TPO peptide response was relatively restricted despite immunization with 109 TPO-Ad particles leading to moderately strong TPO antibody production. As mentioned above, there are no data on TPO peptides recognized by T cells from TPO-DNA-vaccinated DR3-nonobese mouse model of autoimmune diabetes mice (15).
Finally, how do the TPO peptides inducing a splenocyte response in TPO-Ad immunized DR3 mice compare with TPO peptides that stimulate T cells from patients with autoimmune thyroid disease Data on the latter (usually not HLA typed and therefore not necessarily DR3 positive) have been reported in numerous studies (36, 37, 38, 39, 40, 41). TPO peptide 30 (amino acid residues 463–481) used in our study is identical with a peptide reported by Tandon et al. (39) to induce a response in peripheral blood lymphocytes from patients. This peptide is recognized by three of nine TPO-Ad immunized DR3 mice. TPO peptide 7 (amino acid residues 117–136) has partial overlap with a human disease-associated peptide encompassing residues 110–129 (38) and is recognized by only one of nine mice. The dominant TPO peptides inducing responses in four of nine and five of nine DR3 mice (peptides 42 and 47; amino acid residues 642–661 and 717–736, respectively) are not among those presently identified in human disease. However, the relationship between human lymphocyte peptide reactivity and the pathogenesis of autoimmune thyroiditis has not been established. Of interest is peptide 47, which overlaps with a region (residues 713–721) associated with TPO autoantibody binding (34, 35). Whether overlap between potential T cell and B cell TPO epitopes is simply fortuitous or of pathophysiological significance remains to be determined. Finally, a peptide encompassing murine TPO amino acids 540–559 (homologous to human TPO residues 528–547) has recently been found to be immunodominant in an induced murine model of autoimmune thyroiditis in mice (42). This region of TPO, encompassed by human TPO peptides 34 and 35, did not elicit T cell responses in our TPO-Ad BALB/c or DR3 mice.
In conclusion, the present study determines that weak TPO-Ad immunization of BALB/c mice with resultant low TPO antibody titers is required for enhanced epitopic focus on the IDR yet is associated with T cell responses to diverse and variable TPO peptides. In contrast, DR3 transgenic mice, despite stronger TPO-Ad immunization leading to higher titer TPO antibodies, do focus on the autoantibody IDR. Moreover, T cells from the humanized DR3 mice recognize a more limited range of TPO peptides. These data suggest a relationship between MHC class II molecules and the development of antibodies to the IDR, a feature of human thyroid autoimmunity.
Acknowledgments
We thank Dr. Yuji Nagayama (Nagasaki University School of Biomedical Sciences, Nagasaki, Japan) for his generous gift of TPO-Ad and Julie Hansen (Mayo Clinic) for her assistance with DR3 mice. We are also grateful for contributions by Dr. Boris Catz (Los Angeles, CA).
Footnotes
This work was supported by National Institutes of Health Grants DK 36182 (to B.R.) and DK 54684 (to S.M.M.).
Abbreviations: DR3, DRB1*0301; HLA, human leukocyte antigen; IDR, immunodominant region; IFN, interferon; MHC, major histocompatibility complex; TPO, thyroid peroxidase; TPO-Ad, TPO-adenovirus.
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Ng HP, Kung AWC Induction of autoimmune thyroiditis and hypothyroidism by immunization of thyroid peroxidase (TPO) immunodominant peptide in a mouse model. Program of the 87th Annual Meeting of The Endocrine Society, San Diego, CA, 2005, p 155 (Abstract OR54-1)(Jin Guo, Sandra M. McLach)
Department of Immunology (C.S.D.), Mayo Clinic, Rochester, Minnesota 55905
Abstract
We investigated the relationship between thyroid peroxidase (TPO) antibody and T lymphocyte epitopes in TPO-adenovirus (TPO-Ad) immunized BALB/c mice and mice transgenic for the human class II molecule DR3 associated with human thyroid autoimmunity. TPO autoantibodies are largely restricted to an immunodominant region (IDR). BALB/c mice immunized with fewer (107 vs. 109) TPO-Ad particles developed TPO antibodies with lower titers that displayed greater restriction to the IDR. However, as with higher-dose TPO-Ad immunization, T cell epitopes (assessed by splenocyte interferon- response to TPO in vitro) were highly diverse and variable in different animals. In contrast, DR3 mice immunized the higher TPO-Ad dose (109 particles) had high TPO antibody levels that showed relative focus on the IDR. Moreover, T cell epitopes recognized by splenocytes from DR3 mice showed greater restriction than BALB/c mice. Antibody affinities for TPO were higher in DR3 than in BALB/c mice. The present study indicates that weak TPO-Ad immunization of BALB/c mice (with consequent low TPO antibody titers) is required for enhanced IDR focus yet is not associated with T cell epitopic restriction. Humanized DR3 transgenic mice, despite stronger TPO-Ad immunization, develop higher titer TPO antibodies that do focus on the autoantibody IDR with T cells that recognize a more limited range of TPO peptides. These data suggest a relationship between major histocompatibility complex class II molecules and the development of antibodies to the IDR, a feature of human thyroid autoimmunity.
Introduction
AUTOIMMUNE (HASHIMOTO’S) THYROIDITIS, the most common organ-specific autoimmune disease affecting humans, involves loss of tolerance to thyroid peroxidase (TPO), a thyroid cell surface antigen. The immune response to TPO is both humoral and cell-mediated. TPO autoantibodies can activate the complement cascade (1); are mainly associated with complement-fixing IgG1, not IgG4 (2); and can damage thyroid cells in vitro by an antibody-dependent cell cytotoxic mechanism involving NK cells (3, 4, 5, 6). Nevertheless, whether TPO autoantibodies directly cause thyrocyte damage has not been definitively established. In contrast, recent evidence in a humanized T cell transgenic model establishes that T cells alone are sufficient to induce thyroiditis (7).
Even if not directly involved in thyrocyte damage, TPO autoantibodies have two important roles in studying Hashimoto’s disease. First, TPO autoantibodies in patients’ sera are excellent clinical markers of disease, perhaps unrivaled among organ-specific autoimmune diseases. Second, autoantibodies on the B cell surface function as antigen receptors that (unlike macrophages and dendritic cells) capture specific antigen for processing and presentation to T cells. Antibodies (both soluble and membrane associated) can profoundly effect antigen processing within the antigen-presenting cell, introducing bias in antigenic determinants (peptides) presented to T cells (reviewed in Ref.8). In particular, human TPO autoantibodies with different epitopes can influence which TPO peptides are presented by antigen-presenting cell to human T cell clones (9, 10). A remarkable feature of polyclonal human TPO autoantibodies is their relative restriction to epitopes within an immunodominant region (IDR) on the surface of the native antigen (11, 12). Understanding the factors contributing to this epitopic focus of TPO autoantibodies is clearly of value in understanding the pathogenesis of autoimmune thyroiditis with, ultimately, the potential of immunotherapy for this disease.
Animal models are important tools in studying the pathogenesis of autoimmunity. Conventional immunization of mice with soluble TPO protein in adjuvant effectively induces antibodies, but their epitopes, unlike human autoantibodies, are not largely to restricted to the IDR (11, 13). Antibodies with the epitopic profiles of human autoantibodies have been produced by nonconventional immunization using fibroblasts coexpressing TPO and major histocompatibility complex (MHC) class II molecules (13) as well as by TPO-DNA vaccination (14, 15). However, these two approaches have not permitted identification of the TPO T cell epitopes associated with IDR-focused TPO antibodies. The TPO-MHC class II expressing fibroblasts induce very high background responses by splenocytes in vitro, most likely because of high costimulatory molecule (B7) expression (16). On the other hand, TPO plasmid DNA vaccination led to splenocyte responses to a highly diverse array of synthetic TPO peptides (both intra- and interanimal) (17). More recently TPO-adenovirus (TPO-Ad) immunization induced high-titer TPO antibodies in BALB/c mice but without the IDR restriction typical of human autoantibodies (14).
In the present study, we tried two new approaches to facilitate investigation of the relationship between antibody (B cell) and T cell epitopes on TPO. First, we tested the hypothesis that immunizing BALB/c mice with fewer TPO-Ad particles would generate TPO antibodies with greater IDR restriction than observed with high-dose TPO-Ad immunization and whose T cell epitopes would also be more restricted. Second, because human leukocyte antigen (HLA)-DRB1*0301 (DR3) is a class II molecule associated with human thyroid autoimmunity (although more strongly in Graves’ disease than Hashimoto’s thyroiditis; reviewed in Ref.18), we examined whether TPO-Ad immunization of DR3 transgenic mice (19) would present T cell epitopes that could be related to TPO antibodies with greater focus on the IDR.
Materials and Methods
Immunization protocols
Adenovirus expressing human TPO, kindly provided by Dr. Yuji Nagayama (Nagasaki University School of Biomedical Sciences, Nagasaki, Japan), was propagated in 293 HEK cells and purified through two rounds of CsCl density gradient centrifugation (20). The viral particle concentration was determined by measuring the absorbance at 260 nm as described; an absorbance of 1 corresponds to 1.1 x 10 (12) particles per milliliter (21).
Female BALB/c mice (6–7 wk old; Jackson Laboratories, Bar Harbor, ME) were injected in the thigh muscle with 109, 107, or 105 TPO-Ad viral particles in 50 μl PBS per injection (five animals in each group). Mice received three injections at 3-wk intervals. A control group (five mice) was immunized similarly with 109 particles of -galactose-expressing adenovirus. Mice transgenic for DR3 were bred on to mice lacking murine MHC class II genes (IAb knockouts) (22, 23). The background non-MHC genes of these mice, previously used to study TSH receptor-DNA vaccination (24), is 50% C57BL/10 and a 50% contribution from CBA, C57BL6, and 129. Mice were bred in a pathogen-free facility (Cedars-Sinai) and expression of HLA-DR3 was tested by flow cytometry using antibody L227 (American Type Culture Collection, Manassas, VA) and/or PCR. We immunized 10 DR3 animals with 109 TPO-Ad particles and five DR3 animals with 109 control-Ad per injection according to the protocol described above. One of the 10 DR3 mice died before completion of the protocol, leaving nine animals for analysis. Four weeks after the third injection, BALB/c or DR3 mice were euthanized to obtain blood, spleens, and thyroids. Thyroids were fixed in paraformaldehyde and serial sections prepared from paraffin blocks were stained with hematoxylin and eosin. All animal studies were approved by the local Institutional Animal Care and Use Committees and performed with the highest standards of care in pathogen-free facilities.
ELISA for TPO antibodies
TPO produced by Chinese hamster ovary cells was affinity purified from culture supernatants (25). ELISA wells were coated with TPO (1 mg/ml) and incubated with test sera (duplicate aliquots, diluted 1:100). Antibody binding was detected with horseradish peroxidase-conjugated goat antimouse IgG (Sigma Chemical Co., St. Louis, MO), the signal developed with o-phenylenediamine, and the OD read at 490 nm.
Antibody binding of 125I-TPO and affinity for TPO
Purified TPO protein was labeled using Iodogen to a specific activity of approximately 50 mCi/mg) as previously described (26). Duplicate serum aliquots (diluted 1:100) were incubated 1 h at room temperature with 125I-TPO (15,000 cpm). For determination of antibody affinity, purified unlabeled TPO at concentrations indicated in the text was included in the incubation mixture. Antigen-antibody complexes were precipitated with antimouse IgG coupled to a solid phase (Sac Cel; IDS, Boldon, Tyne and Wear, UK) and radiolabeled TPO remaining in the pellets was counted. Nonspecific 125I-TPO binding by normal mouse serum (5% of total cpm) was subtracted in calculating the percent 125I-TPO bound by antibodies in sera from immunized mice.
Recognition of the TPO autoantibody immunodominant region
125I-TPO binding by mouse antibodies was examined in the absence and presence of a pool of four human TPO-specific autoantibody Fab (SP1.4, WR1.7, TR1.8, and TR1.9) (12) that define the immunodominant region recognized by patients’ TPO autoantibodies (reviewed in Ref.27). As previously reported (26), duplicate aliquots of serum were incubated with 125I-TPO (20,000 cpm) alone or with the pool of four Fab (each Fab at 10–8 M final concentration; prepared as previously described (28). After incubation (1 h, room temperature), complexes were precipitated using Sac Cel and radiolabeled TPO remaining in the pellets counted. The human Fab is not precipitated by Sac Cel. Preliminary experiments were performed to determine the murine serum antibody dilutions required to provide binding values of 10–15% in the absence of TPO Fab. Such dilution is necessary to attain maximal inhibition of TPO binding by the addition of an excess concentration of Fab. Nonspecific 125I-TPO binding (3% of total counts per minute) was subtracted to calculate the percent inhibition by the TPO-specific Fab. This value is referred to in the text as the proportion (percent) of serum antibodies in a mouse directed to the human autoantibody IDR.
Spleen cell cultures for T cell responses to TPO protein and TPO synthetic peptides
Spleen cells (duplicate 200-ml aliquots of 4 x 105 cells) were incubated in round-bottomed 96-well plates in the presence and absence of soluble TPO (10 μg/ml) or synthetic TPO peptides. The amino acid composition of these peptides has been reported previously (17). In brief, the panel comprised 55 peptides each 20 amino acids in length with a five-residue overlap. Peptides were numbered sequentially (1–55) and spanned amino acid residues 27–853, corresponding to TPO extracellular region without the 26-residue signal peptide (29). Peptides were synthesized, HPLC purified, and their structures confirmed by mass spectrometry by methodology previously reported (30). The peptides were resuspended in sterile distilled water and used at a final concentration of 10 μg/ml. Concanavalin A (5 μg/ml) (Sigma) was used to determine the maximum responses. Culture medium was RPMI 1640, 10% heat-inactivated fetal bovine serum, 2 mM glutamine, 1 mM sodium pyruvate, 50 μg/gentamicin, 50 μM -mercaptoethanol, and 100 U/ml penicillin. The cells were cultured for 6 d and, after centrifugation, supernatants (100 μl in duplicate) were tested for interferon (IFN)- by ELISA using capture and biotinylated detection antibodies (PharMingen, San Diego, CA). IFN values are reported as picograms per milliliter extrapolated from recombinant IFN standards.
Statistical analysis
Differences between the properties of antibodies in mice immunized by different protocols were tested by ANOVA and Student’s t test, where indicated (SigmaStat; Jandel Scientific Software, San Rafael, CA).
Results
TPO antibodies induced by TPO-Ad immunization
Previously BALB/c mice were immunized with 1011 TPO-Ad particles per injection according to the immunization protocol initially used for TSH receptor adenovirus induction of Graves’ hyperthyroidism in mice (31). These animals developed TPO antibodies but with epitopes largely outside the IDR when compared with human autoantibodies and antibodies in mice injected with TPO-expressing fibroblasts or after TPO-DNA vaccination (14). Because injection of fewer TSH receptor adenovirus particles increased the pathogenicity of induced TSH receptor antibodies (32), in the present study, we injected female BALB/c mice with fewer (109, 107, and 105) TPO-Ad particles. As a control, we injected mice with 109 -galactose-expressing adenovirus. HLA-DR3 transgenic mice (that lack endogenous MHC) were injected with 109 TPO-Ad particles because it has been reported that such mice have a reduced number of CD4+ cells (33).
TPO antibodies were determined in sera from BALB/c and DR3 mice 4 wk after three injections at three weekly intervals. Injection of 109 TPO-Ad particles led to strong TPO antibody responses as measured by ELISA (Fig. 1A) and 125I-TPO immunoprecipitation (Fig. 1B). Injection of fewer (107) TPO-Ad particles generated a much weaker TPO antibody response, although still detectable relative to control adenovirus. The lowest dose of TPO-Ad (105 particles) did not induce detectable TPO antibodies in either assay (data not shown). DR3 mice injected with 109 TPO-Ad particles also developed TPO antibodies detectable at the same serum dilution (1:100) by ELISA (Fig. 1A) and 125I-TPO immunoprecipitation (Fig. 1B). However, TPO reactivity was lower in the DR3 mice than in BALB/c mice injected with a similar TPO-Ad dose (109 particles).
The antigen binding affinity of TPO antibodies in TPO-Ad immunized animals was tested by competition inhibition for binding to radiolabeled TPO. DR3 mice injected with 109 TPO-Ad particles developed TPO antibodies with the highest affinity, affinity constant of approximately 1.2 x 10–9 M (Fig. 2). In contrast, BALB/c mice injected with the same dose of TPO-Ad developed antibodies with a lower affinity (affinity constant 4.5 x 10–9 M) than the DR3 mice. This lower affinity was associated with the higher TPO antibody titer attained in BALB/c than DR3 mice (Fig. 1, A and B).
Antibody recognition of the human immunodominant region in TPO-Ad immunized mice
Four monoclonal human TPO autoantibodies define the IDR on the surface of native TPO (12). Competition by a pool of these four autoantibodies (expressed as Fab) for TPO antibody binding in an individual mouse serum determines the proportion of these polyclonal antibodies directed to the IDR. Previously, injecting BALB/c mice with a high dose of TPO-Ad particles (1011 per injection) induced antibodies whose epitopes lay largely outside the IDR (14). In the present study, BALB/c mice injected with 100-fold fewer (109) TPO-Ad particles also developed antibodies that poorly recognized the autoantibody IDR (Fig. 3, left panel). Indeed, sera from only two of five of these mice exceeded 20% recognition of the IDR. Injection of BALB/c mice with even fewer TPO-Ad particles (107) (Fig. 3, left panel) increased the average IDR recognition from 11 ± 15 to 41 ± 21% (mean ± SD; P = 0.034, Student’s t test).
TPO antibodies in DR3 mice injected with 109 TPO-Ad particles (Fig. 3, right panel) were directed to the IDR to a significantly greater extent than BALB/c mice receiving the same TPO-Ad dose (Fig. 3, left panel), namely 44 ± 21 vs. 11 ± 15%; mean ± SD, P = 0.009). This difference is associated with the lower TPO antibody titer in the DR3 mice (Fig. 1, A and B). Indeed, analysis of the data for all BALB/c and DR3 mice revealed a significant inverse correlation (r = –0.538; P = 0.017) between TPO antibody titer and the proportion of TPO antibodies within an individual serum directed to the IDR (Fig. 4).
Splenocyte responses to challenge with TPO antigen in vitro
Previously after TPO-plasmid DNA vaccination, memory T cell responses were elicited by culturing splenocytes with TPO antigen and measuring secretion of IFN (not IL-4) (17). In the present study, regardless of the TPO-Ad particle dose injected (105, 107, 109), splenocytes from all BALB/c and DR3 mice responded to TPO challenge, unlike splenocytes from control adenovirus-injected mice (Fig. 5). Of note is the IFN response, even in BALB/c mice injected with 105 TPO-Ad, a dose insufficient to induce TPO antibodies. However, consistent with the low antibody levels induced by TPO-Ad in DR3 mice, their splenocytes had a significantly lower IFN response to TPO challenge than BALB/c mice injected with the same number of TPO-Ad particles (109) (Fig. 5).
Next we challenged splenocytes with individual synthetic TPO peptides encompassing the intact antigen used in the foregoing experiments. Peptides were 20-mers overlapping by five amino acid residues, spanning amino acid residues 27–853 and numbered sequentially as 1–55. A positive IFN response to an individual peptide was defined as being greater than 2 SD above the mean of the responses to the entire panel of 55 peptides.
For TPO-Ad injected BALB/c mice, the range of peptides inducing a splenocyte IFN response (between one and three peptides in each animal) was diverse, showing no common pattern among different animals (Fig. 6). Surprisingly, unlike the greater IDR focus of antibodies in animals injected with fewer (107 vs. 109) viral particles (Fig. 3), there was no corresponding restriction in TPO peptides recognized (Fig. 6). As for IFN responses to the intact antigen, splenocytes from 105 TPO-Ad immunized BALB/c mice (with no detectable TPO antibodies) nevertheless responded to TPO peptides. Splenocytes from DR3 transgenic mice immunized with 109 TPO-Ad particles responded to a more restricted set of TPO peptides, namely peptides 5, 7, 30, and a cluster between 40 and 48 (Fig. 6). Moreover, there was minimal overlap in the peptides eliciting IFN responses between BALB/c and DR3 mice.
Discussion
The epitopic specificity of both B cell antigen receptors and soluble antibodies can influence the epitopes (peptides) presented to T cells (8), a phenomenon also demonstrated in thyroid autoimmunity (9, 10). The important question then arises as to why in Hashimoto’s thyroiditis the epitopes of TPO autoantibodies are largely restricted to an IDR and whether there is a relationship between autoantibody epitopic specificity and presentation of particular, potentially pathogenic TPO T cell epitopes. Previous studies in mice on the immune response to TPO, elicited by various immunization approaches, have not permitted study of this relationship. TPO antibodies from conventionally immunized animals (13) and animals injected with adenovirus expressing TPO in vivo (14) do not demonstrate relative restriction to the IDR. In other models involving in vivo TPO expression, splenocytes have not been amenable to study (13), have not been investigated (15), or respond to an excessively broad and random array of TPO peptides (17).
In the present study, we hypothesized that less vigorous TPO-Ad immunization, involving a lower level of in vivo TPO expression and consequently less antibody epitopic spreading, would generate TPO antibodies with greater restriction to the IDR. Support for this hypothesis was that injection of fewer TSH receptor adenoviral particles altered the epitopic balance of TSH receptor antibodies (32). Consistent with this hypothesis, reducing the dose of TPO-Ad injected into BALB/c mice increased the proportion of TPO antibodies directed to the IDR. In addition, we found that TPO-Ad immunization of mice transgenic for DR3 developed TPO antibodies with relative IDR restriction. It should be pointed out that DR3 is more strongly associated with Graves’ disease than with Hashimoto’s thyroiditis (18).
Turning to T cell epitopes, recognition of diverse, random TPO peptides, observed previously after TPO-DNA vaccination (17), was not altered by progressively reducing the number of TPO-Ad particles injected into BALB/c mice. This finding occurred even when immunization with 105 adenovirus particles was insufficient to elicit a detectable TPO antibody response. Remarkably, even when injected with the highest dose (109 TPO-Ad particles), the T cell epitopic response was more restricted in humanized DR3 mice. TPO peptides recognized by DR3 mice were predominantly near the C terminus of the myeloperoxidase-like domain. Perhaps coincidentally, because T cell and B cell epitopes do not necessarily overlap, this C-terminal region of the TPO extracellular region is also an autoantibody epitopic hot spot (34, 35).
Taken together, these data raise the interesting question of whether the greater propensity of DR3 mice than BALB/c mice to develop TPO antibodies to the IDR relates to TPO antibody titer or to different TPO peptides restricted by different MHC class II molecules. On the one hand, our data indicate that focus on the autoantibody IDR is greater at lower TPO antibody titers. This conclusion is supported by our previous findings using other TPO immunization protocols of inverse proportionality between antibody titer and IDR restriction (13, 14). On the other hand, the present data also suggest a relationship between IDR recognition and MHC-dependent TPO peptide restriction. DR3 mice injected with 109 TPO-Ad particles develop higher TPO antibody titers than BALB/c mice receiving only 107 TPO-Ad particles (Fig. 1) without a reduction in IDR recognition (Fig. 3). Therefore, independent of antibody titer, TPO antibodies in DR3 mice are biased toward the IDR. Our data for DR3 mice are consistent with the findings of Flynn et al. (15) that the majority of TPO antibodies induced by TPO-DNA vaccination were to the IDR in HLA-DR3 transgenic mice on the nonobese mouse model of autoimmune diabetes (NOD) background. However, the DR3 mice used in the present and a previous study (24) have mixed background genes (50% C57BL/10 and a 50% contribution from CBA, C57BL6, and 129). For this reason, background genes are unlikely to contribute to antibody IDR recognition.
Support for the notion that DR3 class II restriction of T cell epitopes is related to TPO antibody IDR recognition is the marked difference in TPO peptide responses between antibody IDR resistant BALB/c mice and greater IDR recognition in DR3 mice. The tendency of BALB/c mice to have diverse and variable TPO peptide responses after TPO-DNA vaccination (17) persisted even when TPO-Ad vaccination was so weak (105 particles per injection) that no TPO antibody response could be detected. On the other hand, in DR3 mice the TPO peptide response was relatively restricted despite immunization with 109 TPO-Ad particles leading to moderately strong TPO antibody production. As mentioned above, there are no data on TPO peptides recognized by T cells from TPO-DNA-vaccinated DR3-nonobese mouse model of autoimmune diabetes mice (15).
Finally, how do the TPO peptides inducing a splenocyte response in TPO-Ad immunized DR3 mice compare with TPO peptides that stimulate T cells from patients with autoimmune thyroid disease Data on the latter (usually not HLA typed and therefore not necessarily DR3 positive) have been reported in numerous studies (36, 37, 38, 39, 40, 41). TPO peptide 30 (amino acid residues 463–481) used in our study is identical with a peptide reported by Tandon et al. (39) to induce a response in peripheral blood lymphocytes from patients. This peptide is recognized by three of nine TPO-Ad immunized DR3 mice. TPO peptide 7 (amino acid residues 117–136) has partial overlap with a human disease-associated peptide encompassing residues 110–129 (38) and is recognized by only one of nine mice. The dominant TPO peptides inducing responses in four of nine and five of nine DR3 mice (peptides 42 and 47; amino acid residues 642–661 and 717–736, respectively) are not among those presently identified in human disease. However, the relationship between human lymphocyte peptide reactivity and the pathogenesis of autoimmune thyroiditis has not been established. Of interest is peptide 47, which overlaps with a region (residues 713–721) associated with TPO autoantibody binding (34, 35). Whether overlap between potential T cell and B cell TPO epitopes is simply fortuitous or of pathophysiological significance remains to be determined. Finally, a peptide encompassing murine TPO amino acids 540–559 (homologous to human TPO residues 528–547) has recently been found to be immunodominant in an induced murine model of autoimmune thyroiditis in mice (42). This region of TPO, encompassed by human TPO peptides 34 and 35, did not elicit T cell responses in our TPO-Ad BALB/c or DR3 mice.
In conclusion, the present study determines that weak TPO-Ad immunization of BALB/c mice with resultant low TPO antibody titers is required for enhanced epitopic focus on the IDR yet is associated with T cell responses to diverse and variable TPO peptides. In contrast, DR3 transgenic mice, despite stronger TPO-Ad immunization leading to higher titer TPO antibodies, do focus on the autoantibody IDR. Moreover, T cells from the humanized DR3 mice recognize a more limited range of TPO peptides. These data suggest a relationship between MHC class II molecules and the development of antibodies to the IDR, a feature of human thyroid autoimmunity.
Acknowledgments
We thank Dr. Yuji Nagayama (Nagasaki University School of Biomedical Sciences, Nagasaki, Japan) for his generous gift of TPO-Ad and Julie Hansen (Mayo Clinic) for her assistance with DR3 mice. We are also grateful for contributions by Dr. Boris Catz (Los Angeles, CA).
Footnotes
This work was supported by National Institutes of Health Grants DK 36182 (to B.R.) and DK 54684 (to S.M.M.).
Abbreviations: DR3, DRB1*0301; HLA, human leukocyte antigen; IDR, immunodominant region; IFN, interferon; MHC, major histocompatibility complex; TPO, thyroid peroxidase; TPO-Ad, TPO-adenovirus.
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