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Endocrinology Vol. 145, No. 8 3671-3678
Copyright © 2004 by The Endocrine Society

"Naked" Deoxyribonucleic Acid Vaccination Induces Recognition of Diverse Thyroid Peroxidase T Cell Epitopes

Jin Guo, Pavel N. Pichurin, John C. Morris, Basil Rapoport and Sandra M. McLachlan

Autoimmune Disease Unit, Cedars-Sinai Research Institute and University of California School of Medicine (J.G., P.N.P., B.R., S.M.M.), Los Angeles, California 90048; and Division of Endocrinology and Metabolism, Department of Medicine, Mayo Clinic College of Medicine (J.C.M.), Rochester, Minnesota 55905

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


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Recently, we observed that vaccination of BALB/c mice with thyroid peroxidase (TPO)-DNA in a plasmid is highly effective at inducing antibodies that interact with the immunodominant region recognized by human autoantibodies. We have now analyzed the TPO epitopes recognized by memory T cells in these animals. Splenocytes from TPO-DNA (not control DNA)-vaccinated mice responded to TPO protein antigen, as measured by interferon-{gamma} production. As a group, TPO-immunized mice recognized 35 of 55 overlapping synthetic peptides that encompass the 814-amino acid TPO ectodomain. In individual mice, between five and 10 peptides induced splenocyte responses. Two T cell epitopes were immunodominant, one of which is also recognized by patients with autoimmune thyroid disease. To explore a potential correlation between T and B cell epitopes, we analyzed serum TPO antibody epitopic fingerprints. No relationship was evident. However, the number of T cell epitopes recognized by individual mice was inversely proportional to recognition of an antibody epitopic subdomain. The diversity of TPO T cell epitopes is in striking contrast to the restricted number of TSH receptor (TSHR) peptides (four of 29) recognized by T cells, as is the paucity of antibodies in the same strain of mice vaccinated with TSHR-DNA. In conclusion, our data highlight differences for both antibody and T cell epitopic recognition in TPO- vs. TSHR-DNA-immunized BALB/c mice. These findings provide insight into mechanisms that may be involved in spontaneous immune responses to two major thyroid autoantigens in humans.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
VACCINATING BALB/c MICE with thyroid peroxidase (TPO)-DNA in a plasmid induces antibodies in a high proportion of animals (1). Moreover, unlike the humoral response to conventional immunization with purified TPO protein and adjuvant (2), TPO-DNA-induced antibodies that resemble human autoantibodies in terms of their epitopes and high affinities for antigen (1). Human TPO autoantibodies interact with conformational epitopes that cannot be identified using synthetic peptides (reviewed in Ref.3). Using monoclonal antibodies from humans (or mice) to compete with patients’ sera for binding to TPO, human autoantibodies have been shown to interact with a restricted set of overlapping epitopic domains on TPO (4, 5, 6). Remarkably, antibodies in TPO-DNA-vaccinated mice focus on the human TPO autoantibody-immunodominant region (IDR) (1). The ease of antibody induction by naked TPO-DNA vaccination in BALB/c mice contrasts with the outcome of TSH receptor (TSHR)-DNA vaccination in the same mouse strain. We and others observed low or undetectable antibody levels in BALB/c mice vaccinated with TSHR-DNA (7, 8, 9, 10), although contrary findings were reported by one group (11).

There is presently no information on T cell responses in TPO-DNA-vaccinated mice. In contrast, TSHR-DNA vaccination induces memory T cells that respond to challenge in vitro with TSHR antigen by proliferating and secreting cytokines, such as interferon-{gamma} (IFN-{gamma}) (7, 8). Furthermore, BALB/c splenocytes recognize a restricted number of TSHR T cell epitopes identified using a panel of overlapping synthetic peptides encompassing the TSHR ectodomain (12). Of interest, one of these peptides has previously been defined as a major human T cell epitope (discussed in Ref.12). In the present study we examined the ability of splenocytes from TPO-DNA-vaccinated mice to respond to TPO antigen and to a panel of synthetic peptides encompassing the large ectodomain component of the mature TPO molecule (827 of 906 amino acid residues). Antibodies play an important role in antigen presentation and can also influence the peptides made available to T cells (reviewed in Ref.13). For this reason we also determined the subdomains within the IDR recognized by TPO antibodies in these mice. Our unexpected findings highlight the difference between induced immune responses to TPO and the TSHR, two major thyroid autoantigens in humans.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
DNA vaccination of mice
BALB/c mice (The Jackson Laboratory, Bar Harbor, ME) were vaccinated with the cDNA for human TPO in the vector pcDNA3 (Invitrogen, Carlsbad, CA) as previously described (1). Briefly, female mice, aged 6–7 wk, were pretreated in the anterior tibialis muscle with cardiotoxin (50 µl 10 µM Naja nigricollis; Calbiochem, La Jolla, CA). Five to 7 d later, the mice were injected at the same site with 100 µg (50 µl) TPO-DNA (eight mice) or empty vector DNA (five mice). The vaccination protocol was repeated after 3 and 6 wk. Four weeks after the third vaccination, mice were euthanized to obtain blood and spleens. All animal studies were approved by the institutional animal care and usage committee and were performed in accordance with the highest standards of care in a pathogen-free facility.

TPO protein for antibody measurements and proliferative studies
Recombinant human TPO, secreted by Chinese hamster ovary cells, was affinity-purified from culture medium as previously described (14), and the buffer was changed to PBS using Centricon 50 columns (Amicon, Inc., Beverly, MA). The concentration was determined by spectrophotometry at OD 280 nm (extinction coefficient, 17.9), and purity was determined by PAGE.

TPO peptides
A panel of 55 peptides corresponding to amino acids in the extracellular domain of TPO were synthesized and HPLC-purified, and their structures were confirmed by mass spectrometry using methodology previously reported (15). Each peptide was 20 amino acids in length and overlapped the subsequent peptide by five residues. Peptides were numbered sequentially (1 through 55) beginning with amino acid 27 (Table 1Go), excluding the putative TPO signal peptide that comprises residues 1–26. The peptides were resuspended in sterile distilled water and used at a final concentration of 10 µg/ml.


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  		TABLE 1. Panel of synthetic peptides encompassing the human TPO ectodomain used to study responses in TPO-DNA-vaccinated mice

 
Cytokine response to TPO antigen and peptides
Splenocytes (duplicate 200-µl aliquots of ~5 x 105 cells) were incubated in round-bottom, 96-well plates in the presence or absence of soluble TPO (10 µg/ml), TPO peptides (10 µg/ml), or Con A (Sigma-Aldrich Corp., St. Louis, MO; 5 µg/ml). The culture medium was RPMI 1640, 10% heat-inactivated fetal bovine serum, 2 mM glutamine, 1 mM sodium pyruvate, 50 µg/ml gentamicin, 50 µM ß-mercaptoethanol, and 100 U/ml penicillin. After 5–6 d (37 C, 5% CO2), supernatants were centrifuged to remove cell debris and were stored at –80 C. Supernatants were assayed for IFN-{gamma} and IL-4 by ELISA (100 µl; in duplicate) using capture and biotinylated detection antibodies from BD PharMingen (San Diego, CA). Cytokine values are reported as picograms per milliliter, extrapolated from recombinant standards (BD PharMingen).

Serum TPO antibody analysis
TPO antibodies in mouse sera were analyzed by ELISA as previously described (1). Briefly, ELISA wells were coated with TPO (1 µg/ml) and incubated with mouse sera (duplicate aliquots, diluted 1:1000). Antibody binding was detected with horseradish peroxidase-conjugated mouse anti-IgG (Sigma-Aldrich Corp.), the signal was developed with o-phenylenediamine, and OD was read at 490 µm.

Antibody epitopic domains were determined using four monoclonal human TPO-specific autoantibodies (Fab SP1.4, WR1.7, TR1.8, and TR1.9) (4) to inhibit serum antibody binding. The subdomains recognized by these recombinant Fab, renamed A1, A2, B1, and B2, respectively (16), define the immunodominant region recognized by patients’ autoantibodies (reviewed in Ref.3). Previously, Fab inhibition was measured using binding of radiolabeled TPO by human autoantibodies (17) or murine antibodies (2). For the present study we used an ELISA approach, because antibodies in TPO-DNA-vaccinated mice interact poorly with radiolabeled TPO (1). Duplicate aliquots of mouse sera were preincubated with buffer, individual Fab, or the pool of four Fabs (each Fab at 4 x 10–8 M), prepared as previously described (17). After applying the serum-Fab mixtures to TPO-coated ELISA wells, serum antibody binding was detected with peroxidase-conjugated goat antimouse-IgG-Fc and o-phenylenediamine (as described above). Fab lack the CH2 domain of the Fc region and are not bound by antibodies to the Fc region. To ensure maximal inhibition by an excess concentration of Fab, preliminary studies were performed to determine the appropriate serum dilution required to give TPO antibody OD values of approximately 0.8 in the absence of TPO Fab.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Recognition of TPO and TPO peptides
We first studied the response of splenocytes from DNA-vaccinated BALB/c mice challenged in vitro with TPO protein for approximately 6 d. Splenocytes from TPO-DNA injected animals, but not control DNA-injected mice, produced IFN-{gamma} (but not IL-4) in response to TPO (Fig. 1Go). These findings demonstrate that memory T cells are present in mice 4 wk after the third vaccination with TPO-DNA. Incidentally, vaccination did not result in histological evidence of thyroiditis (1).



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  		FIG. 1. Splenocytes from TPO-DNA-vaccinated BALB/c mice respond to TPO protein. Responses were assessed in terms of IFN-{gamma} production measured by ELISA in supernatants from splenocytes cultured for 5–6 d. The mean IFN-{gamma} (+SEM) concentration in splenocytes from mice vaccinated with TPO-DNA (n = 8) or control-DNA (n = 5) is shown. *, IFN-{gamma} production significantly greater for splenocyte cultures with TPO than in medium only (by paired t test, P = 0.007).

 
Next, we used a panel of 55 overlapping 20-mer peptides encompassing the TPO ectodomain (Table 1Go) to determine the epitopes recognized by these memory T cells. As for the entire TPO antigen, peptide reactivity was assessed by IFN-{gamma} generation. Splenocytes from five control DNA-vaccinated mice were largely quiescent when challenged in vitro with the TPO peptide (Fig. 2Go). Splenocytes from the eight TPO-DNA-immunized mice, in contrast, were generally reactive to a wide range of peptides. Because so many peptides in the latter group generated a response, we focused on the most reactive peptides in an individual mouse (>1 SD above the mean of all values in that particular mouse; Fig. 2Go, dotted lines). On this basis, between five and 10 peptides induced major responses in different animals. These data were subsequently used to investigate a possible relationship between TPO antibody epitopic domains and T cell peptide recognition (see below).



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  		FIG. 2. TPO peptides recognized by TPO-DNA-vaccinated mice. Profiles of IFN-{gamma} production are shown for eight TPO-DNA-vaccinated mice and (in a single panel) for five control-DNA-vaccinated mice. For each profile, the first data point is the value for splenocytes cultured in medium without TPO peptide. The dotted lines represents the mean + SD IFN-{gamma} production in response to all 55 peptides in each of the eight TPO-DNA-vaccinated mice.

 
TPO antibody epitopic fingerprints and T cell epitopes
As mentioned above, antibodies can influence the peptides made available for presentation to T cells (reviewed in Ref.13). In this context we hypothesized that variability in splenocyte peptide recognition by different mice was related to TPO antibody epitopic domains. For this purpose we determined the epitopic patterns, or fingerprints, recognized by serum TPO antibodies in mice whose splenocyte responses to TPO peptides had been studied. Four individual human TPO monoclonal autoantibodies (in the form of recombinant Fab) define subdomains in the TPO-immunodominant region in human autoimmune thyroiditis (4). Mouse serum TPO antibodies were tested for binding to TPO-coated ELISA wells in the absence and presence of each Fab.

Based on the percent inhibition, we found four different antibody epitopic patterns or fingerprints (Fig. 3Go). In three mice (no. 1, 2, and 9), serum TPO antibody binding was strongly inhibited (~100%) by Fab A1 and to a lesser and variable extent by Fab A2, B1, and B2. For three other mice (no. 4, 8, and 7), serum TPO antibodies were strongly inhibited by each monoclonal Fab (A1, A2, B1, and B2), indicating comparable recognition of all four antibody epitopic subdomains. TPO binding by the serum from mouse 5 was inhibited by three of four Fab, but not by the B2 domain Fab. Finally, TPO binding by serum antibodies from mouse 6 was minimally inhibited by all four Fab, indicating poor recognition of the immunodominant region. Using a pool of the four human autoantibody Fab, TPO binding by sera from all mice except animal 6 was strongly inhibited (>75%). These data indicate that, in seven of eight mice, serum TPO antibodies are directed toward the human TPO autoantibody-immunodominant region.



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  		FIG. 3. TPO antibody epitopic fingerprints in TPO-DNA-vaccinated mice. Serum binding to intact TPO was measured by ELISA in the absence and presence of individual Fab (Fab A1, A2, B1, and B2) that define overlapping epitopes in the TPO-immunodominant region (inset). Data are presented for eight mice as the percent inhibition of TPO binding by Fab A1, Fab A2, Fab B1, or Fab B2 individually as well as for all four Fab as a pool. The dashed vertical lines are inserted to emphasize grouping of the mice according to their epitopic profiles.

 
Next, we examined the relationship between T cell epitopes and antibody epitopic fingerprints of individual mice (Fig. 4Go). The panel of TPO peptides is depicted in relation to the structural components of the TPO ectodomain: the myeloperoxidase (MPO)-like, complement control protein-like, and epidermal growth factor-like regions (18, 19, 20). Of the 55 TPO peptides studied, 35 were recognized by TPO-DNA-vaccinated mice; 31 were located in the large MPO-like domain. Some peptides were only recognized by one mouse, e.g. peptide 2 (mouse 6) and peptide 55 (mouse 4). However, seven of the 35 peptides induced responses by splenocytes from three or more mice: peptide 27 (five of eight mice), peptide 45 (four of eight mice), and peptides 5, 14, 20, 41, and 49 (three of eight mice). Quantitatively, considering the group of eight TPO-DNA-vaccinated mice as a whole, only peptides 27 and 45 induced significantly greater IFN-{gamma} production than medium alone (P = 0.011 and P = 0.027, by paired t tests).



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  		FIG. 4. Relationships between peptides recognized by splenocytes from TPO-DNA-vaccinated mice and serum TPO antibody epitopes. Of the 55 peptides, peptides 1–47 encompass the MPO-like portion of the TPO ectodomain, and peptides 48–55 cover the ectodomain region with homology to the complement control protein (CCP) and epidermal growth factor (EGF). The TPO peptides inducing a splenocyte response are listed for each of eight TPO-DNA-vaccinated mice (identified as M1–M9). Mice are categorized into four groups according to their TPO antibody epitopic patterns, or fingerprints, as shown by the bar graphs on the left. The most commonly recognized peptides (no. 27 and 45) are indicated by the dark gray area. Peptides recognized by at least three of the eight animals are shown by the light gray area.

 
No obvious associations between antibody epitopic fingerprints and T cell epitopes were apparent. We then analyzed whether the number of peptides to which splenocytes responded correlated with the antibody titer (ELISA OD value) or recognition of a particular TPO antibody epitopic subdomain (percent inhibition by the relevant subdomain Fab). There was no relationship between the number of TPO peptides recognized and the antibody titer (Fig. 5Go, lower right panel). In contrast, we did observe an inverse correlation between the number of peptides inducing splenocyte responses and recognition of the A2 subdomain (Fig. 5Go, upper left panel). No such correlation was seen for the other TPO antibody epitopic subdomains.



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  		FIG. 5. Relationships between the number of TPO synthetic peptides recognized by splenocytes from TPO-DNA-vaccinated mice and serum antibody epitopic subdomains within the TPO autoantibody IDR. The IDR, containing four subdomains (A1, A2, B1, and B2), was previously mapped on the surface of intact TPO using recombinant human autoantibody Fab (17 ). These Fab were used to compete for mouse serum binding to intact TPO. The percent inhibition by each of the four Fab is indicated on the abscissa (four left panels). Each point represents the serum from an individual mouse. Also shown (lower right panel) is the relationship between the number of T cell peptides recognized and mouse TPO antibody levels measured in serum (1:1000) by ELISA.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The present study indicates that BALB/c mice vaccinated with a plasmid containing the cDNA for human TPO develop memory T cells detected by in vitro challenge of splenocytes with TPO protein. We analyzed the TPO T cell epitopes recognized by these cells using a panel of 55 synthetic, overlapping, 20-mer peptides encompassing the 827-amino acid residue TPO ectodomain. In contrast to splenocytes from control DNA-immunized mice, cells from TPO-DNA-vaccinated mice showed a higher level of general reactivity to numerous, diverse peptides. Thus, 35 of the 55 peptides induced responses in splenocytes from at least one mouse. Individual TPO-DNA-immunized mice recognized five to 10 diverse peptides, although two peptides appeared to be more commonly recognized. Of interest, one of these two immunodominant peptides is also recognized by peripheral blood lymphocytes from patients with autoimmune thyroid disease (discussed below).

To obtain insight into the wide diversity of TPO T cell epitopes in these animals, we first considered a possible relationship between T cell epitopes and serum antibody epitopic domains. Naturally processed T cell epitopes are generated by degradation of intact proteins to form short linear peptides (8–20 amino acids in length) that traffic in major histocompatibility complex (MHC) molecules to the cell surface for presentation to T cells. Antibody complexed to antigen may modify the intracellular processing of the latter and can thereby influence the peptides available for presentation (reviewed in Ref.13). Indeed, antibodies that bind to different epitopic domains can enhance or suppress presentation of particular T cell peptides, as shown for antibodies to tetanus toxoid (21) or to glutamic acid decarboxylase 65 (22, 23).

Previously, we observed that antibodies in TPO-DNA-vaccinated mice interact with the same IDR on TPO recognized by human autoantibodies (1). However, no information was available on the epitopic subdomains within the IDR for these murine antibodies. Dissection of the antibody epitopic domains within the IDR was first necessary to detect any possible correlation with the diverse T cell epitopes present in the TPO-DNA-vaccinated animals. Technically, analysis of these antibody (or B cell) epitopic domains was not straightforward. Unlike T cell epitopes that can be analyzed with synthetic peptides, the epitopic domains recognized by antibodies (such as to TPO) are conformational and typically involve nonlinear/noncontiguous parts of the folded protein (reviewed in Ref.24). For this reason, as used previously for human autoantibodies (4, 17) and mouse antibodies (2), we determined epitopic fingerprints using monoclonal TPO autoantibodies (recombinant Fab) to compete for murine TPO antibody binding to intact TPO.

The epitopic fingerprints in all eight TPO-DNA-vaccinated mice covered the spectrum observed in patients with thyroid autoimmunity (16, 17, 25, 26, 27). Among individual TPO-DNA-vaccinated mice, we observed no clear qualitative association between T cell peptide recognition and a particular B cell epitopic fingerprint. However, we did observe a quantitative, inverse relationship between the number of peptides recognized and the proportion of polyclonal antibodies interacting with the TPO A2 subdomain. The mechanism underlying this association is unknown, but may relate to the influence of A2 subdomain antibodies in suppressing the presentation of a wider spectrum of T cell epitopes.

Despite the diversity of T cell epitopes in the TPO-DNA-vaccinated mice, two peptides stood out in inducing responses in at least half the mice, namely peptides 27 and 45 (Fig. 4Go and Table 2Go). Peptides corresponding to (or overlapping with) peptide 27 (LAAALKALNAHWSADAVYQE) are recognized by T cells from some patients with autoimmune thyroid disease (28, 29, 30). Peripheral or thyroid-derived lymphocytes and T cell clones from humans with thyroid autoimmunity also respond to five other epitopic regions on TPO (summarized in Table 2Go). However, none of these regions overlaps with the second most common peptide recognized by TPO-DNA-vaccinated BALB/c mice (no. 45; EKHSLSRVICDNTGLTRVPM).


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  		TABLE 2. Summary of TPO peptides recognized by T cells in immunized mice and in human disease

 
T cell epitopes have also been analyzed in mice immunized conventionally with TPO protein and adjuvant. A porcine TPO peptide corresponding to human TPO residues 776–790 stimulated lymphocytes in vitro, which, on adoptive transfer, induced thyroid lesions in C57BL/6 mice (31). Moreover, CBA/N mice immunized with human thyroglobulin recognized an epitope (32) corresponding to a hypothetical cross-reactive thyroglobulin peroxidase (TgPO) epitope (33). Neither of these two peptides induced splenocyte responses in TPO-DNA vaccinated BALB/c mice. These differences between mouse strains could relate to different murine MHC molecules (H2-d, H2-b, and H2-k in BALB/c, C57BL/6, and CBA/N mice, respectively). However, recognition of peptide 27 by both BALB/c mice and humans involves xenogeneic MHC molecules. Therefore, our data for TPO add to observations of shared T cell epitopic recognition between mice and humans as we reported for TSHR peptides (12) and as observed for the H+,K+-adenosine triphosphatase in autoimmune gastritis (34). These observations indicate promiscuous binding of the same peptides derived from autoantigens by human and murine MHC molecules.

Finally, our findings for TPO are important because of their striking contrast with observations in the same strain of mouse (BALB/c) vaccinated with TSHR-DNA. TPO-DNA vaccination readily induces TPO antibodies (~80% positivity) (1), whereas TSHR-DNA vaccination rarely induces TSHR antibodies (0–5%) in multiple studies (7, 8, 9, 10), with one exception (11). Despite this difference in antibody production, memory T cell responses to intact antigen challenge were similar for TPO-DNA and TSHR-DNA vaccinations. However, diverse T cell epitopes were recognized after TPO-DNA vaccination (34 of 55 peptides) compared with restricted recognition in TSHR-DNA-vaccinated mice (four of 29 peptides) (12). Moreover, only two of 55 TPO peptides were recognized by T cells from at least half of the TPO-DNA-vaccinated mice compared with two of 29 TSHR peptides recognized by almost all of the TSHR-DNA-vaccinated mice. Our study reveals another difference between TPO-DNA and TSHR-DNA vaccinations. Whereas we observed a relationship between TPO antibody subdomain recognition and T cell epitope number, antibodies do not appear to be related to TSHR T cell epitope recognition (12).

In conclusion, our present study highlights major differences for antibodies as well as for T cell epitopic recognition in TPO-DNA- vs. TSHR-DNA-immunized BALB/c mice. These findings, which suggest an antigen-specific role of B cells in T cell recognition of TPO, but not of the TSHR, shed light on the mechanisms involved in the spontaneous immune response to two major thyroid autoantigens in humans.


   Acknowledgments
 
We thank Dr. Boris Catz (Los Angeles, CA) for his generous support.


   Footnotes
 
This work was supported by National Institutes of Health Grants DK-36182 (to B.R.) and DK-54684 (to S.M.M.), and by a Winnick Family Clinical Research Scholar Award (to S.M.M.).

Abbreviations: IDR, Immunodominant region; IFN-{gamma}, interferon-{gamma}; MHC, major histocompatibility complex; MPO, myeloperoxidase; TPO, thyroid peroxidase; TSHR, TSH receptor.

Received March 9, 2004.

Accepted for publication April 20, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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