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The Journal of Immunology, 2008, 180, 7039 -7046
Copyright © 2008 by The American Association of Immunologists, Inc.
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H2E-Derived E{alpha}52-68 Peptide Presented by H2Ab Interferes with Clonal Deletion of Autoreactive T Cells in Autoimmune Thyroiditis1

Nicholas K. Brown*, Daniel J. McCormick{dagger}, Chella S. David{ddagger} and Yi-chi M. Kong2,*

* Department of Immunology and Microbiology, Wayne State University School of Medicine, Detroit, MI 48201; and {dagger} Department of Biochemistry and Molecular Biology and {ddagger} Department of Immunology, Mayo Clinic College of Medicine, Rochester, MN 55905


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Susceptibility and resistance to experimental autoimmune thyroiditis is encoded by MHC H2A genes. We reported that traditionally resistant B10 (H2b) mice permit thyroiditis induction with mouse thyroglobulin (mTg) after depleting regulatory T cells (Tregs), supporting Ab presentation to thyroiditogenic T cells. Yet, Eak transgenic mice, expressing Ab and normally absent Eb molecules (E+B10 mice), are susceptible to thyroiditis induction without Treg depletion. To explore the effect of Eb expression on mTg presentation by Ab, seven putative Ab-binding, 15–16-mer peptides were synthesized. Five were immunogenic for both B10 and E+B10 mice. The effect of Eb expression was tested by competition with an E{alpha}52-68 peptide, because E{alpha}52-68 occupies ~15% of Ab molecules in E+B10 mice, binding with high affinity. E{alpha}52-68 competitively reduced the proliferative response to mTg, mTg1677, and mTg2342 of lymph node cells primed to each Ag. Moreover, mTg1677 induced mild thyroiditis in Treg-depleted B10 mice, and in E+B10 mice without the need for Treg depletion. E{alpha}52-68 competition with mTg-derived peptides may impede clonal deletion of pathogenic, mTg-specific T cells in the thymus.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Experimental autoimmune thyroiditis (EAT)3 is a murine model for Hashimoto’s thyroiditis, an organ-specific autoimmune disease. Like Hashimoto’s thyroiditis, EAT is characterized by mononuclear cell infiltration of the thyroid, T cell proliferative response, and production of autoAb (1). EAT can be induced in susceptible strains by administration of mouse thyroglobulin (mTg), either in repeated doses (2) or with an adjuvant, such as CFA or LPS (3). Susceptibility or resistance to EAT of various H2 haplotypes is linked to the class II genes (4), and HLA association has been shown with HLA class II-transgenic mice (5). Whereas the H2A gene encodes susceptibility or resistance to EAT, the contribution of the other class II locus, H2E, is less clear. A functional H2E class II molecule is not expressed in every strain, yet it is found in both susceptible and resistant haplotypes. Moreover, there is ample evidence that surface expression of H2E molecules influences susceptibility or resistance to autoimmune diseases. Upon introduction of an Ea transgene resulting in H2E expression, the NOD mouse became resistant to autoimmune diabetes (6), and myasthenia gravis and systemic lupus erythematosus were similarly reduced in H2b mice (7, 8). In EAT, H2Es expression also leads to decreased thyroid infiltration in otherwise susceptible B10.S (A+E–) mice (9). We have further studied the role of H2E on EAT induction, using a transgenic mouse that expresses H2Eb in the absence of endogenous Ab molecules due to a mutation in the Ab gene (10, 11). This E+B10.Ab0 (A–E+) strain is highly susceptible to EAT induction with heterologous Tg, but is not susceptible to mTg-induced thyroiditis (10). The nonresponsiveness to mTg is profound, as not even a humoral response to mTg is detected in the E+B10.Ab0 mouse when immunized with mTg, unlike conventional, resistant B10 (A+E–) mice.

In contrast, the resistant B10 strain can be made permissive for EAT induction by depletion of regulatory T cells (Tregs) (12), demonstrating that the traditional role of H2Ab encoding for EAT resistance can be subverted, and thyroiditogenic T cells can be activated by H2Ab presentation of mTg epitopes. Furthermore, upon introduction of an Ea transgene, the new H2A+E+ strain became permissive for mTg-induced thyroiditis (12). The varied responses of these strains to EAT induction with mTg are summarized in Table I. Thus, instead of reducing susceptibility to autoimmune disease, H2E expression increased susceptibility. We therefore sought to elucidate the mechanism(s) by which H2Eb disrupts resistance to mTg-induced EAT normally encoded by H2Ab.


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  		Table I. Influence of MHC class II expression on response to EAT induction with mTg

 
Mice expressing both H2Eb and H2Ab have been studied by others. Of particular note is the finding that H2Ab class II molecules can bind a peptide derived from the conserved E{alpha}-chain (E{alpha}52-68) with high affinity (13). In transgenic or recombinant mice expressing both H2Ab and H2Eb class II molecules, 10–40% of the H2Ab molecules harbor E{alpha}52-68 in the peptide-binding cleft (E{alpha}52-68/Ab) (13, 14). Moreover, altering the composition of H2Ab-binding peptides by expression of E{alpha} has been shown to affect the presentation of immunogenic epitopes and thus reduce T cell activation (14, 15). One of us has demonstrated that a peptide derived from HLA-DR, the human H2E equivalent, and presented by HLA-DQ, the human H2A equivalent, lowered susceptibility to collagen-induced arthritis (CIA) (16).

Because we have observed the opposite effect that H2E expression increased susceptibility to mTg-induced EAT, we hypothesized that the Eb molecule may be acting via binding of E{alpha}52-68 to H2Ab, thereby altering mTg presentation by H2Ab. To test this hypothesis, we compared H2Ab-mediated EAT in the A+E– and A+E+ strains. The mTg amino acid sequence was scanned for putative Ab-binding peptides. Of five peptides verified to be presented by H2Ab, E{alpha}52-68 competed with two for presentation by H2Ab. This resembled the properties of intact mTg, with which E{alpha}52-68 also competed for presentation by H2Ab. Of the two mTg peptides with which E{alpha}52-68 competed, one, mTg1677, was tested for pathogenicity and found to be thyroiditogenic, demonstrating that E{alpha}52-68 competes with a pathogenic peptide for presentation by H2Ab.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Mice

C57BL/10 (B10, H2A+E–) and transgenic E+B10 (H2A+E+) mice were bred at the Immunogenetics Mouse Core Facility at the Mayo Clinic. The generation of transgenic H2A+E+ mice on B10 background was previously described (10). Briefly, B10 mice were mated with Eak transgenic mice (17) and repeatedly backcrossed to B10 mice. Mice of both sexes were kept on acidified, chlorinated water and used at 10–16 wk of age. All procedures were approved by the institutional animal care and use committee at Wayne State University.

Thyroglobulin and peptides

mTg was obtained from frozen thyroids fractionated on a Sephadex G-200 column as previously described (1). mTg was checked for the presence of LPS by the Limulus amebocyte assay (Associates of Cape Cod, Woods Hole, MA) (a 40-µg dose contained <1 ng of LPS) and diluted in nonpyrogenic saline.

Peptides were synthesized by the Peptide Synthesis Facility of Mayo Proteomics Center using orthogonal solid-phase methods on Rink resin (Novabiochem) and N{alpha}-9-fluorenyl-methoxycaronyl protected L-amino acids (18). Each peptide was then purified to >90% homogeneity by reverse-phase HPLC, and the molecular mass of each peptide was confirmed by electrospray-ionization mass spectroscopy (MSQ single quad instrument, ThermoFisher-Scientific). The lyophilized peptides were reconstituted in nonpyrogenic water.

Hybridomas and mAbs

CD25 mAb (PC61, rat IgG1{lambda}; American Type Culture Collection), used for depletion of Tregs in vivo, was produced and purified either by 2x ammonium sulfate precipitation of supernatants from FiberCell modules (Fiber Cell Systems) or used as ascites fluid from PC61-injected nude mice (Harlan Bioproducts for Science). CD25 mAb concentration was determined by anti-rat{lambda} (BD Pharmingen) ELISA. A different CD25 mAb, PE-7D4 (rat IgM; Southern Biotechnology Associates), and FITC-CD4 (GK1.5, rat IgG2b; eBioscience) were used to label peripheral blood leukocytes from PC61-injected mice to monitor depletion of CD4+CD25+ cells, as previously described (12). For in vitro class II blocking, culture supernatants of H2Ab mAb (Y-3P (19), mouse IgG2a; ATCC), and protein G-purified culture supernatants of H2Eb mAb (Y-17 (20), mouse IgG2b), were used. Hybridoma Y-Ae, kindly provided by the laboratory of Charles Janeway, Jr. (Yale University, New Haven, CT) produces a mouse IgG2b mAb recognizing E{alpha}52-68/Ab (21). For FACS analysis, protein A-purified and biotinylated Y-Ae and Y-3P were used with PE-streptavidin (BD Pharmingen) for detection. B220-FITC (clone RA3–6B2; BD Pharmingen) was used to colabel class II+ APC.

Immunizations

Immunization with mTg was performed by s.c. injection of mTg emulsified in CFA at four sites (hind footpads and inner thighs). Each site received 50 µl of emulsion containing 40 µg mTg and 150 µg Mycobacterium tuberculosis (strain H37Ra; Difco Laboratories). For in vitro proliferative responses to peptides, mice were immunized with peptide-CFA as described above, with 50 µg peptide/each of four sites. To assess Ab titers and thyroid pathology, the four doses of peptide-CFA were divided into two time points, days 0 and 7, and injected into alternate thighs and hind footpads. To augment the immune response in this EAT-resistant haplotype, mice were pretreated with an i.v. injection of 1 mg CD25 mAb on days –14 and –10 (12). CD4+CD25+ T cell depletion was verified on day –4 by FACS analysis of peripheral blood leukocytes as described above.

Adoptive transfer of cells activated with and proliferative response to Tg or peptides

For adoptive transfer, spleen cells (SC), obtained on day 35 after immunization with mTg1677-CFA, were cultured for 3 days with 10 µg/ml mTg1677 in RPMI 1640 (supplemented with 25 mM HEPES buffer, 100 U/ml penicillin, 100 µg/ml streptomycin, 2 mM L-glutamine, and 50 µM 2-ME) and 1% normal mouse serum (NMS). The activated cells (6–7 x 107/recipient) were then transferred by i.v. injection into 137Cs-irradiated (500 rads) recipients.

For in vitro proliferation, SC (6 x 105 cells/well) or lymph node cells (LNC) [6 x 105 cells plus 4 x 105 irradiated (2000 rads) SC/well] were cultured for 5 days in RPMI 1640/1% NMS in flat-bottom 96-well plates in quadruplicate, either with or without 40 µg/ml Tg or 10 µg/ml peptide (1). To block class II presentation, some cultures contained anti-H2Ab mAb or anti-H2Eb mAb. To assess whether E{alpha}52-68 can block presentation of mTg peptides by Ab, irradiated SC were incubated for 1 h at 37°C with 10 µg/ml E{alpha}52-68, washed, re-incubated for 1 h at 37°C with an mTg peptide (10 µg/ml), washed again, and added to cultures (4 x 105/well) of LNC for 5 days. Cells were pulsed with 1 µCi/well [3H]thymidine 18–20 h before harvest onto glass fiber filter paper (Tomtech Mach3Man Cell Harvester; LKB Wallac) and incorporation assessed by a Microbeta Plus 1450 liquid scintillation counter (LKB Wallac). Stimulation index (SI) was calculated as mean cpm ± SE of cells with Ag divided by mean cpm of cells without Ag.

Assessment of E{alpha}52-68/H2Abcomplex

A+E+ SC were labeled with mAb Y-Ae (anti-E{alpha}52-68/Ab) and colabeled with B220 mAb as described above. Gated B220+ cells were FACS analyzed for E{alpha}52-68/Ab expression. To detect E{alpha}52-68 binding in A+E– mice, SC were incubated with 10 µg/ml E{alpha}52-68 for 1 h at 37°C, washed, and labeled with Y-Ae for FACS analysis. To determine whether mTg peptides block binding of E{alpha}52-68 to Ab molecules, A+E– SC were first incubated for 1 h with 10 µg/ml mTg peptide, washed, and subsequently incubated with 10 µg/ml E{alpha}52-68 for 1 h. Cells were washed again and labeled for FACS analysis.

Determination of Abs to Tg and peptides

Mice were bled from the tail artery on the day of sacrifice, and sera were stored at –20°C. Tg or peptide Abs were measured by ELISA, using plate-bound Tg or peptide (1 µg/well in Immulon II microtiter plates) and alkaline phosphatase-labeled goat anti-mouse IgG (Sigma-Aldrich), as described previously (22). The OD405 values were corrected for nonspecific binding by subtracting the OD of NMS.

Evaluation of thyroiditis

Thyroid pathology was assessed by histologic examination of H&E-stained thyroids, sectioned vertically through both thyroid lobes (50–60 sections from 10 to 15 step levels). Mononuclear cell infiltration was scored on an index of 0–4.0: 0, normal thyroid; 0.5 small interstitial foci of infiltration involving >0–10% of the thyroid; 1.0, follicular destruction with >10–20% involvement; 2.0, >20–40% involvement; 3.0, >40–80% involvement; and 4.0, >80% involvement (1).

Statistical analysis

Differences between groups of in vitro proliferation assays were analyzed using the unpaired Student’s t test.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Identification of Ab-restricted peptides derived from mTg

To delineate mTg epitopes pathogenic for the A+E– (B10) and A+E+ (E+B10) strains, which may require prior Treg depletion as an adjunct (see Table I), we synthesized putative H2Ab-presented peptides derived from the amino acid sequence of mTg (23). We used the Ab anchor residues published by Liu et al. (24) to search in silico for sequences that contained the preferred anchor residues at positions 1, 4, 6, and 9. Sequences containing preferred anchor residues at all four positions were lengthened from the N and C termini to 15 or 16 amino acids, based on predicted solubility, and synthesized. The six peptides (mTg691 – mTg2086, designated by the position of the first amino acid in the mTg sequence) are listed in Table II. One other mTg-derived peptide, mTg2342, synthesized as a corresponding peptide of an Eb-restricted human (h)Tg epitope, was found to be Ab-restricted, and was also tested here.


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  		Table II. Location and amino acid sequences of Ab-binding synthetic peptides

 
To first determine the in vitro proliferative responses to mTg in A+E– and A+E+ strains, mice were depleted of Tregs and immunized with mTg-CFA. Depletion of Tregs before immunization was used to generate as strong an mTg response as possible in these EAT-resistant strains. As seen in Fig. 1A, mTg-primed cells from Treg-depleted A+E– mice proliferated to intact mTg, but cells from similarly treated A+E+ mice did not. The lack of a detectable in vitro proliferative response to mTg in the permissive A+E+ strain is surprising because mTg is more pathogenic in A+E+ mice than in A+E– mice (12). When these mTg-primed cells from either strain were incubated with the mTg peptides, no response was seen, even in A+E– mice that responded to intact mTg (Fig. 1A). To determine whether any of the peptides was presented by Ab, mice were immunized with individual mTg peptides. Five of the seven peptides (mTg1677 to mTg2343) stimulated (SI > 3) cells primed with the same peptide in both strains of mice. (Fig. 1B). These results demonstrate that a subset of the synthetic mTg peptides is immunogenic in Ab-expressing mice. Further analysis of these five immunogenic peptides in A+E+ mice showed that all are Ab-restricted; anti-Ab, but not anti-Eb, significantly reduced the peptide-specific proliferative responses (Fig. 2).


Figure 1
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  		FIGURE 1. Five mTg-derived peptides are immunogenic in A+E– and A+E+ mice. A, A+E– or A+E+ mice were depleted of Tregs by injection of 1 mg CD25 mAb 14 and 10 days before immunization with mTg-CFA. LNC (6 x 105 cells plus 4 x 105 irradiated SC/well) were harvested at day 10 and cultured with indicated Ags for 5 days before proliferation was assessed by [3H]thymidine incorporation (background cpm 590-1400 ± 150–850). B, A+E– and A+E+ mice were immunized with the indicated peptide in CFA and lymph nodes were removed on day 10. LNC were cultured with mTg-derived peptides (10 µg/ml) for 5 days and proliferation was determined by [3H]thymidine incorporation (background cpm 1100–3300 ± 30–340).

 

Figure 2
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  		FIGURE 2. The five immunogenic mTg-derived peptides are H2Ab-resticted. A+E+ mice were immunized with the indicated peptide in CFA and lymph nodes were removed on day 10. LNC (6 x 105 cells plus 4 x 105 irradiated SC/well) were cultured with the immunizing peptide (10 µg/ml) in the presence of anti-Ab (Y-3P) or anti-Eb (Y-17) mAb for 5 days before proliferation was assessed (background cpm 1500–6300 ± 250–950). *, p < 0.05; **, p < 0.001.

 
An endogenously presented peptide derived from the E{alpha}-chain competes with mTg and two mTg-derived peptides

E{alpha}52-68, a peptide derived from the Ea gene product (not normally expressed in H2b strains), is known to be presented by Ab and to bind with high affinity (13). Moreover, when the Ea gene is expressed in an H2b strain, anywhere from 10 to 40% of all Ab molecules on APC are occupied by this self peptide (13, 14). We used the mAb Y-Ae, which specifically labels the E{alpha}52-68/Ab complex, to verify that APC from our A+E+ strain, but not from the A+E– strain, contains E{alpha}52-68 (Fig. 3A). We also determined that in our A+E+ strain, ~10–15% of Ab molecules on splenic APC express E{alpha}52-68/Ab (data not shown), similar to other reports (13). Moreover, synthetic E{alpha}52-68 peptide (Table II) bound to Ab on A+E– APC when provided exogenously (Fig. 3B).


Figure 3
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  		FIGURE 3. Detection of the E{alpha}52-68/Ab complex in H2E-expressing A+E+ mice. A, B220-gated APC from A+E– (left) or A+E+ (right) SC were FACS analyzed for expression of E{alpha}52-68/Ab (mAb Y-Ae, thick line) or total Ab (mAb Y-3P, dotted line) as a control. The dashed line shows background fluorescence. B, SC from A+E– mice were incubated with E{alpha}52-68 (thick line) or medium (dotted line) for 1 h and then labeled with mAb Y-Ae. The cells were FACS analyzed as in A.

 
Because we hypothesized that E{alpha}52-68 may be involved in the increased susceptibility of A+E+ mice to mTg-induced EAT, we tested whether E{alpha}52-68 competed with mTg for presentation by Ab. The proliferation of mTg-primed A+E– cells was significantly reduced when E{alpha}52-68 was added with mTg to the culture (Fig. 4A), demonstrating that E{alpha}52-68 competed with naturally processed epitopes on mTg for presentation by Ab. When the five immunogenic mTg peptides were individually tested for competition by E{alpha}52-68 with peptide-primed cells, only the responses to mTg1677 and mTg2342 were decreased in the presence of E{alpha}52-68 (Figs. 4, B and D); no reduction was observed for mTg1744 (Fig. 4C) or for mTg2051 or mTg2086 (data not shown). mTg1677- and mTg2342-specific proliferation was similarly reduced in A+E+ mice upon coincubation with E{alpha}52-68 (data not shown), although E{alpha}52-68 is endogenously present in this strain.


Figure 4
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  		FIGURE 4. E{alpha}52-68 competes with native mTg as well as mTg1677 and mTg2342, but not mTg1744, for presentation by H2Ab. A, A+E– mice were depleted of Tregs by injection of 1 mg CD25 mAb 14 and 10 days before immunization with mTg-CFA. LNC (6 x 105 cells plus 4 x 105 irradiated SC/well) were harvested at day 10 and cultured with mTg (40 µg/ml). E{alpha}52-68 (10 µg/ml) was added to some wells to compete for presentation by Ab, and anti-Ab mAb (Y-3P) was added as a control to block presentation. The cells were cultured for 5 days before proliferation was assessed by [3H]thymidine incorporation (background cpm 1400 ± 850). B–D, A+E– mice were immunized with the indicated peptide in CFA and lymph nodes were removed between days 10 and 12. LNC were cultured with the immunizing peptide (10 µg/ml), and E{alpha}52-68 (10 µg/ml) and Y-3P were added as in A. Proliferation was determined by [3H]thymidine incorporation (background cpm 1400–3100 ± 850-1000). *, p < 0.05; **, p < 0.01.

 
To further address whether E{alpha}52-68 inhibited the proliferation of peptide-primed cells by competitive occupancy of the Ab peptide-binding cleft, mTg1677 was chosen for further study, as it was more immunogenic than mTg2342 (Figs. 1B and 2). The mTg1677-specific proliferative response in A+E– mice was significantly reduced by preincubation of APC with E{alpha}52-68 (Fig. 5A), demonstrating that E{alpha}52-68 occupancy blocked presentation of mTg1677. mTg1744 was used as a control, and preincubation with E{alpha}52-68 did not reduce the response to mTg1744 (Fig. 5B), just as it did not compete with mTg1744 when added to cultures simultaneously.


Figure 5
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  		FIGURE 5. E{alpha}52-68 blocks presentation of mTg1677, but not mTg1744; neither can interfere with binding of E{alpha}52-68 to Ab molecules. A and B, A+E– mice were immunized with the indicated peptide in CFA and lymph nodes were removed on day 10. On day of culture, irradiated SC were incubated for 1 h with or without E{alpha}52-68 (1° incubation, 10 µg/ml) and washed. The SC were subsequently incubated for another hour with the immunizing peptide (2° incubation, 10 µg/ml) and washed. These pulsed APC were then added to cultures (4 x 105/well) along with peptide-primed LNC from immunized mice (6 x 105/well) for 5 days. Proliferation was assessed by [3H]thymidine incorporation (background cpm: A, 1200 ± 160; B, 6800 ± 820). *, p < 0.01. C, A+E– SC were incubated for 1 h with either mTg1677 or mTg1744 (10 µg/ml), or control medium, and washed. The SC were then incubated for 1 h with E{alpha}52-68 (10 µg/ml), washed, and labeled with anti-B220 and anti-E{alpha}52-68/Ab (mAb Y-Ae). B220-gated APC were analyzed by FACS for E{alpha}52-68/Ab expression. The dotted line represents unlabeled APC, the filled histogram represents cells incubated with E{alpha}52-68 only, the solid line represents cells incubated first with mTg1677 and then E{alpha}52-68, and the dashed line represents cells incubated first with mTg1744 and then E{alpha}52-68.

 
Because E{alpha}52-68 preincubation was capable of blocking mTg1677 presentation, a reciprocal experiment was performed to address whether any of the immunogenic mTg peptides was capable of blocking E{alpha}52-68 presentation by Ab. A+E– APC were incubated with mTg1677, mTg1744, mTg2051, mTg2086, or mTg2342 before incubation with E{alpha}52-68. Preincubation with any of these peptides failed to reduce the fluorescence intensity of anti-E{alpha}52-68/Ab, as compared with APC incubated with E{alpha}52-68 alone (Fig. 5C). It appears that none of the immunogenic mTg peptides can block the presentation of the high affinity E{alpha}52-68 peptide; in contrast, E{alpha}52-68 can block the response to mTg1677.

mTg1677 pathogenicity is subject to Treg influence in A+E– and A+E+ mice

As the Ab gene traditionally codes for EAT resistance, it was surprising that mTg1677 was immunogenic in A+E– and A+E+ mice and was one of only two mTg peptides competed by E{alpha}52-68 for Ab binding. Because we have reported that mTg-induced EAT in A+E– mice is controlled by Tregs, requiring prior Treg depletion, and the more permissive A+E+ strain becomes more susceptible to mTg-induced EAT after Treg depletion (12), we tested the role of Tregs in mTg1677 pathogenicity. As shown in Fig. 6A, A+E– and A+E+ mice, depleted of CD25+ Tregs and subsequently immunized with mTg1677-CFA, had significantly higher ex vivo proliferative responses to mTg1677 than mice treated with control rat IgG. Similarly, the ex vivo proliferative responses to all of the other immunogenic peptides (mTg1744-mTg2342) were increased after Treg depletion in both strains (data not shown).


Figure 6
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  		FIGURE 6. The immune response to mTg1677 is influenced by Tregs. A, A+E– or A+E+ mice were depleted of Tregs by injection of 1 mg CD25 mAb 14 and 10 days before immunization; control mice were injected with 1 mg rat IgG. On day 0, mice were immunized with mTg1677-CFA, and on day 10 mice were killed. LNC (6 x 105 cells plus 4 x 105 irradiated SC/well) were cultured with mTg1677 for 5 days before proliferation was assessed by [3H]thymidine incorporation (background cpm 410-1600 ± 70–110). *, p < 0.05; **, p < 0.0001. B–D, Representative sections of thyroids from Treg-depleted, mTg1677-immunized mice (original magnification x200 and x400 for boxed inlays connected to areas by arrows). A+E– or A+E+ mice were depleted of Tregs as described above and immunized with mTg1677-CFA on days 0 and 7. Thyroids were removed on day 35. B, Section from an A+E– mouse without mononuclear cell infiltration. C, Section from an A+E– mouse with destruction involving 15% of the thyroid. D, Section from an A+E+ mouse with destruction involving 30% of the thyroid.

 
To determine whether mTg1677 was thyroiditogenic for mice expressing Ab, A+E– mice were immunized with mTg1677-CFA. As can be seen in Table III, normal Ab mice immunized with mTg1677 did not develop thyroiditis, as expected. After Treg depletion, mild thyroiditis was inducible in 3/19 mice (16%). Thyroiditogenicity was further amplified by in vitro expansion of SC from Treg-depleted, mTg1677-immunized mice and subsequent transfer into irradiated recipients, resulting in thyroiditis development in 4/10 mice (40%) with up to 20–40% thyroid involvement. In contrast, mTg1677 immunization resulted in detectable thyroid infiltration in the permissive A+E+ strain either with or without prior Treg depletion, with respectively 4/10 (40%) and 3/6 (50%) developing thyroiditis (Table III). Fig. 6, B and C, presents representative thyroid sections, depicting zero mononuclear cell infiltration to follicular destruction, from Treg-depleted A+E– and A+E+ mice 35 days after immunization.


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  		Table III. mTg1677 is pathogenic in A+E– and A+E+ mice

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
The resistance to EAT normally encoded by H2Ab can be subverted either by peripheral depletion of Tregs or expression of the normally nonfunctional Ea gene (Table I) (12). Because we had previously shown that the Eb molecule is incapable of supporting mTg-induced thyroiditis (10), we explored the effects of Eb expression on Ab-mediated thyroiditis. We began by defining epitopes of mTg involved in Ab-mediated EAT. After searching the mTg sequence for epitopes predicted to bind to Ab, we synthesized six peptides (Table II). Of the six peptides, four (mTg1677, mTg1744, mTg2051, and mTg2086) were immunogenic and Ab-restricted (Fig. 2). mTg1677 was further shown to be pathogenic. We also tested a peptide, mTg2342, synthesized as a corresponding epitope of the Eb-restricted hTg peptide hTg2344 (25). We found it to be immunogenic in A+E– and A+E+ mice, and Ab-restricted. Interestingly, an hTg-homologue of this peptide, p2340, was recently shown to be immunogenic and mildly pathogenic in H2b mice (26). Because mTg2342 is presented in Ab-expressing mice, it is likely that those p2340 (hTg peptide)-primed T cells responded to endogenously presented mTg2342 to induce thyroid destruction.

In this study, we used Ab anchor residues previously published (24) to identify sequences that contained a preferred residue at each of the MHC binding sites. After our search for Ab-binding peptides was complete, an online MHC epitope search engine, RANKPEP, became available (27). Using RANKPEP to search for Ab-binding epitopes on mTg, we found that 4/5 peptides we identified as immunogenic (mTg1744, mTg2051, mTg2086, and mTg2342) were scored by RANKPEP as Ab-binders, and both of the peptides we determined to be nonimmunogenic (mTg691 and mTg1646) were scored as nonbinders. However, surprisingly, the only discrepancy between our empirical data and the in silico scoring by RANKPEP was for mTg1677. Although RANKPEP scored mTg1677 as a nonbinder for Ab, we found mTg1677 to be immunogenic as well as pathogenic, and to be one of only two peptides (along with mTg2342) with which E{alpha}52-68 competed for presentation by Ab, mirroring native mTg (Fig. 4). Epitope search algorithms such as RANKPEP do not, to our knowledge, include the ability to search for epitopes that would compete with self MHC peptides, an important consideration that would need to be rectified.

Competition with E{alpha}52-68 may provide an explanation for the increased susceptibility to mTg in the presence of the Ea gene. E{alpha}52-68, derived from the conserved {alpha}-chain of H2E, is not normally expressed in H2b mice. But, upon expression of Ea gene, E{alpha}52-68 binds with high affinity to Ab (13) and occupies the peptide-binding cleft of up to 40% of Ab molecules (14). Reports of MHC-derived peptide presentation are not uncommon. Benichou et al. (28) demonstrated in 1990 that both class I and class II MHC molecules are capable of presenting peptides derived from self MHC. Studies with HLA-DQ8-transgenic mice later showed that peptides derived from HLA-DR can be presented by DQ8 class II molecules (29). The E{alpha}52-68 peptide stands out, however, not only because it binds with high affinity to Ab and occupies a significant proportion of these molecules, but also because it binds to class II molecules of different haplotypes, including Ag7 (30) and Ad (31); an example is BALB/c mice which naturally express the Ea gene. Thus, E{alpha}52-68 was a good candidate for the study of the immunomodulatory effects of the Ea transgene.

We showed here that E{alpha}52-68 is indeed presented by Ab in our transgenic A+E+ mouse and, similar to other reports (13), occupies ~10–15% of Ab molecules on APC. Moreover, E{alpha}52-68 competed with naturally processed epitopes of mTg for presentation by Ab (Fig. 4A). Of the immunogenic mTg epitopes synthesized, E{alpha}52-68 was found to compete only with two, mTg1677 and mTg2342, reducing the peptide-specific proliferation of primed cells when added to cultures (Fig. 4). This rules out an MHC-independent mode of suppression for E{alpha}52-68, unlike a report of an MHC-derived peptide that antagonized an intracellular kinase involved in IL-2 signaling (32). Rather, E{alpha}52-68 appears to specifically compete with certain Ab-bound mTg epitopes, such as mTg1677 and mTg2342. In addition, we showed that exogenously supplied E{alpha}52-68 reduced mTg1677- and mTg2342-specific responses in A+E+ mice, which harbor endogenously derived E{alpha}52-68. Although our A+E+ mice already express E{alpha}52-68/Ab, it only constitutes ~10–15% of all of the Ab molecules, the lower limit of E{alpha}52-68 occupancy. Mice expressing high levels of the Ea transgene may have up to 40% E{alpha}52-68/Ab expression (14). Presumably, the remaining ~85% of Ab molecules are capable of presenting either E{alpha}52-68 or an mTg peptide, and similar competition can take place as in A+E– mice in the presence of E{alpha}52-68. When we analyzed the competitive effect of E{alpha}52-68, we found that preincubation with E{alpha}52-68 blocked the binding of mTg1677, but not control mTg1744 (Fig. 5). These results suggest that the binding characteristics of mTg1677 and E{alpha}52-68 to Ab are similar, but the conformation taken by Ab when bound to mTg1744, mTg2051, or mTg2086 (Fig. 4) does not allow for replacement by E{alpha}52-68. This phenomenon of a high-affinity peptide replacing prebound peptide has been well-studied (33, 34, 35).

As one of two studied peptides competed by E{alpha}52-68, mTg1677 was found to be pathogenic in A+E– mice only after Treg depletion. However, mTg1677 was pathogenic in either normal or Treg-depleted A+E+ mice, mirroring the results observed for intact mTg (12), and underscoring the increased permissiveness of A+E+ mice for EAT. It should be noted, however, that we could not test whether mTg1677 was capable of expanding mTg-primed cells to transfer thyroiditis to naive mice, as mTg-primed cells did not respond to mTg1677 (Fig. 1A). Thus, mTg1677 should be classified as a nonimmunodominant epitope of mTg. The lack of response of mTg-primed cells to a pathogenic epitope is not surprising, as, to date, an immunodominant peptide has not been uncovered for EAT even in susceptible strains (36). The Tg molecule, a homodimer with ~2700 aa, is the largest autoantigen known, and mTg1677 is most likely only one of the epitopes on mTg pathogenic in the A+E– and A+E+ strains. This may also explain why mTg1677 was not capable of inducing a substantially higher incidence of thyroiditis in Treg-depleted A+E+ mice, compared with normal A+E+ mice (40–50%, Table III); much greater thyroiditis incidences approaching 100% likely would require immunization with several pathogenic peptides. Nevertheless, these results demonstrate that E{alpha}52-68 competes with a pathogenic epitope for presentation by Ab. The ability of MHC-derived peptides to alter autoimmune disease induction has been studied in several models. Using HLA-transgenic mice, Das et al. (16) showed that a peptide derived from HLA-DRB1*0402, a human H2E homologue, is presented by HLA-DQ8, a human H2A homologue, in DQ8-transgenic mice, and that this presentation prevented CIA. In EAT, the presence of HLA-DQ8 also reduces severity in susceptible DR3-transgenic mice (37). Similarly, in NOD mice, other class II-derived peptides that bind to Ag7 can prevent autoimmune diabetes (30, 38). Interestingly, although E{alpha}52-68 also binds to Ag7, a different peptide, E{alpha}65-77, was found associated with the suppressive effects in Ea-transgenic NOD mice (30).

Our study differs from these reports, however, in that E{alpha}52-68/Ab expression alters the resistance phenotype of the H2b haplotype. The mechanism of raised susceptibility induced by E{alpha}52-68 presentation cannot then be due to the generation of Tregs specific for the MHC-derived peptides, as described in the CIA (16) and NOD (38) reports. Rather, the model of peptide-specific Ag competition by E{alpha}52-68, and subsequent reduced T cell activation of the Ag-specific T cells, proposed by Martinez-Soría et al. (14) for murine lupus, better describes our data. Applying this model to our EAT system, we would expect to see reduced activation of mTg-specific T cells in the periphery of our A+E+ mice, which express E{alpha}52-68/Ab. Indeed, mTg immunization of A+E– mice leads to a measurable ex vivo proliferation to mTg, whereas the same immunization of A+E+ mice yields no mTg-specific proliferation (Fig. 1A). Because mTg-specific T cells exist in both strains, as mTg is capable of inducing thyroiditis in both A+E– and A+E+ mice, especially after Treg depletion of A+E– mice (12), we hypothesize that the competitive inhibition of mTg peptide presentation by E{alpha}-derived peptides (Fig. 4) sufficiently reduces mTg-specific T cell proliferation to very low levels (SI < 3). It is likely that this effect is mediated by presentation of E{alpha}52-68, or other E{alpha}-derived peptides, acting on mTg1677, mTg2342, or other unidentified mTg peptides. Had Ab been a susceptible haplotype with stronger pathogenic epitopes for mTg-induced EAT, the blocking effect of E{alpha}52-68, or other E{alpha}-derived peptides, on mTg1677 might not have been manifested.

Because it has been shown that both Tg (39, 40) and E{alpha}52-68 (41) are presented by thymic cells, it follows that E{alpha}52-68 could also block the binding of mTg peptides, such as mTg1677 and mTg2342, in the thymus of A+E+ mice. This blockage could interfere with clonal deletion of mTg-specific T cells, allowing more autoreactive T cells to reach the periphery of A+E+ mice, compared with A+E– mice. Indeed, subtle changes in thymic expression of Tg have been shown to result in increased numbers of autoreactive T cells in the periphery (42). The increased frequency of autoreactive mTg-specific T cells in A+E+ mice could account for the permissiveness for EAT induction of this Ea-transgenic strain, wherein thyroiditis is mediated by traditionally resistant Ab genes. As the E{alpha}52-68 sequence is conserved in humans, as well as in other mouse strains, this peptide (or other similar conserved peptides on self MHC), could potentially participate in editing the TCR repertoire, thereby affecting susceptibility to autoimmune diseases.


   Acknowledgments
 
We thank Julie Hanson and staff for the breeding and care of mice and Renee Wilder for histologic preparation of thyroids. We also thank Dr. C. Jeffries for S. enteritidis LPS and the laboratory of Dr. Charles Janeway, Jr. for the Y-Ae hybridoma.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by National Institutes of Health, National Institute of Diabetes and Digestive and Kidney Diseases Grant 45960 (to Y.M.K.). Back

2 Address correspondence and reprint requests to Dr. Yi-chi M. Kong, Department of Immunology and Microbiology, Wayne State University School of Medicine, 540 East Canfield Avenue, Detroit, MI 48201. E-mail address: ykong{at}med.wayne.edu Back

3 Abbreviations used in this paper: EAT, experimental autoimmune thyroiditis; mTg, mouse thyroglobulin; h, human; Treg, regulatory T cell; SC, spleen cell; LNC, lymph node cell; SI, stimulation index; CIA, collagen-induced arthritis; NMS, normal mouse serum. Back

Received for publication December 12, 2007. Accepted for publication March 13, 2008.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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