SEARCH

SEARCH BY CITATION

Keywords:

  • experimental autoimmune thyroiditis;
  • high/low responders;
  • pathogenic thyroglobulin peptides;
  • resistant/susceptible strains;
  • T-cell epitopes

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosures
  9. References

Experimental autoimmune thyroiditis (EAT) is commonly induced by thyroglobulin (Tg) or Tg peptides in mice genetically susceptible to thyroiditis. In the present study, we investigated the immunogenic and pathogenic potential of a novel 20mer human Tg peptide, p2208 (amino acids 2208–2227), in mouse strains classified as low (LR) or high (HR) responders in EAT. The peptide was selected for its content in overlapping binding motifs for MHC class II products, associated with either resistance (Ab), or susceptibility (As, Ek) to EAT. We therefore immunized LR BALB/c (H-2d) and C57BL/6 (H-2b) strains, as well as HR CBA/J (H-2k) and SJL/J (H-2s) mice with 100 nmol of p2208 in adjuvant and collected their sera, lymph nodes and thyroid glands for further analysis. The p2208 peptide was found to contain B-cell and cryptic T-cell epitope(s) in two of the four strains examined, one LR and one HR. Specifically, it elicited direct EAT in C57BL/6 mice (two of seven mice, infiltration index 1–3), as well as in SJL/J mice (two of six mice, infiltration index 1–2). Such an EAT model could provide insights into the immunoregulatory cascades taking place in resistant hosts.


Abbreviations
aa

amino acid

DMEM

Dulbecco's modified Eagles’ medium

EAT

experimental autoimmune thyroiditis

HR

high responder

hTg

human Tg

IFN-γ

interferon-γ

I.I.

infiltration index

IL

interleukin

LNC

lymph node cell

LR

low responder

mTg

mouse Tg

S.I.

stimulation index

Tg

thyroglobulin

Th

T helper

TNF-α

tumour necrosis factor-α

Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosures
  9. References

Εxperimental autoimmune thyroiditis (EAT) is a T-cell-mediated disease animal model that resembles Hashimoto's thyroiditis in humans. EAT can be induced in mice upon immunization with intact thyroglobulin (Tg) [1] or with Tg peptides in the presence of adjuvant.[2, 3] EAT can also be elicited by adoptive transfer of Tg-primed lymph node cells (LNCs) into naive hosts.[4] Traditionally, the relative intensity of the salient EAT features, i.e. mononuclear cell infiltration of the thyroid gland and anti-Tg antibody titres, have classified mouse strains as high responders (HR) (susceptible) or low responders (LR) (resistant). Various H-2 haplotypes were shown in early studies to associate with the response status, e.g. H-2k, H-2s were classified as HR haplotypes and H-2b, H-2d or H-2v as LR.[5]

Over the last 20 years, studies on the pathogenic mechanisms of EAT have been carried out almost exclusively in HR mouse strains. Out of the 34 Tg peptides tested for pathogenic potential in HR strains, 17 were found to be thyroiditogenic. In contrast, only one of ten tested Tg peptides, the 20mer p2340 [amino acids (aa) 2340–2359)], was previously identified in our laboratory to induce EAT in LR strains.[6-18] This peptide was pathogenic in LR strains C57BL/6 (H-2b) and BALB/c (H-2d) only via the adoptive transfer of syngeneic peptide-primed LNCs and in HR strains SJL/J (H-2s) and CBA/J (H-2k) by direct challenge.[18] Identification of defined Tg peptides with pathogenic potential in LR mouse strains could facilitate studies of immunoregulation of EAT through the use of an expanded list of available hosts, such as recombinant inbred strains and knock-in or knock-out mice, currently available only in LR strain backgrounds. In addition, Tg peptides with established pathogenic activity in both HR and LR strains might serve as model antigens in studies of disease-promoting factors beyond the level of determinant selection afforded by H-2 loci.

In this study, we continued the search to identify pathogenic Tg peptides in LR hosts by assessing the presence of LR and HR MHC class II-binding motifs in 48 20mer peptides spanning the C-terminal end of Tg (aa 2172–2749), which had attracted our interest in our previous studies.[18-23] The peptide p2208 (aa 2208–2227) met our criteria and was subsequently analysed in terms of its T-cell and B-cell immunogenicity, as well as the ability to induce EAT in LR and HR mouse strains.

Materials and methods

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosures
  9. References

Animals and antigens

BALB/c (H-2d), C57BL/6 (H-2b), CBA/J (H-2k) and SJL/J (H-2s) mice were kept under specific pathogen-free conditions in the Experimental Animal Unit of the Hellenic Pasteur Institute. All animal procedures were approved by National Authorities and conformed to European Community guidelines. The human thyroglobulin (hTg) peptide QFLGVPYAAPPLAERRFQAP (p2208) (aa 2208–2227) was synthesized by JPT Peptide Technologies GmbH (Berlin, Germany) and the control hTg peptide PYEFSRKVPTFATPWPDFVP (p2652) (aa 2652–2671) was synthesized by Genosys Biotechnologies (Cambridge, UK). Both peptides were synthesized in >80% purity, as verified by HPLC, with their N- and C-terminal ends either blocked by acetyl and amide groups, respectively, for use in immunizations and assays, or, unblocked for immunoadsorbent preparation. The amino acid sequences are assigned in accordance with the revised hTg sequence by Van de Graaf et al.[24] Human Tg was purified from surgically removed human thyroid glands as previously described.[25]

MHC-binding prediction analysis of Tg peptides

The ability of a given Tg peptide to bind to MHC class II molecules was determined via the use of algorithms searching for specific aa sequence motifs described by Altuvia et al.,[26] as well as the online database RANKPEP.[27]

T-cell proliferation assays

Proliferation assays of LNCs were performed as previously described.[18] Briefly, 6- to 8-week-old female mice were immunized subcutaneously at both the tail base and the back with 100 nmol p2208, or with 75 μg hTg emulsified in complete Freund's adjuvant (CFA) (Sigma, St Louis, MO). Nine days later, their inguinal, brachial and axillary lymph nodes (pooled from three mice) were aseptically collected and single-cell suspensions were prepared in Dulbecco's modified Eagle's minimal essential medium (DMEM) (Biochrom, Cambridge, UK) supplemented with 10% heat-inactivated fetal bovine serum (Gibco, Paisley, UK) (complete medium – cDMEM), 20 mm HEPES buffer (Biochrom, Berlin, Germany), 2 mm l-glutamine (Gibco), 0·5 mm sodium pyruvate (Gibco), 0·2% sodium bicarbonate (Gibco), 5 × 105 IU streptomycin (KOPER, Athens, GR), 5 × 105 IU penicillin (KOPER), 5 × 10−5 m β-mercaptoethanol (Sigma). Cells were centrifuged, washed and subsequently cultured (4 × 105 cells/well in 200 μl cDMEM) with serial twofold dilutions (9–0·14 μm) of p2208, control peptide or equimolar concentrations of whole hTg in flat-bottomed 96-well plates (Orange Scientific, Braine-l’ Alleud, Belgium) for 3 days at 37° in a 5% CO2, 95% air humidified incubator, to assess their proliferation. Eighteen hours before the 3-day incubation, 1 μCi [3H]thymidine (25 Ci/mmol; Amersham Pharmacia, Stockholm, Sweden) in 20 μl cDMEM was added per well. The incorporated radioactivity was measured using the 1450 microbeta Trilux counter (Wallac, Turku, Finland). The Stimulation Index (S.I.) was defined as counts per minute (c.p.m) in the presence of antigen divided by the c.p.m in the absence of antigen, and values ≥3 were regarded as positive.

Measurement of cytokines by ELISA

Cytokine production in harvested supernatants of p2208-primed LNCs (2 × 106 cells/well in 1 ml cDMEM) cultured with 4·4 μm of antigen (p2208, or control p2652) in 24-well culture plates for 48 hr, was determined by ELISA. The levels of interleukin-2 (IL-2), IL-4, interferon-γ (IFN-γ) and tumour necrosis factor-α (TNF-α) were determined by the ELISA-Ready-SET-Go kit (eBioscience, San Diego, CA) according to the manufacturer's protocol.

Induction and evaluation of EAT

Mice were challenged subcutaneously at the tail base with 100 nmol of p2208 in CFA emulsion, and boosted 21 days later with 50 nmol of peptide in incomplete Freund's adjuvant (Sigma). Thirty-five days after the initial immunization, antisera were collected for analysis by ELISA, and the thyroid glands were removed for EAT assessment, along with the kidney, liver and spleen. Organs were fixed in 10% formalin fixative for 24 hr at room temperature and then placed to a fluid-transfer tissue processor (Leica, Solms, Germany). Samples were then dehydrated in increasing concentrations of ethanol solutions, cleaned in xylene (Surgipath, Richmond, UK) and embedded in paraffin (Paraplast, Tyco Healthcare Group LP, Mansfield, MA) for sectioning. Serial 4-μm sections were produced across the entire gland with a microtome blade RM2135 (Leica) and stained with haematoxylin Harris and eosin (BDH, Poole, UK) for observation under the optical microscope. The mononuclear cell infiltration index (II) of the thyroid gland was scored as follows: 0, no infiltration; 1, interstitial accumulation of cells between two or three follicles; 2, one or two foci of cells at least the size of one follicle; 3, extensive infiltration 10–40% of total area; 4, 40–80% of total area; and 5, >80% of total area.

Detection of peptide-specific antibodies by ELISA

Sera collected from p2208-immunized mice were tested with ELISA for their reactivity against the immunizing antigen, intact hTg and control p2652 peptide. Briefly, the wells of polystyrene microtitre plates (Nunc, Roskilde, Denmark) were coated with 5 μg/ml of p2208 and control peptide or 10 μg/ml of intact hTg in carbonate–bicarbonate buffer 0·1 m, pH 9·3, (incubated for 1 hr at 37° and overnight at 4°), washed with PBS and blocked for 1 hr with 0·1% BSA in PBS. Samples were incubated at threefold dilutions (1/50, 1/150, 1/450) for 2 hr at 37°, the wells were washed and then 100 μl of an alkaline phosphatase-conjugated goat anti-mouse IgG secondary antibody (0·5 μg/ml in PBS-BSA-Tween) (Merck, Hohenbrunn, Germany) was added per well, followed by 1 hr incubation at 37°. The chromogen substrate p-nitrophenyl phosphate (Sigma) was added and the light absorbance at 405 nm of the p-nitrophenolate product was measured on an Expert Plus microplate reader (Asys, Eugendorf, Austria).

Depletion of p2208-specific antibodies from antisera

To examine the hTg reactivity of antisera derived from the p2208-immunized LR C57BL/6 mice, a p2208 immunoadsorbent was produced, according to an established protocol.[28-30] Briefly, 1 ml of glutaraldehyde-activated polyacrylamide-agarose beads Ultrogel AcA3·4 (IBF biotechnics, Villeneuve-la-Garenne, France) were incubated with 800 μg of p2208, for 3 days, at room temperature. The final amount of bound peptide (0·5 mg of peptide per ml beads) was indirectly evaluated by optical density (OD) measurement of the resulting supernatant at 280 nm (ε = 1280/m/cm). Three hundred microlitres of pooled sera from four p2208-immunized C57BL/6 mice were incubated with the immunoadsorbent, under gentle stirring for 2 hr at room temperature. The bound anti-p2208 antibodies were eluted by 0·2 n HCl-glycine pH 2·8 treatment (Merck), until no significant OD (<0·040) was detected. The whole procedure was repeated four times to ensure that all anti-p2208 antibodies were removed from the pooled antisera. Pooled sera, depleted of p2208-specific antibodies, were tested for their reactivity against intact hTg and the autologous peptide as a negative control, by ELISA.

Statistical analysis

The Mann–Whitney U-test was used to identify significant differences in cellular proliferation between experimental and control wells. A P-value ≤0·05 was considered statistically significant.

Results

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosures
  9. References

Algorithmic searches for the identification of p2208

Two computational analyses were conducted on the sequences of 48 Tg peptides of interest, located on the C-terminal end of the molecule, to identify peptides with potential immunogenicity in LR and HR mouse strains. From the in silico analysis, peptide p2208 (aa 2208–2227) was selected because it contained three Ab-binding motifs and one As-binding motif according to the online RANKPEP database (Table 1). There was significant overlap among the various motifs, most notable of which was the seven aa sequence overlap between the 9mer As-binding motif and one of the Ab-binding motifs (sequence VPYAAPP, Table 1). On the other hand, the algorithms described by Altuvia et al. indicated the existence of four Ek-binding motifs, three of which are almost identical to each other, but no Ak-binding motifs. Overall, the two analyses indicated that the 20mer peptide p2208 might bind to class II MHC molecules of both HR and LR strains.

Table 1. MHC binding motifs
AlgorithmBinding motifMHC molecule
  1. 1Predicts MHC-binding motifs based on their similarity to a set of protein sequences known to bind to a given MHC molecule. Similarity is scored using the Position Specific Scoring Matrices derived from the aligned peptides known to bind to that MHC molecule.

  2. 2The prediction is based on the analysis of a given sequence for the identification of specific amino acids among a known set that are able to bind to each position of the MHC-cleft. Bold and underlined sequences represent the predicted binding motif.

RankpepQFLGVPYAAPPLAERRFQAPAs
RankpepQFLGVPYAAPPLAERRFQAPAb
RankpepQFLGVPYAAPPLAERRFQAPAb
RankpepQFLGVPYAAPPLAERRFQAPAb
Altuvia motif aQFLGVPYAAPPLAERRFQAPEk
Altuvia motif cQFLGVPYAAPPLAERRFQAPEk
Altuvia motif cQFLGVPYAAPPLAERRFQAPEk
Altuvia motif dQFLGVPYAAPPLAERRFQAPEk

Peptide p2208 is immunogenic at the T-cell level

Based on the above findings, 100 nmol p2208 was administered subcutaneously to the SJL/J (H-2s), C57BL/6 (H-2b) and CBA/J (H-2k) strains. To further validate the predictive value of the search algorithms, p2208 was also administered in BALB/c (H-2d) control mice because the peptide does not carry H-2d-binding motifs. LNCs were cultured in vitro in the presence of serial dilutions of p2208 and control peptide p2652. The p2208 was found to induce a significant dose-dependent proliferation of LNCs only from SJL/J and C57BL/6 mice (Fig. 1a,b) (9 μm of p2208 elicited an SI of 32·49 in SJL/J mice and 6·63 in C57BL/6 mice), whereas no response was detected in LNCs from CBA/J and BALB/c mice (data not shown). Τhe proliferation was specific for p2208, as no response was detected in the presence of the control peptide (maximum S.I. = 1·8 from both mouse strains). The LNCs from p2208-primed mice did not proliferate in the presence of hTg (Fig. 1a,b) and similarly, hTg-primed LNCs did not proliferate in the presence of p2208 (data not shown). These data demonstrated that p2208 is immunogenic in both HR and LR mouse strains, bearing the H-2s and H-2b haplotypes, respectively, and suggested that this peptide sequence does not encompass immunodominant epitope(s).

image

Figure 1. Assessment of p2208 immunogenicity. (a, b) Proliferative responses of peptide-primed lymph node cells (LNCs) from SJL/J (a) and C57BL/6 (b) mice to the antigen shown were evaluated according to their Stimulation indices (SI) and were regarded as positive when ≥ 3. Mice were challenged subcutaneously with 100 nmol of peptide, LNCs were collected 9 days later and were cultured in vitro for 3 days in the presence or absence of the antigens. Background counts per minute (c.p.m.) ranged from 300 to 1000 and the results were statistically significant (< 0·05). (c) Interleukin-2 (IL-2) and interferon-γ (IFN-γ) levels (pg/ml) in the supernatants of p2208-primed LNCs from SJL/J and C57BL/6 mice, cultured in the presence of the respective peptide. Mice were immunized with 100 nmol of p2208 and 9 days later their LNCs were collected and cultured for 48 hr in the presence of 4·4 μm of p2208. The cytokine concentration was calculated according to the standard curve of the respective cytokine in the manufacturer's protocol. Error bars represent SD of duplicate wells and the results are representative of three independent experiments.

Download figure to PowerPoint

p2208 elicits a T helper type 1 response

To elucidate the T helper type 1 (Th1)/Th2 subset of the in vitro proliferating T cells, LNCs from p2208-primed SJL/J and C57BL/6 mice were cultured in the presence or absence of p2208 or control peptide p2652. The levels of IL-2, IL-4, IFN-γ and TNF-β in culture supernatants were determined 48 hr later by ELISA. Interleukin-2 and IFN-γ were detected on both strains examined, whereas no IL-4 and TNF-β was measured (Fig. 1c), as expected, because the onset of the disease is defined by a Th1 response.[31] The levels of IL-2 and IFN-γ were 515·26 pg/ml and 60·73 pg/ml in the SJL/J mice, respectively, compared with 202·42 pg/ml and 163·45 pg/ml in C57BL/6 mice. No clear correlation between HR and LR mouse strains and their p2208-induced cytokine production was shown.

p2208 can induce direct EAT in HR and LR mouse strains

The immunogenicity of p2208 prompted us to further test its pathogenicity in HR (SJL/J, CBA/J), as well as in LR (C57BL/6, BALB/c) mice. Six to seven mice per strain were challenged with p2208 and mononuclear cell infiltration of the thyroid was assessed 5 weeks after the initial challenge. The p2208 conferred direct EAT not only on the HR SJL/J (2/6, highest I.I. = 2, mean I.I. = 0·5), but also on the LR C57BL/6 (2/7, highest I.I. = 3, mean I.I. = 0·54) (Table 2). However, minimal EAT was observed in the strains with I.I. = 1. Representative thyroid gland sections are shown in Fig. 2. The pathology was organ-specific because of the absence of any mononuclear infiltrating cells in kidney, liver and spleen (data not shown).

Table 2. Experimental autoimmune thyroiditis (EAT) induction by the thyroglobulin peptide p2208
Mouse strainInfiltration indexMice with EAT
0123
  1. 1Mice were subcutaneously challenged with 100 nmol of p2208 in complete Freund's adjuvant, and boosted 21 days later with 50 nmol in incomplete Freund's adjuvant. Five weeks after the initial challenge, experimental autoimmune thyroiditis was assessed and their thyroid gland infiltration was scored as described in 'Materials and methods'.

SJL/J41102/6
CBA/J70000/7
C57BL/651012/7
BALB/c70000/7
image

Figure 2. Representative appearance of experimental autoimmune thyroiditis severity after challenge of high responder and low responder mouse strains with p2208. (a) Normal thyroid gland from C57BL/6 or SJL/J mice [infiltration index (I.I.) = 0]; (b) focal infiltration by mononuclear cells in C57BL/6 or SJL/J thyroids (I.I. = 1); (c) SJL/J thyroid (I.I. = 2); (d) C57BL/6 thyroid (I.I. = 3). Magnification: × 40.

Download figure to PowerPoint

p2208 is immunogenic at the B-cell level

Antisera from p2208-immunized mice collected on day 0 and day 35 of EAT induction were analysed by ELISA in terms of their antibody reactivity against p2208 and hTg. Specific anti-p2208 antibodies were detected in five (mice 1–5) of the seven C57BL/6 mice (Fig. 3c), and in two (mice 3 and 5) of the six SJL/J mice (Fig. 3a) inspected, although the latter presented with lower reactivity. Pre-immune sera and antisera did not react with the control peptide (data not shown). Noticeably, two of the two positive antisera from SJL/J mice (mice 3 and 5) and two of the five from the LR C57BL/6 mice (mice 1 and 2) also exhibited reactivity against the whole hTg molecule (Fig. 3b,d). The above findings suggest the existence of B-cell epitopes within the p2208 sequence, which are probably expressed on the surface of intact Tg.

image

Figure 3. IgG responses against p2208 and human thyroglobulin (hTg) in immunized mice. Sera were collected from p2208-immunized SJL/J and C57BL/6 mice and their reactivity was individually assessed (mice 1–6 or 7) against the immunizing peptide (a, c) and hTg (b, d). Samples were incubated at threefold serial dilutions (1/50 to 1/450) and in duplicate wells. The reactivity of all pre-immune sera against hTg or p2208 was undetectable. Data are representative of three independent experiments.

Download figure to PowerPoint

p2208 does not induce B-cell epitope spreading

The Tg-specific reactivity of anti-p2208 antibodies in peptide-immunized mice suggests recognition of either the same sequence on intact Tg, or recognition of other Tg sequences due to epitope spreading. To test the two hypotheses, antisera from C57BL/6 mice were pooled and depleted of p2208-specific antibodies using a peptide-immunoadsorbent. After exhaustive passages, the antisera lost reactivity against hTg and Tg-specific titres were similar to those of the pre-immune sera (Fig. 4). This finding supported the view that p2208-specific IgG recognize the exposed epitope on intact Tg.

image

Figure 4. The p2208-specific antibodies reacted with intact human thyroglobulin (hTg). Antibody reactivity against hTg was assessed by ELISA using: (a) pooled antisera from four p2208-immunized mice (●); (b) pooled antisera depleted of anti-p2208 antibodies by passing through an immunoadsorbent column (○); (c) pre-immune pooled sera from the same mice used as controls (×).

Download figure to PowerPoint

Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosures
  9. References

The present study was initiated when scanning of the C-terminal end of the hTg molecule identified that the peptide p2208, bearing 80% aa sequence homology with the human analogue, carries Ab-, As- and Ek-binding motifs, i.e. it could be potentially immunopathogenic in mouse strains conventionally considered as HR and LR strains in the field of EAT. The results confirmed the predictive value of the search algorithms, to the extent that p2208 induced EAT of the same severity in both LR (C57BL/6) and HR (SJL/J) mouse strains. On the other hand, despite the presence of Ek-binding motifs within p2208, the peptide was not immunogenic in CBA/J (HR) mice, suggesting lack of binding to MHC or lack of recognition of the MHC–peptide complex by T cells in these hosts. Overall, computerized algorithms searching for immunogenic epitopes on the basis of potential MHC-binding ability have yielded significant results when applied to the large (2748 aa) Tg molecule sequence because they have so far identified 22 EAT-inducing peptides out of a total of 75 tested.

So far, only one other hTg peptide (p2340, aa 2340–2359) has been known to induce EAT in LR mouse strains but its pathogenicity was demonstrable only after adoptive transfer of peptide-specific syngeneic LNCs into naive hosts.[18] The capacity of p2208 to induce direct EAT in C57BL/6 mice may further facilitate the design of future studies on immunoregulation of the response against this pathogenic sequence as many knock-in or knock-out strains exist on the B6 background. Induction of EAT has also been reported via the use of the mouse thyroglobulin peptides mTg409 (aa 409–423) and mTg1677 (aa 1677–1692) tested in H2A E+ transgenic mice with a C57BL/10 (LR) genetic background.[32] However, these mice carry the otherwise not normally expressed Eb MHC molecule and are not as widely available as C57BL/6. In the present study, we have not examined whether the pathogenicity of the 20mer p2208 in both H-2b and H-2s mice can be attributed to recognition of the same minimal epitope within p2208 and the potential role flanking residues might play in the recognition process. In this regard, previous work from our laboratory has shown that while the hTg peptide p2340 elicits EAT in both HR and LR mice, a minimal 9mer epitope within this peptide sequence (aa 2344–2352) was immunopathogenic only in HR strains.[23]

The failure of p2208-primed or Tg-primed LNCs to proliferate in vitro in the presence of Tg or peptide, respectively, supports a cryptic role of this peptide in both C57BL/6 and SJL/J hosts. Such a finding is in agreement with several studies in the EAT field that have failed to identify a dominant epitope in Tg.[6-16, 32-35] The p2208 must be expressed intrathyroidally to be recognized by effector T cells and as has been suggested before,[36] this ‘crypticity paradox’ may be explained on the basis that either Tg is processed differently in vivo than in vitro, or Tg as a substrate, is taken-up by antigen-presenting cells in vivo in the form of fragments rather than as intact antigen, resulting in alteration of the immunodominance hierarchy.

At the B-cell level, p2208 elicited production of IgG antibodies recognizing both the immunizing peptide as well as intact Tg, strongly suggesting that the pathogenic p2208 peptide also served as a helper T-cell epitope for specific B-cell responses in SJL/J and C57BL/6 mice. Depletion of anti-p2208 antibodies from the pooled antisera of four peptide-immunized C57BL/6 mice resulted in a dramatic decrease of reactivity against the intact hTg molecule. This finding confirmed that the induced Tg-specific reactivity was not a secondary event developing during the course of EAT, as a result of the inflammatory response. Rather it suggested strongly that p2208 is accessible on the surface of the quarternary structure of Tg by specific IgG antibodies. Similar findings have also been described for other pathogenic Tg peptides used as immunogens by other research teams.[9, 16, 37, 38, 18] Such antibodies can be useful tools in mapping the antigenic topography of B-cell epitopes on Tg and in the construction of diagnostic kits for the detection of Tg in serum.

Acknowledgements

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosures
  9. References

This work was financially supported by the Greek General Secretariat of Research and Technology (ARISTEIA-HPI). We wish to thank Dr George Carayanniotis for valuable discussions throughout this study.

References

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosures
  9. References