A novel pathogenic peptide of thyroglobulin (2208–2227) induces autoreactive T‐cell and B‐cell responses in both high and low responder mouse strains

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 (A^b), or susceptibility (A^s, E^k) to EAT. We therefore immunized LR BALB/c (H‐2^d) and C57BL/6 (H‐2^b) strains, as well as HR CBA/J (H‐2^k) and SJL/J (H‐2^s) 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.     

Pathogenicity of a human thyroglobulin peptide (2340-2359) in mice with high or low genetic susceptibility to thyroiditis

We have previously identified a 20-mer peptide of human thyroglobulin (hTg), p2340 (aa2340-2359), which induced experimental autoimmune thyroiditis (EAT) in AKR/J (H-2(k)) and HLA-DR3 transgenic mice. In this study, we investigated the thyroiditogenic potential of p2340 in 'high responder' CBA/J (H-2(k)) and SJL/J (H-2(s)) or 'low responder' C57BL/6 (H-2(b)) and BALB/c (H-2(d)) mice. Mice were immunized subcutaneously with 100 nmol of p2340 in complete Freund's adjuvant (CFA) and both the proliferative capacity of their lymph node cells in the presence of p2340 or intact Tg and the production of peptide-specific antibodies were investigated. The p2340 peptide was found to contain B-cell and non-dominant T-cell epitope(s) in all strains tested. Moreover, it elicited EAT in CBA/J (2/6, infiltration index (I.I.) 1) and SJL/J (5/5, I.I. 1-3) mice after direct challenge and in BALB/c (4/7, I.I. 1) and C57BL/6 (1/5, I.I. 1) after adoptive transfer of p2340-primed lymph node cells. P2340 is the first Tg peptide found to be pathogenic in low as well as high responder mouse strains and thus will allow us to investigate mechanisms of EAT induction in a genetically resistant host.     

Human thyroglobulin peptide p2340 induces autoimmune thyroiditis in HLA-DR3 transgenic mice

In a previous study we demonstrated that the human thyroglobulin (hTg) peptide p2340 (aa 2340-2359) can stimulate a T cell response and elicit experimental autoimmune thyroiditis (EAT) in AKR/J (H-2(k)) mice. In the present study we examined whether p2340 can induce EAT in single HLA class II DR3 transgenic mice. This peptide was found to be immunogenic at the T cell level in DR3 mice, since it induced specific proliferative responses, as well as IL-2 and IFN-gamma secretion in secondary cultures of peptide-primed lymph node cells (LNC). Immunization of HLA-DR3 mice with p2340 in CFA elicited EAT (infiltration index of 1 to 2) in eight of nine mice. Peptide-primed LNC responded to intact hTg, whereas, hTg-primed LNC did not respond to p2340 in culture, suggesting that p2340 contains subdominant T cell epitope(s). P2340 was also found to be immunogenic at the B cell level, since strong p2340-specific IgG response was detected in all transgenic mice tested. Thus, we provide evidence for a pathogenic role of an hTg peptide in HLA-DR3 transgenic mice. Therefore, p2340 could be presented by DR3 molecule in patients with Hashimoto's thyroiditis and participate in the development of the disease.     

Induction of murine thyroiditis by a non dominant E(k)-restricted peptide of human thyroglobulin

We have previously shown that the human thyroglobulin (hTg) 20-mer peptide p2340 (aa 2340-2359) contains an epitope recognized by Tg-reactive B cells in patients with Graves' disease. The presence of several Ek-binding motifs within p2340 prompted us to examine whether this peptide can stimulate a T-cell response and elicit experimental autoimmune thyroiditis (EAT) in AKR/J (H-2k) mice. The peptide was found to be immunogenic at the T-cell level since it induced specific proliferative responses as well as interleukin-2 and interferon-gamma secretion in secondary cultures of peptide-primed lymph node cells (LNC). The p2340-specific proliferation was blocked almost completely by an Ek-specific monoclonal antibody (mAb) but was unaffected by a control Ak-specific mAb. Peptide-primed LNC did not respond to intact hTg and conversely, LNC primed in vivo with hTg did not respond to p2340 in culture, suggesting that p2340 contains non-dominant T-cell epitope(s). Direct subcutanaeous challenge of AKR/J mice (n = 9) with p2340 in adjuvant, elicited mild to moderate EAT (infiltration index of 1-2) and strong p2340-specific immunoglobulin G responses in all mice tested. These data delineate a new thyroiditogenic sequence within the carboxyl terminal region of hTg.     

Contrasting immunopathogenic properties of highly homologous peptides from rat and human thyroglobulin.

The current lack of amino acid sequence data for mouse thyroglobulin (Tg) necessitates mapping of pathogenic T-cell epitopes on heterologous Tg in mouse experimental autoimmune thyroiditis (EAT). A prevailing assumption has been that epitopes sharing a high degree of amino acid homology among heterologous Tg are likely to exhibit the same immunopathogenic properties in the same host. In this report, we have examined this concept while working with the 18-mer rat(r)Tg(2695-13) peptide that was previously shown to elicit 'A'-restricted T cells and EAT in SJL mice. A major immunopathogenic T-cell epitope was localized within the 12-mer rTg(2695-06). It was found that the human 12-mer homologue that carries two Ser substitutions at Glu2703 and Thr2704 exhibited contrasting properties: it failed to activate Th1 cells in lymphokine and proliferation assays; it did not cross-react with rTg(2695-06) at the T-cell level; and it induced only focal thyroiditis following adoptive transfer of specific lymph node cells. These data highlight the caveat involved in extrapolating results of pathogenic T-cell epitope mapping across heterologous Tgs, even when such epitopes share a high degree of amino acid homology.     

Major T cell epitope-containing peptides can elicit strong antibody responses

Peptides containing major T cell epitopes have the capacity to induce T cell anergy and have therefore been proposed for the treatment of allergic and autoimmune diseases. Such peptides should not be immunogenic, i. e. should not contain a B cell recognition site. We have evaluated in BALB/c mice the therapeutic potential of a 15-mer peptide (p21 - 35) derived from Der p2, a major allergen of the house dust mite Dermatophagoides pteronyssinus, which contains a dominant T cell epitope but is not recognized by antibodies to Der p2. Unexpectedly, p21 - 35 elicited strong immune responses, suggesting the presence of a cryptic B cell epitope. Similar results were obtained with mice of three additional MHC haplotypes. A core sequence of four amino acids (Ile-Ile-His-Arg) corresponding to residues 28 - 31 was shared by the B and T cell epitopes. Critical residues for B cell recognition were Arg31 and Lys33, while Ile28 was essential for T cell recognition. A Lys33Ala mutant of p21 - 35 still activated T cells but had much reduced immunogenic properties, making it a suitable alternative peptide for T cell anergy induction. Careful investigation of the immunogenic potential of peptides used to induce T cell anergy should be carried out prior to their clinical application.     

Experimental autoimmune thyroiditis (EAT) induced by the thyroglobulin peptide (2596-2608): influence of H-2 and non H-2 genes

We have previously identified five thyroglobulin (Tg) peptides with Ak-binding motifs that induce experimental autoimmune thyroiditis (EAT) in CBA/J (H-2k) mice. In this study, we have examined whether H-2 or non H-2 genes can influence the immunopathogenicity of peptide p2596 (a.a. 2596-2608), which earlier elicited considerable pathology in CBA/J hosts. The p2596 peptide induced mild EAT--(infiltration index range=1-2)-- in H-2-compatible AKR/J, B10.BR, and C3H/HeJ mice. Moreover, p2596-primed LNC from these mice exhibited peptide-specific proliferative responses and secreted significant amounts of IL-2 and IFN-gamma in recall in vitro assays. Priming and boosting of these strains with p2596 resulted in the generation of specific IgG responses five weeks after the initial challenge. In contrast, s.c. challenge of H-2-incompatible strains such as DBA/1J (H-2q), SJL (H-2s), DBA/2J (H-2d) and C57BL/6 (H-2b) with the same peptide dose did not elicit EAT pathology and peptide-specific B- or T-cell responses. These data demonstrate the thyroiditogenic potential of p2596 in H-2k strains of diverse non-H-2 backgrounds but not in mice carrying H-2b, d, q or s haplotypes.     

Fine-specificity of the anti-CENP-A B-cell autoimmune response

The major targets recognized by anti-centromere autoantibodies are the three centromere-associated proteins (CENPs) A, B, and C, with apparent molecular masses of 19, 80, and 140 kDa, respectively. Previously a major epitope region on the 19-kDa CENP-A antigen was identified by synthesis of a soluble synthetic 15-mer peptide (amino acids 3-17) to be used in enzyme-linked immunosorbent assay and western blot competition assays. However, no systematic experimental scanning for epitope regions on the CENP-A autoantigen has yet been performed. In this study we scanned the complete CENP-A amino acid sequence for epitopes using 19 previously characterized autoimmune-sera. Overlapping peptides 15 amino acids in length and offset by three amino acids were synthesized on activated membranes, covering the whole CENP-A autoantigen. Probing of the membranes with various anti-centromere sera showed that all epitopes are clustered in the N-terminal 45 amino acids. For fine-mapping of this autoreactive region the N-terminus of CENP-A (amino acids 1-45) was scanned again by probing overlapping 15-mer, 12-mer, 10-mer, 8-mer, 7-mer, 6-mer, and 5-mer peptides, all offset by one amino acid, with anti-centromere sera. In this way we localized two epitope core regions within the N-terminal 45 amino acids, one covering amino acids 2-17, recognized by 17 sera, and the other covering amino acids 22-38, recognized by 18 sera. One serum did not react with CENP-A at all. Several sera seem to recognize overlapping individual epitopes within these two epitope core regions. All sera, however, recognize a sequence motif G/A-P-R/S-R-R.     

Experimental Autoimmune Thyroiditis (EAT) Induced by the Thyroglobulin Peptide (2596–2608): Influence of H-2 and Non H-2 Genes

We have previously identified five thyroglobulin (Tg) peptides with A^k-binding motifs that induce experimental autoimmune thyroiditis (EAT) in CBA/J (H-2^k) mice. In this study, we have examined whether H-2 or non H-2 genes can influence the immunopathogenicity of peptide p2596 (a.a. 2596–2608), which earlier elicited considerable pathology in CBA/J hosts. The p2596 peptide induced mild EAT—(infiltration index range=1–2)— in H-2-compatible AKR/J, B10.BR, and C3H/HeJ mice. Moreover, p2596-primed LNC from these mice exhibited peptide-specific proliferative responses and secreted significant amounts of IL-2 and IFN-γ in recall in vitro assays. Priming and boosting of these strains with p2596 resulted in the generation of specific IgG responses five weeks after the initial challenge. In contrast, s.c. challenge of H-2-incompatible strains such as DBA/1J (H-2^q), SJL (H-2^s), DBA/2J (H-2^d) and C57BL/6 (H-2^b) with the same peptide dose did not elicit EAT pathology and peptide-specific B- or T-cell responses. These data demonstrate the thyroiditogenic potential of p2596 in H-2^k strains of diverse non-H-2 backgrounds but not in mice carrying H-2^{b, d, q?or?s} haplotypes.     

Identification of minimal CD8+ and CD4+ T cell epitopes in the Plasmodium yoelii hepatocyte erythrocyte protein 17kDa

Immunization of mice with subunit vaccines based on the Plasmodium yoelii 17kDa hepatocyte erythrocyte protein (PyHEP17), orthologue of Plasmodium falciparum exported protein 1 (PfExp1), induces antigen-specific immune responses and protects against sporozoite challenge. To aid in the characterization of candidate subunit vaccines based on this antigen, we have mapped the immunodominant and subdominant CD8+ and CD4+ T cell epitopes on PyHEP17. Using a panel of 29 15-mer synthetic peptides representing the complete sequence of PyHEP17 (amino acids 1-153), and overlapping each other by 10 residues, we identified an immunogenic region between amino acids 61-85. To define the minimal CD4+ and CD8+ T cell epitopes within this region, we synthesized 25 9-mer peptides overlapping each other by one residue. We screened the capacity of the 15-mer and 9-mer peptides to be recognized by splenocytes and lymph node cells from mice immunized with PyHEP17 plasmid DNA or peptides in Freund's adjuvant, as assessed by cytokine secretion, lymphoproliferation, and cytotoxicity. The profile of response to the T cell epitopes varied depending upon the immunization regimen. Antigen-specific T cell responses were detected to three 15-mer peptides (residues 61-75, 66-80 and 71-85) representing two 10-mer epitopes mapping to residues 66-75 (LTKNKKSLRK) and 71-80 (KSLRKINVAL). IFN-gamma responses after DNA immunization predominantly mapped to two overlapping 9-mer peptides (residues 73-81 and 74-82) sharing an eight amino acid overlap (residues 74-81, RKINVALA), whereas CTL responses predominantly mapped to four 9-mer peptides (residues 61-69, 70-78, 76-84, and 84-92). In addition, a subdominant 10-mer CD8+ T cell epitope recognized by peptide immunization but not DNA immunization mapped to residues 31-40 (GKYGSQNVIK). The identification of these epitopes will allow the evaluation of delivery systems for malaria vaccine candidates as well as the delineation of protective immune mechanisms.     

The lifespan of major histocompatibility complex class I/peptide complexes determines the efficiency of cytotoxic T-lymphocyte responses

Major histocompatibility complex (MHC)/peptide association and stability are determined by specific amino acid interactions between peptide antigens and the MHC groove, and are regarded as a critical feature in ensuring efficient monitoring by T cells. In this investigation we examined the relationship between MHC/peptide stability and the immunostimulatory capacity of MHC/peptide complexes. For this purpose we compared synthetic peptide analogues derived from the immunodominant HLA-A11-presented IVTDFSVIK (IVT) epitope, for their capacity to reactivate IVT-specific memory cytotoxic T-lymphocyte (CTL) responses. The analogues differentiated from the wild-type epitope by single amino acid substitution at position 2. All peptides showed similar affinity for HLA-A11 molecules and were recognized by IVT-specific CTL clones, but induced HLA-A11 complexes at the cell surface with different lifespan. This model offered the possibility of comparing the capacity of an immunogenic epitope to stimulate a unique population of T-cell precursors depending on the lifespan of its presentation at the cell surface. We demonstrated that stable HLA-A11/peptide complexes efficiently stimulate IVT-specific CTL responses, while HLA-A11/peptide complexes with short lifespan do not. The precise identification of the role of amino acid residues in the formation of stable MHC/peptide complexes may be relevant for the design of wild-type-derived epitopes with high immunogenicity. These analogues may have important applications in the immunotherapy of infectious diseases and immunogenic tumours.     

Minute quantities of a single immunodominant foreign epitope are presented as large nested sets by major histocompatibility complex class II molecules

The processing and presentation of immunogenetic peptides is an obligate event in the generation of an immune response. However, the degree of complexity with which an immunogenic foreign epitope is presented is still unclear. This question was addressed by analyzing the naturally processed peptides generated from exogenously-derived hen egg white lysozyme (HEL) bound to the murine major histocompatibility complex (MHC) class II molecule, H-2A^k. Using reversed-phase chromatography (RPC), T cell hybridomas and mass spectrometry, 16 peptides were identified that contain the minimal MHC binding epitope 52–61. These peptides exhibited substantial N- and C-terminal extensions and ranged from 13–28 amino acids in length. In contrast, MHC class I molecules present peptides of 8–11 residues and each foreign epitope appears to be represented by only a single peptide. The data here also show that only ～ 0.8% of the total bound peptide was derived from this single HEL epitope. These findings provide direct evidence that relatively small amounts of processed peptide are required to stimulate an effective T cell response.     

Immunisation with phage displaying peptides representing single epitopes of the glycoprotein G can give rise to partial protective immunity to HSV-2

Filamentous phage displaying peptides representing single epitopes of the glycoprotein G of HSV-2 (gG2) were used as immunogens via the subcutaneous route in Balb/c mice without additional adjuvant. The phage were isolated from a random phage peptide display library and contain 15-mer peptide inserts that mimic epitopes of gG2. In each case, an antibody response to gG2 was generated that was dependent on the dose of phage administered and on the presence of the peptide insert. Phage displaying epitopes of gG2, which map to amino acids 551-570, were the most immunogenic; interestingly, this region of gG2 is frequently recognised by patients infected with HSV-2. The data also provide interesting information as regards choice of peptide mimics for use as immunogens because, surprisingly, the most antigenic of the individual clones was the least immunogenic. In two of the experiments, mice immunised with phage displaying a single epitope of gG2 were protected against challenge with a lethal dose of whole HSV-2. This suggests a possible role for phage-displayed peptides in inducing protective immunity against pathogens and provides a model system for investigating the underlying mechanisms.     

An N-glycosylated tyrosinase epitope associates with newly synthesized MHC class I molecules in melanoma cells

Endogenous antigenic epitopes are presented to CD8⁺ T cells by MHC class I molecules. Many endogenous antigens are glycoproteins, and it is not clear what effect the attachment of carbohydrate to potential immunogenic epitopes has on their processing and presentation (i.e., is the carbohydrate moiety removed prior to presentation, or is it presented along with the peptide to T cells?). A major question in this regard is whether natural antigenic epitopes that possess N-linked carbohydrate can associate with class I molecules during assembly in the endoplasmic reticulum (ER). One such antigenic epitope, corresponding to amino acids 369–377 of the enzyme tyrosinase, possesses an N-linked glycosylation site. We have studied the transport and loading of this epitope in streptolysin O-permeabilized melanoma cells. We show here that that the glycosylated epitope is capable of loading onto newly synthesized HLA-A2 molecules in the ER of two melanoma cell lines. The results are discussed in respect to the processing and presentation of the tyrosinase epitope.     

Efficacy of HLA-DRB1∗03:01 and H2E transgenic mouse strains to correlate pathogenic thyroglobulin epitopes for autoimmune thyroiditis

Thyroglobulin (Tg), a homodimer of 660 kD comprising 2748 amino acids, is the largest autoantigen known. The prevalence of autoimmune thyroid disease, including Hashimoto’s thyroiditis and Graves’ disease, has provided the impetus for identifying pathogenic T cell epitopes from human Tg over two decades. With no known dominant epitopes, the search has long been a challenge for investigators. After identifying HLA-DRB1∗03:01 (HLA-DR3) and H2E^b as susceptibility alleles for Tg-induced experimental autoimmune thyroiditis in transgenic mouse strains, we searched for naturally processed T cell epitopes with MHC class II-binding motif anchors and tested the selected peptides for pathogenicity in these mice. The thyroiditogenicity of one peptide, hTg2079, was confirmed in DR3 transgenic mice and corroborated in clinical studies. In H2E^b-expressing transgenic mice, we identified three T cell epitopes from mouse Tg, mTg179, mTg409 and mTg2342, based on homology to epitopes hTg179, hTg410 and hTg2344, respectively, which we and others have found stimulatory or pathogenic in both DR3- and H2E-expressing mice. The high homology among these peptides with shared presentation by DR3, H2E^b and H2E^k molecules led us to examine the binding pocket residues of these class II molecules. Their similar binding characteristics help explain the pathogenic capacity of these T cell epitopes. Our approach of using appropriate human and murine MHC class II transgenic mice, combined with the synthesis and testing of potential pathogenic Tg peptides predicted from computational models of MHC-binding motifs, should continue to provide insights into human autoimmune thyroid disease.     

Permissive recognition of a mycobacterial T-cell epitope: localization of overlapping epitope core sequences recognized in association with multiple major histocompatibility complex class II I-A molecules.

Most T-cell epitopes are recognized in the context of a single or limited number of major histocompatibility complex (MHC) class II molecules. We have shown previously, however, that the immunodominant p61-80 epitope from the Mycobacterium tuberculosis 19,000 MW protein is recognized in a genetically permissive manner. In this study, permissive recognition of p61-80 was analysed in three murine MHC haplotypes (H-2b,d and k) with respect to: (i) T-cell-epitope core structure; (ii) I-A/I-E class II MHC restriction; and (iii) the identification of critical amino acid residues within the core region. Overlapping epitope core sequences composed of 6 to 8 amino acids were identified for each of the three H-2 haplotypes by T-cell epitope scanning (PEPSCAN) using peptide-specific T-cell lines. The epitope core sequences recognized by peptide and 19,000 MW protein-specific T cells were similar. In all three haplotypes, responses to p61-80 were restricted by class II MHC I-A molecules. To identify residues within the epitope core critically required for recognition, single substitution (alanine or leucine) analogue peptides were tested for their capacity to stimulate p61-80-specific T-cell hybridomas. A heterogeneous pattern of reactivity was observed, even among individual hybridomas derived from the same H-2 haplotype. Although every core residue could be defined as critical for at least one hybridoma, only one critical substitution (74Val-->Ala) was common to all hybridomas. The identification and structural analysis of genetically permissive epitopes of mycobacteria may be a useful strategy for the rational design of peptide-based vaccines for tuberculosis.     

T cell mapping of one epitope from thyroglobulin inducing experimental autoimmune thyroiditis (EAT).

These data collect the advance made in the last few years in our laboratory in defining one epitope from the thyroglobulin (Tg) molecule (660 KDa) inducing Experimental Autoimmune thyroiditis (EAT) in CBA/J mice. We achieved the characterization of one EAT-inducer Tg peptide by combining "in vitro" biochemical and immunological approaches and "in vivo" studies. Since T cells recognize degraded forms of the antigen and since endogenous antigens preferentially activate class I-restricted T cells, we hypothesized that one cytotoxic T cell hybridoma, named HTC2, which prevents further EAT induction in mice injected with Tg would be specific for one EAT inducer peptide. In order to identify one Tg epitope inducing EAT, enzymatic treatment of the protein by trypsin, HPLC purification and sequence analysis were performed. Simultaneously, tryptic digests were used to pulse CBA/J macrophages and tested for their ability to be recognized by HTC2 cells. Lastly, when digests were recognized by HTC2 cells their capacity to induce EAT in CBA/J mice was evaluated. To further assess the pathogenicity of the sequenced Tg peptide, one synthetic peptide was made and its capacity to induce EAT verified. By this procedure we identified for the first time one 40 amino-acid peptide from human thyroglobulin inducing EAT in CBA/J mice.     

A linear peptide containing minimal T- and B-cell epitopes of Plasmodium falciparum circumsporozoite protein elicits protection against transgenic sporozoite challenge

An effective malaria vaccine is needed to address the public health tragedy resulting from the high levels of morbidity and mortality caused by Plasmodium parasites. The first protective immune mechanism identified in the irradiated sporozoite vaccine, the "gold standard" for malaria preerythrocytic vaccines, was sporozoite-neutralizing antibody specific for the repeat region of the surface circumsporozoite (CS) protein. Previous phase I studies demonstrated that a branched peptide containing minimal T- and B-cell epitopes of Plasmodium falciparum CS protein elicited antirepeat antibody and CD4(+)-T-cell responses comparable to those observed in volunteers immunized with irradiated P. falciparum sporozoites. The current study compares the immunogenicity of linear versus tetrabranched peptides containing the same minimal T- and B-cell epitopes, T1BT*, comprised of a CS-derived universal Th epitope (T*) synthesized in tandem with the T1 and B repeats of P. falciparum CS protein. A simple 48-mer linear synthetic peptide was found to elicit antisporozoite antibody and gamma interferon-secreting T-cell responses comparable to the more complex tetrabranched peptides in inbred strains of mice. The linear peptide was also immunogenic in outbred nonhuman primates (Aotus nancymaae), eliciting antibody titers equivalent to those induced by tetrabranched peptides. Importantly, the 48-mer linear peptide administered in adjuvants suitable for human use elicited antibody-mediated protection against challenge with rodent malaria transgenic sporozoites expressing P. falciparum CS repeats. These findings support further evaluation of linear peptides as economical, safe, and readily produced malaria vaccines for the one-third of the world's population at risk of malaria infection.     

Inhibition of T cell proliferation specific for acetylcholine receptor epitopes related to myasthenia gravis with antibody to T cell receptor or with competitive synthetic polymers.

Long-term T cell lines and clones of C3H.SW origin specific to synthetic peptides representing immunogenic epitopes of the human aetylcholine receptor alpha-subunit were established. Using these lines and clones, it was possible to characterize the T cell recognition process of myasthenic epitopes. Testing a panel of N-and/or C-terminal truncated peptides it could be demonstrated that the deletion of the two C-terminal amino acids of peptides p195-212 and p259-271 resulted in a loss or reduction of the stimulatory capacity of the peptides towards the specific T cell lines. In contrast, no substantial effect on the stimulation of the line could be observed by shortening peptide p195-212 by up to five amino acids at the N-terminal end. The proliferation of T cell lines and clones specific to peptide p195-212 was inhibited by a mAb directed against the V beta 8 region of the T cell receptor. Furthermore, it was possible to block the peptide-specific proliferative responses of the lines and clones by the I-Ab restricted synthetic polypeptide (T, G)-A--L but not by the I-Ak restricted polypeptide antigen (H,G)-A--L. Similarly, p195-212 inhibited the proliferative response of the TCSW259-271 T cell line and p259-271 inhibited the specific proliferative response of the TCSW195-212 line. Moreover, the C-terminal shortened peptides inhibited significantly the in vitro stimulation of the T cell lines by the immunogenic peptides p195-212 and p259-271. The inhibition by the synthetic peptides or by (T,G)-A--L may be due to competitive blockade of the MHC binding site for the T cell line stimulating AChR peptides.     

IgE and IgG4 epitope mapping by microarray immunoassay reveals the diversity of immune response to the peanut allergen, Ara h 2

BACKGROUND: Detailed assessment of antibody responses to allergens reveals clinically relevant information about both host response and antigen structure. Microarray technology offers advantages of scale and parallel design over previous methods of epitope mapping. OBJECTIVE: We designed a redundant peptide microarray for IgE and IgG4 epitope mapping of the previously characterized peanut allergen, Ara h 2. METHODS: Six complete sets of overlapping peptides were commercially synthesized and site-specifically bound to epoxy-derivatized glass slides in triplicate. Peptides were 10, 15, or 20 amino acids in length with an offset of either 2 or 3 amino acids. A total of 10 control and 45 peanut-allergic sera were assayed. Specific IgE and IgG4 were detected by using fluorochrome-labeled monoclonal secondary antibodies. RESULTS: By using 15-mer and 20-mer peptides, we could define 11 antigenic regions, whereas only 5 were identifiable using 10-mers. Controls and patients produced IgG4 recognizing a comparable number of Ara h 2 peptides, although the dominant epitopes were distinct. As expected, patient IgE bound a larger number of Ara h 2 peptides (9.4% vs 0.9%). IgE and IgG4 epitopes recognized by patients were largely the same, and there was a positive association between IgE and IgG(4) signal, suggesting coordinate regulation. Cluster analysis of peptide binding patterns confirmed the specificity of antibody-peptide interactions and was used to define 9 core epitopes ranging from 6 to 16 residues in length-7 of which (78%) agreed with previous mapping. CONCLUSION: Epitope mapping by microarray peptide immunoassay and cluster analysis reveals interpatient heterogeneity and a more detailed map.