Beatrice Jahn-Schmid

OLIGODEOXYNUCLEOTIDES CONTAINING CPG MOTIFS INDUCE IL-12, IL-18 AND IFN-GAMMA PRODUCTION IN CELLS FROM ALLERGIC INDIVIDUALS AND INHIBIT IGE SYNTHESIS IN VITRO

The effects of phosphorothioate oligonucleotides containing CpG motifs (CpG‐ODN) on cultured cells from allergic patients and non‐atopic individuals were investigated. In peripheral blood mononuclear cells (PBMC) CpG‐ODN led to a significant increase of IFN‐γ. By intracellular cytokine staining, IFN‐γ production could be attributed to NK cells and inhibition experiments indicated an IL‐12‐dependent mechanism. Moreover, CpG‐ODN increased mRNA expression of IL‐12 and IL‐18 in PBMC. In this respect, no significant difference between allergic and non‐atopic individuals was observed. Monocyte‐derived dendritic cells were identified as one IL‐12‐ and IL‐18‐producing source. In addition, stimulation of PBMC derived from atopic patients with CpG‐ODN led to a considerable increase of polyclonal IgG and IgM synthesis. In contrast, the production of total IgE was suppressed. CpG‐ODN induced a significant rise of IgG and IgM specific for allergens to which the patients were sensitized, whereas allergen‐specific IgE levels remained unchanged. Our data suggest that CpG‐ODN display a strong influence on the ongoing immune response and might represent potential adjuvants for specific immunotherapy of type I allergy.

Allergen microarray: comparison of microarray using recombinant allergens with conventional diagnostic methods to detect allergen‐specific serum immunoglobulin E

Background The availability of recombinant allergens and recent advances in biochip technology led to the development of a novel test system for the detection of allergen‐specific IgE.

Bet v 1, the major birch pollen allergen, initiates sensitization to Api g 1, the major allergen in celery: evidence at the T cell level.

Due to IgE cross‐reactivity, birch pollen‐allergic individuals frequently develop typeu2004I hypersensitivity reactions to celery tuber. We evaluated the Tu2004cell response to the major allergen in celeriac, Apiu2004gu20041, and the cellular cross‐reactivity with its homologous major allergen in birch pollen, Betu2004vu20041. Apiu2004gu20041‐specific Tu2004cell lines (TCL) and clones (TCC) were established from peripheralblood mononuclear cells of allergic patients. Epitope mapping of Apiu2004gu20041 with overlapping Apiu2004gu20041‐derived peptides revealed one dominant Tu2004cell‐activating region, Apiu2004gu20041109–126. TCL and TCC generated with Apiu2004gu20041 cross‐reacted with the birch pollen allergen and, although initially stimulated with the food allergen, cellular responses to Betu2004vu20041 were stronger than to Apiu2004gu20041. Epitopemapping with Betu2004vu20041‐derived peptides revealed that Tu2004cells specific for several distinct epitopes distributed over the complete Betu2004vu20041 molecule could be activated by Apiu2004gu20041. Betu2004vu20041109–126 was identified as the most important Tu2004cell epitope for cross‐reactivity with Apiu2004gu20041. This epitope shares 72% amino acid sequence similarity with the major Tu2004cell‐activating region of the food allergen, Apiu2004gu20041109–126. Our data provide evidence that humoral as well as cellular reactivity to the major celery allergen is predominantly based on cross‐reactivity with the major birch pollen allergen. The activation of Betu2004vu20041‐specific Th2 cells by Apiu2004gu20041, in particular outside the pollen season, may have consequences for birch pollen‐allergic individuals.

Modulation of the allergic immune response in BALB/c mice by subcutaneous injection of high doses of the dominant T cell epitope from the major birch pollen allergen Bet v 1.

Several in vitro and in vivo studies indicate that application of high doses of dominant T cell epitopes can induce a state of antigen‐specific non‐responsiveness (anergy). In the present study, we developed a murine model of an allergic immune response to Bet v 1, the major birch pollen allergen. Mice were sensitized by injection of rBet v 1 and the allergic state was proven by the presence of allergen‐specific IgE and positive immediate‐type skin tests to Bet v 1. In epitope mapping experiments, an immunodominant T cell epitope of Bet v 1 in BALB/c mice was identified by the use of overlapping peptides. This peptide (BV139) was subsequently employed for treatment. Two tolerization protocols were used: in one approach, the peptide was administered to naive mice before immunization (group BV139‐S), in the second, already sensitized mice were treated (S‐BV139). The results demonstrated that administering high doses of the dominant T cell epitope of Bet v 1 profoundly diminished T cell proliferation to the peptide in the BV139‐S group, and to the peptide as well as to the whole protein in the S‐BV139 group. Skin test reactivity to Bet v 1 was reduced in the BV139‐S group. However, no differences in terms of specific antibody production between treated and untreated mice could be observed. This study provides evidence that administration of dominant T cell epitopes can down‐regulate the allergen‐specific T cell response. Proceeding on the assumption that the T lymphocyte response to allergens is crucial for the induction and maintenance of the allergic disease, a modulation of the immune response to allergens by treatment with T cell epitope peptides could represent a promising concept for immunotherapy in the future.

The T Cell Response to Art v 1, the Major Mugwort Pollen Allergen, Is Dominated by One Epitope

Mugwort (Artemisia vulgaris) pollen allergens represent the main cause of pollinosis in late summer in Europe. At least 95% of sera from mugwort pollen-allergic patients contain IgE against a highly glycosylated 24- to 28-kDa glycoprotein. Recently, this major allergen, termed Art v 1, was characterized, cloned in Escherichia coli, and produced in recombinant form. In the present study we characterized and compared the T cell responses to natural (nArt v 1) and recombinant Art v 1 (rArt v 1). In vitro T cell responses to nArt v 1 and rArt v 1 were studied in PBMC, T cell lines (TCL), and T cell clones (TCC) established from PBMC of mugwort-allergic patients. Stimulation of PBMC or allergen-specific TCL with either nArt v 1 or rArt v 1 resulted in comparable proliferative T cell responses. Eighty-five percent of the TCC reactive with rArt v 1 cross-reacted with the natural protein. The majority of the CD4+CD8−TCR αβ+ Art v 1-specific TCC, obtained from 10 different donors, belonged to the Th2 phenotype. Epitope mapping of TCL and TCC using overlapping peptides revealed a single immunodominant T cell epitope recognized by 81% of the patients. Inhibition experiments demonstrated that the presentation of this peptide is restricted by HLA-DR molecules. In conclusion, the T cell response to Art v 1 is characterized by one strong immunodominant epitope and evidently differs from the T cell responses to other common pollen allergens known to contain multiple T cell epitopes. Therefore, mugwort allergy may be an ideal candidate for a peptide-based immunotherapy approach.

Oligodeoxynucleotides containing CpG motifs modulate the allergic TH2 response of BALB/c mice to Bet v 1, the major birch pollen allergen

BACKGROUNDnThe use of adequate adjuvants to modulate the allergic T(H2)-type immune response is a promising concept for future immunotherapy of type I allergy. Bacterial DNA or oligodeoxynucleotides containing CpG motifs (CpG-ODNs) have been demonstrated to foster T(H1)-type immune responses.nnnOBJECTIVEnWe investigated the adjuvanticity of CpG-ODNs and their capability to modulate the allergic T(H2) response in a mouse model.nnnMETHODSnBALB/c mice were treated with CpG-ODNs and Bet v 1, the major birch pollen allergen, in different experimental setups. Allergen-specific antibody responses, T(H) cytokines, and eosinophilic infiltration of the airways were investigated.nnnRESULTSnIntraperitoneal administration of Bet v 1 together with aluminium hydroxide led to a typical T(H2) response. In contrast, coadminstration of CpG-ODNs with Bet v 1 in aluminium hydroxide resulted in markedly increased T(H1) activities (high IgG2a levels) and subsequently to reduced airway inflammation. The T(H1)-like immune response indicated by these humoral findings was also reflected by decreased IL-5 and increased IFN-gamma levels in cell cultures. CpG-ODNs as sole adjuvants with Bet v 1 did not lead to measureable Ig responses after subcutaneous or intraperitoneal immunizations; after intranasal application, 3 of 10 mice reacted. Nevertheless, a prophylactic effect was obtained with all routes tested; that is, mice treated subsequently with an established aerosol sensitization protocol displayed altered immune responses characterized by drastically elevated levels of Bet v 1-specific IgG2a, indicating a T(H1)/T(H0)-like immunity. Application of CpG-ODNs after aerosol sensitization also induced IgG2a.nnnCONCLUSIONnBy inducing T(H1)/T(H0)-biased immune responses to allergens, the use of CpG-ODNs as adjuvants may have important impacts for new forms of specific immunotherapy in type I hypersensitivity.

Immunoreactivity of allergen (Bet v 1) conjugated to crystalline bacterial cell surface layers (S-layers)

BACKGROUNDnCrystalline cell surface layers (S-layers) from Gram-positive eubacteria had been demonstrated as carrier/adjuvants for chemically synthesized tumor-associated oligosaccharide haptens and capsular polysaccharide antigens of Streptococcus pneumoniae strains.nnnOBJECTIVESnThe applicability of S-layers as vaccine carrier for treatment of Type I allergy was investigated.nnnSTUDY DESIGNnNative or cross-linked S-layer self-assembly products and cell wall preparations from Bacillus sphaericus CCM 2177 and Thermoanaerobacter thermohydrosulfuricus L111-69 and L110-69 were used for immobilization of recombinant major birch pollen allergen Bet v 1.nnnRESULTS AND CONCLUSIONSnDepending on the carrier used, amounts of approximately 20-40 micrograms allergen per mg conjugate could be immobilized. By application of L-glutamic acid dimethyl ester as a spacer, this value could be increased approximately 10-fold. The functionality of the rBet v 1-conjugates was assessed in immunological systems. (i) The presence of intact B-cell epitopes was demonstrated in inhibition experiments using human Bet v 1-specific IgE. (ii) The rBet v 1-S-layer conjugates were immunogenic in mice. (iii) The proliferation of rBet v 1-specific T-cell clones suggested that the peptides created by processing of immobilized Bet v 1 were similar to those derived from natural allergen. (iv) Stimulation of human allergen-specific TH2 lymphocytes with S-layer-conjugated Bet v 1 led to a modulation of the cytokine production pattern from TH2 to TH0/TH1. This study indicates that S-layers may be suitable carriers for few immunotherapeutical vaccines for Type 1 hypersensitivity.

B and T Cell Responses to the Spliceosomal Heterogeneous Nuclear Ribonucleoproteins A2 and B1 in Normal and Lupus Mice

Autoantibodies directed against spliceosomal heterogeneous nuclear ribonucleoproteins (hnRNPs) are a typical feature of rheumatoid arthritis, systemic lupus erythematosus, and mixed-connective tissue disease. With the aim of investigating a potential pathogenic role of these Abs, we have studied the Ab response to A2/B1 hnRNPs in different murine models of lupus. The specificity of anti-A2/B1 Abs was tested with a series of 14 overlapping synthetic peptides covering the region 1–206 of A2 that contains most of the epitopes recognized by patients’ Abs. A major epitope recognized very early during the course of the disease by Abs from most of MRL lpr/lpr mice but not from other lupus mice and from mice of different MHC haplotypes immunized against B1 was identified in residues 50–70. This peptide contains a highly conserved sequence RGFGFVTF also present in other hnRNPs and small nuclear ribonucleoproteins. Abs reacting with a second A2 epitope identified in residues 35–55 were detectable several weeks later, suggesting an intramolecular B cell epitope spreading during the course of the disease. We identified several T cell epitopes within the region 35–175 that generated an effective Th cell response with IL-2 and IFN-γ secretion in nonautoimmune CBA/J mice sharing the same MHC haplotype H-2k as MRL/lpr mice. None of the peptides stimulated T cells primed in vivo with B1. Because Abs to peptide 50–70 were detected significantly earlier than Abs reacting with other A2 peptides and the protein itself, it is possible that within the protein, this segment contains residues playing an initiator role in the induction of the anti-A2/B1 and antispliceosome Ab response.

Modulation of an Allergic Immune Response via the Mucosal Route in a Murine Model of Inhalative Type–I Allergy

A murine model of aerosol inhalation, leading to sensitization to birch pollen (BP) and its major allergen Bet v 1, was established in order to try to influence type–I allergic immune responses via the mucosal route. We previously demonstrated that simultaneous inhalation of BP and cholera toxin, a potent mucosal adjuvant, induced a Th1–like immune response to the allergen in naive mice and modulated allergic immune responses in sensitized mice. In contrast to cholera holotoxin, mucosal application of the cholera B subunit (CTB) conjugated to antigen has been shown to induce peripheral tolerance in certain models of Th1–based autoimmune diseases. In the present study we investigated the potential of such an antigen delivery system to suppress Th2–based, allergic immune responses. Mucosal administration of CTB/Bet v 1 conjugates prior to sensitization led to significantly increased allergen–specific IgE/IgG1 and IgG2a antibody levels and cytokine production (IL–5, IFN–γ) in vitro. Thus, CTB coupled to Bet v 1 acted as an adjuvant rather than a tolerogen. On the other hand we noted that mucosal application of CTB coupled to ovalbumin led to marked suppression of antigen–specific IgE antibody levels and IL–5 production in vitro and thereby restricted allergic sensitization. These results indicated that the effects of CTB/antigen conjugates depended on the nature of the antigen. In contrast to Bet v 1 coupled to CTB, nasal as well as oral application of low doses of unconjugated, Bet v 1 prior to aerosol sensitization inhibited allergen–specific antibody responses of all isotypes, cutaneous type–I skin tests in vivo as well as allergen–specific lymphoproliferative responses and cytokine production (IL–4, IL–5, IL–10, IFN–γ) in vitro, suggesting that both T– and B–cell tolerance to the allergen were induced. Taken together, mucosal tolerance induction as well as the use of certain transmucosal antigen delivery systems might be promising new strategies to modulate type–I allergic immune responses

Characterization of autoreactive T cells to the autoantigens hnRNP-A2/RA33 and filaggrin in patients with rheumatoid arthritis and controls

In an attempt elucidate the role of autoimmune processes in the pathogenesis of rheumatoid arthritis (RA) we investigated the T cell responses to two autoantigens targeted by autoantibodies of patients with RA, (i) the heterogeneous nuclear ribonucleoprotein (hnRNP) A2/RA33 and (ii) filaggrin which is one of the target structures recognized by anti-citrulline antibodies. n nStimulation assays were performed with peripheral blood mononuclear cells of 50 RA patients, 20 patients with osteoarthritis and 21 healthy control individuals using recombinant hnRNP-A2/RA33 as well as some fragments thereof and recombinant filaggrin both in unmodified and citrullinated form. Antigen-specific T cell clones (TCC) were obtained by cultivating T cell lines in the presence of antigen and IL-2 followed by limiting-dilution cloning. n nProliferative responses to hnRNP-A2/RA33 were seen in 60% of the RA patients with a mean stimulation index (SI) of 3.5 ± 2.8 and were significantly higher than those observed in the control group (mean SI=1.7 ± 1, P < 0.00005). There was no correlation with the presence of anti-A2/RA33 autoantibodies nor with MHC genes, although more than 60% of the responsive patients carried the shared epitope. Results obtained with recombinant fragments indicated a major T cell epitope to be located in the N-terminal first RNA binding domain of the protein. Anti-A2/RA33 specific TCC (n = 16) derived from RA patients were almost exclusively CD4?, whereas only 7 of 12 TCC derived from controls showed this phenotype, and secreted high amounts of IFNg upon antigen stimulation as did all TCC derived from controls. Proliferative responses to filaggrin in either form were seen in only 25% of the RA patients tested and did not differ from those observed in the control group indicating that filaggrin-reactive T cells do presumably not drive the autoantibody response to citrullinated antigens. n nTaken together, a Th1 type autoimmune response to hnRNP-A2/RA33 was commonly observed in RA patients suggesting this nuclear protein to constitute a major T cell autoantigen which might be fuelling one of the pathological autoimmune reactions that drive the destructive processes effective in RA.