SummaryA cDNA encoding an allergenic protein was isolated from an Aspergillusfumigatus (/1. fumigatus) cDNA library displayed on the surface of filamentous phage. Serum immunoglobulin E (IgE) from A. fumigatus--sensitized individuals was used to enrich phage-expressing gene products binding to IgE. One of the cDNAs encoded a 26.7-kD protein that was identified as a manganese superoxide dismutase (MnSOD) sharing 51.5% identity and 67.2% homology to the corresponding human enzyme. Both human and A. fumigatus MnSOD coding sequences were expressed in Escherichia coli as [His]6-tagged fusion proteins and purified by Ni2+-chelate affinity chromatography. The two recombinant MnSODs were both recognized by IgE antibodies from subjects allergic to the A. fumigatus MnSOD and elicited specific immediate type allergic skin reactions in these individuals. Moreover, both human and A. fumigatus MnSOD induced proliferation in peripheral blood monouuclear cells of A. fumigatus-allergic subjects who showed specific IgE responses and reacted in skin tests to MnSOD. These observations provide evidence for autoreactivity to the human MnSOD in allergic persons sensitized to an environmental allergen from A.fumigatus who share a high degree of sequence homology to the corresponding human enzyme.

IL‐10 induces T cell anergy in numerous mouse models and specific immunotherapy of allergy in humans. Here, we demonstrate that IL‐10 directly acts on T cells which are stimulated via CD28 by efficiently blocking proliferation and cytokine production. T cells tolerized by IL‐10 showed high viability and the unresponsive state was reversed by anti‐CD3 monoclonal antibody (mAb) stimulation and IL‐2, but not by anti‐CD28 mAb stimulation. Signal transduction via CD28 requires CD28 tyrosine phosphorylation and binding of phosphatidylinositol 3‐kinase. IL‐10 inhibited tyrosine phosphorylation of CD28; thus, the phosphatidylinositol 3‐kinase binding to CD28 was blocked. Consequently, IL‐10 inhibited the antigen‐induced secretion of both Th1 and Th2 cytokines, including IL‐2, IFN‐γ, IL‐4, IL‐5 and IL‐13. Furthermore, neutralization of endogenously produced IL‐10 significantly increased T cell proliferation and both Th1 and Th2 cytokine production in vitro. Using superantigen stimuli, T cell suppression by IL‐10 was merely induced at low doses when co‐stimulation by CD28 was essential. Together, these data demonstrate that IL‐10 directly acts on the CD28 signaling pathway and this represents an important T cell suppression mechanism leading to anergy.

Specific immune suppression and induction of anergy in T cells are essential processes in regulation and circumvention of immune defense. IL‐10, a suppressor cytokine of T cell proliferative and cytokine responses, plays a key regulatory role in tolerizing exogenous antigens during specific immunotherapy and natural exposure. The present study demonstrates that IL‐10 induces T cell suppression by blocking the CD28 costimulatory signal. T cell receptor counting and T cell proliferation studies by anti‐CD3 and anti‐CD28 stimulation in the presence or absence of IL‐10 revealed that IL‐10 only inhibits T cells stimulated by low numbers of triggered T cell receptors and that depend on CD28 costimulation. T cells receiving a strong signal by the T cell receptor alone and that do not require CD28 costimulation are therefore not affected by IL‐10. Coprecipitation experiments demonstrated that CD28 and the IL‐10 receptor are associated in activated T cells. IL‐10 inhibited CD28 tyrosine phosphorylation, the initial step of the CD28 signaling pathway. In consequence, phosphatidylinositol 3‐kinase p85 binding to CD28 was inhibited. Thus, IL‐10‐induced selective inhibition of the CD28 costimulatory pathway demonstrates a decisive mechanism in determining whether a T cell will contribute to an immune response or become anergic.

Enhanced production of T helper (Th)2 cytokines by allergen-specific Th cells plays a major role in the induction and maintenance of IgE-mediated allergic disorders. The mechanism that triggers this type of response in atopic individuals is not fully understood. Allergen-specific human Th cell clones produce interleukin (IL)-4 and low or undetectable levels of interferon (IFN)-gamma after stimulation with low concentrations of antigen. However, these Th cell clones are capable of generating significant amounts of IFN-gamma after optimal activation through their T cell receptor (TcR). Allergen-specific Th cell clones isolated from allergic individuals required higher doses of antigen to reach the plateau of proliferation and to generate Th0 cytokine responses than their counterparts isolated from nonallergic subjects. On the other hand, if allergen was replaced by anti-CD3 monoclonal antibody (mAb), both allergic and nonallergic Th cell clones attained the highest level of proliferation and significant IFN-gamma production in response to equivalent concentrations of anti-CD3 mAb. These results indicate that the strength of T cell ligation, which can be modulated by the availability of the TcR ligand, controls the balance of Thl/Th2 cytokines produced by memory Th cells in vitro. In the particular case of bee venom phospholipase A2, it is shown that the expression of allergen-specific surface Ig on antigen-presenting B cells has little influence on antigen uptake and therefore in determining the levels of T cell activation and cytokine production. Alternatively, the affinity of particular major histocompatibility complex class II molecules on antigen-presenting cells for allergen-derived peptides might determine the amount of specific ligand presented to the Th cells and play a decisive role skewing the Th cell cytokine production towards Th1 or Th2 phenotypes. These findings, which are consistent with the changes in cytokine patterns observed following clinical hyposensitization, suggest that polarized human Th2 cell subsets still retain the capacity to modulate their cytokine pattern.

Greater clinical benefit in controlling the symptoms of asthma is frequently observed through combining moderate doses of inhaled glucocorticoids together with long-acting beta(2)-agonists, as compared with increasing glucocorticoid dosage alone. To address in vitro whether glucocorticoids plus beta(2)-agonists, compared with glucocorticoids alone, have greater inhibitory activity on CD4+ T cell responses to allergen, peripheral blood CD4+ T cell responses to allergen were compared in the presence or absence of the glucocorticoid fluticasone proprionate and the short- and long-acting beta(2)-agonists salbutamol and salmeterol, respectively. Fluticasone proprionate inhibited interleukin (IL)-5 and IL-13 and enhanced IL-10 synthesis in allergen-stimulated cultures in a concentration-dependent manner. Salmeterol, but not salbutamol, inhibited IL-5 and IL-13 and enhanced IL-10 synthesis in these cultures. When used in combination the two drugs demonstrated an additive effect on this pattern of cytokine production. Allergen-specific T cell lines induced in the presence of salmeterol and fluticasone proprionate inhibited IL-5 and IL-13 production by allergen-specific Th2 cell lines in an IL-10-dependent manner. Thus fluticasone proprionate and salmeterol increased IL-10 and reduced Th2 cytokine synthesis additively in allergen stimulated human CD4+ T cells.

Background: Manganese superoxide dismutase (MnSOD) from Aspergillus fumigatus has been demonstrated to be an allergen showing a high degree of homology with phylogenetically distant MnSODs. We describe cloning, production and characterization of MnSODs from different species. Methods: MnSODs were cloned by PCR, expressed as inclusion body protein in Escherichia coli, purified by Ni²⁺-chelate affinity chromatography and characterized. Results: The MnSODs from A. fumigatus, man, yeast, Drosophila melanogaster and E. coli show about 50% identity and 70% similarity on the primary structure. All proteins were produced at a high level in E. coli and refolded to achieve enzymatic activity. The proteins were able to bind IgE from sera of individuals sensitized to the A. fumigatus MnSOD. Conclusions: MnSOD represents a novel pan-allergen restricted to individuals sensitized to A. fumigatus.

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The elicitation of an immune response to protein antigens depends on the specific recognition of antigenic determinants (epitopes) by T and B lymphocytes. Bee venom phospholipase A₂ (PLA) represents the major antigen/allergen of honey bee venom. It displays three dominant immunogenic peptide and one glycopeptide T cell recognition sites. These epitopes are equally recognized by both allergic and nonallergic individuals. A mixture of the three epitope containing peptides was successfully used in specific immunotherapy of bee venom–allergic patients. Both peptide and whole bee venom immunotherapy induced a state of specific anergy in T cells. The production of specific IgE and IgG4 antibodies directly correlated with the secreted interleukin–4:γ–interferon (IL–4:IFNγ) ratio, which itself depended on the concentration of available antigen and the strength of the T cell–activating signal. This signal comprises accumulated molecular interactions delivered by engagement of the antigenic peptide/MHC class II complex with the T cell receptor (TcR). Indeed the thermodynamic laws of chemical equilibrium reactions reveal that the antigen concentration, together with the equilibration constant K_i and the related Gibbs standard free energy ΔG° of the MHC–II/Ag/TcR complex reaction, may govern the secreted IL–4:IFNγ ratio, and in consequence, differential IgE and IgG4 antibody formation. K_i includes epitope and MHC–II haplotype variability and therefore represents a measure of immunological individuality. A major B cell epitope was determined by using point–mutated PLA. Specific antigen recognition by B cells can trigger distinct cytokine profiles in T cells and contribute to the differential regulation of specific IgE and IgG4 antibodies. Our results indicate that distinct cytokine profiles inducing allergic and nonallergic responses can be attributed to thresholds of T cell activation generated by the specific binding properties of individual MHC–II molecules to immunogenic T cell epitopes and their presentation to TcR.