Non-eosinophilic airway hyper-reactivity in mice, induced by IFN-γ producing CD4(+) and CD8(+) lung T cells, is responsive to steroid treatment.

Non-eosinophilic asthma is characterized by infiltration of neutrophils into the lung and variable responsiveness to glucocorticoids. The pathophysiological mechanisms have not been characterized in detail. Here, we present an experimental asthma model in mice associated with non-eosinophilic airway inflammation and airway hyper-responsiveness (AHR). For this, BALB/c mice were sensitized by biolistic DNA immunization with a plasmid encoding the model antigen ß-galactosidase (pFascin-ßGal mice). For comparison, eosinophilic airway inflammation was induced by subcutaneous injection of ßGal protein (ßGal mice). Intranasal challenge of mice in both groups induced AHR to a comparable extent as well as recruitment of inflammatory cells into the airways. In contrast to ßGal mice, which exhibited extensive eosinophilic infiltration in the lung, goblet cell hyperplasia and polarization of CD4(+) T cells into Th2 and Th17 cells, pFascin-ßGal mice showed considerable neutrophilia, but no goblet cell hyperplasia and a predominance of Th1 and Tc1 cells in the airways. Depletion studies in pFascin-ßGal mice revealed that CD4(+) and CD8(+) cells cooperated to induce maximum inflammation, but that neutrophilic infiltration was not a prerequisite for AHR induction. Treatment of pFascin-ßGal mice with dexamethasone before intranasal challenge did not affect neutrophilic infiltration, but significantly reduced AHR, infiltration of monocytes and lymphocytes as well as content of IFN-γ in the bronchoalveolar fluid. Our results suggest that non-eosinophilic asthma associated predominantly with Th1/Tc1 cells is susceptible to glucocorticoid treatment. pFascin-ßGal mice might represent a mouse model to study pathophysiological mechanisms proceeding in the subgroup of asthmatics with non-eosinophilic asthma that respond to inhaled steroids.

Antigen dose-dependent suppression of murine IgE responses is mediated by CD4(-)CD8(-) double-negative T cells.

BACKGROUND: The IgE response against protein antigens is profoundly influenced by the dose used for sensitization. OBJECTIVE: The aim of the study was to identify immune cells that are involved in antigen dose-dependent regulation of IgE formation. METHODS: Wild-type mice as well as T helper (Th)1-deficient IL-12p40(-/-) and IFN-gamma(-/-) mice were immunized by repeated intraperitoneal injection of either low doses (K01 mice) or high doses (K100 mice) of keyhole limpet haemocyanin adsorbed to aluminium hydroxide. Splenocytes of immunized mice were restimulated in vitro and antigen-dependent T cell proliferation and cytokine production were measured. The frequency of regulatory T cell subsets among splenocytes from K01 and K100 mice was compared using fluorocytometry and RT-PCR analysis. Splenocytes or T cell subpopulations were transferred into naïve mice and the effect of lymphocyte transfer on IgE production after priming of recipients with low antigen doses was determined. RESULTS: Specific IgE production was considerably impaired in K100 mice. Antigenic restimulation revealed hypoproliferation of K100 splenocytes and reduced production of Th2 cytokines IL-4, IL-5 and IL-13, but no induction of IFN-gamma production. Moreover, lymphocytes from K01 and K100 mice did not show significant differences in the expression of molecules associated with the phenotype or activity of conventional regulatory T cells. Transfer of splenocytes or purified T cells from K100 mice substantially suppressed the induction of IgE production in the recipients in an antigen- and isotype-specific manner. Neither CD4(+) nor CD8(+) T cells from K100 mice were able to inhibit IgE formation; instead, we identified CD4(-)CD8(-) double-negative T cells (dnT cells) as the principal T cell population, which potently suppressed IgE production. CONCLUSION: Our data demonstrate that CD4(-)CD8(-) dnT cells play a major role in the regulation of IgE responses induced by high antigen doses.

Efficacy of recombinant adenovirus as vector for allergen gene therapy in a mouse model of type I allergy.

DNA-based immunization represents an attractive alternative approach to the current treatment of allergic diseases by specific immunotherapy with allergen extracts. In this study, we used a replication-deficient adenovirus vector (AdCMV), to examine the in vivo efficacy of preventive and therapeutic genetic immunization in a mouse model of type I allergy. Primary immunization with a recombinant adenovirus expressing the model antigen beta-galactosidase (AdCMV-(beta)gal) induced a Th1 immune response (predominance of IgG2a antibodies, high frequency of IFN-gamma producing T cells) and large numbers of cytotoxic T lymphocytes. Prophylactic vaccination with AdCMV-(beta)gal abolished the production of specific IgE following subsequent immunization with (beta)gal-protein, and skewed the Th2-biased immune response to a Th1-orientated response. In contrast, therapeutic administration of AdCMV-(beta)gal after priming with (beta)gal-protein neither significantly inhibited ongoing IgE production nor modulated a manifest Th2 immune response. Thus, allergen gene transfer via recombinant adenovirus represents an effective method to establish protection against the development of allergic disorders, but does not qualify as a therapeutic tool to interfere with ongoing high IgE production.

Deficient cytokine response of human allergen-specific T lymphocytes from humanized SCID mice and reconstitution by professional antigen-presenting cells.

BACKGROUND: Hu-PBL-SCID mice generated by the transfer of PBMCs from atopic individuals may provide a physiologic in vivo model for investigating human responses to allergens and potential approaches toward immunotherapy. OBJECTIVE: This study was undertaken to investigate the functional activity and cytokine profile of human allergen-reactive T lymphocytes isolated from hu-PBL-SCID mice. METHODS: PBMCs from allergic individuals were coinjected with allergen into SCID mice. Human lymphocyte migration and phenotype were established by reverse transcription-PCR and immunohistochemistry, IgE levels in sera were determined, and the frequency of allergen-reactive cytokine-producing T lymphocytes was established. RESULTS: After immunization with allergen, specific IgE levels in hu-PBL-SCID sera were comparable with levels in donor sera. Although the majority of lymphocytes remained in the peritoneum, significant numbers of T lymphocytes were located in the spleen, where human IL-4, IL-5, and IFN-gamma messenger RNA expression was detected after stimulation with PHA and phorbol myristate acetate. Failure to induce cytokine production by human T lymphocytes isolated from the peritoneum and spleen of hu-PBL-SCID mice by allergen was reversed by stimulating with allergen in the presence of exogenously added IL-2 and antigen-presenting cells (APC), particularly CD14(+) monocytes. Under these conditions, allergen-reactive T cells expressed a T(H)2-like phenotype. CONCLUSIONS: These data suggest that, after initial activation and induction of antibody production, human T lymphocytes enter a state of unresponsiveness, arising from a loss of human professional APC, in hu-PBL-SCID mice. The use of hu-PBL-SCID mouse models in studies on therapeutic approaches for allergy may benefit from the additional transfer of human professional APC.

Inhibition of murine IgE and immediate cutaneous hypersensitivity responses to ovalbumin by the immunomodulatory agent leflunomide.

Leflunomide has been identified as an immunoregulatory and anti-inflammatory compound. Allergic disease is characterized by elevated serum IgE levels, production of allergen-specific IgE and the release of inflammatory mediators from mast cells and granulocytes. Here we demonstrate, using an in vivo murine model, the ability of leflunomide to down-regulate levels of total and allergen-specific serum IgE production. Mice receiving leflunomide (45 mg/kg) orally at the time of primary immunization with ovalbumin adsorbed to aluminium hydroxide adjuvant, showed a reduction in total serum IgE levels of 95%, 41% and 32% following primary, secondary and tertiary immunizations, respectively (P < 0.05). When leflunomide was administered both at the time of primary and subsequent immunizations, reductions in total and specific serum IgE levels of > 80% and > 38%, respectively, were observed (P < 0.05). Administration of leflunomide to mice which had already developed an IgE response resulted in reductions in total and specific serum IgE levels of > 80% and > 45%, respectively (P < 0.05). Following leflunomide treatment, animals failed to develop immediate cutaneous hypersensitivity responses when challenged intradermally with allergen. Down-regulation of immunoglobulin production was not restricted to IgE, since levels of allergen-specific IgG1 and IgG2a in serum were also reduced. The finding of significant reductions in total and allergen-specific IgM suggests that the mechanism of action does not involve selective inhibition of immunoglobulin class switching. A loss in production of the T helper cell-derived B cell differentiation factor IL-5 may account for the reduction in immunoglobulin levels. In adoptive transfer experiments leflunomide did not induce tolerance in allergen-reactive Th2 populations, contrary to animal disease models of transplantation and autoimmunity, where leflunomide was shown to induce tolerance in the effector T cell population.

Responses of human birch pollen allergen-reactive T cells to chemically modified allergens (allergoids).

BACKGROUND: Allergoids are widely used in specific immunotherapy for the treatment of IgE-mediated allergic diseases. OBJECTIVE: The aim of this study was to analyse whether a modification of birch pollen allergens with formaldehyde affects the availability of T-cell epitopes. METHODS: Efficient modification of the allergens was verified by determining IgE and IgG binding activity using ELISA inhibition tests. T-cell responses to birch pollen allergoids were analysed in polyclonal systems, using peripheral blood mononuclear cells (PBMC) of five birch pollen-allergic individuals, as well as birch pollen extract-reactive T-cell lines (TCL), established from the peripheral blood of 14 birch pollen-allergic donors. To determine whether the modification of natural (n)Bet v 1 with formaldehyde or maleic anhydride results in epitope-specific changes in T-cell reactivities, 22 Bet v 1-specific T-cell clones (TCC), established from nine additional birch pollen-allergic individuals, were tested for their reactivity with these products. RESULTS: The majority of PBMC and TCL showed a reduced response to the birch pollen extract allergoid. Bet v 1-specific TCC could be divided into allergoid-reactive and -non-reactive TCC. No simple correlation between possible modification sites of formaldehyde in the respective T-cell epitopes and the stimulatory potential of the allergoid was observed. Mechanisms of suppression or of anergy induction were excluded as an explanation for the non-reactivity of representative TCC. All TCC could be stimulated by maleylated and unmodified nBet v 1 to a similar extent. CONCLUSION: These results demonstrate differences in the availability of T-cell epitopes between allergoids and unmodified allergens, which are most likely due to structural changes within the allergen molecule.

Heterogeneity in the polyclonal T cell response to birch pollen allergens.

BACKGROUND: Immunodominant epitopes of Bet v 1a had been identified before, using recombinant (r) Bet v 1a-reactive T cell clones generated from peripheral blood mononuclear cells of patients allergic to birch pollen. This study aimed at evaluating the T cell-stimulating capacity of immunodominant Bet v 1a-derived peptides in a polyclonal system corresponding more closely to the situation in patients. METHODS: Short-term T cell lines (TCL) were established in presence of a protein extract of birch pollen (BP extract). TCL proliferation induced by the BP extract, by natural Bet v 1, rBet v 1a, rBet v 2 or 5 selected immunodominant Bet v 1a-derived peptides was determined. RESULTS: Consistent with the knowledge that Bet v 1 is the major IgE-binding allergen of birch pollen, we found comparable T cell reactivity to natural Bet v 1 and the BP extract within the majority of the TCL. Accordingly, the response to rBet v 2 was low compared with the reactivity to the BP extract. The response of the TCL to rBet v 1a proved to be highly heterogeneous. Furthermore, the TCL response to the 5 immunodominant Bet v 1a-derived peptides showed considerable diversity. The proliferative responses of most TCL (with one exception) following stimulation by these peptides were low, in relation to the expansion induced by the BP extract. CONCLUSION: These findings argue against the use of selected peptides derived from Bet v 1a in specific immunotherapy of patients with birch allergy.