Long-term tolerance after allergen immunotherapy is accompanied by selective persistence of blocking antibodies.

BACKGROUND: Grass pollen immunotherapy for allergic rhinitis is a disease-modifying treatment that results in long-term clinical tolerance lasting years after treatment discontinuation. Active treatment is associated with generation of inhibitory grass pollen-specific IgG antibodies capable of blocking allergen-IgE interactions. OBJECTIVES: We sought to investigate the involvement of IgG-associated inhibitory antibodies with long-term clinical tolerance after discontinuation of grass pollen immunotherapy. METHODS: We conducted a 4-year study in which patients who had moderate-to-severe allergic rhinitis underwent a randomized, double-blind, placebo-controlled discontinuation of subcutaneous grass pollen immunotherapy. All subjects received grass pollen immunotherapy injections for 2 years (n = 13), followed by a further 2 years of either active (n = 7) or placebo (n = 6) injections. Clinical outcomes included seasonal symptoms and use of rescue medication. Serum specimens were collected at baseline and after 2 and 4 years for quantification of allergen-specific IgG antibodies. Sera were also tested for IgG-dependent inhibitory bioactivity against IgE-allergen binding in cellular assays by using flow cytometry and confocal microscopy to detect binding of IgE-grass pollen allergen complexes to B cells. RESULTS: Clinical improvement was maintained after 2 years of discontinuation. Although immunotherapy-induced grass pollen-specific IgG1 and IgG4 levels decreased to near-preimmunotherapy levels during discontinuation, inhibitory bioactivity of allergen-specific IgG antibodies was maintained unchanged. CONCLUSION: Grass pollen immunotherapy induces a subpopulation of allergen-specific IgG antibodies with potent inhibitory activity against IgE that persists after treatment discontinuation and that could account for long-term clinical tolerance.

The IgE-facilitated allergen binding (FAB) assay: validation of a novel flow-cytometric based method for the detection of inhibitory antibody responses.

The IgE-facilitated allergen binding (IgE-FAB) assay represents an in vitro model of facilitated allergen presentation. Allergen-IgE complexes are incubated with an EBV-transformed B cell line and complexes bound to CD23 on the surface of cells are detected by flow cytometry. The addition of serum from patients who have received allergen-specific immunotherapy has been shown previously to inhibit allergen-IgE complex binding to CD23 on B cells. In this study, we describe the characterisation and analytical validation of the grass pollen-specific IgE-FAB assay according to guidelines from the International Conference on Harmonisation. We established the intra- and inter-assay variability of IgE-FAB and have defined the detection limits of this assay. We have also demonstrated assay linearity and robustness. Using the results from a randomised double-blind placebo-controlled trial of grass pollen immunotherapy (n=33), we have defined the clinical sensitivity and specificity of the IgE-FAB assay using ROC curve analysis. In conclusion, the IgE-FAB assay is reproducible, robust, sensitive and a specific method suitable as a tool for monitoring inhibitory antibody function from patients receiving allergen immunotherapy.

Detection of Allergen-Specific IgE Antibody Responses.

Allergen-specific IgE production is the central event in the pathogenesis of atopic disorders and increases in specific IgE serum antibodies are an indicator of immediate hypersensitivity responses in humans and in animal models of allergy. Consequently, accurate and user-friendly methods are needed to measure serum levels of allergen-specific IgE. This review examines historical and recent developments in in vivo and in vitro methods for the detection of allergen-specific IgE in humans and in animal models. Routinely, in vitro methods such as enzyme-linked immunosorbant assays or radioallergosorbant tests and in vivo methods such as the skin prick test (SPT) for humans and the passive cutaneous anaphylaxis assay (PCA) used in animals are utilized to detect allergen-specific IgE. While in vivo assays are usually more accurate than in vitro assays since they provide a functional readout of IgE activity, they are relatively costly and require considerable expertise. On the other hand in vitro assays are limited by the fact that the amount of allergen-specific serum IgG exceeds IgE antibody by several orders of magnitude, resulting in competition for allergen binding. Consequently, methods that use allergen as a direct capture step are limited by the availability of free allergen binding sites for IgE. In order to circumvent this problem, in vitro methods usually require prior depletion of IgG or use high amounts of allergen in order to facilitate availability of free binding sites for IgE detection. Clearly, these approaches are limited for small sample volumes and allergens that are in short supply. New methods such as protein microarray could potentially overcome this problem by providing high allergen concentrations in a relatively small reaction volume. Currently, in vitro methods are rarely used in isolation for prognosis but are used primarily to complement the information obtained from in vivo assays. With the emergence of new technologies it is conceivable that in vitro assays may in the future replace in vivo assays, however until then in vivo assays remain the gold standard of allergen-specific IgE detection.

Mechanisms of immunotherapy: IgG revisited.

PURPOSE OF REVIEW: This paper will review historical and recent evidence for the induction of 'blocking' IgG antibodies during successful specific immunotherapy. RECENT FINDINGS: Specific immunotherapy is frequently associated with a rise in allergen-specific IgG4 antibodies and a modest reduction in specific IgE titres, although this does not always correlate with clinical efficacy. There is accumulating evidence that specific immunotherapy also influences the blocking activity on IgE-mediated responses by IgG4, and cellular assays are commonly used to investigate these changes. Recently, a novel assay, which detects allergen-IgE binding using flow cytometry, has been used to detect 'functional' specific immunotherapy-induced changes in IgG antibody activity. Results suggest that successful specific immunotherapy is associated with an increase in IgG blocking activity that is not solely dependent on the quantity of IgG antibodies. SUMMARY: Successful immunotherapy is associated with quantitative and qualitative changes in the allergen-specific IgG antibody response. The induction of IgG antibodies with blocking activity may have a protective role not only through the inhibition of allergen-induced, IgE-mediated release of inflammatory mediators from mast cells and basophils, but also through the inhibition of IgE-facilitated antigen presentation to T cells. Qualitative changes in the allergen-specific IgG antibody response may possibly be an important mechanism underlying the clinical efficacy of specific immunotherapy. Monitoring changes in blocking activity using cellular assays may give an early indication of the potential success of treatment.

Grass pollen immunotherapy induces mucosal and peripheral IL-10 responses and blocking IgG activity.

T regulatory cells and IL-10 have been implicated in the mechanism of immunotherapy in patients with systemic anaphylaxis following bee stings. We studied the role of IL-10 in the induction of clinical, cellular, and humoral tolerance during immunotherapy for local mucosal allergy in subjects with seasonal pollinosis. Local and systemic IL-10 responses and serum Ab concentrations were measured before/after a double-blind trial of grass pollen (Phleum pratense, Phl P) immunotherapy. We observed local increases in IL-10 mRNA-positive cells in the nasal mucosa after 2 years of immunotherapy, but only during the pollen season. IL-10 protein-positive cells were also increased and correlated with IL-10 mRNA(+) cells. These changes were not observed in placebo-treated subjects or in healthy controls. Fifteen and 35% of IL-10 mRNA signals were colocalized to CD3(+) T cells and CD68(+) macrophages, respectively, whereas only 1-2% of total CD3(+) cells and 4% of macrophages expressed IL-10. Following immunotherapy, peripheral T cells cultured in the presence of grass pollen extract also produced IL-10. Immunotherapy resulted in blunting of seasonal increases in serum allergen Phl p 5-specific IgE, 60- to 80-fold increases in Phl p 5-specific IgG, and 100-fold increases in Phl p 5-specific IgG4. Post-immunotherapy serum exhibited inhibitory activity, which coeluted with IgG4, and blocked IgE-facilitated binding of allergen-IgE complexes to B cells. Both the increases in IgG and the IgG "blocking" activity correlated with the patients' overall assessment of improvement. Thus, grass pollen immunotherapy may induce allergen-specific, IL-10-dependent "protective" IgG4 responses.

Inhibition of allergen-IgE binding to B cells by IgG antibodies after grass pollen immunotherapy.

BACKGROUND: Among atopic individuals, levels of allergen-specific IgG antibodies have been inversely associated with the degree of allergen sensitization. Additionally, allergen-specific IgG antibodies are markedly increased by allergen injection immunotherapy. These observations have led to proposals that allergen-specific IgG antibodies might have protective properties in atopic individuals. OBJECTIVE: We hypothesized that after grass pollen immunotherapy, these antibodies disrupt IgE-dependent allergen processing by antigen-presenting cells. METHODS: We have developed a novel flow cytometric assay based on detection of allergen-IgE binding to the low-affinity IgE receptor on B cells to examine the blocking effects of sera collected from 18 patients who participated in a double-blind, controlled trial of grass pollen immunotherapy for 1 year. RESULTS: In all 10 patients who received active therapy, there was induction of activity that inhibited allergen-IgE binding to B cells (P =.02, vs placebo subjects), as well as subsequent allergen presentation to T cells. This activity copurified with IgG and was allergen specific, because sera taken from patients treated with grass pollen immunotherapy but who were also birch pollen sensitive did not inhibit IgE-birch pollen allergen binding to B cells. CONCLUSION: We conclude that allergen-specific IgG antibodies induced by immunotherapy can disrupt formation of allergen-IgE complexes that bind to antigen-presenting cells and facilitate allergen presentation.

Grass pollen immunotherapy for hayfever is associated with increases in local nasal but not peripheral Th1:Th2 cytokine ratios.

Grass pollen immunotherapy is the only treatment for hayfever that is both effective and confers long-term benefit. Immunotherapy may act by altering the local nasal mucosal T helper type 2 (Th2) to type 1 (Th1) cytokine balance either by down-regulation and/or immune deviation of T-lymphocyte responses. There is controversy as to whether these changes are detectable in peripheral blood. We therefore examined both local nasal and peripheral T-cell responses to allergen exposure in the same subjects before and after immunotherapy. In a double-blind trial of grass pollen immunotherapy, nasal biopsies were obtained at baseline and during the peak pollen season following 2 years of immunotherapy. Placebo-treated patients showed a seasonal increase in CD3(+) T cells (P = 0.02) and in interleukin-5 (IL-5) mRNA(+) cells (P = 0.03) and no change in interferon-gamma (IFN-gamma ) mRNA(+) cells (P = 0.2) in the nasal mucosa. In contrast, in the immunotherapy-treated group, there were no changes in the number of CD3(+) T cells (P = 0.3) and IL-5 mRNA+ cells (P = 0.2) but a significant increase in the number of IFN-gamma mRNA(+) cells (P = 0.03). Furthermore, clinical improvement in the immunotherapy-treated group was accompanied by a seasonal increase in the ratio of IFN-gamma to IL-5 mRNA(+) cells in the nasal mucosa (P = 0.03). In contrast, there were no significant changes in peripheral T-cell proliferative responses or cytokine production for IFN-gamma or IL-5 in response to grass pollen either within or between the two treatment groups. We conclude that successful grass pollen immunotherapy was associated with an increase in the ratio of IFN-gamma to IL-5 mRNA(+) cells in the nasal mucosa, whereas these changes were not reflected by alterations in peripheral blood T-cell proliferative responses or cytokine production before/after treatment.