Lars Harder Christensen

Facilitated antigen presentation and its inhibition by blocking IgG antibodies depends on IgE repertoire complexity

BACKGROUND The antibody repertoires of allergic subjects are characterized by the presence of allergen-specific IgE antibodies. We have previously shown that the composition of the IgE repertoire is critical for allergen-mediated activation of human effector cells. Activation of CD4(+) T cells in allergic subjects is highly potentiated by the process of facilitated antigen presentation (FAP), in which allergen in complex with IgE is taken up by B cells through the low-affinity IgE receptor CD23 and presented to T cells. OBJECTIVE We sought to investigate the influence of IgE repertoire complexity on the formation of IgE/allergen/CD23 complexes on B cells and subsequent T-cell activation. METHODS Using defined allergen-specific recombinant IgE and IgG antibodies, we investigated the influence of individual IgE affinity, IgE clonality, specific IgE concentration, and the ratio between IgE specificities on IgE/allergen/CD23 complex formation in vitro. RESULTS Although IgE affinity is an important factor, IgE clonality seems to be governing complex formation, especially with medium- and low-affinity IgE antibodies. We demonstrate that differences in allergen-specific IgE affinity correlate with the efficiency of subsequent T-cell activation. In addition, we show that the complexity of an IgE repertoire also affects the ability of allergen-specific IgG antibodies to block FAP. CONCLUSION The composition of allergen-specific IgE repertoires in individual patients, especially with respect to IgE clonality, might play an important role in the manifestation of allergic disease not only for the immediate allergic reaction through activation of basophils and mast cells but also for the exacerbation of allergic inflammation through recurring activation of allergen-specific T cells by FAP.

Antibody repertoire complexity and effector cell biology determined by assays for IgE-mediated basophil and T-cell activation.

Effector cell activation and T-cell activation, the latter mediated by facilitated antigen presentation, are immunological mechanisms that play crucial roles in the manifestation and maintenance of allergic disease. In addition to their relevance for the pathogenesis of allergy in-vivo, in-vitro assays based on these immunological mechanisms have been established and used for diagnostics, for monitoring the progression of disease and for the effect of specific immunotherapy as well as for basic research purposes. Here we review different parameters that affect effector cell activation and facilitated antigen uptake and presentation, including assay designs, readout parameters and critical experimental conditions. Central to the two immunological mechanisms is complex formation between allergen-specific IgE, allergen, and cell surface-anchored immunoglobulin receptor; the high affinity IgE-receptor FcεRI on basophils and mast cells, and the low affinity IgE-receptor FcεRII (CD23) on B-cells. Accordingly, the effect of IgE repertoire complexity and allergen diversity on effector cell and facilitated antigen presentation is discussed in detail.

The complexity of allergic patients' IgE repertoire correlates with serum concentration of allergen‐specific IgE

The governing factor of both effector‐cell activation and facilitated antigen presentation is IgE‐repertoire complexity (IgE‐clonality, ‐affinity and ‐concentration). Yet, the compositions of individual IgE repertoires and correlation between IgE‐repertoire complexity and establishment of allergic sensitization remain to be determined.

Cultured human mast cells are heterogeneous for expression of the high-affinity IgE receptor FcεRI.

Objective: We determined the density of FcΕRI on mast cells cultured from cord (CBMC) and peripheral blood (PBMC) and studied the kinetics of the response through FcΕRI. Methods: Mast cells were cultured from CD133+ progenitors from peripheral or cord blood. FcΕRI was stabilized by culture with 2 µg/ml IgE. Cells were activated by addition of anti-FcΕRI antibody (1 ng/ml–10 µg/ml). Maximal activation, sensitivity, and cooperativity were determined. Results: All cultures were homogeneous for tryptase and metachromasy. All cells expressing FcΕRI could be activated by cross-linking FcΕRI to upregulate CD63. PBMC bind 203,000 molecules of IgE/cell. Stabilization of FcΕRI with IgE doubled the number of CD63+ cells (p = 0.0001) and increased the sensitivity (from 0.083 to 0.013 µg/ml anti-FcΕRI) and the slope factor (from 10.8 to 68) of PBMC but not of CBMC. Anti-IgE reversed these effects (p = 0.0002) but did not reduce activation levels below that of cell lines not stabilized with IgE. Conclusion: Baseline expression of FcΕRI is independent of anti-IgE. The fraction of PBMC that binds high levels of IgE can be activated through FcΕRI.

Immunoproteomic analysis of house dust mite antigens reveals distinct classes of dominant T cell antigens according to function and serological reactivity

House dust mite (HDM) allergens are a common cause of allergy and allergic asthma. A comprehensive analysis of proteins targeted by T cells, which are implicated in the development and regulation of allergic disease independent of their antibody reactivity, is still lacking.

Short ragweeds is highly cross-reactive with other ragweeds

BACKGROUND The most widespread ragweed (Ambrosia) species in North America are short ragweed (Ambrosia artemisiifolia; Amb a), giant ragweed (Ambrosia trifida; Amb t), and western ragweed (Ambrosia psilostachya; Amb p). Varied geographic distributions of ragweed species raise questions regarding the need for ragweed species-specific allergen immunotherapy. OBJECTIVE To determine allergenic cross-reactivity among ragweed species by immunologic analyses of sera from subjects allergic to ragweed from North America and Europe. METHODS Sera were collected from 452 subjects allergic to ragweed who participated in Amb a sublingual immunotherapy tablet clinical trials. All subjects had positive skin prick test and serum IgE against Amb a. Ragweed-specific IgE (pre treatment) and IgG4 (post treatment) were measured by ImmunoCAP. IgE inhibition studies among Amb a, Amb t, and Amb p were conducted. Using pooled sera from another ragweed-allergic population, IgE inhibition studies of 7 less widespread Ambrosia species also were conducted. RESULTS A strong correlation between Amb a vs Amb p and Amb t serum IgE levels was observed. In the vast majority of pretreatment sera, Amb a inhibited Amb a, Amb p, and Amb t IgE reactivity by more than 90%. Strong correlations were observed between Amb a vs Amb p and Amb t post-treatment IgG4 levels. In pooled sera, Amb a extract inhibited the binding of serum IgE to all 10 ragweed species by 98%-100%. CONCLUSION In a population of subjects allergic to Amb a, substantial allergenic cross-reactivity among Amb a, Amb p, and Amb t was demonstrated. These in vitro data suggest that an Amb a-based single-species ragweed allergen immunotherapy may be therapeutically active in patients exposed to diverse ragweed pollens. TRIAL REGISTRY Clinicaltrials.gov, NCT00770315, NCT00783198, and NCT00330083.

Strong and frequent T-cell responses to the minor allergen Phl p 12 in Spanish patients IgE-sensitized to Profilins

Profilins are dominant pan‐allergens known to cause cross‐sensitization, leading to clinical symptoms such as pollen‐food syndrome. This study aimed to determine the T‐cell response to Phl p 12 in profilin‐sensitized patients, by measuring the prevalence, strength and cross‐reactivity to clinically relevant profilins.