Christian Möbs

Birch Pollen Immunotherapy Leads to Differential Induction of Regulatory T Cells and Delayed Helper T Cell Immune Deviation

Correction of an imbalance between allergen-specific T cell subsets is considered a critical event in establishing allergen tolerance by specific immunotherapy (SIT). In a comprehensive, longitudinal study, distinct T cell populations and Ig subtypes were analyzed in subjects allergic to birch pollen during decisive time points of SIT (i.e., induction and maintenance phase), as well as in and out of birch pollen season. An increase in Bet v 1-specific, IL-10–secreting T cells, fulfilling the criteria of inducible type 1 regulatory T (Tr1) cells, was observed by the end of the induction phase; this resulted in a decreased ratio of allergen-specific IL-5+ Th2/Tr1 cells. In contrast, CD4+CD25+CD127low regulatory T cell numbers did not change. Furthermore, enhanced concentrations of allergen-specific IgG Abs were observed, whereas allergen-specific IgE and IgA levels remained unchanged. After 1 y of SIT, a reduced ratio of allergen-specific Th2/IFN-γ+ Th1 cells was apparent. Although untreated and SIT-treated allergic subjects developed enhanced Th2 cell responses during birch pollen season, only SIT-treated patients experienced elevated numbers of allergen-specific Tr1 cells, which were associated with reduced skin prick test reactivity and diminished clinical symptoms. In coculture assays, allergen-specific Tr1 cells showed an IL-10– and dose-dependent inhibition of CD4+CD25− T effector cells. Thus, SIT has differential effects on regulatory T cell subsets, resulting in an early induction of allergen-specific Tr1 cells associated with an increase in allergen-specific IgG, and it leads to a delayed shift from an allergen-specific Th2- to a Th1-dominated immune response.

Specific immunotherapy modifies allergen-specific CD4+ T-cell responses in an epitope-dependent manner

BACKGROUND Understanding the mechanisms by which the immune system induces and controls allergic inflammation at the T-cell epitope level is critical for the design of new allergy vaccine strategies. OBJECTIVE We sought to characterize allergen-specific T-cell responses linked with allergy or peripheral tolerance and to determine how CD4(+) T-cell responses to individual allergen-derived epitopes change over allergen-specific immunotherapy. METHODS Timothy grass pollen (TGP) allergy was used as a model for studying grass pollen allergies. The breadth, magnitude, epitope hierarchy, and phenotype of the DR04:01-restricted TGP-specific T-cell responses in 10 subjects with grass pollen allergy, 5 nonatopic subjects, and 6 allergy vaccine-treated subjects was determined by using an ex vivo peptide-MHC class II tetramer approach. RESULTS CD4(+) T cells in allergic subjects are directed to a broad range of TGP epitopes characterized by defined immunodominance hierarchy patterns and with distinct functional profiles that depend on the epitope recognized. Epitopes that are restricted specifically to either TH2 or TH1/TR1 responses were identified. Allergen-specific immunotherapy was associated with preferential deletion of allergen-specific TH2 cells and without a significant change in the frequency of TH1/TR1 cells. CONCLUSIONS Preferential allergen-specific TH2 cell deletion after repeated high-dose antigen stimulation can be another independent mechanism to restore tolerance to allergen during immunotherapy.

Birch pollen immunotherapy results in long-term loss of Bet v 1–specific TH2 responses, transient TR1 activation, and synthesis of IgE-blocking antibodies

BACKGROUND Early events of specific immunotherapy (SIT) are induction of allergen-specific IL-10-producing T(R)1 cells and production of IgG antibodies, but there is little knowledge about the long-term immune mechanisms responsible for sustained allergen tolerance. OBJECTIVE Bet v 1-specific immune responses of 16 patients with birch pollen allergy were characterized up to 54 months at defined time points before, during, and after a 3-year period of SIT. METHODS We sought to analyze allergen-specific T- and B-cell responses. Bet v 1-specific IL-5-, IFN-γ-, and IL-10-secreting T cells were quantified in peripheral blood, and birch pollen-specific IgE and IgG antibody levels were determined in serum. Furthermore, the inhibitory capacity of SIT-induced IgG was evaluated by blocking allergen binding to IgE and inhibition of facilitated allergen presentation. RESULTS Seasonal increases in Bet v 1-specific T(H)2 cell numbers ceased to appear after the first year of SIT without deviation to a T(H)1-dominated immune response. Furthermore, the frequency of IL-10-producing T(R)1 cells, which had increased during the first year of SIT, returned to pretreatment levels in the second year. In contrast, allergen-specific IgG antibody concentrations continuously increased during SIT but started to decrease after cessation of treatment. Functional analysis confirmed the ability of the IgG antibodies to inhibit IgE-allergen interactions, which peaked at the end of SIT but then slowly started to decrease. CONCLUSION Long-term allergen tolerance achieved by SIT is associated with the development of peripheral T-cell tolerance characterized by decreased reactivity of Bet v 1-specific T(H)2 cells and enriched allergen-specific IgG competing with IgE antibodies for allergen binding.

Kinetics, cross-reactivity, and specificity of Bet v 1-specific IgG4 antibodies induced by immunotherapy with birch pollen

IgE antibodies specific for the major birch pollen allergen frequently cross‐react with Bet v 1 homologous food proteins, for example Cor a 1 in hazelnut and Mal d 1 in apple. Specific immunotherapy with birch pollen (BP‐SIT) induces IgG4 antibodies that inhibit IgE binding to Bet v 1. However, information on cross‐reactivity of BP‐SIT‐induced Bet v 1‐specific IgG4 antibodies with food allergens is limited. In this study, we investigated the kinetics of production, cross‐reactivity, and IgE‐blocking activity of Bet v 1‐specific IgG4 antibodies emerging during conventional BP‐SIT and whether IgG4‐epitopes overlapped with IgE epitopes.

Cellular and Humoral Mechanisms of Immune Tolerance in Immediate-Type Allergy Induced by Specific Immunotherapy

The management of immediate-type allergy (ITA) is based on allergen avoidance, symptomatic pharmacological therapy and specific immunotherapy (SIT). Among these, SIT presents the only curative treatment. The efficacy of SIT in the treatment of IgE-mediated ITA has been proven in numerous clinical studies and is well established. This review discusses the relevance of immunoregulative humoral and cellular mechanisms leading to immune tolerance in ITA. Special focus is placed on the role of antibodies potentially interfering with the IgE-mediated immune reaction and regulatory T (Treg) cells including their immunosuppressive cytokines, which play a critical role in shifting the T helper 2 cell-driven allergic immune response towards allergen tolerance. Distinct subsets of constitutive and inducible Treg cells have been identified inhibiting the activation of allergen-specific effector T cells via cell contact- or cytokine-dependent suppression. Current research suggests that both inducible interleukin-10-producing CD4+ Treg cells and naturally occurring CD4+CD25+ Treg cells actively control allergic responses and that the disturbance of their function or number may contribute to the development or progression of allergy. Thus, the fine balance between allergen-specific T helper 2 and Treg cells constitutes a critical factor for the successful treatment of ITA by SIT.

T and B cells target identical regions of the non-collagenous domain 1 of type VII collagen in epidermolysis bullosa acquisita.

Epidermolysis bullosa acquisita (EBA) is a severe immunobullous disease and is caused by IgG against type VII collagen (Col VII) of anchoring fibrils. In this study, utilizing ELISA and immunoblot, 13/15 EBA sera but 0/20 bullous pemphigoid sera and 0/30 healthy control sera showed IgG reactivity with distinct recombinant subregions of the non-collagenous domain 1 (NC1) of Col VII. In two EBA patients, IgG titers against Col VII-NC1 were grossly correlated to clinical disease activity. Moreover, Col VII-reactive T cells were identified in a representative EBA patient which recognized identical subdomains of Col VII-NC1. These findings strongly suggest that (1) the Col VII-NC1 ELISA is a powerful tool for making the diagnosis of EBA, (2) Col VII-specific IgG grossly relates to disease activity and (3) IgG reactivity is associated with T cell recognition of identical subdomains of Col VII-NC1.

Decline of Ves v 5‐specific blocking capacity in wasp venom‐allergic patients after stopping allergen immunotherapy

While allergen‐specific immunotherapy (AIT) is very efficient in hymenoptera venom (HV)‐allergic patients, long‐term outcome after finishing AIT is not well investigated, especially regarding mechanisms that are suggested to contribute to allergen‐specific tolerance. Here, we analyse the Ves v 5‐inhibitory activity of sera from wasp venom‐allergic patients using the novel cell‐free enzyme‐linked immunosorbent facilitated antigen binding (ELIFAB) assay. Compared to pre‐AIT, sera from patients undergoing AIT displayed an increased ability to inhibit Ves v 5 binding by IgE antibodies. In contrast, this inhibitory activity was reduced in patients having finished AIT 5–12 years ago. Allergen‐blocking capacity correlated with serum concentrations of Ves v 5‐specific IgG4 which rose during AIT but almost reached pretreatment levels in patients who had stopped AIT more than 5 years ago. These data raise questions about how long allergen tolerance is maintained in AIT‐treated HV‐allergic patients and suggest that the ELIFAB assay might be an easy‐to‐use tool assessing long‐term tolerance in patients treated with HV‐AIT.

Monitoring the epitope recognition profiles of IgE, IgG1, and IgG4 during birch pollen immunotherapy

To the Editor: It is well established that allergen-specific IgG antibodies induced during allergen-specific immunotherapy (AIT) can block the IgE-mediated cascade of allergic inflammation. Although this role of IgG has been intensively studied, little is known about the epitope diversities of immunoglobulin subclasses induced during AIT. Thus far, analyses of IgG4-binding patterns to major cow’s milk and peanut allergens during oral AIT revealed discrepant results. The overall diversity of IgG4 specific for cow’s milk allergens changed little, whereas the IgG4 repertoire specific for peanut allergens was broadly expanded in a polyclonal fashion, including de novo–generated specificities different from IgE. Binding of IgE to Bet v 1, the major birch pollen allergen, is dependent on the protein’s native conformation. We have previously assessed the diversity of IgG4 antibodies specific for Bet v 1 during subcutaneous AIT with birch pollen using competitive immunoscreening of phage-displayed peptides and found that the Bet v 1–specific IgG4 repertoire did not broadly expand in most patients. 5 However, competitive immunoscreening is laborious and can be performed only with a limited number of serum samples. Therefore our previous work was restricted to the analysis of IgG4 diversity at 2 time points (before and after 3 years) of AIT by using sera of 5 patients. Here, we applied a chimera-based approach to monitor development of the Bet v 1–specific IgE, IgG1, and IgG4 repertoires in narrow time intervals during 3 years of AIT in more subjects. Four chimeras were generated by grafting 4 different nonoverlapping, contiguous, Bet v 1–derived surface areas onto the Bet v 1 homolog from celeriac, Api g 1, by replacing Api g 1–specific surface residues by corresponding residues from Bet v 1 (see Fig E1 in this article’s Online Repository at www. jacionline.org). Api-Bet-1 contains the P-loop, Api-Bet-2 contains the region opposite the P-loop, Api-Bet-3 contains the C-terminus and surrounding residues, and Api-Bet-4 contains the C-terminala-helix of Bet v 1. Sera from 11 patients with birch pollen allergy with improved symptom and medication scores were collected before (time point 0) and after 1, 3, 6, 12, 18, 24, 30, and 36 months of AIT. Patients received weekly doses of birch pollen extract (ALK-depot SQ 108; ALK-Abell o, Hørsholm, Denmark) subcutaneously until a maintenance dose of 100,000 standard quality units per injection was reached, followed by monthly maintenance injections for 3 years. Antibody binding to Bet v 1, the 4 Bet v 1–specific areas on the chimeras, and Api g 1 (scaffold protein control) was assessed by means of ELISA. Sera were not available from all time points from 3 subjects (p1, p5, p8). Proteins (2 mg/mL) were coated onto 96-well microtiter plates (MaxiSorp; Nalge Nunc International, Roskilde, Denmark) in carbonate buffer (50 mmol/L, pH 9.6). Nonspecific binding sites were saturated for 2 hours at room temperature with 1% human serum albumin in Tris-buffered saline containing 0.5%

Increased Frequency of T Follicular Helper Cells and Elevated Interleukin-27 Plasma Levels in Patients with Pemphigus

Pemphigus is an autoimmune disease in which IgG auto-antibodies (auto-ab) against the desmosomal cadherins desmoglein (Dsg) 3 and Dsg1 cause loss of epidermal keratinocyte adhesion. Aim of this study was to investigate cytokines derived from antigen-presenting cells (APC) and their relation to CD4+ T cell subpopulations and to the auto-ab response in pemphigus. In this regard, patients with pemphigus were compared to patients with myasthenia gravis (MG), an unrelated auto-ab–mediated autoimmune disease, and healthy controls. In pemphigus and MG, the plasma concentrations of the APC-derived immunomodulatory cytokine IL-27 were highly increased. Strikingly, IL-27 strongly correlated with Dsg-specific IgG auto-ab titers. T helper (Th) 17 cells were augmented in both pemphigus and MG patients while T follicular helper (Tfh) cells, which are essential in providing B cell help, were increased only in pemphigus along with increasing plasma concentrations of IL-21, a cytokine produced by Th17 and Tfh cells. Moreover, we could detect Dsg3-specific autoreactive T cells producing IL-21 upon ex vivo stimulation with Dsg3. These findings suggest that IL-27 and IL-21-producing T cells, are involved in the pathogenesis of pemphigus. The further characterization of IL-21-producing T cells and of the role of IL-27 will lead to a more defined understanding of the auto-ab response in pemphigus.

Cellular in vitro diagnosis of adverse drug reactions

Adverse drug reactions can manifest clinically in a variety of ways. Whilst the majority can be explained by dose-dependent side effects, there is group of unpredictable immunological or non-immunological intolerances that represent a particular diagnostic challenge. Skin tests are frequently negative, whilst challenge tests are time-consuming and often hazardous. Against this backdrop, cellular in vitro tests play a particularly important role in the identification of the causative drug. Whilst basophil tests can be used in the case of immunoglobulin E (IgE)- as well as non-IgE-mediated hypersensitivity reactions, T-cellular test methods assist in the diagnosis of drug eruptions. The reliability of individual tests can be affected by a variety of parameters, such as the pathomechanism underlying the drug reaction, the causative medication, or the point in time of testing. Not only is a sound knowledge of the basic principles of the individual assays an essential prerequisite for correctly indicating and interpreting this test method, but also an awareness of these additional factors.