Trained immunity and tolerance in innate lymphoid cells, monocytes, and dendritic cells during allergen-specific immunotherapy.

BACKGROUND: Despite the efficacy of allergen-specific immunotherapy (AIT), the role of trained immunity and tolerance in this process has not been elucidated. OBJECTIVE: Here, we have performed a comprehensive longitudinal analysis of the systemic innate immune cell repertoire during the course of AIT. METHODS: Patients with allergy received standard preseasonal subcutaneous AIT with allergoids to birch and/or grass. Healthy controls were monitored without any intervention. Flow cytometry of innate lymphoid cell (ILC), natural killer cell, monocyte cell, and dendritic cell (DC) subsets was performed at baseline, 3 months (birch season), 6 months (grass seasons), and 12 months after the therapy in patients or at similar seasonal time points in controls. Additional analyses were performed in the third-year birch and grass season. RESULTS: We observed a durable decrease in group 2 ILCs and an increase of group 1 ILCs after AIT, with dynamic changes in their composition. We found that an expansion of CD127+CD25++ clusters caused observed shifts in the heterogeneity of group 1 ILCs. In addition, we observed development of CD127+CD25++c-Kit+ group 3 ILC clusters. Moreover, we found an increase in the number of intermediate monocytes in parallel with a reduction in nonclassical monocytes during the first year after AIT. Classical and intermediate monocytes presented significant heterogeneity in patients with allergy, but AIT reduced the HLA-DR++ clusters. Finally, an increase in plasmacytoid DCs and CD141+ myeloid DCs was observed in individuals with allergy, whereas the number of CD1c+ myeloid DCs was reduced during the first year of AIT. CONCLUSION: AIT induces changes in the composition and heterogeneity of circulating innate immune cells and brings them to the level observed in healthy individuals. Monitoring of ILCs, monocytes, and DCs during AIT might serve as a novel biomarker strategy.

Crystal structure and immunologic characterization of the major grass pollen allergen Phl p 4.

BACKGROUND: Phl p 4 is a major pollen allergen but exhibits lower allergenicity than other major allergens. The natural protein is glycosylated and shows cross-reactivity with related and structurally unrelated allergens. OBJECTIVE: We sought to determine the high-resolution crystal structure of Phl p 4 and to evaluate the immunologic properties of the recombinant allergen in comparison with natural Phl p 4. METHODS: Different isoallergens of Phl p 4 were expressed, and the nonglycosylated mutant was crystallized. The specific role of protein and carbohydrate epitopes for allergenicity was studied by using IgE inhibition and basophil release assays. RESULTS: The 3-dimensional structure was determined by using x-ray crystallography at a resolution of 1.9 Å. The allergen is a glucose dehydrogenase with a bicovalently attached flavin adenine dinucleotide. Glycosylated and nonglycosylated recombinant Phl p 4 showed identical inhibition of IgE binding, but compared with natural Phl p 4, all recombinant isoforms displayed a reduced IgE-binding inhibition. However, the recombinant protein exhibited an approximately 10-fold higher potency in basophil release assays than the natural protein. CONCLUSION: The crystal structure reveals the compact globular nature of the protein, and the observed binding pocket implies the size of the natural substrate. Plant-derived cross-reactive carbohydrate determinants (CCDs) appear to reduce the allergenicity of the natural allergen, whereas the Pichia pastoris-derived glycosylation does not. Our results imply yet undescribed mechanism of how CCDs dampen the immune response, leading to a novel understanding of the role of CCDs.

Hypoallergenic mutants of the Timothy grass pollen allergen Phl p 5 generated by proline mutations.

BACKGROUND: Phl p 5 is a major allergen of Timothy grass (Phleum pratense). A recombinant native Phl p 5 has already been used in clinical trials of allergen-specific immunotherapy as a component of a cocktail of allergens. Recombinant hypoallergenic allergens should further improve the treatment by reducing the risk of anaphylactic reactions at an increased therapeutic dosage. Native Phl p 5 is formed by α-helical regions separated by regions containing prolines. In order to generate hypoallergenic mutants, we studied the effect of proline mutations in single and multiple regions. METHODS: All mutants were analyzed by IgE inhibition assays and size exclusion chromatography with on-line mass determination. Selected mutants were additionally analyzed by field-flow fractionation, dynamic light scattering, circular dichroism spectroscopy, basophil activation and T-cell proliferation assays. RESULTS: Variants lacking prolines in a single region were obtained as soluble monomers. Six of eight molecules showed a slightly reduced IgE-binding capacity. Mutants carrying proline deletions in multiple regions formed monomers, dimers or insoluble aggregates. The mutant MPV.7 with five proline deletions and a substitution of proline 211 to leucine is monomeric, shows a strongly diminished IgE binding and maintains T-cell reactivity. The hydrodynamic radius and the content of the α-helical structure of MPV.7 are well comparable with the wild-type allergen. CONCLUSIONS: The hypoallergenic Phl p 5 variant MPV.7 combines multiple proline deletions with a substitution of proline 211 to leucine and meets basic demands for a pharmaceutical application. MPV.7 is a promising candidate for grass pollen immunotherapy with a cocktail of recombinant hypoallergens.

Plasma-derived extracellular vesicles discriminate type-1 allergy subjects from non-allergic controls.

BACKGROUND: Allergies are on the rise globally, with an enormous impact on affected individuals' quality of life as well as health care resources. They cause a wide range of symptoms, from slightly inconvenient to potentially fatal immune reactions. While allergies have been described and classified phenomenologically, there is an unmet need for easily accessible biomarkers to stratify the severity of clinical symptoms. Furthermore, biomarkers marking the success of specific immunotherapy are urgently needed. OBJECTIVES: Plasma extracellular vesicles (pEV) play a role in coordinating the immune response and may be useful future biomarkers. A pilot study on differences in pEV content was carried out between patients with type I allergy, suffering from rhinoconjunctivitis with or without asthma, and voluntary non-allergic donors. METHODS: We examined pEV from 38 individuals (22 patients with allergies and 16 controls) for 38 chemokines, cytokines, and soluble factors using high-throughput data mining approaches. RESULTS: Patients with allergies had a distinct biomarker pattern, with 7 upregulated (TNF-alpha, IL-4, IL-5, IL-6, IL-17F, CCL2, and CCL17) and 3 downregulated immune mediators (IL-11, IL-27, and CCL20) in pEV compared to controls. This reduced set of 10 factors was able to discriminate controls and allergic patients better than the total array. CONCLUSIONS: The content of pEV showed potential as a target for biomarker research in allergies. Plasma EV, which are readily measurable via blood test, may come to play an important role in allergy diagnosis. In this proof-of-principle study, it could be shown that pEV's discriminate patients with allergies from controls. Further studies investigating whether the content of pEVs may predict the severity of allergic symptoms or even the induction of tolerance to allergens are needed.

Production of T-cell lines.

Allergen-specific T-cell lines established from allergic patients provide the opportunity of investigating T-cell functions at the poly- or oligoclonal level. T-cell lines are useful in determining the presence or absence of antigen-specific T-cell reactivity. However, to obtain detailed knowledge of the action of T cells with clearly defined features, for example epitope specificity or phenotype, T-cell clones are necessary.The frequency of allergen-specific T cells in peripheral blood mononuclear cells (PBMC) tends to be low and so stimulation of PBMC with single allergens often results in low allergen-specific reactivity or requires high doses of the allergen. In contrast, the stimulation of PBMC with whole allergen extract results in stronger reactivity because a greater spectrum of T-cell specificities is addressed. Therefore, for the investigation of polyclonal reactivity toward single allergens it is useful to establish T-cell lines, which represent an allergen-specific enrichment of T cells from the respective individual. These T cells are poly- or oligoclonal and might possess different epitope specificities. The method described here is based on experiences with human T-cell lines and clones specific for several allergens from grass pollens and tree pollens.

Monoclonal antibodies.

Monoclonal antibodies (mabs) are powerful tools for the quantification, detection, and targeting of specific molecules. Allergen-specific mabs are important for the quantification of major allergens in allergen preparations used for allergen-specific immunotherapy and allergy diagnosis. Indeed, progress in the understanding of the mechanisms of the immunological responses underlying allergic disease would not have been possible without the use of mabs. Quantification assays are also important in the assessment of environmental allergen exposure and monitoring of avoidance procedures.Mabs against human IgE provide the basis for various test systems for the detection of specific and nonspecific IgE. Mabs raised against IgE or defined cytokines or cytokine receptors have potential as neutralizing reagents in vivo for the treatment of allergic diseases.Allergen-specific mabs are also valuable tools for the localization of allergens within their source material and the characterization of allergens derived from natural sources and by recombinant technologies. Furthermore they are often used for the isolation of allergens from complex extracts by affinity chromatography. The procedure described in this chapter has been used successfully to produce mabs against numerous allergens from house dust mites, insect venoms, cat, hens egg white, tree-, grass-, and herb pollens, and fungi, with the ultimate aim of obtaining matched antibody pairs to establish two-site binding assays for the quantification of major allergens. The method has also been used successfully to generate mabs against human IgE.

Strategies for recombinant allergen vaccines and fruitful results from first clinical studies.

Recombinant DNA technology has delivered the prospect of a new generation of preparations for allergen-specific immunotherapy. The first clinical studies with recombinant allergens have yielded encouraging results, suggesting that there is a good chance that such preparations will become available for use in the routine management of allergic disease.

Primary structure, recombinant expression, and molecular characterization of Phl p 4, a major allergen of timothy grass (Phleum pratense).

Grass pollen allergy is one of the most important allergic diseases world-wide. Several meadow grasses, like timothy grass and rye grass, contribute to allergic sensitizations, but also allergens from extensively cultivated cereals, especially rye, make a profound contribution. The group 4 allergens are well known as important major allergens of grasses. We have cloned for the first time group 4 sequences from Phleum pratense, Lolium perenne, Secale cereale, Triticum aestivum, and Hordeum vulgare, and investigated the IgE-reactivity of recombinant Phl p 4 as a candidate for allergy diagnostic and therapeutic applications.

Transition of recombinant allergens from bench to clinical application.

The cloning and production of an increasing number of allergens through the use of DNA technology has provided the opportunity to use these proteins instead of natural allergen extracts for the diagnosis and therapy of IgE-mediated allergic disease. For diagnostic purposes, it is essential that the molecules exhibit IgE-reactivity comparable with that of the natural wild-type molecules, whereas T cell reactivity and immunogenic activity may be more important for allergen-specific immunotherapy. In relation to the latter, the development of hypoallergenic recombinant allergen variants is an approach which shows great promise. Clinical application of the proteins requires that they must be produced under conditions of Good Manufacturing Practice and meet the specifications set down in the appropriate Regulatory Guidelines, principally the ICH-Guidelines. Special consideration has to be given to the choice of expression system, the design of the expression vectors, and the purification strategy to obtain a pure product free from toxins and contamination. The availability of the pure recombinant molecules provides the opportunity to formulate preparations that are free from the non-allergenic ballast proteins present in natural allergen extracts and which contain relative concentrations of the allergens in clinically appropriate proportions.

Mutants of the major ryegrass pollen allergen, Lol p 5, with reduced IgE-binding capacity: candidates for grass pollen-specific immunotherapy.

More than 400 million individuals are sensitized to grass pollen allergens. Group 5 allergens represent the most potent grass pollen allergens recognized by more than 80 % of grass pollen allergic patients. The aim of our study was to reduce the allergenic activity of group 5 allergens for specific immunotherapy of grass pollen allergy. Based on B- and T-cell epitope mapping studies and on sequence comparison of group 5 allergens from different grasses, point mutations were introduced by site-directed mutagenesis in highly conserved sequence domains of Lol p 5, the group 5 allergen from ryegrass. We obtained Lol p 5 mutants with low IgE-binding capacity and reduced allergenic activity as determined by basophil histamine release and by skin prick testing in allergic patients. Circular dichroism analysis showed that these mutants exhibited an overall structural fold similar to the recombinant Lol p 5 wild-type allergen. In addition, Lol p 5 mutants retained the ability to induce proliferation of group 5 allergen-specific T cell lines and clones. Our results demonstrate that a few point mutations in the Lol p 5 sequence yield mutants with reduced allergenic activity that represent potential vaccine candidates for immunotherapy of grass pollen allergy.