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.

Perspectives in allergen immunotherapy: 2019 and beyond.

The seventh "Future of the Allergists and Specific Immunotherapy (FASIT)" workshop held in 2019 provided a platform for global experts from academia, allergy clinics, regulatory authorities and industry to review current developments in the field of allergen immunotherapy (AIT). Key domains of the meeting included the following: (a) Biomarkers for AIT and allergic asthma; (b) visions for the future of AIT; (c) progress and data for AIT in asthma and the updates of GINA and EAACI Asthma Guidelines (separated for house dust mite SCIT, SLIT tablets and SLIT drops; patient populations) including a review of clinically relevant endpoints in AIT studies in asthma; (d) regulatory prerequisites such as the "Therapy Allergen Ordinance" in Germany; (e) optimization of trial design in AIT clinical research; (f) challenges planning and conducting phase III (field) studies and the future role of Allergen Exposure Chambers (AEC) in AIT product development from the regulatory point of view. We report a summary of panel discussions of all six domains and highlight unmet needs and possible solutions for the future.

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.

Triggering of specific Toll-like receptors and proinflammatory cytokines breaks allergen-specific T-cell tolerance in human tonsils and peripheral blood.

BACKGROUND: The generation and maintenance of allergen-specific T-cell tolerance is a key step in healthy immune responses to allergens and successful allergen-specific immunotherapy. Breaking of peripheral T-cell tolerance to allergens can lead to the development of allergies, but the mechanisms are not completely understood. OBJECTIVE: We sought to identify molecular mechanisms that break allergen-specific T-cell tolerance in human subjects. METHODS: Proliferative responses of allergen-specific T cells from tonsils and peripheral blood were measured by using tritiated thymidine incorporation and carboxyfluorescein succinimidyl ester (CFSE) dilution experiments. Cytokine levels in cell-free supernatants were quantified by using the cytometric bead array, and mRNA expression of transcription factors and cytokines was determined by using quantitative PCR. Myeloid dendritic cells (DCs) were characterized by using flow cytometry. RESULTS: In allergic patients the immune profile of the tonsils represents the atopic status of patients, with low expression of the TH1 cell-specific transcription factor T-bet and the cytokine IFN-γ, as well as IL-10. Human tonsils show very low levels of allergen-induced T-cell proliferation, thus representing a very suitable in vivo model to assess mechanisms of breaking allergen-specific T-cell tolerance. Triggering of Toll-like receptor (TLR) 4 or TLR8 and the proinflammatory cytokines IL-1ß or IL-6 break allergen-specific T-cell tolerance in human tonsils and peripheral blood through a mechanism dependent on the adaptor molecule myeloid differentiation primary response gene (88) (MyD88). In particular, myeloid DCs and stimulations that activate them broke the tolerance of allergen-specific CD4(+) T cells, whereas plasmacytoid DCs and stimulations that activate them, such as TLR7 and TLR9, did not have any effect. Tolerance-breaking conditions induced by different molecular mechanisms were associated with a mixed cytokine profile with a tendency toward increased levels of IL-13 and IL-17, which are TH2 and TH17 cytokines, respectively. CONCLUSION: Certain innate immune response signals and proinflammatory cytokines break allergen-specific CD4(+) T-cell tolerance in normally unresponsive subjects, which might lead to the development or exacerbation of allergic diseases after encountering microbes or inflammatory conditions.

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.

Recombinant allergens for specific immunotherapy.

Recombinant DNA technology provides the means for producing allergens that are equivalent to their natural counterparts and also genetically engineered variants with reduced IgE-binding activity. The proteins are produced as chemically defined molecules with consistent structural and immunologic properties. Several hundred allergens have been cloned and expressed as recombinant proteins, and these provide the means for making a very detailed diagnosis of a patient's sensitization profile. Clinical development programs are now in progress to assess the suitability of recombinant allergens for both subcutaneous and sublingual immunotherapy. Recombinant hypoallergenic variants, which are developed with the aim of increasing the doses that can be administered while at the same time reducing the risks for therapy-associated side effects, are also in clinical trials for subcutaneous immunotherapy. Grass and birch pollen preparations have been shown to be clinically effective, and studies with various other allergens are in progress. Personalized or patient-tailored immunotherapy is still a very distant prospect, but the first recombinant products based on single allergens or defined mixtures could reach the market within the next 5 years.

WHO/IUIS Allergen Nomenclature: Providing a common language.

A systematic nomenclature for allergens originated in the early 1980s, when few protein allergens had been described. A group of scientists led by Dr. David G. Marsh developed a nomenclature based on the Linnaean taxonomy, and further established the World Health Organization/International Union of Immunological Societies (WHO/IUIS) Allergen Nomenclature Sub-Committee in 1986. Its stated aim was to standardize the names given to the antigens (allergens) that caused IgE-mediated allergies in humans. The Sub-Committee first published a revised list of allergen names in 1986, which continued to grow with rare publications until 1994. Between 1994 and 2007 the database was a text table online, then converted to a more readily updated website. The allergen list became the Allergen Nomenclature database (www.allergen.org), which currently includes approximately 880 proteins from a wide variety of sources. The Sub-Committee includes experts on clinical and molecular allergology. They review submissions of allergen candidates, using evidence-based criteria developed by the Sub-Committee. The review process assesses the biochemical analysis and the proof of allergenicity submitted, and aims to assign allergen names prior to publication. The Sub-Committee maintains and revises the database, and addresses continuous challenges as new "omics" technologies provide increasing data about potential new allergens. Most journals publishing information on new allergens require an official allergen name, which involves submission of confidential data to the WHO/IUIS Allergen Nomenclature Sub-Committee, sufficient to demonstrate binding of IgE from allergic subjects to the purified protein.

Bacterially expressed and optimized recombinant Phl p 1 is immunobiochemically equivalent to natural Phl p 1.

Recombinant production in bacteria of soluble and monomeric Phl p 1, a major allergen of Timothy grass pollen, has proved to be very problematic. In order to facilitate expression and purification of this allergen, a recombinant variant was designed with a single amino acid substitution. Several comparative analyses with natural counterparts using electrophoretic and HPLC separations, together with immunological assays, demonstrated high equivalence. This is the first description of an approach aiming at an improvement of a natural like recombinant allergen.

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.

Promyelocytic leukaemia zinc finger protein (PLZF) is a glucocorticoid- and progesterone-induced transcription factor in human endometrial stromal cells and myometrial smooth muscle cells.

The promyelocytic leukaemia zinc finger (PLZF) protein belongs to the family of Krüppel-like zinc finger proteins. It is a transcriptional repressor involved in cell cycle control and has been implicated in limb development, differentiation of myeloid cells, and spermatogenesis. Little is known about the regulation of PLZF expression. In search for mediators of progesterone signalling in the female reproductive tract, we discovered induction of PLZF mRNA in primary cultures of human endometrial stromal cells and myometrial smooth muscle cells (SMC) in response to progesterone. Surprisingly, dexamethasone was a more potent inducer of PLZF expression than progesterone and elicited a sustained up-regulation of PLZF mRNA levels within 2 h. Immunofluorescence showed localization of PLZF to the nuclei of dexamethasone-treated SMC. In uterine biopsies, nuclear staining for PLZF was found in myometrial cells and endometrial stromal cells of the secretory phase. The transcriptional start site of the PLZF gene was located to position -5801 in SMC. Transfected promoter constructs containing up to 4.1 kb of 5'-flanking DNA were not induced by activated glucocorticoid or progesterone receptor. In contrast, co-transfection of c-jun and c-fos expression vectors resulted in stimulation of reporter gene activity, indicating an involvement of AP-1 transcription factors in PLZF expression.