Despite the efficacy of allergen-specific immunotherapy (AIT), the role of trained immunity and tolerance in this process has not been elucidated.
Here, we have performed a comprehensive longitudinal ...analysis of the systemic innate immune cell repertoire during the course of AIT.
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.
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.
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.
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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.
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.
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.
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.
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.
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.
The major birch (Betula alba L.) pollen allergen, Bet v 1, has been shown to be homologous to pathogenesis-related proteins in a number of plants. Recently, it was demonstrated that a ginseng protein ...with high homology to an intracellular pathogenesis-related protein of parsley and to Bet v 1 is a ribonuclease (RNase). Birch pollen extract was separated in an RNase activity gel. Four major RNase bands were excised from the gel, reseparated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and identified by Western blotting with a specific Bet v 1 monoclonal antibody and patient's serum. Thus the monomer and the dimer of Bet v 1 showed RNase activity. Purified recombinant Bet v 1 was shown to degrade plant RNA. The RNase activity of recombinant Bet v 1 was 180 units·mg-1.
...it seems advisable to select adjuvants for new AIT protocols1,E25,E26 for their ability to promote sialylated IgG(4) antibody responses. Peak identity was confirmed by analyzing the collected peak ...fractions by means of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, as previously described.E35,E38 Glycans with human or murine sialic acids (human N-acetylneuraminic acid or murine N-glycolylneuraminic acid) had different retention times. Mouse IgG antibodies rarely have a bisecting GlcNAc, whereas 10% to 15% of human IgG antibodies have a bisecting GlcNAc. Because more Fc glycans of total serum IgG from untreated C57BL/6 mice are sialylated than pooled serum IgG from healthy human donors (IVIG), percentages of sialylation of murine and human IgG antibodies cannot be directly compared. In all experiments normal distribution was assumed. 1 C.A. Akdis, M. Akdis, Advances in allergen immunotherapy: aiming for complete tolerance to allergens, Sci Transl Med, Vol. 7, 2015, 280ps6 2 F.D. Finkelman, M.V. Khodoun, R. Strait, Human IgE-independent systemic anaphylaxis, J Allergy Clin Immunol, Vol. 137, 2016, 1674-1680 3 R.T. Strait, S.C. Morris, F.D. Finkelman, IgG-blocking antibodies inhibit IgE-mediated anaphylaxis in vivo through both antigen interception and Fc gamma RIIb cross-linking, J Clin Invest, Vol. 116, 2006, 833-841 4 H. Beutier, C.M. Gillis, B. Iannascoli, O. Godon, P. England, R. Sibilano, IgG subclasses determine pathways of anaphylaxis in mice, J Allergy Clin Immunol, Vol. 139, 2017, 269-280.e7 5 C.M. Oefner, A. Winkler, C. Hess, A.K. Lorenz, V. Holecska, M. Huxdorf, Tolerance induction with T cell-dependent protein antigens induces regulatory sialylated IgGs, J Allergy Clin Immunol, Vol. 129, 2012, 1647-1655 6 C. Hess, A. Winkler, A.K. Lorenz, V. Holecska, V. Blanchard, S. Eiglmeier, T cell-independent B cell activation induces immunosuppressive sialylated IgG antibodies, J Clin Invest, Vol. 123, 2013, 3788-3796 7 M. Collin, M. Ehlers, The carbohydrate switch between pathogenic and immunosuppressive antigen-specific antibodies, Exp Dermatol, Vol. 22, 2013, 511-514 8 A. Pincetic, S. Bournazos, D.J. DiLillo, J. Maamary, T.T. Wang, R. Dahan, Type I and type II Fc receptors regulate innate and adaptive immunity, Nat Immunol, Vol. 15, 2014, 707-716 9 C. Möbs, H. Ipsen, L. Mayer, C. Slotosch, A. Petersen, P.A. Würtzen, Birch pollen immunotherapy results in long-term loss of Bet v 1-specific TH2 responses, transient TR1 activation, and synthesis of IgE-blocking antibodies, J Allergy Clin Immunol, Vol. 130, 2012, 1108-1116.e6
Production of T-cell lines Kahlert, Helga
Methods in molecular medicine,
2008, Letnik:
138
Journal Article
Recenzirano
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.
Summary
Background
The assessment of the basophil‐activating potential is an important aspect in the development of improved preparations for specific immunotherapy. The aim of the study was to ...evaluate the suitability of CD203c expression as a measure of basophil activation to compare allergoids with original allergen extracts, and recombinant hypoallergenic allergen derivatives with recombinant wild‐type and natural allergens.
Methods
Heparinized whole blood samples from grass pollen allergic subjects were stimulated with grass pollen allergens and allergen derivatives followed by labelling of the basophils with PE‐conjugated anti‐CD203c. After lysis of the erythrocytes and fixation, the basophils were detected by flow cytometry. In some experiments, histamine release was determined simultaneously.
Results
Grass pollen allergoids revealed a 10–10 000‐fold reduction of basophil‐activating capacity measured by CD203c expression. The deletion mutant DM4 of rPhl p 5b showed stronger hypoallergenic characteristics in a range of 50–10 000‐fold reduction, whereas a combination mutant of rPhl p 5b and Phl p 6 revealed less hypoallergenic features. Histamine release experiments led to a similar outcome as CD203c measurement.
Conclusions
The measurement of CD203c expression on basophils by flow cytometry provides a rapid and sensitive method for the estimation of the allergic or hypoallergenic features of allergen preparations. The results demonstrated the hypoallergenicity of grass pollen allergoids and of the rPhl p 5b variant DM4, which may be a candidate in future preparations for specific immunotherapy.
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.
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.
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.
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.
Human monocytes (Mo) consist of a major subset of Fcγ‐receptor I (CD64)‐positive typical low accessory phagocytes, and a minor CD64– DC‐like subset with high T cell‐accessory and IFN‐α‐releasing ...activity. Both populations also differentially express CD16 (Fcγ‐receptor III). Double labeling with anti‐CD64 and anti‐CD16 mAb, as performed here, identified four different subsets. The CD64– subset consists of CD64– / 16+ cells with high antigen‐presenting cell (APC) function and macrophage‐like phenotype, and a CD64– / 16– subset of less active APC but which exhibits a higher mixed lymphocyte reaction (MLR) stimulating and IFN‐α‐producing capacity, possibly resembling plasmacytoid dendritic cell type II (DC2) blood precursors. As well as the majority of CD64+ cells that appeared CD64+ / 16– and represent typical low‐accessory, CD14high Mo, we could identify and describe a novel minor subset of CD64+ / 16+ cells which is unique in combining typical DC and Mo characteristics in the same cell. These are high IL‐12 production, high accessory capacity for antigen‐ or allogen‐activated lymphocytes, and high expression of HLA‐DR, CD86, and CD11c.
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