Regulatory T Cell Specificity Directs Tolerance versus Allergy against Aeroantigens in Humans.

FOXP3+ regulatory T cells (Tregs) maintain tolerance against self-antigens and innocuous environmental antigens. However, it is still unknown whether Treg-mediated tolerance is antigen specific and how Treg specificity contributes to the selective loss of tolerance, as observed in human immunopathologies such as allergies. Here, we used antigen-reactive T cell enrichment to identify antigen-specific human Tregs. We demonstrate dominant Treg-mediated tolerance against particulate aeroallergens, such as pollen, house dust mites, and fungal spores. Surprisingly, we found no evidence of functional impairment of Treg responses in allergic donors. Rather, major allergenic proteins, known to rapidly dissociate from inhaled allergenic particles, have a generally reduced capability to generate Treg responses. Most strikingly, in individual allergic donors, Th2 cells and Tregs always target disparate proteins. Thus, our data highlight the importance of Treg antigen-specificity for tolerance in humans and identify antigen-specific escape from Treg control as an important mechanism enabling antigen-specific loss of tolerance in human allergy.

On the critical exponents of generalized ballot sequences in three dimensions and large tandem walks.

We answer some questions on the asymptotics of ballot walks raised in [S. B. Ekhad and D. Zeilberger, April 2021] and prove that these models are not D-finite. This short note demonstrates how the powerful tools developed in the last decades on lattice paths in convex cones help us to answer some challenging problems that were out of reach for a long time. On the way we generalize tandem walks to the family of large tandem walks whose steps are of arbitrary length and map them bijectively to a generalization of ballot walks in three dimensions.

The ground of ground, essence, and explanation.

This paper is about the so-called meta-grounding question, i.e. the question of what grounds grounding facts of the sort 'ϕ is grounded in Γ'. An answer to this question is pressing since some plausible assumptions about grounding and fundamentality entail that grounding facts must be grounded. There are three different accounts on the market which each answer the meta-grounding question differently: Bennett's and deRosset's "Straight Forward Account" (SFA), Litland's "Zero-Grounding Account" (ZGA), and "Grounding Essentialism" (GE). I argue that if grounding is to be regarded as metaphysical explanation (i.e. if unionism is true), (GE) is to be preferred over (ZGA) and (SFA) as only (GE) is compatible with a crucial consequence of the thought that grounding is metaphysical explanation. In this manner the paper contributes not only to discussions about the ground of ground but also to the ongoing debate concerning the relationship between ground, essence, and explanation.

The epistemology of modality and the problem of modal epistemic friction.

There are three theories in the epistemology of modality that have received sustained attention over the past 20 years (1998-2018): conceivability-theory, counterfactual-theory, and deduction-theory. In this paper we argue that all three face what we call the problem of modal epistemic friction (PMEF). One consequence of the problem is that for any of the three accounts to yield modal knowledge, the account must provide an epistemology of essence. We discuss an attempt to fend off the problem within the context of the internalism versus externalism debate about epistemic justification. We then investigate the effects that the PMEF has on reductive and non-reductive theories of the relation between essence and modality.

A hybrid of two major Blomia tropicalis allergens as an allergy vaccine candidate.

INTRODUCTION: Allergen-specific immunotherapy (AIT) represents a curative approach for treating allergies. In the tropical and subtropical regions of the world, Blomia tropicalis (Blo t 5 and Blo t 21) is the likely dominant source of indoor allergens. AIM: To generate a hypoallergenic Blo t 5/Blo t 21 hybrid molecule that can treat allergies caused by B tropicalis. METHODS: Using in silico design of B tropicalis hybrid proteins, we chose two hybrid proteins for heterologous expression. Wild-type Blo t 5/Blo t 21 hybrid molecule and a hypoallergenic version, termed BTH1 and BTH2, respectively, were purified by ion exchange and size exclusion chromatography and characterized by physicochemical, as well as in vitro and in vivo immunological, experiments. RESULTS: BTH1, BTH2 and the parental allergens were purified to homogeneity and characterized in detail. BTH2 displayed the lowest IgE reactivity that induced basophil degranulation using sera from allergic rhinitis and asthmatic patients. BTH2 essentially presented the same endolysosomal degradation pattern as the shortened rBlo t 5 and showed a higher resistance towards degradation than the full-length Blo t 5. In vivo immunization of mice with BTH2 led to the production of IgG antibodies that competed with human IgE for allergen binding. Stimulation of splenocytes from BTH2-immunized mice produced higher levels of IL-10 and decreased secretion of IL-4 and IL-5. In addition, BTH2 stimulated T-cell proliferation in PBMCs isolated from allergic patients, with secretion of higher levels of IL-10 and lower levels of IL-5 and IL-13, when compared to parental allergens. CONCLUSIONS AND CLINICAL RELEVANCE: BTH2 is a promising hybrid vaccine candidate for immunotherapy of Blomia allergy. However, further pre-clinical studies addressing its efficacy and safety are needed.

Effect of structural stability on endolysosomal degradation and T-cell reactivity of major shrimp allergen tropomyosin.

BACKGROUND: Tropomyosins are highly conserved proteins, an attribute that forms the molecular basis for their IgE antibody cross-reactivity. Despite sequence similarities, their allergenicity varies greatly between ingested and inhaled invertebrate sources. In this study, we investigated the relationship between the structural stability of different tropomyosins, their endolysosomal degradation patterns, and T-cell reactivity. METHODS: We investigated the differences between four tropomyosins-the major shrimp allergen Pen m 1 and the minor allergens Der p 10 (dust mite), Bla g 7 (cockroach), and Ani s 3 (fish parasite)-in terms of IgE binding, structural stability, endolysosomal degradation and subsequent peptide generation, and T-cell cross-reactivity in a BALB/c murine model. RESULTS: Tropomyosins displayed different melting temperatures, which did not correlate with amino acid sequence similarities. Endolysosomal degradation experiments demonstrated differential proteolytic digestion, as a function of thermal stability, generating different peptide repertoires. Pen m 1 (Tm 42°C) and Der p 10 (Tm 44°C) elicited similar patterns of endolysosomal degradation, but not Bla g 7 (Tm 63°C) or Ani s 3 (Tm 33°C). Pen m 1-specific T-cell clones, with specificity for regions highly conserved in all four tropomyosins, proliferated weakly to Der p 10, but did not proliferate to Bla g 7 and Ani s 3, indicating lack of T-cell epitope cross-reactivity. CONCLUSIONS: Tropomyosin T-cell cross-reactivity, unlike IgE cross-reactivity, is dependent on structural stability rather than amino acid sequence similarity. These findings contribute to our understanding of cross-sensitization among different invertebrates and design of suitable T-cell peptide-based immunotherapies for shrimp and related allergies.

Counting and sampling gene family evolutionary histories in the duplication-loss and duplication-loss-transfer models.

Given a set of species whose evolution is represented by a species tree, a gene family is a group of genes having evolved from a single ancestral gene. A gene family evolves along the branches of a species tree through various mechanisms, including-but not limited to-speciation ([Formula: see text]), gene duplication ([Formula: see text]), gene loss ([Formula: see text]), and horizontal gene transfer ([Formula: see text]). The reconstruction of a gene tree representing the evolution of a gene family constrained by a species tree is an important problem in phylogenomics. However, unlike in the multispecies coalescent evolutionary model that considers only speciation and incomplete lineage sorting events, very little is known about the search space for gene family histories accounting for gene duplication, gene loss and horizontal gene transfer (the [Formula: see text]-model). In this work, we introduce the notion of evolutionary histories defined as a binary ordered rooted tree describing the evolution of a gene family, constrained by a species tree in the [Formula: see text]-model. We provide formal grammars describing the set of all evolutionary histories that are compatible with a given species tree, whether it is ranked or unranked. These grammars allow us, using either analytic combinatorics or dynamic programming, to efficiently compute the number of histories of a given size, and also to generate random histories of a given size under the uniform distribution. We apply these tools to obtain exact asymptotics for the number of gene family histories for two species trees, the rooted caterpillar and complete binary tree, as well as estimates of the range of the exponential growth factor of the number of histories for random species trees of size up to 25. Our results show that including horizontal gene transfers induce a dramatic increase of the number of evolutionary histories. We also show that, within ranked species trees, the number of evolutionary histories in the [Formula: see text]-model is almost independent of the species tree topology. These results establish firm foundations for the development of ensemble methods for the prediction of reconciliations.

Harmonization of the Genetic Code Effectively Enhances the Recombinant Production of the Major Birch Pollen Allergen Bet v 1.

BACKGROUND: Enhancing the quality and yield of protein production in heterologous expression systems is an important issue for developing new biopharmaceuticals. It has been shown that the dynamics of protein folding is influenced by codon frequencies. As codon usage frequencies are species specific, this can affect heterologous protein expression. In this respect, "codon harmonization," that is, the usage of synonymous codons with usage frequencies in the host resembling the usage frequencies in the native organism, is a promising strategy. As recombinant proteins are important tools in the area of allergy research, we investigated in this study the influence of codon harmonization on the production of the major birch pollen allergen Bet v 1.0101. METHODS: To accomplish this task, parallel production of several batches of rBet v 1, BWT, together with a harmonized variant, BH, was applied. The expression yield of soluble and insoluble protein was assayed via densitometric analysis of -SDS-PAGEs for every batch. The quality of purified proteins was assessed with a variety of physicochemical methods including mass spectrometry, circular dichroism, dynamic light scattering, Fourier transform infrared spectroscopy, in vitro degradation, and 1-anilino-8-naphthalene sulfonate-binding assays. Patients' IgE reactivity was tested in enzyme-linked immunosorbent assays and rat basophil mediator release experiments. RESULTS: No significant differences in the ligand-binding capacity and secondary structure elements, as well as, in immunological assays could be found; however, the production yield was drastically increased for BH. CONCLUSION: We could show that codon harmonization is a powerful method to enhance protein yields in heterologous expression systems and should be considered especially for difficult-to-express proteins.

Tackling Bet v 1 and associated food allergies with a single hybrid protein.

BACKGROUND: Allergy vaccines should be easily applicable, safe, and efficacious. For Bet v 1-mediated birch pollen and associated food allergies, a single wild-type allergen does not provide a complete solution. OBJECTIVE: We aimed to combine immunologically relevant epitopes of Bet v 1 and the 2 clinically most important related food allergens from apple and hazelnut to a single hybrid protein, termed MBC4. METHODS: After identification of T cell epitope-containing parts on each of the 3 parental allergens, the hybrid molecule was designed to cover relevant epitopes and evaluated in silico. Thereby a mutation was introduced into the hybrid sequence, which should alter the secondary structure without compromising the immunogenic properties of the molecule. RESULTS: MBC4 and the parental allergens were purified to homogeneity. Analyses of secondary structure elements revealed substantial changes rendering the hybrid de facto nonreactive with patients' serum IgE. Nevertheless, the protein was monomeric in solution. MBC4 was able to activate T-cell lines from donors with birch pollen allergy and from mice immunized with the parental allergens. Moreover, on immunization of mice and rabbits, MBC4 induced cross-reactive IgG antibodies, which were able to block the binding of human serum IgE. CONCLUSION: Directed epitope rearrangements combined with a knowledge-based structural modification resulted in a protein unable to bind IgE from allergic patients. Still, properties to activate specific T cells or induce blocking antibodies were conserved. This suggests that MBC4 is a suitable vaccine candidate for the simultaneous treatment of Bet v 1 and associated food allergies.

Expression and Characterization of Functional Recombinant Bet v 1.0101 in the Chloroplast of Chlamydomonas reinhardtii.

BACKGROUND: Allergen immunotherapy (AIT) still plays a minor role in the treatment of allergic diseases. To improve the acceptance of AIT by allergic patients, the treatment has to become more convenient and efficacious. One possibility is the oral application of allergens or derivatives thereof. Therefore, we sought to produce a recombinant allergen in the green alga Chlamydomonas reinhardtii as a novel production platform. METHODS: The major birch pollen allergen Bet v 1 was selected as candidate molecule, and a codon-optimized gene was synthesized and stably integrated into the microalga C. reinhardtii FUD50. Positive transformants were identified by PCR, cultured, and thereafter cells were disrupted by sonication. Bet v 1 was purified from algal total soluble protein (TSP) by affinity chromatography and characterized physicochemically as well as immunologically. RESULTS: All transformants showed expression of the allergen with yields between 0.01 and 0.04% of TSP. Algal-derived Bet v 1 displayed similar secondary structure elements as the Escherichia coli-produced reference allergen. Moreover, Bet v 1 produced in C. reinhardtii showed binding comparable to human IgE as well as murine Bet v 1-specific IgG. CONCLUSION: We could successfully produce recombinant Bet v 1 in C. reinhardtii. As microalgae are classified as GRAS (generally recognized as safe), the pilot study supports the development of novel allergy treatment concepts such as the oral administration of allergen-containing algal extracts for therapy.

Allergens of Blomia tropicalis: An Overview of Recombinant Molecules.

Allergic diseases are considered a major problem for healthcare systems in both developed and developing countries. House dust mites are well-known triggers of allergic manifestations. While the Dermatophagoides genus is widely distributed globally, Blomia tropicalis is the most prominent mite species in the tropical and subtropical regions of the world. Over the last decades, an increase in sensitization rates to B. tropicalis has been reported, leading to increased research efforts on Blomia allergens. In fact, 8 new allergens have been identified and characterized to different degrees. Here, we provide an overview of recent developments concerning the identification and production of recombinant Blomia allergens, as well as their structural and immunological characterization. Although considerable progress has been achieved, detailed molecule-based studies are still needed to better define the clinical relevance of Blomia allergens. Thus, the establishment of a well-standardized and fully characterized panel of allergens remains a challenge for the development of better diagnosis and therapy of allergic diseases induced by B. tropicalis.

T-cell epitope conservation across allergen species is a major determinant of immunogenicity.

BACKGROUND: Patients with pollen allergies are frequently polysensitized. Pollens contain epitopes that are conserved across multiple species. OBJECTIVE: We sought to demonstrate that cross-reactive T cells that recognize conserved epitopes show higher levels of expansion than T cells recognizing monospecific epitopes because of more frequent stimulation. METHOD: RNA was sequenced from 9 pollens, and the reads were assembled de novo into more than 50,000 transcripts. T-cell epitopes from timothy grass (Phleum pratense) were examined for conservation in these transcripts, and this was correlated to their ability to induce T-cell responses. T cells were expanded in vitro with P pratense-derived peptides and tested for cross-reactivity to pollen extracts in ELISpot assays. RESULTS: We found that antigenic proteins are more conserved than nonimmunogenic proteins in P pratense pollen. Additionally, P pratense epitopes that were highly conserved across pollens elicited more T-cell responses in donors with grass allergy than less conserved epitopes. Moreover, conservation of a P pratense peptide at the transcriptomic level correlated with the ability of that peptide to trigger T cells that were cross-reactive with other non-P pratense pollen extracts. CONCLUSION: We found a correlation between conservation of peptides in plant pollens and their T-cell immunogenicity within P pratense, as well as their ability to induce cross-reactive T-cell responses. T cells recognizing conserved epitopes might be more prominent because they can be stimulated by a broader range of pollens and thereby drive polysensitization in allergic donors. We propose that conserved peptides could potentially be used in diagnostic or immunomodulatory approaches that address the issue of polysensitization and target multiple pollen allergies.

Comparing Proteolytic Fingerprints of Antigen-Presenting Cells during Allergen Processing.

Endolysosomal processing has a critical influence on immunogenicity as well as immune polarization of protein antigens. In industrialized countries, allergies affect around 25% of the population. For the rational design of protein-based allergy therapeutics for immunotherapy, a good knowledge of T cell-reactive regions on allergens is required. Thus, we sought to analyze endolysosomal degradation patterns of inhalant allergens. Four major allergens from ragweed, birch, as well as house dust mites were produced as recombinant proteins. Endolysosomal proteases were purified by differential centrifugation from dendritic cells, macrophages, and B cells, and combined with allergens for proteolytic processing. Thereafter, endolysosomal proteolysis was monitored by protein gel electrophoresis and mass spectrometry. We found that the overall proteolytic activity of specific endolysosomal fractions differed substantially, whereas the degradation patterns of the four model allergens obtained with the different proteases were extremely similar. Moreover, previously identified T cell epitopes were assigned to endolysosomal peptides and indeed showed a good overlap with known T cell epitopes for all four candidate allergens. Thus, we propose that the degradome assay can be used as a predictor to determine antigenic peptides as potential T cell epitopes, which will help in the rational design of protein-based allergy vaccine candidates.

Tiam1/Rac1 signals contribute to the proliferation and chemoresistance, but not motility, of chronic lymphocytic leukemia cells.

Signals from the tumor microenvironment promote the migration, survival, and proliferation of chronic lymphocytic leukemia (CLL) cells. Rho GTPases control various signaling pathways downstream of microenvironmental cues. Here, we analyze the function of Rac1 in the motility and proliferation of CLL cells. We found decreased transcription of the Rac guanine nucleotide exchange factors Tiam1 and Vav1 in unstimulated peripheral blood CLL cells with almost complete loss of Tiam1 but increased transcription of the potential Rac antagonist RhoH. Consistently, stimulation of CLL cells with the chemokine CXCL12 induced RhoA but not Rac1 activation, whereas chemokine-induced CLL cell motility was Rac1-independent. Coculture of CLL cells with activated T cells induced their activation and subsequent proliferation. Here, Tiam1 expression was induced in the malignant cells in line with increased Ki-67 and c-Myc expression. Rac1 or Tiam1 knockdown using siRNA or treatment with the Tiam1/Rac inhibitor NSC-23766 attenuated c-Myc transcription. Furthermore, treatment of CLL cells with NSC-23766 reduced their proliferation. Rac inhibition also antagonized the chemoresistance of activated CLL cells toward fludarabine. Collectively, our data suggest a dynamic regulation of Rac1 function in the CLL microenvironment. Rac inhibition could be of clinical use by selectively interfering with CLL cell proliferation and chemoresistance.

Pollen Allergens for Molecular Diagnosis.

Pollen allergens are one of the main causes of type I allergies affecting up to 30% of the population in industrialized countries. Climatic changes affect the duration and intensity of pollen seasons and may together with pollution contribute to increased incidences of respiratory allergy and asthma. Allergenic grasses, trees, and weeds often present similar habitats and flowering periods compromising clinical anamnesis. Molecule-based approaches enable distinction between genuine sensitization and clinically mostly irrelevant IgE cross-reactivity due to, e. g., panallergens or carbohydrate determinants. In addition, sensitivity as well as specificity can be improved and lead to identification of the primary sensitizing source which is particularly beneficial regarding polysensitized patients. This review gives an overview on relevant pollen allergens and their usefulness in daily practice. Appropriate allergy diagnosis is directly influencing decisions for therapeutic interventions, and thus, reliable biomarkers are pivotal when considering allergen immunotherapy in the context of precision medicine.

Ligand binding modulates the structural dynamics and compactness of the major birch pollen allergen.

Pathogenesis-related plant proteins of class-10 (PR-10) are essential for storage and transport of small molecules. A prominent member of the PR-10 family, the major birch pollen allergen Bet v 1, is the main cause of spring pollinosis in the temperate climate zone of the northern hemisphere. Bet v 1 binds various ligand molecules to its internal cavity, and immunologic effects of the presence of ligand have been discussed. However, the mechanism of binding has remained elusive. In this study, we show that in solution Bet v 1.0101 is conformationally heterogeneous and cannot be represented by a single structure. NMR relaxation data suggest that structural dynamics are fundamental for ligand access to the protein interior. Complex formation then leads to significant rigidification of the protein along with a compaction of its 3D structure. The data presented herein provide a structural basis for understanding the immunogenic and allergenic potential of ligand binding to Bet v 1 allergens.