Single-cell characterization of dog allergen-specific T cells reveals TH2 heterogeneity in allergic individuals.

BACKGROUND: Allergen-specific type 2 CD4+ TH2 cells are critically involved in the pathogenesis of IgE-mediated allergic diseases. However, the heterogeneity of the TH2 response has only recently been appreciated. OBJECTIVE: We sought to characterize at the single-cell level the ex vivo phenotype, transcriptomic profile, and T-cell receptor (TCR) repertoire of circulating CD4+ T cells specific to the major dog allergens Can f 1, Can f 4, and Can f 5 in subjects with and without dog allergy. METHODS: Dog allergen-specific memory CD4+ T cells were detected ex vivo by flow cytometry using a CD154-based enrichment assay and single-cell sorted for targeted gene expression analysis and TCR sequencing. RESULTS: Dog allergen-specific T-cell responses in allergic subjects were dominantly of TH2 type. TH2 cells could be phenotypically further divided into 3 subsets, which consisted of TH2-like (CCR6-CXCR3-CRTH2-), TH2 (CCR6-CXCR3-CRTH2+CD161-), and TH2A (CCR6-CXCR3-CRTH2+CD161+CD27-) cells. All these subsets were nonexistent within the allergen-specific T-cell repertoire of healthy subjects. Single-cell transcriptomic profiling confirmed the TH2-biased signature in allergen-specific T cells from allergic subjects and revealed a TH1/TH17 signature in nonallergic subjects. TCR repertoire analyses showed that dog allergen-specific T cells were diverse and allergic subjects demonstrated less clonality compared to nonallergic donors. Finally, TCR and transcriptomic analyses revealed a close relationship between TH2-like, TH2, and TH2A cells, with the last ones representing the most terminally differentiated and highly polarized subtype. CONCLUSIONS: Our study demonstrates heterogeneity within allergen-specific TH2 cells at the single-cell level. The results may be utilized for improving immune monitoring after allergen immunotherapy and for designing targeted immunomodulatory approaches.

Inhalant Mammal-Derived Lipocalin Allergens and the Innate Immunity.

A major part of important mammalian respiratory allergens belongs to the lipocalin family of proteins. By this time, 19 respiratory mammalian lipocalin allergens have been registered in the WHO/IUIS Allergen Nomenclature Database. Originally, lipocalins, small extracellular proteins (molecular mass ca. 20 kDa), were characterized as transport proteins but they are currently known to exert a variety of biological functions. The three-dimensional structure of lipocalins is well-preserved, and lipocalin allergens can exhibit high amino acid identities, in several cases more than 50%. Lipocalins contain an internal ligand-binding site where they can harbor small principally hydrophobic molecules. Another characteristic feature is their capacity to bind to specific cell-surface receptors. In all, the physicochemical properties of lipocalin allergens do not offer any straightforward explanations for their allergenicity. Allergic sensitization begins at epithelial barriers where diverse insults through pattern recognition receptors awaken innate immunity. This front-line response is manifested by epithelial barrier-associated cytokines which together with other components of immunity can initiate the sensitization process. In the following, the crucial factor in allergic sensitization is interleukin (IL)-4 which is needed for stabilizing and promoting the type 2 immune response. The source for IL-4 has been searched widely. Candidates for it may be non-professional antigen-presenting cells, such as basophils or mast cells, as well as CD4+ T cells. The synthesis of IL-4 by CD4+ T cells requires T cell receptor engagement, i.e., the recognition of allergen peptides, which also provides the specificity for sensitization. Lipocalin and innate immunity-associated cell-surface receptors are implicated in facilitating the access of lipocalin allergens into the immune system. However, the significance of this for allergic sensitization is unclear, as the recognition by these receptors has been found to produce conflicting results. As to potential adjuvants associated with mammalian lipocalin allergens, the hydrophobic ligands transported by lipocalins have not been reported to enhance sensitization while it is justified to suppose that lipopolysaccharide plays a role in it. Taken together, type 2 immunity to lipocalin allergens appears to be a harmful immune response resulting from a combination of signals involving both the innate and adaptive immunities.

Associations Between Neonatal Cry Acoustics and Visual Attention During the First Year.

It has been suggested that early cry parameters are connected to later cognitive abilities. The present study is the first to investigate whether the acoustic features of infant cry are associated with cognitive development already during the first year, as measured by oculomotor orienting and attention disengagement. Cry sounds for acoustic analyses (fundamental frequency; F0) were recorded in two neonatal cohorts at the age of 0-8 days (Tampere, Finland) or at 6 weeks (Cape Town, South Africa). Eye tracking was used to measure oculomotor orienting to peripheral visual stimuli and attention disengagement from central stimuli at 8 months (Tampere) or at 6 months (Cape Town) of age. Only a marginal positive correlation between fundamental frequency of cry (F0) and visual attention disengagement was observed in the Tampere cohort, but not in the Cape Town cohort. This correlation indicated that infants from the Tampere cohort with a higher neonatal F0 were marginally slower to shift their gaze away from the central stimulus to the peripheral stimulus. No associations between F0 and oculomotor orienting were observed in either cohort. We discuss possible factors influencing the current pattern of results suggesting a lack of replicable associations between neonatal cry and visual attention and suggest directions for future research investigating the potential of early cry analysis in predicting later cognitive development.

Improving competing voices segregation for hearing impaired listeners using a low-latency deep neural network algorithm.

Hearing aid users are challenged in listening situations with noise and especially speech-on-speech situations with two or more competing voices. Specifically, the task of attending to and segregating two competing voices is particularly hard, unlike for normal-hearing listeners, as shown in a small sub-experiment. In the main experiment, the competing voices benefit of a deep neural network (DNN) based stream segregation enhancement algorithm was tested on hearing-impaired listeners. A mixture of two voices was separated using a DNN and presented to the two ears as individual streams and tested for word score. Compared to the unseparated mixture, there was a 13%-point benefit from the separation, while attending to both voices. If only one output was selected as in a traditional target-masker scenario, a larger benefit of 37%-points was found. The results agreed well with objective metrics and show that for hearing-impaired listeners, DNNs have a large potential for improving stream segregation and speech intelligibility in difficult scenarios with two equally important targets without any prior selection of a primary target stream. An even higher benefit can be obtained if the user can select the preferred target via remote control.

Immunotherapy for pet allergies.

Allergic diseases compose a serious challenge for modern societies. Their individual, medical and economical burden is large. As humans spend most of their time indoors, exposure to indoor allergens is a significant contributor to the development of allergic sensitization and respiratory allergies, such as allergic rhinoconjunctivitis and asthma. One important source of indoor allergens are pets, in particular cats and dogs. Allergens from these and other mammals spread effectively and they are encountered widely in public places. If patient education, allergen avoidance and pharmacotherapy do not suffice for controlling the symptoms of pet allergy, allergen immunotherapy can be a treatment option. Current information on allergen immunotherapy in pet allergy suggests that it can be effective in reducing allergic symptoms. However, the low number of high-quality randomized controlled trials of allergen immunotherapy in pet allergy warrants for further investigations.

Dimerization of lipocalin allergens.

Lipocalins are one of the most important groups of inhalant animal allergens. The analysis of structural features of these proteins is important to get insights into their allergenicity. We have determined two different dimeric crystal structures for bovine dander lipocalin Bos d 2, which was earlier described as a monomeric allergen. The crystal structure analysis of all other determined lipocalin allergens also revealed oligomeric structures which broadly utilize inherent structural features of the ß-sheet in dimer formation. According to the moderate size of monomer-monomer interfaces, most of these dimers would be transient in solution. Native mass spectrometry was employed to characterize quantitatively transient dimerization of two lipocalin allergens, Bos d 2 and Bos d 5, in solution.

Fluctuations of hi-hat timing and dynamics in a virtuoso drum track of a popular music recording.

Long-range correlated temporal fluctuations in the beats of musical rhythms are an inevitable consequence of human action. According to recent studies, such fluctuations also lead to a favored listening experience. The scaling laws of amplitude variations in rhythms, however, are widely unknown. Here we use highly sensitive onset detection and time series analysis to study the amplitude and temporal fluctuations of Jeff Porcaro's one-handed hi-hat pattern in "I Keep Forgettin'"-one of the most renowned 16th note patterns in modern drumming. We show that fluctuations of hi-hat amplitudes and interbeat intervals (times between hits) have clear long-range correlations and short-range anticorrelations separated by a characteristic time scale. In addition, we detect subtle features in Porcaro's drumming such as small drifts in the 16th note pulse and non-trivial periodic two-bar patterns in both hi-hat amplitudes and intervals. Through this investigation we introduce a step towards statistical studies of the 20th and 21st century music recordings in the framework of complex systems. Our analysis has direct applications to the development of drum machines and to drumming pedagogy.

Characterization of human memory CD4(+) T-cell responses to the dog allergen Can f 4.

BACKGROUND: The recently identified dog lipocalin allergen Can f 4 is an important respiratory allergen. OBJECTIVE: We sought to comprehensively characterize the memory CD4(+) T-cell responses of allergic and nonallergic subjects to Can f 4. METHODS: Can f 4-specific CD4(+)CD45RO(+) T-cell lines (TCLs) from allergic and healthy subjects were established and characterized by their functional and phenotypic properties. The epitope specificity of the TCLs was tested with 48 overlapping 16-mer peptides spanning the sequence of Can f 4. HLA restriction of the specific TCLs and the binding capacity of the epitope-containing peptides to common HLA class II molecules were studied. RESULTS: Can f 4-specific memory CD4(+) TCLs were obtained at an 8-fold higher frequency from allergic than from nonallergic subjects. Functionally, the TCLs of allergic subjects exhibited a higher T-cell receptor avidity and expression of CD25 and predominantly produced IL-4 and IL-5. The TCLs of nonallergic subjects mostly secreted IFN-γ and IL-10, with high CXCR3 expression. Several distinct T-cell epitope regions along the allergen were identified. Importantly, the peptides from the region between amino acids 43 and 67 showed promiscuous HLA-binding capacity and induced memory CD4(+) T-cell responses in 90% of the allergic donors. CONCLUSION: Productive TH2-deviated memory T-cell responses to Can f 4 are observed in allergic but not nonallergic subjects. A 19-mer peptide sequence covering the core of the immunodominant region of the allergen is a potential target for the development of peptide-based allergen immunotherapy.

IgE Reactivity of the Dog Lipocalin Allergen Can f 4 and the Development of a Sandwich ELISA for Its Quantification.

PURPOSE: Divergent results on the IgE reactivity of dog-allergic subjects to Can f 4 have been reported. The aim of this study was to evaluate the significance of Can f 4 in dog allergy and to develop an immunochemical method for measuring Can f 4 content in environmental samples. METHODS: We purified the natural dog allergen Can f 4 from a dog dander extract by monoclonal antibody-based affinity chromatography and generated its variant in a recombinant form. Sixty-three dog-allergic patients and 12 nonallergic control subjects were recruited in the study. The IgE-binding capacity of natural Can f 4 and its recombinant variant was assessed by ELISA, immunoblotting, and skin prick tests (SPT). RESULTS: Eighty-one percent of the dog-allergic patients showed a positive result to the immunoaffinity-purified natural Can f 4 in IgE ELISA, but only 46% in IgE immunoblotting. Respective results with the recombinant Can f 4 variant were 54% and 49%. SPT results reflected those obtained in ELISA and immunoblotting. The overall IgE reactivity of the immunoaffinity-purified natural Can f 4 was found to depend strongly on the integrity of the allergen's conformation. A sandwich ELISA based on monoclonal antibodies was found to be functional for measuring Can f 4 in environmental samples. CONCLUSIONS: Can f 4 is a major allergen of dog together with Can f 1 and Can f 5. In combination with other dog allergens, it improves the reliability of allergy tests in dog allergy.

Structural aspects of dog allergies: the crystal structure of a dog dander allergen Can f 4.

Four out of six officially recognized dog allergens are members of the lipocalin protein family. So far, a three-dimensional structure has been determined for only one dog allergen, Can f 2, which is a lipocalin protein. We present here the crystal structure of a second lipocalin allergen from dog, a variant of Can f 4. Moreover, we have compared and analyzed the structures of these two weakly homologous (amino acid identity 21%) dog allergens. The size and the amino acid composition of the ligand-binding pocket indicate that Can f 4 is capable of binding only relatively small hydrophobic molecules which are different from those that Can f 2 is able to bind. The crystal structure of Can f 4 contained both monomeric and dimeric forms of the allergen, suggesting that Can f 4 is able to form transient (weak) dimers. The existence of transient dimers in solution was confirmed by use of native mass spectrometry. The dimeric structure of Can f 4 is formed when the ends of four ß-strands are packed against the same strands from the second monomer. The residues in the interface are mainly hydrophobic and the formation of the dimer is similar to the major horse allergen Equ c 1. Interestingly, the crystal structure of dog Can f 2 has been reported to show a different type of dimer formation. The capability of these allergens to form dimers may be important for the development of immediate allergic reaction (mast cell activation) because oligomeric allergens can effectively present multivalent epitopes.

Human CD4+ T cell responses to the dog major allergen Can f 1 and its human homologue tear lipocalin resemble each other.

Lipocalin allergens form a notable group of proteins, as they contain most of the significant respiratory allergens from mammals. The basis for the allergenic capacity of allergens in the lipocalin family, that is, the development of T-helper type 2 immunity against them, is still unresolved. As immunogenicity has been proposed to be a decisive feature of allergens, the purpose of this work was to examine human CD4+ T cell responses to the major dog allergen Can f 1 and to compare them with those to its human homologue, tear lipocalin (TL). For this, specific T cell lines were induced in vitro from the peripheral blood mononuclear cells of Can f 1-allergic and healthy dog dust-exposed subjects with peptides containing the immunodominant T cell epitopes of Can f 1 and the corresponding TL peptides. We found that the frequency of Can f 1 and TL-specific T cells in both subject groups was low and close to each other, the difference being about two-fold. Importantly, we found that the proliferative responses of both Can f 1 and TL-specific T cell lines from allergic subjects were stronger than those from healthy subjects, but that the strength of the responses within the subject groups did not differ between these two antigens. Moreover, the phenotype of the Can f 1 and TL-specific T cell lines, determined by cytokine production and expression of cell surface markers, resembled each other. The HLA system appeared to have a minimal role in explaining the allergenicity of Can f 1, as the allergic and healthy subjects' HLA background did not differ, and HLA binding was very similar between Can f 1 and TL peptides. Along with existing data on lipocalin allergens, we conclude that strong antigenicity is not decisive for the allergenicity of Can f 1.

Differential CD4+ T-cell responses of allergic and non-allergic subjects to the immunodominant epitope region of the horse major allergen Equ c 1.

The responses of allergen-specific CD4(+) T cells of allergic and healthy individuals are still incompletely understood. Our objective was to investigate the functional and phenotypic properties of CD4(+) T cells of horse-allergic and healthy subjects specific to the immunodominant epitope region of the major horse allergen Equ c 1. Specific T-cell lines (TCLs) and clones were generated from peripheral blood mononuclear cells with Equ c 1(143-160), the peptide containing the immunodominant epitope region of Equ c 1. The frequency, proliferative response, cytokine production and HLA restriction of the cells were examined. The frequency of Equ c 1-specific CD4(+) T cells was low (approximately 1 per 10(6) CD4(+) T cells) in both allergic and non-allergic subjects. The cells of allergic subjects had a stronger proliferative capacity than those of non-allergic subjects, and they predominantly emerged from the memory T-cell pool and expressed the T helper type 2 cytokine profile, whereas the cells of non-allergic subjects emerged from the naive T-cell pool and produced low levels of interferon-γ and interleukin-10. T-cell response to Equ c 1(143-160) was restricted by several common HLA class II molecules from both DQ and DR loci. As the phenotypic and functional properties of Equ c 1-specific CD4(+) T cells differ between allergic and non-allergic subjects, allergen-specific T cells appear to be tightly implicated in the development of diseased or healthy outcome. Restriction of the specific CD4(+) T-cell response by multiple HLA alleles suggests that Equ c 1(143-160) is a promising candidate for peptide-based immunotherapy.

[Update on current care guidelines: Allergen specific immunotherapy]. / Siedätyshoito.

Immunotherapy involves the specific treatment of IgE-mediated allergic diseases, indicated for allergic rhinitis and conjunctivitis, allergic asthma, insect sting (bee and wasp) allergy, and food allergy (especially cow's milk, egg and wheat). Subcutaneous injection immunotherapy with pollens (both trees and grass), animal danders, insect venoms and house dust mite preparations for allergic rhinitis and asthma is effective for both adults and children. Sublingual immunotherapy indicated for allergic rhinitis caused by grass pollens (especially timothy), is effective and appears to be a safe route of administration. Specific oral tolerance induction is used in children over five years of age with severe food allergy.

Association of HLA class II alleles with sensitization to cow dander Bos d 2, an important occupational allergen.

Allergic sensitization results from a complex interaction between genetic and environmental factors. Earlier studies have shown that highly polymorphic HLA genes are associated with a variety of allergies. Several important respiratory allergens belong to the family of lipocalin proteins. These include occupational sensitizers, such as cow Bos d 2 or rat Rat n 1, and prevalent indoor sensitizers, such as dog Can f 1 or cockroach Bla g 4. HLA associations with sensitization to lipocalin allergens are incompletely known. In the present study we have investigated an association between HLA alleles and sensitization to the major cow allergen Bos d 2. The HLA-DR/DQ genotypes of 40 Bos d 2-sensitized subjects having occupational asthma were determined by polymerase chain reaction (PCR) and the results were compared with the genotypes of 151 unrelated Finnish subjects. The frequencies of HLA class II alleles DRB1*0101, DRB1*0404, DQB1*0302, and DQB1*0501 were significantly higher among Bos d 2-sensitized than among control subjects. In addition, the allergic subjects expressed significantly lower frequencies of HLA-DRB1*0301 and DQB1*0201 alleles than did the control subjects. These data suggest that the HLA class II alleles DRB1*0101, DRB1*0404, DQB1*0302, and DQB1*0501, and the haplotypes that include them, are associated with sensitization to the major cow allergen Bos d 2, whereas HLA-DRB1*0301 and DQB1*0201 are dissociated with it. Amino acid analysis provides a biologically plausible explanation for the HLA associations.

Allergen-specific naïve and memory CD4+ T cells exhibit functional and phenotypic differences between individuals with or without allergy.

Although allergen-specific CD4(+) T cells are detectable in the peripheral blood of both individuals with or without allergy, their frequencies and phenotypes within the memory as well as naïve repertoires are incompletely known. Here, we analyzed the DRB1*0401-restricted responses of peripheral blood-derived memory (CD4(+)CD45RO(+)) and naïve (CD4(+)CD45RA(+)) T cells from subjects with or without allergy against the immunodominant epitope of the major cow dander allergen Bos d 2 by HLA class II tetramers in vitro. The frequency of Bos d 2(127-142)-specific memory T cells in the peripheral blood-derived cultures appeared to be higher in subjects with allergy than those without, whereas naïve Bos d 2(127-142)-specific T cells were detectable in the cultures of both groups at nearly the same frequency. Surprisingly, the TCR avidity of Bos d 2(127-142)-specific T cells of naïve origin, as assessed by the intensity of HLA class II tetramer staining, was found to be higher in individuals with allergy. Upon restimulation, long-term Bos d 2(127-142)-specific T-cell lines generated from both memory and naïve T-cell pools from individuals with allergy proliferated more strongly, produced more IL-4 and IL-10, and expressed higher levels of CD25 but lower levels of CXCR3 than the T-cell lines from individuals without allergy, demonstrating differences also at the functional level. Collectively, our current results suggest that not only the memory but also the naïve allergen-specific T-cell repertoires differ between individuals with or without allergy.

Suboptimal recognition of a T cell epitope of the major dog allergen Can f 1 by human T cells.

We have previously proposed that mammalian lipocalin allergens are recognized suboptimally by the human immune system due to their homology with endogenous lipocalins. Here, we have characterized in detail the human T cell recognition of one of the previously identified T cell epitopes of the major dog allergen Can f 1, contained in peptide p105-120. A panel of peptide analogues (altered peptide ligands, APLs) of p105-120 was tested on two specific T cell clones restricted by different human leukocyte antigen (HLA) alleles. Interestingly, we identified for both of the clones several heteroclitic APLs that were capable of stimulating them at 10-30-fold lower concentrations than the natural peptide. Moreover, one of the heteroclitic APLs identified with the T cell clones, L115F, was observed to induce a stronger polyclonal T cell response than the natural allergen peptide from the peripheral blood mononuclear cells (PBMCs) of six Can f 1-allergic subjects studied. The heteroclitic APLs bound with the same affinity as p105-120 to common HLA-DR- and HLA-DP-alleles, suggesting that their improved stimulatory capacity is attributable to a more efficient T cell receptor (TCR) recognition rather than increased HLA binding. Collectively, our data suggest that p105-120 is recognized suboptimally by human T cells. This may contribute to the allergenicity of Can f 1.