Dog-allergic Patients Research Articles

Albumins represent highly cross-reactive animal allergens.

Albumins from animals are highly cross-reactive allergens for patients suffering from immunoglobulin E (IgE)-mediated allergy. Approximately 20-30% of cat and dog allergic patients show IgE reactivity and mount IgE-mediated allergic reactions to cat and dog albumin. It is astonishing that allergic patients can develop specific IgE responses against animal albumins because these proteins exhibit a more than 70% sequence identity to human serum albumin (HSA) which is the most abundant protein in the blood of the human body. The sequence identity of cat albumin (Fel d 2) and dog albumin (Can f 3) and HSA are 82% and 80%, respectively. Given the high degree of sequence identity between the latter two allergens and HSA one would expect that immunological tolerance would prohibit IgE sensitization to Fel d 2 and Can f 3. Here we discuss two possibilities for how IgE sensitization to Fel d 2 and Can f 3 may develop. One possibility is the failed development of immune tolerance in albumin-allergic patients whereas the other possibility is highly selective immune tolerance to HSA but not to Fel d 2 and Can f 3. If the first assumption is correct it should be possible to detect HSA-specific T cell responses and HSA-containing immune complexes in sensitized patients. In the latter scenario few differences in the sequences of Fel d 2 and Can f 3 as compared to HSA would be responsible for the development of selective T cell and B cell responses towards Fel d 2 as well as Can f 3. However, the immunological mechanisms of albumin sensitization have not yet been investigated in detail although this will be important for the development of allergen-specific prevention and allergen-specific immunotherapy (AIT) strategies for allergy to albumin.

Immunological mechanisms involved in the human response to a dog dander allergoid

BackgroundDog allergens are a common cause of allergic sensitisation and trigger respiratory symptoms worldwide. However, clinical evidence regarding dog immunotherapy is limited. Therefore, the aim of this study was to analyse the immunomodulatory properties of a new allergoid from dog dander, thereby deepening the understanding of the molecular mechanisms involved in the reestablishment of the tolerogenic response. MethodsThree independent batches of dog dander native and allergoid allergen extracts were manufactured and characterised. Allergenic profiles were analysed by the identification of all dog allergens and quantification of the major allergens Can f 1 and Can f 5. The allergenicity profile of the allergoid was studied using biological potency and basophil activation tests. In vitro immunomodulatory parameters was evaluated as the capacity of the allergoid to induce IgG antibodies that block IgE binding to the allergen and cytokine promotion (IFN-γ, IL-4, IL-6, IL-10, IL-13, and TNF-α) in PBMCs from allergic donors. ResultsThe presence of all dog allergens, including Can f 1 and Can f 5, was confirmed in both types of extracts. The new allergoid showed a low IgE binding capacity, which significantly affected the activation of effector cells, such as basophils. The IgG antibodies induced by the allergoid in rabbits blocked human IgE binding epitopes on the dog native extract and induced Th1 and Treg responses by increasing IFN-γ and IL-10 levels in PBMCs from allergic donors. ConclusionThis new dog dander allergoid containing Can f 1 and Can f 5 showed a low capacity to bind IgE and to activate basophils in dog allergic patients. Furthermore, it showed potent activation of Th1 mediators and induction of tolerance through Treg activation. This allergoid could offer a safer profile than the native extract and could be an effective immunotherapy treatment for dog allergic patients.

Structure-based prediction of the IgE epitopes of the major dog allergen Can f 1.

Allergy to dogs has become increasingly prominent worldwide. Seven dog allergens have been identified, including Canis familiaris allergen 1-7 (Can f 1-7). Although Can f 1 is a major dog allergen sensitized to 50-75% of dog-allergic subjects, its IgE epitopes have not been identified. The structural analysis of an allergen is important to identify conformational epitopes. In this study, we generated a recombinant Can f 1 protein and determined its crystal structure using X-ray crystallography. Can f 1 had a typical lipocalin fold, which is composed of an eight-stranded β-barrel and α-helix, and has high similarity to Can f 2, Can f 4, and Can f 6 in overall structure. However, the localizations of surface charges on these proteins were quite different. Based on sequence alignment and tertiary structure, we predicted five critical residues (His86, Glu98, Arg111, Glu138, and Arg152) for the IgE epitopes. The relevance of these residues to IgE reactivity was assessed by generating Can f 1 mutants with these residues substituted for alanine. Although the effects of the mutation on IgE binding depended on the sera of dog-allergic patients, H86A and R152A mutants showed reduced IgE reactivity compared with wild-type Can f 1. These results suggest that Can f 1 residues His86 and Arg152 are candidates for the IgE conformational epitope.

Component-resolved diagnosis using guinea-pig allergens elucidates allergen sensitization profiles in allergy to furry animals.

BackgroundFurry animals are an important source of indoor allergens. Diagnosis of allergy to small pets such as guinea‐pigs still relies on animal dander extracts which do not allow to define the primary sensitization source.ObjectiveTo identify major guinea‐pig allergens and to evaluate their potential as marker allergens for in vitro IgE‐diagnosis in comparison with dander extracts.MethodsA group of patients allergic to guinea‐pig (n = 29) and a group of patients allergic to cat and dog (n = 30) were recruited for the study. A panel of four guinea‐pig lipocalin allergens was expressed as recombinant proteins in E. coli. Specific IgE were quantified by ImmunoCAP and ELISA.ResultsThe combination of 4 guinea‐pig lipocalin allergens, including 2 new lipocalins, Cav p 1.0201 and Cav p 6.0101, and the previously characterized lipocalins Cav p 2 and Cav p 3, enabled the identification of 90% of all patients allergic to guinea‐pig. The vast majority had specific IgE to Cav p 1 (83%). Cav p 6 shares 54% sequence identity with Fel d 4 and Can f 6 and was found to be IgE‐cross‐reactive with these allergens. In the group of cat‐ and dog‐allergic patients, 73% had also specific IgE to guinea‐pig dander. However, only 27% of the cat /dog‐allergic patients had specific IgE to any of the non‐cross‐reactive guinea‐pig allergens Cav p 1, Cav p 2 or Cav p 3. The high prevalence of IgE to guinea‐pig dander could be explained by IgE‐cross‐reactivity among serum albumins and certain lipocalins.Conclusions and clinical relevanceThe availability of specific allergen markers is essential for the assessment of primary sensitization, especially in polysensitized patients. The proposed panel of guinea‐pig allergens Cav p 1, Cav p 2 and Cav p 3 is a first step to component‐resolved IgE‐diagnosis of allergy to small furry pets.

Subcutaneous Immunotherapy With High-Dose Cat and Dog Extracts: A Real-life Study

Background: Data on the efficacy of immunotherapy administered to patients with cat or dog allergy are scarce. Objective: We aimed to evaluate the safety and efficacy of subcutaneous immunotherapy (SCIT) in patients with allergy to cat and dog dander. Consecutive patients with rhinitis and/or asthma related to sensitization to cat or dog dander were included in a pragmatic, real-life, prospective, observational study. All patients had specific IgE to cat, dog, or both. SCIT was administered using an infusion pump over 3 sessions as part of a rush protocol, followed by monthly administration over 12 months. We recorded adverse events, clinical outcomes, pulmonary function, FeNO, symptoms of rhinitis and asthma, quality of life (QoL), Asthma Control Test (ACT) score, and visual analog scale (VAS) score at baseline, 6 months, and 12 months. The study population comprised 66 patients (38 females, 46 allergic to cat and 20 to dog), with ages ranging from 9 to 59 years. During the up-dosing phase, in which the infusion pump was used, 8.1% of doses elicited a systemic reaction and 5.4% caused a local reaction, while 9.3% of doses administered during the maintenance phase (ie, without an infusion pump) induced a systemic reaction. No local reactions were recorded. A significant improvement in FEV1, symptoms of rhinitis and asthma, QoL, use of medication, VAS score, and ACT score was observed at 6 months and continued at 12 months. Clinical improvement with cat extract was significantly better than with dog extract. High-dose SCIT has substantial clinical value in many cat- and dog-allergic patients.

Frequency of allergic sensitization to Can f 5 in North East Italy. An analysis of 1403 ISACs 112 (Component Resolved Diagnosis) collected retrospectively

Recent studies have shown the increasing relevance of allergic sensitization to Can f 5, a prostatic kallicrein expressed in the prostate and detectable only in male dogs. The aim of the present study was to establish the frequency, level of sensitization and association with other dog allergens of Can f 5, as assessed by Component Resolved Diagnosis (CRD- ISAC 112, ThermoFisher Scientific, Uppsala, Sweden), in North East Italy. A total of 1403 CRD ISAC 112 were examined retrospectively. Five-hundred twenty subjects (37 %) had a positive IgE response to at least one of the available animal allergens. Among these 520 subjects, 268 (51.5 %) showed at least one sensitization to dog allergens. Among dog-sensitized individuals, 183 (69.02%) showed IgE against Can f 5, and 106 (57.92%) were sensitized exclusively against Can f 5. The average Can f 5 specific IgE was 8.810 ISU-E, with 77.6 % of individuals showing medium or high values of specific IgE according to manufacturer's specifications. In conclusions, our data confirmed that there is a high number of sensitized patients to Can f 5, which have a high degree of allergic sensitization. These results should be taken into account by allergists managing dog allergic patients. In fact, clinical consequences of this sensitization regard respiratory allergy (burden of rhinitis/asthma), systemic reactions (anaphylaxis during sexual intercourse from cross-reaction with human prostatic antigen), allergen immunotherapy-AIT (likely ineffective in patients with exclusive sensitization), and preventive measures (possibility to own a female dog and a likely reduction of allergen passive transport). Further studies are needed to better explore these aspects in "real life".

Dog allergy: can a prevalent or exclusive sensitization to Can f 5 be considered a lucky or negative event in real life?

Recent studies have shown the increasing relevance of allergic sensitization to Can f 5 (a prostatic kallikrein), which is an androgen-regulated protein expressed in the prostate and detectable only in male dogs. Can f 5 can be a prevalent or exclusive sensitizing agent in a considerable percentage of dog-allergic patients. Its specific allergenic characteristics are able to induce possible negative as well as positive clinical effects in individuals sensitized to dogs. In the present article we pointed out the possible pros or cons of sensitization to this allergen in real life. Further studies should be carried out to correctly assess some peculiar characteristics of Can f 5, in order to support the most of positive aspects and remedy at best the negative effects.

Canis familiaris allergen Can f 6: expression, purification and analysis of B-cell epitopes in Chinese dog allergic children.

Dog allergy is common worldwide. However, the allergenicity of dog allergy is still unclear in China as well as in special group, such as children. In this study, we chose Can f 6, a major dog allergen which belongs to the lipocalin to study its allergenicity in Chinese dog allergic children. Can f 6 gene was subcloned into pET-28a vector and transformed into E. coli BL21 (DE3) cells for expression. The recombinant Can f 6 was purified by nickel affinity chromatography, identified by SDS-PAGE, and tested for its allergenicity by Western blot with sera and basophil activation test. Secondary structures, B cell epitopes and homology modeling of Can f 6 were predicted by using a series of bioinformatical approaches. And the verification of B cell epitopes was detected by ELISA. The recombinant allergen showed an explicit band with the molecular weight of 20 kDa by SDS-PAGE. Sera from 56.3 % (18/32) of dog-allergic children patients reacted with Can f 6. The induction of the expression of CD63 and CCR3 of dog allergic children in passively sensitized basophils was up to approximately 5.0 times higher than healthy subjects. The secondary structure of Can f 6 contains 3 α-helices, 9 β-sheets and random coils. Five B cell epitopes of Can f 6 were predicted and were confirmed successfully by ELISA. The results showed Can f 6 is a major allergen in Chinese children, which provides a basis for further study of Can f 6 in diagnosis and treatment of symptoms in children in China. The structural information of Can f 6 will help to form a foundation for the future design of vaccines and therapies for Can f 6 related allergies.

Clustering of conformational IgE epitopes on the major dog allergen Can f 1

Immunoglobulin E (IgE)-associated allergy affects more than 25% of the population. Can f 1 is the major dog allergen associated with respiratory symptoms but the epitopes recognized by allergic patients IgE on Can f 1 are unknown. To characterize IgE epitopes of Can f 1 recognized by dog allergic patients, six overlapping peptides spanning the Can f 1 sequence were synthesized. In direct IgE epitope mapping experiments peptides were analyzed for IgE reactivity by dot blot and Enzyme-linked immunosorbent assay (ELISA) with sera from dog allergic patients. For indirect epitope-mapping, rabbits were immunized with the peptides to generate specific IgG antibodies which were used to inhibit allergic patients’ IgE binding to Can f 1. IgE binding sites were visualized on a model of the Can f 1 three-dimensional structure. We found that Can f 1 does not contain any relevant sequential IgE epitopes. However, IgE inhibition experiments with anti-peptide specific IgGs showed that Can f 1 N- and C-terminal portion assembled a major conformational binding site. In conclusion, our study is the first to identify the major IgE epitope-containing area of the dog allergen Can f 1. This finding is important for the development of allergen-specific treatment strategies.

The cat lipocalin Fel d 7 and its cross‐reactivity with the dog lipocalin Can f 1

We investigated the prevalence of sensitization to the cat lipocalin Fel d 7 among 140 cat-sensitized Swedish patients and elucidated its allergenic activity and cross-reactivity with the dog lipocalin Can f 1. Sixty-five of 140 patients had IgE to rFel d 7 whereof 60 also had IgE to rCan f 1. A moderate correlation between IgE levels to rFel d 7 and rCan f 1 was found. rFel d 7 activated basophils in vitro and inhibited IgE binding to rCan f 1 in 4 of 13 patients, whereas rCan f 1 inhibited IgE binding to rFel d 7 in 7 of 13 patients. Fel d 7 and Can f 1 showed high similarities in protein structure and epitopes in common were found using cross-reactive antisera. Fel d 7 is a common allergen in a Swedish cat-sensitized population that cross-reacts with Can f 1, and may contribute to symptoms in cat- but also in dog-allergic patients.

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

PurposeDivergent 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.MethodsWe 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).ResultsEighty-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.ConclusionsCan 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.

Designing a multimer allergen for diagnosis and immunotherapy of dog allergic patients.

BackgroundDog dander extract used for diagnosis and allergen-specific immunotherapy is often of variable and of poor quality.ObjectiveTo assemble four well-established dog allergen components into one recombinant folded protein for improved diagnosis and vaccination of allergy to dog.MethodsA linked molecule, comprising the four dog lipocalin allergens Can f 1, Can f 2, Can f 4 and Can f 6 was constructed. The tetrameric protein was structurally characterized by small angle X-ray scattering, and compared with each single recombinant lipocalin allergen or an equimolar mix of the four allergens by analytical size exclusion chromatography, circular dichroism, allergen-specific IgE in serum by ELISA and allergen-dependent capacity to activate basophils. The immunogenicity of the fusion protein was evaluated in immunized mice by assessing splenocyte proliferation and antibody production.ResultsThe linked tetrameric construct was produced as a soluble fusion protein, with the specific folds of the four individual allergens conserved. This multi-allergen molecule was significantly more efficient (p<0.001) than each single recombinant allergen in binding to dog-specific IgE, and the epitope spectrum was unaffected compared to an equimolar mix of the four allergens. Basophil degranulation revealed that the biologic activity of the linked molecule was retained. Immunization of mice with the linked construct induced comparable allergen-specific IgG responses with blocking capacity towards all included allergens and generated comparably low T-cell responses.ConclusionWe provide the first evidence for a linked recombinant molecule covering the major dog allergens for potential use in diagnostics and allergy vaccination of dog allergic patients.

Microarrayed dog, cat, and horse allergens show weak correlation between allergen-specific IgE and IgG responses⋆

Microarrayed dog, cat, and horse allergens show weak correlation between allergen-specific IgE and IgG responses⋆

Dog saliva – an important source of dog allergens

BackgroundAllergy to dog (Canis familiaris) is a worldwide common cause of asthma and allergic rhinitis. However, dander extract in routine diagnostics is not an optimal predictor of IgE-mediated dog allergy. Our objective was to evaluate saliva as an allergen source for improved diagnostics of allergy to dog.MethodsIgE-binding proteins in dog saliva and dander extract were analysed by immunoblot and mass spectrometry (LC-MS/MS) using pooled or individual sera from dog-allergic patients (n = 13). Sera from 59 patients IgE positive to dander and 55 patients IgE negative to dander but with symptoms to dog were analysed for IgE against saliva and dander by ELISA. Basophil stimulation with dog saliva and dander extract was measured by flow cytometry among three dog-allergic patients. Additionally, IgE-binding protein profiles of saliva from different breeds were investigated by immunoblot.ResultsGreater number and diversity of IgE-binding proteins was found in saliva compared to dander extract and varied among dog breeds. In saliva, Can f 1, 2, 3 and 6 were identified but also four new saliva allergen candidates. The majority of the 59 dog dander–positive sera (n = 44) were IgE positive to dog saliva. Among patients IgE negative to dander, but with symptoms to dog, 20% were IgE positive to saliva. The biological activity of saliva was confirmed by basophil degranulation.ConclusionsDog saliva is an allergen source for improved diagnostics of dog allergy. The IgE-binding protein profile of saliva from different dogs varies.

Characterization of the dog lipocalin allergen Can f 6: the role in cross‐reactivity with cat and horse

Allergy to the domestic dog (Canis familiaris) affects 5-10% of the population in affluent countries. Three of four patients are allergic to more than one pet, which can only partially be explained by cross-reactivity between serum albumins. The lipocalin protein family harbours allergens in mammalian species. We set out to clone and characterize a novel dog allergen, and investigate its potential role in cross-sensitization between dog, cat and horse. The gene encoding Can f 6 was amplified from dog skin and bladder cDNA libraries. The corresponding allergen was produced and purified by recombinant techniques and evaluated by SDS-PAGE, size exclusion chromatography, circular dichroism spectra, ELISA and basophil activation test. IgE antibodies to Can f 6 were found in serum from 38% of dog-sensitized subjects. Sequence similarities between the lipocalin allergens Can f 6, Fel d 4 (cat) and Equ c 1 (horse) suggested a probability for cross-reactivity, which was demonstrated by competitive ELISA. The biological relevance of Can f 6 was confirmed by basophil activation test in dog-allergic patients. Can f 6 is a new lipocalin dog allergen that cross-reacts with lipocalins from horse and cat. Can f 6 and homologous allergens may contribute to multisensitization and symptoms in individuals allergic to mammals.

Pet dander and difficult-to-control asthma: Therapeutic options

The prevalence of sensitization to cat and dog allergens is high in the general population and poses a challenge to the physician managing allergic asthma. Adequate allergen avoidance is difficult to achieve because of the physical characteristics of airborne animal allergens and patient noncompliance. Allergen-specific high-dose subcutaneous immunotherapy has shown benefit in cat-allergic patients with asthma and rhinoconjunctivitis, whereas the data for dog-allergic patients are not as convincing. Alternative immunotherapy approaches including the sublingual route or allergen-derived peptide-based immunotherapy remain experimental. Pharmacotherapy of pet-allergic asthmatic patients requires a stepwise approach following established asthma management guidelines. In addition to short-acting beta-agonists and inhaled corticosteroids, prophylactic antihistamines before anticipated pet exposure, the use of intranasal steroids, and the use of leukotriene antagonists may also be considered as adjunctive therapy in pet-allergic patients with asthma and/or allergic rhinitis. Omalizumab appears to have particular efficacy in pet allergen-induced asthma. Novel therapies such as Fcgamma-Fel d 1 chimeric proteins still have to be evaluated in the human setting.

Crystal Structure of the Dog Lipocalin Allergen Can f 2: Implications for Cross-reactivity to the Cat Allergen Fel d 4

The dog lipocalin allergen Can f 2 is an important cause of allergic sensitization in humans worldwide. Here, the first crystal structure of recombinant rCan f 2 at 1.45 Å resolution displays a classical lipocalin fold with a conserved Gly-Xaa-Trp motif, in which Trp19 stabilizes the overall topology of the monomeric rCan f 2. Phe38 and Tyr84 localized on the L1 and L5 loops, respectively, control access to the highly hydrophobic calyx. Although the rCan f 2 calyx is nearly identical with the aero-allergens MUP1, Equ c 1 and A2U from mouse, horse and rat, respectively, no IgE cross-reactivity was found using sera from five mono-sensitized subjects. However, clear IgE cross-reactivity was demonstrated between Can f 2 and the cat allergen Fel d 4, although they share less than 22% sequence identity . This suggests a role for these allergens in co-sensitization between cat- and dog-allergic patients.

Animal‐derived lipocalin allergens exhibit immunoglobulin E cross‐reactivity

Although knowledge of the IgE cross-reactivity between allergens is important for understanding the mechanisms of allergy, the regulation of the allergic immune response and the development of efficient modes of allergen immunotherapy, the cross-reactivity of animal allergens is poorly known. The aim of this study was to characterize IgE cross-reactivities between lipocalin proteins, including five animal-derived lipocalin allergens and one human endogenous lipocalin, tear lipocalin (TL). The recombinant proteins were validated by chromatography and mass spectrometry. The IgE-binding capacity of the allergens was confirmed by IgE. immunoblotting and IgE immunoblot inhibition. IgE ELISA was performed with sera from 42 atopic patients and 21 control subjects. The IgE cross-reactivities between the lipocalin proteins were determined by ELISA inhibition. ELISA inhibition revealed IgE cross-reactivities between Can f 1 and human TL, between Can f 1 and Can f 2, and between Equ c 1 and Mus m 1. Low levels of IgE to human TL were found in the sera of seven dog-allergic patients of whom six were IgE-positive for Can f 1. Several lipocalins exhibited IgE cross-reactivity, probably due to the sequential identity of the proteins and also due to similarities in their three-dimensional structures. The clinical significance of the findings needs to be elucidated. Low-level IgE cross-reactivity can play a role in regulating immune response to lipocalin allergens.

Immunological Response to Administration of Standardized Dog Allergen Extract at Differing Doses

RATIONALE: The purpose of this study was to examine the immunologic response to three doses of Acetone Precipitated (AP) dog extract (Hollister Stier) expressed as Can f 1 content. METHODS: Twenty-eight dog-allergic patients were randomly assigned to one of four maintenance treatment arms: placebo, 0.6 μg, 3.0 μg, or 15.0 μg of Can f 1. Studies included the response to titrated skin prick tests, late cutaneous response to dog extract, serum allergen-specific IgE and IgG4, titrated nasal challenges, and measurement of secreted and intracellular cytokines. RESULTS: After 5 weeks of immunotherapy, there was a statistically significant dose response in suppression of prick skin tests (p=0.01) with the high-dose group exhibiting the largest change from baseline. There were statistically significant differences when the high-dose group was compared to the placebo (p=0.0003), when the medium-dose group was compared to the placebo (p= 0.006), and when the high-dose group was compared to the low-dose group (p=0.05). There was an overall significant change from baseline in suppression of the late cutaneous response (p=0.03) and significant differences from baseline when the high-dose group was compared to the placebo group (p= <0.05). There was statistically significant differences from baseline in the high and medium-dose groups (p=0.03 for both), an increase in dog-specific IgG4 in both the high and low-dose groups (p=0.03, 0.02, respectively) and suppression in secreted IL-4 in the high-dose group (p=0.05). CONCLUSIONS: The dose response to immunotherapy with dog extract displayed the greatest and most consistent response with a dose containing 15 mcg of the major allergy.

The DR4–DQ8 haplotype and a specific T cell receptor Vβ T cell subset are associated with absence of allergy to Can f 1

The significance of specific T cell receptor (TCR) Vbeta subtypes and human leucocyte antigen (HLA) class II alleles for the development of allergy to lipocalin allergens such as the major dog allergen Can f 1 is not clear at present. To characterize the TCR Vbeta usage in the Can f 1-specific T cell lines and the HLA class II genotypes of Can f 1-allergic and non-allergic subjects. T cell lines were induced with recombinant Can f 1 from the peripheral blood mononuclear cells of 12 non-atopic dog owners and 26 dog-allergic patients. Thirteen of the dog-allergic subjects were sensitized to Can f 1. Expression of the TCR Vbeta subtypes on CD4(+) T cells in the T cell lines was measured by flow cytometry. The subjects were HLA genotyped for DRB1, DQB1 and DPB1 loci. Can f 1-specific T cell lines were obtained from 18 subjects, with either positive (n=8) or negative (n=10) skin prick tests (SPTs) to recombinant Can f 1. The frequency of TCR Vbeta5.1(+) T cells was significantly higher in the T cell lines of subjects with negative SPTs to the allergen. Moreover, DR4-DQ8 haplotype was over-represented among these subjects. The DR4-DQ8 haplotype and the TCR Vbeta5.1(+) CD4(+) T cells may be protective against allergy to Can f 1.