Marja Rytkönen-Nissinen

Assessment of recombinant dog allergens Can f 1 and Can f 2 for the diagnosis of dog allergy

Background The use of recombinant allergens for the diagnosis and immunotherapy of allergy may offer several advantages over allergen extracts.

Animal‐derived lipocalin allergens exhibit immunoglobulin E cross‐reactivity

Background 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.

Mammalian lipocalin allergens – insights into their enigmatic allergenicity

Most of the important mammal‐derived respiratory allergens, as well as a milk allergen and a few insect allergens, belong to the lipocalin protein family. As mammalian lipocalin allergens are found in dander, saliva and urine, they disperse effectively and are widely present in the indoor environments. Initially, lipocalins were characterized as transport proteins for small, principally hydrophobic molecules, but now they are known to be involved in many other biological functions. Although the amino acid identity between lipocalins is generally at the level of 20–30%, it can be considerably higher. Lipocalin allergens do not exhibit any known physicochemical, functional or structural property that would account for their allergenicity, that is, the capacity to induce T‐helper type 2 immunity against them. A distinctive feature of mammalian lipocalin allergens is their poor capacity to stimulate the cellular arm of the human or murine immune system. Nevertheless, they induce IgE production in a large proportion of atopic individuals exposed to the allergen source. The poor capacity of mammalian lipocalin allergens to stimulate the cellular immune system does not appear to result from the function of regulatory T cells. Instead, the T cell epitopes of mammalian lipocalin allergens are few and those examined have proved to be suboptimal. Moreover, the frequency of mammalian lipocalin allergen‐specific CD4+ T cells is very low in the peripheral blood. Importantly, recent research suggests that the lipocalin allergen‐specific T cell repertoires differ considerably between allergic and healthy subjects. These observations are compatible with our hypothesis that the way CD4+ T‐helper cells recognize the epitopes of mammalian lipocalin allergens may be implicated in their allergenicity. Indeed, as several lipocalins exhibit homologies of 40–60% over species, mammalian lipocalin allergens may be immunologically at the borderline of self and non‐self, which would not allow a strong anti‐allergenic immune response against them.

T Cell Epitope-Containing Peptides of the Major Dog Allergen Can f 1 as Candidates for Allergen Immunotherapy

One prerequisite for developing peptide-based allergen immunotherapy is knowing the T cell epitopes of an allergen. In this study, human T cell reactivity against the major dog allergen Can f 1 was investigated to determine peptides suitable for immunotherapy. Seven T cell epitope regions (A–G) were found in Can f 1 with specific T cell lines and clones. The localization of the epitope regions shows similarities with those of the epitopes found in Bos d 2 and Rat n 1. On average, individuals recognized three epitopes in Can f 1. Our results suggest that seven 16-mer peptides (p15–30, p33–48, p49–64, p73–88, p107–122, p123–138, and p141–156), each from one of the epitope regions, show widespread T cell reactivity in the population studied, and they bind efficiently to seven HLA-DRB1 molecules (DRB1*0101, DRB1*0301, DRB1*0401, DRB1*0701, DRB1*1101, DRB1*1301, and DRB1*1501) predominant in Caucasian populations. Therefore, these peptides are potential candidates for immunotherapy of dog allergy.

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 2127–142‐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 2127–142‐specific T cells were detectable in the cultures of both groups at nearly the same frequency. Surprisingly, the TCR avidity of Bos d 2127–142‐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 2127–142‐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.

The major horse allergen Equ c 1 contains one immunodominant region of T cell epitopes.

Background Despite the fact that most significant mammalian respiratory allergens are lipocalin proteins, information on the human T cell reactivity to these allergenic proteins is largely missing.

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.

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

Background The significance of specific T cell receptor (TCR) Vβ 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.

Use of multiple peptides containing T cell epitopes is a feasible approach for peptide‐based immunotherapy in Can f 1 allergy

We have previously shown that the major dog allergen Can f 1 contains seven T cell epitope regions, none of which was preferentially recognized. To identify the immune characteristics of Can f 1 epitopes and to verify their suitability for peptide‐based allergen immunotherapy, short‐term T cell lines were generated with epitope‐containing peptides from peripheral blood mononuclear cells of Can f 1 skinprick test‐positive allergic and healthy control subjects. The lines were examined for their proliferative capacity and cytokine production upon stimulation with the allergen peptide, a homologous peptide from human tear lipocalin (TL) and Can f 1 and TL proteins. Can f 1 peptides induced proliferation of T cells and gave rise to T cell lines with comparable efficiencies. In particular, the T cell lines of allergic subjects induced with p33–48 and p107–122 favoured the production of interferon‐γ and interleukin‐10, respectively. A greater number of Can f 1‐specific T cell lines were generated from allergic than from healthy individuals. Two p107–122‐induced Can f 1‐specific T cell lines also reacted to a homologous peptide of human TL. Our results suggest that several T cell epitope‐containing peptides should be used in combination for specific immunotherapy in Can f 1 allergy.

Threshold levels of purified natural Bos d 2 for inducing bronchial airway response in asthmatic patients

Background Provocation tests are invaluable in establishing threshold levels and a causal relationship between atopic asthma and a certain allergen source, especially in relation to work‐associated exposure. Purified major allergens open possibilities for a more accurate assessment of sensitization.