Soili Saarelainen

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.

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.

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.

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 allergens 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.

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.

Immunotherapeutic potential of the immunodominant T-cell epitope of lipocalin allergen Bos d 2 and its analogues.

Lipocalin allergens, which contain most of the important animal‐derived respiratory sensitizers, induce T helper type 2 (Th2) deviation, but the reasons for this are not clear. To explore the prospects for peptide‐based allergen immunotherapy and to elucidate the characteristics of the immunodominant epitope of Bos d 2, BALB/c mice were immunized with a peptide containing the epitope, peptides containing its analogues, peptides from the corresponding regions of other lipocalin proteins, and peptides with a homologous sequence. We observed that murine spleen cells recognized the immunodominant epitope of Bos d 2, p127–142, in almost the same way as human Bos d 2‐specific T cells did. Enzyme‐linked immunosorbent spot‐forming cell assay (ELISPOT) analyses showed that p127–142 and a corresponding peptide from horse Equ c 1 induced a Th2‐deviated cellular response, whereas a homologous bacterial peptide from Spiroplasma citri induced a Th0‐type response. Interestingly, the spleen cell response to the bacterial peptide and p127–142 was cross‐reactive, that is, able to induce reciprocally the proliferation and cytokine production of primed spleen cells in vitro. More importantly, the peptides were able to skew the phenotype of T cells primed with the other peptide. Our results suggest that modified peptides can be useful in allergen immunotherapy.