T cell reactivity with allergoids: influence of the type of APC.

The use of allergoids for allergen-specific immunotherapy has been established for many years. The characteristic features of these chemically modified allergens are their strongly reduced IgE binding activity compared with the native form and the retained immunogenicity. T cell reactivity of chemically modified allergens is documented in animals, but in humans indirect evidence of reactivity has been concluded from the induction of allergen-specific IgG during immunotherapy. Direct evidence of T cell reactivity was obtained recently using isolated human T cells. To obtain further insight into the mechanism of action of allergoids, we compared the Ag-presenting capacity of different APC types, including DC and macrophages, generated from CD14+ precursor cells from the blood of grass pollen allergic subjects, autologous PBMC, and B cells. These APC were used in experiments together with Phl p 5-specific T cell clones under stimulation with grass pollen allergen extract, rPhl p 5b, and the respective allergoids. Using DC and macrophages, allergoids exhibited a pronounced and reproducible T cell-stimulating capacity. Responses were superior to those with PBMC, and isolated B cells failed to present allergoids. Considerable IL-12 production was observed only when using the DC for Ag presentation of both allergens and allergoids. The amount of IL-10 in supernatants was dependent on the phenotype of the respective T cell clone. High IL-10 production was associated with suppressed IL-12 production from the DC in most cases. In conclusion, the reactivity of Th cells with allergoids is dependent on the type of the APC.

"Allergen engineering": variants of the timothy grass pollen allergen Phl p 5b with reduced IgE-binding capacity but conserved T cell reactivity.

One problem of conventional allergen-specific immunotherapy is the risk of anaphylactic reactions. A new approach to make immunotherapy safer and more efficient might be the application of engineered allergens with reduced IgE-binding capacity but retained T cell reactivity. Using overlapping dodeca-peptides, the dominant T cell epitopes of the timothy grass pollen allergen Phl p 5b were identified. By site-directed mutagenesis outside these regions, point and deletion mutants were generated. Allergen variants were analyzed for IgE-binding capacity with sera of different grass pollen allergic patients by Western blotting, Dot blotting, and EAST inhibition test, and for histamine releasing capacity with peripheral blood basophils from different patients. The deletion mutants revealed significantly reduced IgE reactivity and histamine releasing capacity, compared with the wild-type Phl p 5b. Furthermore, in vivo skin prick tests showed that the deletion mutants had a significantly lower potency to induce cutaneous reactions than the wild-type Phl p 5b. On the other hand, T cell clones and T cell lines from different allergic patients showed comparable proliferation after stimulation with allergen variants and wild-type Phl p 5b. Considering their reduced anaphylactogenic potential together with their conserved T cell reactivity, the engineered allergens could be important tools for efficient and safe allergen-specific immunotherapy.

Mapping of T-cell epitopes of Phl p 5: evidence for crossreacting and non-crossreacting T-cell epitopes within Phl p 5 isoallergens.

BACKGROUND: Group 5 allergens represent major grass pollen allergens because of their high sensitization indices. The identification of T-cell epitopes of these allergens is a prerequisite for the design of immunotherapeutic strategies based on peptide vaccination or modified allergens with conserved T-cell epitopes. OBJECTIVE: This study was undertaken to determine T-cell epitopes on Phl p 5 major pollen allergen of timothy grass (Phleumn pratense). METHODS: T-cell lines (TCLs) and T-cell clones (TCCs), specific to Phl p 5, were established from the peripheral blood of 18 patients allergic to grass pollen. All TCCs were mapped for epitope specificities using 178 overlapping dodecapeptides representing the primary structures of two isoforms of Phl p 5 (Phl p 5a and Phl p 5b). Phenotype and cytokine production profiles of TCCs were tested. Selected TCCs were analysed for HLA class II restriction. RESULTS: A total of 82 TCCs were isolated. All TCCs displayed the helper cell (TH) phenotype. Their reactivity with two recombinant expressed isoforms of Phl p 5a and Phl p 5b was heterogeneous. The epitope specificity of the TCCs was then revealed. Nineteen T-cell epitopes could be identified on Phl p 5. Eighty-one percent of mapped TCCs recognized three T-cell reactive regions on the Phl p 5 allergen. Some TCCs were reactive with isoepitopes presenting on Phl p 5a as well as Phl p 5b. Allergen-specific stimulation induced a TH0-like type of cytokine production in 25 of 50 TCCs. Almost all TCCs secreted high concentrations of interleukin-13. CONCLUSION: Phl p 5, a major grass pollen allergen, contains several T-cell epitopes. Some epitope regions were recognized by several patients. Epitope recognition pattern could not be correlated with special HLA class II haplotypes. T-cell stimulating isoepitopes were found at corresponding regions of Phl p 5a and Phl p 5b isoforms.

Propolis allergy (IV). Studies with further sensitizers from propolis and constituents common to propolis, poplar buds and balsam of Peru.

26 different compounds have been investigated experimentally for their sensitizing capacity in guinea pigs. 19 of these occur in propolis as well as in poplar bud exudates, and 14 of them are also found in balsam of Peru. 4 caffeates and benzyl isoferulate were found to be strong sensitizers. 7 compounds were moderate, and 13 compounds showed only weak sensitizing potency. Methyl cinnamate was negative. Patch tests in 11 propolis-sensitive patients once more revealed 3-methyl-2-butenyl caffeate and phenylethyl caffeate as the major sensitizers. In addition to the 8 compounds already known to occur in propolis as well as in balsam of Peru, we detected 5 further substances that both materials have in common. Among these, benzyl isoferulate is considered a noteworthy sensitizer. Coniferyl benzoate, which was shown to be a moderate sensitizer, is present in fresh samples of balsam of Peru, while in propolis it has been detected only once so far. The flavonoid aglycones occurring in poplar bud exudates, and hence also in propolis, are weak sensitizers which play only a minor role in propolis hypersensitivity.