IgE Abs drive the symptoms of allergic disease upon cross-linking allergens on mast cells or basophils. If the IgE binding sites on the allergens could be identified, it may be useful for creating new forms of immunotherapy. However, direct knowledge of the human IgE (hIgE) epitopes is limited because of the very low frequency of IgE-producing B cells in blood. A new hybridoma technology using human B cells from house dust mite-allergic patients was used to identify four Der p 2-specific hIgE mAbs. Their relative binding sites were assessed and compared by immunoassays with three previously studied murine IgG mAbs. Immunoassays showed that the recognition of Der p 2 by the first three hIgE was inhibited by a single murine IgG, but the fourth hIgE recognized a different epitope from all the other mAbs. The functional ability of the hIgE that bind different epitopes to cross-link Der p 2 was demonstrated in a mouse model of passive systemic anaphylaxis. Nuclear magnetic resonance analyses of Der p 2 in complex with IgG and IgE Abs were used to identify specific residues in the epitopes. To our knowledge, the combination of immunoassays to distinguish overlapping epitopes and nuclear magnetic resonance analyses to identify specific residues involved in Ab binding provided the first epitope mapping of hIgE mAbs to an allergen. The technologies developed in this study will be useful in high-resolution mapping of human epitopes on other Ags and the design of improved therapeutics.

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Four recombinant (r) allergens (rAmb a 8.0101, rArt v 4.0101, rBet v 2.0101, and rPhl p 12.0101) were successfully produced and used for sensitization studies. The allergens belong to the profilin family which is one of the most numerous allergen families. These four proteins represent allergens originating from pollen of weeds (rAmb a 8.0101 and rArt v 4.0101), tree (rBet v 2.0101) and grass (rPhl p 12.0101). The recombinant allergens were characterized using various biochemical and biophysical methods and tested for their ability to bind patient-derived antibodies. One hundred patients aged 2 to 50 years sensitized to pollen and plant-derived food allergens (IgE > 0.35 kU/L) were included. Sensitization to individual allergen sources and components of birch and timothy pollens was evaluated using multiparameter immunoblots. The presence of IgE to pollen-derived recombinant profilins rAmb a 8.0101, rArt v 4.0101, rBet v 2.0101, and rPhl p 12.0101 in serum was evaluated using ELISA method. The presence of IgE against pollen profilins was detected in 20 out of 100 studied patients. High correlation was seen between IgE ELISA results with individual pollen profilins. In summary, it was shown that the recombinant versions of the four allergenic profilins can be used for sensitization studies and for component-resolved allergy diagnostics.

Timothy grass pollen is a source of potent allergens. Among them, Phl p 1 and Phl p 5 are thought to be the most important, as a majority of timothy grass-allergic individuals have IgE antibodies directed against these two allergens. The profilin from timothy grass (Phl p 12) has been registered as a minor allergen, with up to 35% of individuals in populations of grass pollen allergic patients showing IgE binding to Phl p 12. Profilins are primarily minor allergens and are known for a high likelihood of co-sensitization as well as cross-reactivity situations caused by their sequence and structure similarity. The crystal structure of Phl p 12.0101 was determined and it revealed that this allergen may form an unusual dimer not previously observed among any profilins. For example, the Phl p 12 dimer has a completely different geometry and interface when compared with the latex profilin (Hev b 8) dimer that has its crystal structure determined. The structure of Phl p 12.0101 is described in the context of allergenic sensitization and allergy diagnostics. Moreover, the structure of the Phl p 12.0101 dimer is discussed, taking into account the production of recombinant allergens and their storage.

RATIONALE: CBX7 is a H3K27 methyl reader and a polycomb repressor complex-1 (PRC1) member. Its role in cytokine gene expression is unknown. METHODS: ChIP, Flow cytometry, gene knockdown, and mouse model of asthma RESULTS: CBX7 was identified as a steroid resistant gene in a functional genomic study. Airway lymphoid cells from a subgroup of asthmatic patients manifest steroid resistance. For this reason we examined CBX7 regulation of cytokine production. The pharmacologic inhibitor of CBX7 -MS37452 blocked expression of cytokines-IL4, IL5, IL13, and IFN-g by human blood lymphocytes and innate lymphoid cells suggesting an activating but not repressive function of CBX7. Likewise, shRNAmediated knockdown of CBX7 inhibited cytokine expression by lymphocytes. To delineate the molecular mechanism we performed chromatin-Immunoprecipitation (ChIP) of activated lymphocytes, which showed binding of CBX7 to the proximal promoters, conserved non-coding sequences (CNS), and the enhancer region of the cytokine genes and GATA-3. CBX7 physically interacted with the transcriptional co-activators (histone acetyl transferase P300 and casein kinase-2) and polymerase-II in co-precipitation studies, which explained its co-activator function. To define its in vivo function we sensitized and challenged Rag1-/-mice with Alternaria. Pretreatment of mice with MS37452 but not with DMSO abrogated Alternaria-induced airway hyperreactivity and eosinophilic inflammation. MS37452 inhibited IL5 and IL13 production by steroidresistant ILCs and lymphocytes from bronchoalveolar lavage obtained from asthma patients. CONCLUSIONS: CBX7, unexpectedly, functions as a transcriptional coactivator of cytokine genes. CBX7 binds to the promoter and enhancer of cytokines, and recruits transcriptional co-activators to facilitate gene transcription. CBX7 inhibitors are likely to benefit steroid-resistant asthma.Abstracts AB183 SUNDAY