Figure 9.

Stabilization of Phl p 6 shifts immune responses toward T helper 1 reactions. For assessment of in vivo immunogenicity, BALB/c mice were immunized three times at 2-week intervals by intradermal injections and sacrificed 2 weeks after the last immunization (A). Phl p 6-specific serum IgG1 (B) and IgG2a (C) were measured by luminometric ELISA at serum dilutions of 1:100,000 and 1:100, respectively, lying within the linear range of the assay. Data are shown as means ± SEM (n = 10) of relative light units (RLU). IgE crosslinking capacity of the different proteins was tested using RBL cells loaded with high titered Phl p 6 WT specific IgE. Increasing concentrations of the different proteins were added and cross-linking induced beta-hexosaminidase (b-hex) release is presented as percentage of maximum release induced by Triton-X100 cell lysis (n = 3, means ± SD) (D). Biologically active IgE in sera from immunized mice (WT, L89G, S46Y) were measured by incubating RBL cells with serum (n = 10, means ± SEM) at indicated dilutions, followed by cross-linking with WT Phl p 6 (E). Cell bound IgE in blood from immunized (WT, L89G, S46Y) and naïve control mice (n = 10) was measured by basophil activation test after in vitro stimulation of whole blood samples with 10 ng of an equimolar mixture of WT, S46Y, and L89G protein. IgE cross-linking induced upregulation of activation marker CD200R was measured by flow cytometry. Data are shown as means ± SEM of stimulation indices (SI) (F). **P < 0.01, *P < 0.05.