Inhibition of melanoma cell proliferation <i>in vitro</i>.

<p>[³H]thymidine proliferation assay demonstrating effects of purified mouse IgG on HMW-MAA expressing melanoma cells. 518A2 or M14 cells were incubated with increasing concentrations of purified IgG from mice immunized with 15/3/6-KLH conjugate or KLH for 72 h and pulsed with [³H]thymidine. Data are presented as percentage of inhibition of proliferation compared to untreated cells. Percentage of inhibition was calculated as follows: 100−(cpm (treated)/cpm (untreated)×100). Values represent the mean of three independent experiments.</p

Antigen-specific immune response of immunized BALB/c mice.

<p>Serum samples from day 0 (preimmune serum; white bars), day 22 (grey bars), and day 41 (black bars) were tested in ELISA. The mean value of the individual responses of each group is shown and standard deviations are indicated by error bars. Peptide specificity of sera from mice either immunized with 15/3/6- or 43.12p3-KLH conjugate was assessed using the respective peptide-BSA conjugates. Sera from KLH immunized mice were tested for specificity to KLH.</p

Inhibition of mAb binding to HMW-MAA by synthetic peptides.

<p>Microtiter plates were coated with mAb TP61.5 and incubated with microsomal preparation of 518A2 melanoma cells to catch HMW-MAA. Biotinylated mAbs were preincubated with increasing concentrations of synthetic peptides, followed by incubation with HMW-MAA. (<b>A</b>) Biotinylated mAb VT80.12 was preincubated with peptide 15/3/6 (•) or an irrelevant control peptide (▴). (<b>B</b>) Biotinylated mAb VF1-TP43 was preincubated with peptide 43.12p3 (▪) as well as the control peptide (▴). Binding of biotinylated mAbs was measured using an AP-conjugated streptavidin. Percentage of inhibition was calculated as follows: 100−(OD (inhibited)/OD (uninhibited)×100).</p

HMW-MAA-specific Ab response determined by FACS and IHC.

<p>(<b>A</b>) The human melanoma cell lines 518A2 and M14 were each incubated with 1 µg mAb VT80.12 (as positive control to detect HMW-MAA) or 100 µg purified rabbit IgG from immunizations with 15/3/6-KLH conjugate or KLH. Bound IgG were detected with FITC-labeled goat anti-mouse IgG or FITC-labeled goat anti-rabbit IgG. Histogram overlays show unstained (white) vs. stained cells (grey). (<b>B</b>) IHC on 518A2 tumor tissues of C.B.17 SCID/SCID mice <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0019383#pone.0019383-Wagner2" target="_blank">[13]</a>. HMW-MAA staining was done with PBS buffer (negative control), mAb VT80.12 (positive control), and biotinylated IgG purified from rabbits immunized with 15/3/6-KLH conjugate (anti-15/3/6) or KLH (anti-KLH). Bound Abs were detected with StrepAB/HRP and visualized by DAB-chromogen solution and subsequent hematoxylin counterstaining.</p

The Major Birch Pollen Allergen Bet v 1 Induces Different Responses in Dendritic Cells of Birch Pollen Allergic and Healthy Individuals

<div><p>Dendritic cells play a fundamental role in shaping the immune response to allergens. The events that lead to allergic sensitization or tolerance induction during the interaction of the major birch pollen allergen Bet v 1 and dendritic cells are not very well studied. Here, we analyzed the uptake of Bet v 1 and the cross-reactive celery allergen Api g 1 by immature monocyte-derived dendritic cells (iMoDCs) of allergic and normal donors. In addition, we characterized the allergen-triggered intracellular signaling and transcriptional events. Uptake kinetics, competitive binding, and internalization pathways of labeled allergens by iMoDCs were visualized by live-cell imaging. Surface-bound IgE was detected by immunofluorescence microscopy and flow cytometry. Allergen- and IgE-induced gene expression of early growth response genes and Th1 and Th2 related cytokines and chemokines were analyzed by real-time PCR. Phosporylation of signaling kinases was analyzed by Western blot. Internalization of Bet v 1 by iMoDCs of both donor groups, likely by receptor-mediated caveolar endocytosis, followed similar kinetics. Bet v 1 outcompeted Api g 1 in cell surface binding and uptake. MoDCs of allergic and healthy donors displayed surface-bound IgE and showed a pronounced upregulation of Th2 cytokine- and NFκB-dependent genes upon non-specific Fcε receptor cross-linking. In contrast to these IgE-mediated responses, Bet v 1-stimulation increased transcript levels of the Th2 cytokines IL-4 and IL-13 but not of NFκB-related genes in MoDCs of BP allergic donors. Cells of healthy donors were either unresponsive or showed elevated mRNA levels of Th1-promoting chemokines. Moreover, Bet v 1 was able to induce Erk1/2 and p38 MAPK activation in BP allergics but only a slight p38 activation in normal donors. In conclusion, our data indicate that Bet v 1 favors the activation of a Th2 program only in DCs of BP allergic individuals.</p></div

Gene expression induced by Bet v 1 and MFs in iMoDCs of BP allergic donors.

<p>Cells were sham-treated or treated with Bet v 1 (squares), control stimulus (MF, circles), or a combination of Bet v 1 and control stimulus (diamonds) for indicated time periods. mRNA levels of EGR-1 and-3, Th2 cytokines IL-4 and IL-13, Th1 chemokines CXCL10 and 11, and pro-inflammatory cytokines IL-1β and IL-6 were analyzed by real-time PCR. Expression levels, normalized to the average of housekeeping genes, are shown relative to unstimulated cells. Data for donor AD2 are shown, which are representative of independent experiments with mRNA from cells of three donors each performed in duplicates.</p

Phosphorylation of MAP kinases after activation by allergens.

<p>Cells of BP allergic (A) and normal donors (B) were stimulated with the allergens (each 20 μg/ml) or control stimulus (MFs) for the indicated time points and then lysed. Cell extracts were analyzed by Western blot using antibodies specific for the phosphorylated forms of PKCα, Erk1/2, and p38 MAPKs. Sample amounts were determined with antibodies to Erk1/2 and PKCα. The blots shown are representative of independent experiments with cells of three donors for each group yielding similar results (shown for donors AD2 and ND1).</p

Kinetics of allergen uptake into iMoDCs of BP allergic and normal donors.

<p>Internalization of labeled Bet v 1 (Bet v 1–488) and labeled Api g 1 (Api g 1–610) by iMoDCs of allergic (A) and normal donors (B) was followed by live-cell fluorescence imaging. Results are representative of independent experiments of three different donors for each group (shown for donors AD1 and ND3). Exocytosis of fluorescent markers (panel A) and the absence of spillover of fluorescence (panel B) are depicted by framed display details.</p

Gene expression of iMoDCs after Bet v 1 stimulation or cross-linking of cell-bound IgE.

<p>iMoDCs of BP allergic (A) and normal donors (B) were stimulated with Bet v 1 (squares), anti-IgE (diamonds), or loaded with IgE before anti-IgE treatment (IgE+anti-IgE, circles). mRNA levels of IL-4, IL-13 and the NFκB related genes IL-1β and IL-6 were analyzed by real-time PCR. Error bars indicate SD. Results are representative of two independent experiments for each donor group (shown for donors AD1 and ND1). (C) Activation of NFκB was evaluated by detection of its inhibitory protein IκBα. The Western blots shown are representative of independent experiments for three donors of each group yielding similar results (shown for donors AD2 and ND2).</p

Competitive binding of allergens to iMoDCs of BP allergic (AD) and normal donors (ND).

<p>(A) Uptake of labeled Api g 1 (20 μg/ml) in the presence of unlabeled Bet v 1 (shown for donors AD3 and ND3). (B) Cross-competition of labeled Bet v 1 with Api g 1 (Bet v 1*/Api g 1) and self-competition of Bet v 1 (Bet v 1*/Bet v 1) using 20μg/ml of labeled allergen and 200 μg/ml of competitor (donor AD3). Results in (A) and (B) show uptakes after 45 minutes of chase and are representative of four independent experiments with similar outcomes for both donor groups. (C) Densitometric quantification of the competition assays measuring a 1 mm² area.</p