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. 2011 Jun;127(6):1571-8.e9.
doi: 10.1016/j.jaci.2011.01.064. Epub 2011 Mar 21.

Reshaping the Bet v 1 Fold Modulates T(H) Polarization

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Free PMC article

Reshaping the Bet v 1 Fold Modulates T(H) Polarization

Michael Wallner et al. J Allergy Clin Immunol. .
Free PMC article

Abstract

Background: Several alternative mechanisms have been proposed to explain why some proteins are able to induce a T(H)2-biased and IgE-mediated immune response. These include specific interactions with receptors of the innate immune system, proteolytic activities, allergen-associated carbohydrate structures, and intrinsic structural determinants.

Objectives: Available data suggest that a fold-dependent allergy-promoting mechanism could be a driving force for the T(H)2-polarization activity of Bet v 1, the major birch pollen allergen.

Methods: Computer-aided sequence and fold analysis of the Bet v 1 family identified a short stretch susceptible for mutations inducing an altered fold of the entire molecule. With this knowledge, 7 consecutive amino acids of Bet v 1 were replaced with the homologous Mal d 1 sequence, creating the derivative BM4.

Results: The minimal changes of the sequence led to a loss of the Bet v 1-like fold and influenced the immunologic behavior. Compared to wild-type Bet v 1, BM4 induced elevated T-cell proliferation of human PBMCs. In the mouse model, immunization with Bet v 1 absorbed to aluminum hydroxide triggered strong T(H)2 polarization, whereas BM4 immunization additionally recruited T(H)1 cells. Furthermore, the fold variant BM4 showed enhanced uptake by dendritic cells and a decreased susceptibility to endo-/lysosomal proteolysis.

Conclusion: Modifications in the 3-dimensional structure of Bet v 1.0101 resulted in a change of its immunologic properties. We observed that the fold alteration led to a modified crosstalk with dendritic cells and a shift of the immune response polarization toward a mixed T(H)1/T(H)2 cytokine production.

Conflict of interest statement

Disclosure of potential conflict of interest: M. Hauser receives research support from CK-Care AG, Switzerland. M. Himly receives research support from the Austrian Science Fund, Biomay AG, and the Austrian Research Promotion Agency. R. van Ree is a consultant for and receives research support from HAL Allergy BV. J. Thalhamer has consultant arrangements with Biomay AG and receives research support from Biomay AG, Christian Doppler Forschungsgesellschaft, and the Austrian Science Fund. B. Bohle receives research support from the Austrian Science Fund and Christian Doppler Forschungsgesellschaft. F. Ferreira receives research support from Biomay AG, the Austrian Science Fund, the Christian Doppler Research Association, and the European Union and has provided consultation for the AllergenOnline Database. The rest of the authors have declared that they have no conflict of interest.

Figures

Fig 1
Fig 1
A, Circular dichroism spectra of Bet v 1 and BM4 at 20°C. Spectra are baseline corrected and presented as mean residue molar ellipticity. deg, Degree; MRW, mean residue weight. B, The FTIR spectra of Bet v 1 and BM4 are presented as second derivatives. Typical regions of secondary structure elements are highlighted (α-helix, blue; intramolecular β-sheet, red; intermolecular β-sheet, green).
Fig 2
Fig 2
Aggregation behavior of Bet v 1 and BM4 were analyzed by DLS (small panel) and gel filtration (large panel). Hydrodynamic radius and molecular weight of both proteins were determined by light scattering. HP-SEC, High performance-size exclusion chromatography; MW, molecular weight; RALS, Right-angle light scattering; RH, hydrodynamic radius; RI, refractive index.
Fig 3
Fig 3
Proliferative responses of PBMCs from donors with birch pollen allergy stimulated with increasing amounts of Bet v 1 or BM4. Symbols represent individual patients, bars medians. P values were calculated by paired-sample t test (*P < .05).
Fig 4
Fig 4
A, BALB/c mice were immunized 4 times with either Bet v 1 (filled circles) or BM4 (open circles; n = 8 per group). Bet v 1–specific antibody levels were determined by ELISA. B, ELISPOT analysis of splenocytes from immunized animals expressed as mean cytokine-secreting cells per 105 spleen cells ± SEM. P values were calculated with t tests (**P < .01; ***P < .001).
Fig 5
Fig 5
BMDCs were pulsed with FITC or pHrodo-labeled Bet v 1 or BM4, stained for CD11c and CD86, and gated for CD11c expression. Representative fluorescence-activated cell-sorting profiles of 6 experiments per time point and antigen are shown. Numbers in the panels represent percentage values of analyzed cells (left panel). A time course analysis is presented as a line chart ± SEM (right panel). P values were calculated with t tests (*P < .05; **P < .01).
Fig 6
Fig 6
A, SDS-PAGE analysis of time-dependent endo-/lysosomal degradation of Bet v 1 and BM4. B, Percent degradation was determined densitometrically from SDS-PAGE bands and is presented as a line chart. C, Proteolytic fragments of Bet v 1 (black lines) or BM4 (red lines) were analyzed by mass spectrometry. Dominant T cell epitopes of Bet v 1 are indicated by vertical blue bars.

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