Reshaping the Bet v 1 fold modulates T(H) polarization

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.

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

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

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

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

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

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.