Mutations affecting the actin regulator WD repeat-containing protein 1 lead to aberrant lymphoid immunity.

BACKGROUND: The actin-interacting protein WD repeat-containing protein 1 (WDR1) promotes cofilin-dependent actin filament turnover. Biallelic WDR1 mutations have been identified recently in an immunodeficiency/autoinflammatory syndrome with aberrant morphology and function of myeloid cells. OBJECTIVE: Given the pleiotropic expression of WDR1, here we investigated to what extent it might control the lymphoid arm of the immune system in human subjects. METHODS: Histologic and detailed immunologic analyses were performed to elucidate the role of WDR1 in the development and function of B and T lymphocytes. RESULTS: Here we identified novel homozygous and compound heterozygous WDR1 missense mutations in 6 patients belonging to 3 kindreds who presented with respiratory tract infections, skin ulceration, and stomatitis. In addition to defective adhesion and motility of neutrophils and monocytes, WDR1 deficiency was associated with aberrant T-cell activation and B-cell development. T lymphocytes appeared to develop normally in the patients, except for the follicular helper T-cell subset. However, peripheral T cells from the patients accumulated atypical actin structures at the immunologic synapse and displayed reduced calcium flux and mildly impaired proliferation on T-cell receptor stimulation. WDR1 deficiency was associated with even more severe abnormalities of the B-cell compartment, including peripheral B-cell lymphopenia, paucity of B-cell progenitors in the bone marrow, lack of switched memory B cells, reduced clonal diversity, abnormal B-cell spreading, and increased apoptosis on B-cell receptor/Toll-like receptor stimulation. CONCLUSION: Our study identifies a novel role for WDR1 in adaptive immunity, highlighting WDR1 as a central regulator of actin turnover during formation of the B-cell and T-cell immunologic synapses.

High-density IgE recognition of the major grass pollen allergen Phl p 1 revealed with single-chain IgE antibody fragments obtained by combinatorial cloning.

The timothy grass pollen allergen Phl p 1 belongs to the group 1 of highly cross-reactive grass pollen allergens with a molecular mass of ∼25-30 kDa. Group 1 allergens are recognized by >95% of grass pollen allergic patients. We investigated the IgE recognition of Phl p 1 using allergen-specific IgE-derived single-chain variable Ab fragments (IgE-ScFvs) isolated from a combinatorial library constructed from PBMCs of a grass pollen-allergic patient. IgE-ScFvs reacted with recombinant Phl p 1 and natural group 1 grass pollen allergens. Using synthetic Phl p 1-derived peptides, the binding sites of two ScFvs were mapped to the N terminus of the allergen. In surface plasmon resonance experiments they showed comparable high-affinity binding to Phl p 1 as a complete human IgE-derived Ab recognizing the allergens' C terminus. In a set of surface plasmon resonance experiments simultaneous allergen recognition of all three binders was demonstrated. Even in the presence of the three binders, allergic patients' polyclonal IgE reacted with Phl p 1, indicating high-density IgE recognition of the Phl p 1 allergen. Our results show that multiple IgE Abs can bind with high density to Phl p 1, which may explain the high allergenic activity and sensitizing capacity of this allergen.

Passive immunization with allergen-specific antibodies.

The induction of allergen-specific IgG antibodies has been identified as a major mechanism responsible for the reduction of allergic inflammation in allergic patients treated by allergen-specific immunotherapy. Several studies suggest that allergen-specific IgG antibodies induced by vaccination with allergens block mast cell and basophil degranulation, IgE-facilitated allergen presentation to T cells and IgE production. The availability of recombinant allergens and technologies for the production of recombinant human antibodies allows engineering of allergen-specific antibodies which can be used for passive immunization (i.e., therapy) and eventually for the prevention of allergy (i.e., prophylaxis). This chapter summarizes data supporting the possible use of allergen-specific antibodies for treatment and prophylaxis. Finally, concrete approaches for the treatment and prevention of allergy based on blocking antibodies are envisioned.

High-affinity IgE recognition of a conformational epitope of the major respiratory allergen Phl p 2 as revealed by X-ray crystallography.

We report the three-dimensional structure of the complex between the major respiratory grass pollen allergen Phl p 2 and its specific human IgE-derived Fab. The Phl p 2-specific human IgE Fab has been isolated from a combinatorial library constructed from lymphocytes of a pollen allergic patient. When the variable domains of the IgE Fab were grafted onto human IgG1, the resulting Ab (huMab2) inhibited strongly the binding of allergic patients' IgE to Phl p 2 as well as allergen-induced basophil degranulation. Analysis of the binding of the allergen to the Ab by surface plasmon resonance yielded a very low dissociation constant (K(D) = 1.1 x 10(-10) M), which is similar to that between IgE and Fcepsilon;RI. The structure of the Phl p 2/IgE Fab complex was determined by x-ray crystallography to 1.9 A resolution revealing a conformational epitope (876 A(2)) comprised of the planar surface of the four-stranded anti-parallel beta-sheet of Phl p 2. The IgE-defined dominant epitope is discontinuous and formed by 21 residues located mostly within the beta strands. Of the 21 residues, 9 interact directly with 5 of the 6 CDRs (L1, L3, H1, H2, H3) of the IgE Fab predominantly by hydrogen bonding and van der Waals interactions. Our results indicate that IgE Abs recognize conformational epitopes with high affinity and provide a structural basis for the highly efficient effector cell activation by allergen/IgE immune complexes.