ovalbumin and Wiskott-Aldrich-Syndrome

Rearrangement of the cytoskeleton in T cells plays a critical role in the organization of a complex signaling interface referred to as immunologic synapse (IS). Surprisingly, the contribution of antigen presenting cells, in particular dendritic cells (DCs), to the structure and function of the IS has not been investigated in as much detail. We have used a natural model of cytoskeletal dysfunction caused by deficiency of the Wiskott-Aldrich syndrome protein (WASp) to explore the contribution of the DC cytoskeleton to IS formation and to T-cell priming. In an antigen-specific system, T-DC contacts were found to be less stable when DCs alone lacked WASp, and associated with multiple defects of IS structure. As a consequence, DCs were unable to support normal IL-12 secretion, and events downstream of TCR signaling were abrogated, including increased calcium flux, microtubule organizing center (MTOC) polarization, phosphorylation of ZAP-70, and T-cell proliferation. Formation of an effective signaling interface is therefore dependent on active cytoskeletal rearrangements in DCs even when T cells are functionally competent. Deficiency of DC-mediated activities may contribute significantly to the varied immunodysregulation observed in patients with WAS, and also in those with limited myeloid reconstitution after allogeneic hematopoietic stem cell transplantation.

Topics: Animals; Bacterial Proteins; Calcium Signaling; Cell Movement; Crosses, Genetic; Cytoskeleton; Dendritic Cells; Genes, Reporter; Genetic Complementation Test; Humans; Immunological Synapses; Luminescent Proteins; Lymphocyte Activation; Mice; Mice, Inbred C57BL; Mice, Knockout; Mice, Transgenic; Ovalbumin; Receptors, Antigen, T-Cell; Recombinant Fusion Proteins; T-Lymphocytes; Wiskott-Aldrich Syndrome; Wiskott-Aldrich Syndrome Protein

B cells and dendritic cells, lacking functional Wiskott-Aldrich syndrome protein (WASP), have aberrant formation of membrane protrusions. We hypothesized that protrusions may play a role in antigen presentation, and consequently, that impaired antigen presentation may be an underlying factor of the immune deficiency in patients with Wiskott-Aldrich syndrome. In this paper, we investigated the antigen presentation capacity of B cells and dendritic cells from WASP knockout mice, using soluble and particulate antigen, to CD4+ T cells from T-cell receptor transgenic DO11.10 mice. As antigen we used soluble ovalbumin (OVA), a peptide thereof (amino acids 323-339) or bacteria expressing OVA. We found that WASP-deficient B cells and dendritic cells efficiently processed and presented soluble OVA protein as well as its peptide in vitro, inducing proliferation and cytokine production from CD4+ T cells. Antigen presentation of soluble protein was efficient also in vivo, because immunization of WASP-deficient mice with OVA elicited proliferation of transferred, fluorescent-labelled, CD4+ T cells. Although we could detect uptake of bacteria in dendritic cells, processing and presentation of bacterial-expressed OVA was impaired in WASP-deficient dendritic cells. In conclusion, our data suggest that WASP is not needed for processing and presentation of soluble antigen, but that efficient presentation of particulate antigen require WASP.

Topics: Animals; Antigen Presentation; Antigens, Bacterial; B-Lymphocytes; CD4-Positive T-Lymphocytes; Cells, Cultured; Cytokines; Dendritic Cells; Female; Immunity, Cellular; Male; Mice; Mice, Transgenic; Ovalbumin; Peptide Fragments; Proteins; Solubility; Wiskott-Aldrich Syndrome; Wiskott-Aldrich Syndrome Protein

Wiskott-Aldrich syndrome (WAS) is an X-linked recessive disorder characterized by thrombocytopenia with small platelets, severe eczema, and recurrent infections due to defects in the immune system. The disease arises from mutations in the gene encoding the WAS protein (WASP), which plays a role as an adaptor molecule in signal transduction accompanied by cytoskeletal rearrangement in T cells. To investigate the functional domain of WASP, we developed transgenic mice overexpressing the WASP N-terminal region (exon 1-5) including the Ena/VASP homology 1 (pleckstrin homology/WASP homology 1) domain, in which the majority of mutations in WAS patients have been observed. WASP transgenic mice develop and grow normally under the specific pathogen-free environment, and showed normal lymphocyte development. However, proliferative responses and cytokine production induced by TCR stimulation were strongly inhibited in transgenic mice, whereas Ag receptor capping and actin polymerization were normal. These findings suggest that overexpressed Ena/VASP homology 1 (pleckstrin homology/WASP homology 1) domain of WASP inhibits the signaling from TCR without coupling of cytoskeletal rearrangement. WASP transgenic mice shown here could be valuable tools for further understanding the WASP-mediated processes.

Topics: Actins; Animals; B-Lymphocytes; Codon, Nonsense; Cytokines; Cytoskeleton; Immunosuppressive Agents; Lymphocyte Activation; Mice; Mice, Transgenic; Mutation, Missense; Ovalbumin; Peptide Fragments; Platelet Count; Protein Structure, Tertiary; Proteins; Receptor Aggregation; Receptors, Antigen, T-Cell; Spleen; T-Lymphocytes; Vaccination; Wiskott-Aldrich Syndrome; Wiskott-Aldrich Syndrome Protein