HAX1 deletion impairs BCR internalization and leads to delayed BCR-mediated apoptosis.

Deletion of HAX1 in mice causes a severe reduction in the numbers of lymphocytes in the bone marrow and in the spleen. Additionally, B220(+) B progenitor cells in the bone marrow are reduced, suggesting an important function of HAX1 in B cell development. HAX1 is thought to play a protective role in apoptotic processes; therefore, we investigated the role of HAX1 in bone marrow B progenitor cells and splenic B cells. We did not observe an effect on the survival of Hax1(-/-) bone marrow cells but detected enhanced survival of splenic Hax1(-/-) B cells upon in vitro starvation/growth-factor withdrawal. To explain this apparent inconsistency with previous reports of HAX1 function, we also studied the B cell receptor (BCR)-induced apoptosis of IgM-stimulated splenic naïve B cells and found that apoptosis decreased in these cells. We further found impaired internalization of the BCR from Hax1(-/-) splenic B cells after IgM crosslinking; this impaired internalization may result in decreased BCR signaling and, consequently, decreased BCR-mediated apoptosis. We measured HAX1 binding to the cytoplasmic domains of different Ig subtypes and identified KVKWI(V)F as the putative binding motif for HAX1 within the cytoplasmic domains. Because this motif can be found in almost all Ig subtypes, it is likely that HAX1 plays a general role in BCR-mediated internalization events and BCR-mediated apoptosis.

Generation of hypoallergenic DNA vaccines by forced ubiquitination: preventive and therapeutic effects in a mouse model of allergy.

BACKGROUND: Hypoallergenic immunotherapy of type I allergies aims at inducing T-cell immunity while avoiding cross-linking of pre-existing IgE. DNA-based immunotherapy depends on the recruitment of antigen-specific T(H)1 cells and therefore has to provide the whole repertoire of T-cell epitopes. Ubiquitination offers a general approach for the production of hypoallergenic DNA vaccines. OBJECTIVE: A DNA-based vaccine encoding the major birch pollen allergen Bet v 1 stably linked to ubiquitin was evaluated for its antiallergic potential in a BALB/c mouse model of allergy. METHODS: Plasmid DNA was applied to mice before (preventive) or after (therapeutic) sensitization with recombinant Bet v 1. In the preventive setting, mice were exposed to aerosolized allergen in addition. Cytokine production was monitored via ELISPOT and Luminex. IgG(1), IgG(2a), and IgE subclass antibody titers were determined by ELISA. In vitro antigen-specific cross-linking of IgE was measured in a degranulation assay. Bronchoalveolar lavages were analyzed for leukocyte subsets as well as for IFN-gamma and IL-5, and paraffin sections of lungs were examined for mucus production and endothelial damage. RESULTS: Prevaccination with ubiquitinated Bet v 1-stimulated T(H)1-biased immune responses with concomitant suppression of functional IgE, reduction of eosinophil counts in bronchoalveolar lavages, and alleviation of lung pathology, and could also suppress an ongoing IgE response in a therapeutic setting. CONCLUSION: The data clearly demonstrate that hypoallergenic DNA vaccines encoding ubiquitin fusion constructs induce effective antiallergic immune responses. CLINICAL IMPLICATIONS: Ubiquitination of allergen gene vaccines eliminates the risk of IgE cross-linking, thereby meeting the safety requirements for clinical applications.