Gertrude Achatz-Straussberger

Inefficient processing of mRNA for the membraneform of IgE is a genetic mechanism to limit recruitment of IgE-secreting cells

Immunoglobulin E (IgE) is the key effector element in allergic diseases ranging from innocuous hay fever to life‐threatening anaphylactic shock. Compared to other Ig classes, IgE serum levels are very low. In its membrane‐bound form (mIgE), IgE behaves as a classical antigen receptor on B lymphocytes. Expression of mIgE is essential for subsequent recruitment of IgE‐secreting cells. We show that in activated, mIgE‐bearing B cells, mRNA for the membrane forms of both murine and human epsilon (ϵ) heavy chains (HC) are poorly expressed compared to mRNA for the secreted forms. In contrast, in mIgG‐bearing B cells, mRNA for the membrane forms of murine gamma‐1 (γ1) and the corresponding human γ4 HC are expressed at a much higher level than mRNA for the respective secreted forms. We show that these findings correlate with the presence of deviant polyadenylation signal hexamers in the 3′ untranslated region (UTR) of both murine and human ϵ genes, causing inefficient processing of primary transcripts and thus poor expression of the proteins and poor recruitment of IgE‐producing cells in the immune response. Thus, we have identified a genetic steering mechanism in the regulation of IgE synthesis that represents a further means to restrain potentially dangerous, high serum IgE levels.

Targeting the extracellular membrane-proximal domain of membrane-bound IgE by passive immunization blocks IgE synthesis in vivo.

The classical allergic reaction starts seconds or minutes after Ag contact and is committed by Abs produced by a special subset of B lymphocytes. These Abs belong to the IgE subclass and are responsible for Type I hyperreactivity reactions. Treatment of allergic diseases with humanized anti-IgE Abs leads primarily to a decrease of serum IgE levels. As a consequence, the number of high-affinity IgE receptors on mast cells and basophils decreases, leading to a lower excitability of the effector cells. The biological mechanism behind anti-IgE therapy remains partly speculative; however, it is likely that these Abs also interact with membrane IgE (mIgE) on B cells and possibly interfere with IgE production. In the present work, we raised a mouse mAb directed exclusively against the extracellular membrane-proximal domain of mIgE. The interaction between the monoclonal anti-mIgE Ab and mIgE induces receptor-mediated apoptosis in vitro. Passive immunization experiments lead to a block of newly synthesized specific IgEs during a parallel application of recombinant Bet v1a, the major birch pollen allergen. The decrease of allergen-specific serum IgE might be related to tolerance-inducing mechanisms stopping mIgE-displaying B cells in their proliferation and differentiation.

Migration of antibody secreting cells towards CXCL12 depends on the isotype that forms the BCR

Truncation of the cytoplasmic tail of membrane‐bound IgE in vivo results in lower serum IgE levels, decreased numbers of IgE‐secreting plasma cells and the abrogation of specific secondary immune responses. Here we present mouse strain KN1 that expresses a chimeric ε‐γ1 BCR, consisting of the extracellular domains of the ε gene and the transmembrane and cytoplasmic domains of the γ1 gene. Thus, differences in the IgE immune response of KN1 mice reflect the influence of the “γ1‐mediated signalling” of mIgE bearing B cells. KN1 mice show an increased serum IgE level, resulting from an elevated number of IgE‐secreting cells. Although the primary IgE immune response in KN1 mice is inconspicuous, the secondary response is far more robust. Most strikingly, IgE‐antibody secreting cells with “γ1‐signalling history” migrate more efficiently towards the chemokine CXCL12, which guides plasmablasts to plasma cell niches, than IgE‐antibody secreting cells with WT “ε‐signalling history”. We conclude that IgE plasmablasts have an intrinsic, lower chance to contribute to the long‐lived plasma cell pool than IgG1 plasmablasts.

Somatic diversity of the immunoglobulin repertoire is controlled in an isotype‐specific manner

We have studied two aspects of the IgE immune response. First, we have compared the kinetics of the IgE response to the T cell‐dependent antigen ph‐Ox coupled to ovalbumin with that of the IgG1 response and we have assessed the quality of the IgE response. Second, we have studied the generation of somatic diversity, understood as the combined effect of somatic mutation and the selection of D(iversity) and J(oining) elements, in germinal center B cells at the molecular level, using the germ‐line sequence of the prototype anti‐ph‐Ox heavy chain variable element VHOx1 as reference. We evaluated sequences derived from μ‐, γ1‐ and ϵ‐variable elements and showed that somatic diversification was different for all isotypes studied. We further compared the IgE responses of wild‐type mice with those of mice expressing a truncated cytoplasmic IgE tail (IgEKVKΔtail). IgEKVKΔtail mice showed a more diverse sequence pattern. We corroborated previous results suggesting that short CDR3 regions are indicative for high‐affinity antibodies by measuring relative affinities of phage‐expressed Fab fragments with prototype long and short CDR3 regions. Therefore, the composition of the antigen‐receptor is responsible for the selection process and the expansion of antigen‐specific cells, leading to an isotype‐specific antibody repertoire.

HS1-Associated Protein X-1 Interacts with Membrane-Bound IgE: Impact on Receptor-Mediated Internalization

Engagement of the BCR triggers signals that control affinity maturation, memory induction, differentiation, and various other physiological processes in B cells. In previous work, we showed that truncation of the cytoplasmic tail of membrane-bound Ig (mIg)E in vivo resulted in lower serum IgE levels, decreased numbers of IgE-secreting plasma cells, and the abrogation of specific secondary responses correlating with a defect in the selection of high-affinity Abs during the germinal center reaction. We concluded that the Ag receptor is necessary at all times during Ab responses not only for the maturation process, but also for the expansion of Ag-specific B cells. Based on these results, we asked whether the cytoplasmic tail of mIgE, or specific proteins binding the cytoplasmic tail in vivo commit a signal transduction accompanying the B cell along its differentiation process. In this study, we present the identification of HS1-associated protein X-1 as a novel protein interacting with the cytoplasmic tail of mIgE. ELISA, surface plasmon resonance analysis, and coimmunoprecipitation experiments confirmed the specific interaction in vitro. In functional assays, we clearly showed that HS1-associated protein X-1 expression levels influence the efficiency of BCR-mediated Ag internalization.

HAX1 deficiency: impact on lymphopoiesis and B-cell development.

HAX1 was originally described as HS1‐associated protein with a suggested function in receptor‐mediated apoptotic and proliferative responses of lymphoid cells. Recent publications refer to a complex and multifunctional role of this protein. To investigate the in vivo function of HAX1 (HS1‐associated protein X1) in B cells, we generated a Hax1‐deficient mouse strain. Targeted deletion of Hax1 resulted in premature death around the age of 12 wk accompanied by a severe reduction of lymphocytes in spleen, thymus and bone marrow. In the bone marrow, all B‐cell populations were lost comparably. In the spleen, B220+ cells were reduced by almost 70%. However, as investigated by adoptive transfer experiments, this impairment is not exclusively B‐cell intrinsic and we hypothesize that a HAX1‐deficient environment cannot sufficiently provide the essential factors for proper lymphocyte development, trafficking and survival. Hax1−/− B cells show a significantly reduced expression of CXCR4, which might have an influence on the observed defects in B‐cell development.

Phage Display Based Cloning of Proteins Interacting with the Cytoplasmic Tail of Membrane Immunoglobulins

The reduced quantity and quality of serum immunoglobulins (sIgs) in mutant mice expressing truncated cytoplasmic tails of IgE and IgG1 indicate an active role for the cytoplasmic domains of mIgG1 and mIgE. We used phage display technology to identify candidate proteins able to interact with the cytoplasmic tail of mIgE. Using a murine cDNA B cell library displayed on the surface of phage as prey and the 28 amino acid long cytoplasmic tail of IgE as bait, we isolated phage encoding the murine hematopoietic progenitor kinase 1 (HPK1). Surface plasmon resonance analysis measurements confirmed affinity of HPK1 to the mIgE cytoplasmic tail and revealed association to other immunoglobulin isotypes as well. Immunoprecipitation experiments, using lysates from two B cell lines expressing nitrophenyl (NP) specific mIgE molecules showed co-precipitation of IgE and HPK1. The interaction of HPK1 with the cytoplasmic domains of membrane immunoglobulins indicate an active role of the tails as part of an isotype specific signal transduction, independent from the Igα/Igβ heterodimers, and may represent a missing link to upstream regulatory elements of HPK1 activation.

The IgE Antigen Receptor: A Key Regulator for the Production of IgE Antibodies

Immunoglobulins in general form a substantial component of serum proteins, and play a role in homeostatic mechanisms, a first line of defense against pathogenic organisms and in immunological memory. In the secreted form, immunoglobulins represent the effector arm of the humoral immune system. However, immunoglobulins are not only secreted, but can also be expressed on the surface of a B lymphocyte (membrane immunoglobulin), and, in this physical state, most likely convey signals to steer the B cell along its differentiation pathway. A step forward in the understanding of the role of membrane immunoglobulins other than membrane IgM or IgD was achieved with two mouse lines with mutations in the epsilon heavy chain gene. In IgEΔM1M2 mice serum IgE is reduced to less than 10% of normal mice, while IgEKVKΔtail mice show a reduction of 50%, reflecting a serious impairment of the IgE-mediated immune response. We think that the cytoplasmic tail of IgE is involved in a signal transduction which leads to the expression of high quantities and qualities of secreted IgE immunoglobulins.

The Biology of IgE: Molecular Mechanism Restraining Potentially Dangerous High Serum IgE Titres In Vivo

Our knowledge about the regulation of the expression of IgE and its biological function is at best limited. We do, however, know that the production of IgE is tightly regulated which is reflected by the fact that the steady-state serum levels of IgE in mice and humans are 3–4 orders of magnitude lower if compared to IgG1, which is an immunoglobulin isotype expressed in response to the same cytokine milieu. What are the rate-limiting steps responsible for this discrepancy? In the following chapter six molecular mechanisms restraining IgE levels will be discussed in detail. The understanding of these mechanisms, combined with the analysis of the biological function of the IgE molecule during an immune response, is the prerequisite for the establishment of new systemic IgE-targeted therapeutic strategies in the future.

Cutting Edge: IgE Plays an Active Role in Tumor Immunosurveillance in Mice

Exogenous IgE acts as an adjuvant in tumor vaccination in mice, and therefore a direct role of endogenous IgE in tumor immunosurveillance was investigated. By using genetically engineered mice, we found that IgE ablation rendered mice more susceptible to the growth of transplantable tumors. Conversely, a strengthened IgE response provided mice with partial or complete resistance to tumor growth, depending on the tumor type. By genetic crosses, we showed that IgE-mediated tumor protection was mostly lost in mice lacking FcεRI. Tumor protection was also lost after depletion of CD8+ T cells, highlighting a cross-talk between IgE and T cell–mediated tumor immunosurveillance. Our findings provide the rationale for clinical observations that relate atopy with a lower risk for developing cancer and open new avenues for the design of immunotherapeutics relevant for clinical oncology.