Elke Luger

CD38 low IgG-secreting cells are precursors of various CD38 high-expressing plasma cell populations

Despite the important role immunoglobulin G (IgG)‐secreting plasma cells play in memory immune responses, the differentiation and homeostasis of these cells are not completely understood. Here, we studied the differentiation of human IgG‐secreting cells ex vivo and in vitro, identifying these cells by the cellular affinity matrix technology. Several subpopulations of IgG‐secreting cells were identified among the cells isolated from tonsils and bone marrow, particularly differing in the expression levels of CD9, CD19, and CD38. CD38 low IgG‐secreting cells were present exclusively in the tonsils. A major fraction of these cells appeared to be early plasma cell precursors, as upon activation of B cells in vitro, IgG secretion preceded up‐regulation of CD38, and on tonsillar sections, IgG‐containing, CD38 low cells with a plasmacytoid phenotype were found in follicles, where plasma cell differentiation starts. A unitary phenotype of migratory peripheral blood IgG‐secreting cells suggests that all bone marrow plasma cell populations share a common precursor cell. These data are compatible with a multistep model for plasma cell differentiation and imply that a common CD38 low IgG‐secreting precursor gives rise to a diverse plasma cell compartment.

Induction of long-lived allergen-specific plasma cells by mucosal allergen challenge

BACKGROUND Allergen-specific IgE antibodies are responsible for the pathogenesis of type I hypersensitivity. In patients with allergy, IgE titers can persist in the apparent absence of allergen for years. Seasonal allergen exposure triggers clinical symptoms and enhances allergen-specific IgE. Whether allergen-specific plasma cells originating from seasonal allergen exposures can survive and become long-lived is so far unclear. OBJECTIVE We analyzed the localization and lifetimes of allergen-specific IgE-secreting, IgA-secreting, and IgG(1)-secreting plasma cells after allergen inhalation in an ovalbumin-induced murine model of allergic asthma. METHODS Ovalbumin-specific IgG(1)-secreting, IgA-secreting, and IgE-secreting cells in lungs, spleen, and bone marrow were isolated and tested for antibody secretion by the ELISpot technique. Longevity of ovalbumin-specific plasma cells was determined by cyclophosphamide treatment, which depletes proliferating plasmablasts but leaves plasma cells untouched. Ovalbumin aerosol-induced infiltrates in lungs were localized by confocal microscopy. RESULTS Long-lived ovalbumin-specific plasma cells were generated by systemic sensitization and survived in bone marrow and spleen, maintaining systemic ovalbumin-specific titers of IgG, IgA, and IgE. On inhalation of ovalbumin-containing aerosol, sensitized mice developed airway inflammation and more ovalbumin-specific IgG(1)-secreting, IgA-secreting, and IgE-secreting cells in the lungs and in secondary lymphoid organs. These plasma cells joined the pool of ovalbumin-specific plasma cells in the bone marrow and became long-lived-that is, they are resistant to cyclophosphamide. Termination of ovalbumin inhalation depleted ovalbumin-specific plasma cells from the lungs, but they persisted in spleen and bone marrow. CONCLUSION Our results show that inhalation of aerosolized allergen generates long-lived, allergen-specific IgG(1)-secreting, IgA-secreting, and IgE-secreting plasma cells that survive cytostatic treatment.

Adaptation of humoral memory.

Summary:  Immunological memory, as provided by antibodies, depends on the continued presence of antibody‐secreting cells, such as long‐lived plasma cells of the bone marrow. Survival niches for these memory plasma cells are limited in number. In an established immune system, acquisition of new plasma cells, generated in response to recent pathogenic challenges, requires elimination of old memory plasma cells. Here, we review the adaptation of plasma cell memory to new pathogens. This adaptation is dependent upon the influx of plasmablasts, generated in a secondary systemic immune reaction, into the pool of memory plasma cells, the efficiency of competition of new plasmablasts with old plasma cells, and the frequency of infection with novel pathogens. To maintain old plasma cells at frequencies high enough to provide protection and to accommodate as many specificities as possible, an optimal influx rate per infection exists. This optimal rate is approximately three times higher than the minimal number of plasma cells providing protection. Influx rates of plasmablasts generated by vaccination approximately match this optimum level. Furthermore, the observed stability of serum concentrations of vaccine‐specific antibodies implies that the influxing plasmablasts mobilize a similar number of plasma cells and that competitive infectious challenges are not more frequent than once per month.

Nerve Growth Factor and Neurotrophin-3 Mediate Survival of Pulmonary Plasma Cells during the Allergic Airway Inflammation

Allergen-specific Abs play a pivotal role in the induction and maintenance of allergic airway inflammation. During secondary immune responses, plasma cell survival and Ab production is mediated by extrinsic factors provided by the local environment (survival niches). It is unknown whether neurotrophins, a characteristic marker of allergic airway inflammation, influence plasma cell survival in the lung. Using a mouse model of allergic asthma, we found that plasma cells from the lung and spleen are distinct subpopulations exhibiting differential expression patterns of neurotrophins and their receptors (Trks). In vitro, the nerve growth factor (NGF) and neurotrophin-3 (NT3) led to a dose-dependent increase in viability of isolated pulmonary plasma cells due to up-regulation of the antiapoptotic Bcl2 pathway. In parallel, the expression of transcription factors that stimulate the production of immunoglobulins (X-box binding protein 1 and NF-κB subunit RelA) was enhanced in plasma cells treated with NGF and NT3. These findings were supported in vivo. When the NGF pathway was blocked by intranasal application of a selective TrkA inhibitor, sensitized mice showed reduced numbers of pulmonary plasma cells and developed lower levels of allergen-specific and total serum IgE in response to OVA inhalation. This suggests that in the allergic airway inflammation, NGF/TrkA-mediated pulmonary IgE production contributes significantly to serum-IgE levels. We conclude that the neurotrophins NGF and NT3 act as survival factors for pulmonary plasma cells and thus are important regulators of the local Ab production in the allergic airway disease.

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.

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.

Construction of an sIgE:FLAG-mIgE:GFP Reporter Mouse Strain

Like all other immunoglobulins, IgE can be secreted into the blood or expressed as a membrane receptor on the surface of B lymphocytes. Secreted immunoglobulins trace the antigen and contribute to its destruction. Membrane immunoglobulins accompany the B cell along its differentiation pathway, regulating processes like the induction and maintenance of immunological memory and differentiation of plasma cells. The regulation of the expression of IgE is very complex. A lot of positive and negative regulators influence the synthesis of IgE. In previous publications, we were able to show that the membrane IgE (mIgE) antigen receptor itself controls the quantity and quality of serum IgE produced. However, the knowledge about the regulatory function of the antigen receptor on these processes is at best limited. In the present paper, we present the construction of a reporter mouse strain, which will help us to follow an mIgE-bearing B cell during the immune response more precisely.