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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., (Copyright © 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

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 epsilon-gamma1 BCR, consisting of the extracellular domains of the epsilon gene and the transmembrane and cytoplasmic domains of the gamma1 gene. Thus, differences in the IgE immune response of KN1 mice reflect the influence of the "gamma1-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 "gamma1-signalling history" migrate more efficiently towards the chemokine CXCL12, which guides plasmablasts to plasma cell niches, than IgE-antibody secreting cells with WT "epsilon-signalling history". We conclude that IgE plasmablasts have an intrinsic, lower chance to contribute to the long-lived plasma cell pool than IgG1 plasmablasts.

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 V(H)Ox1 as reference. We evaluated sequences derived from mu-, gamma 1- and epsilon-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 (IgE(KVK Delta tail)). IgE(KVK Delta tail) 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.

We have studied the induction and maintenance of tolerance in the B lymphocyte compartment. Neonatal and adult transgenic mice which expressed either surface IgM (sIgM) or sIgM and sIgD anti-2,4,6-trinitrophenyl (TNP) were treated with soluble mono- and multivalent forms of TNP-modified carriers. We compared the B cell compartment of mice treated with antigen and of littermates injected with phosphate-buffered saline. Antigen-mediated cross-linking of membrane-bound IgM (sIgM) caused deletion of B cells both in neonatal and adult mice with mu and kappa transgenes. Deletion was the result of apoptosis. In mice that carried an additional delta transgene sIgD interfered with tolerance induction. The stage in which the cells were sensitive to deletion was characterized as a transitional stage between immature (sIgMdull, heat-stable antigenbright, B220dull, sIgD-) and more mature (IgMbright, heat-stable antigendull, B220bright, sIgD-) B cells. Surviving cells were functional as measured by receptor-mediated changes in the intracellular free Ca2+ concentration. We propose that when the immature B cells have reached the final stages of maturation IgM always transmits negative signals in the absence of T cell help. When B cells need to be screened against self reactivity IgM is the only antigen receptor expressed. The presence of sIgD protects resting B cells from deletion and allows them to initiate an effective immune response.

We have generated and examined transgenic mice carrying a rearranged immunoglobulin transgene coding for the heavy chain of an IgE antibody. These mice produce the secreted form of the recombinant epsilon heavy chain. Serum IgE levels were increased at least 100-fold over control values. Transgenic epsilon mRNA was detected in spleen and thymus, not in liver and heart. Transgenic epsilon production in vitro was slightly up-regulated by T cells, but not affected by interleukin 4 in vitro or Nippostrongylus infestation in vivo. The B cell and T cell compartments and antigen-specific IgE, IgG1 and IgM responses as well as the increase in endogenous IgE after Nippostrongylus infestation in transgenic mice were normal. These data indicate that the presence of high levels of transgenic IgE did not induce class-specific suppressive mechanisms. Transgenic IgE bound to Fc epsilon receptor type I and Fc epsilon receptor type II and mediated histamine release from mast cells in vitro and an allergic skin reaction in vivo. It inhibited an ovalbumin-specific skin reaction in ovalbumin-immunized transgenic mice only during the initial phases of the immune response. This result has a bearing on the feasibility of immune therapy of allergic diseases with substances that block binding of IgE to its receptors.

We have examined the immune repertoire and immune response of a mouse that carries transgenes for a mu heavy chain and kappa light chain. The expression of these genes is under the regulation of their own controlling elements. The transgenes are expressed early in ontogeny and are easily detectable from day 13 of gestation onwards. The pre-B cells seem to function normally as they generate IgM-secreting colonies at normal frequencies. Colonies show predominantly the transgenic specificity. Expression of the transgenes is not limited to B cells since around 10%-20% of peripheral T cells and 50% of thymocytes express the mu transgene as an intracellular protein. Ectopic expression of kappa was not seen. The spleen size of the transgenic mouse is decreased by around 20%; this reduction is largely caused by a reduction of the B cell pool. Almost all B cells express the transgenes, only 30% co-express endogenous heavy chain genes and all co-express endogenous light chain genes. Serum Ig levels for IgM and IgA were normal, 20% of the IgM consist of the transgenic product. Serum IgG levels were decreased. T cell functions (helper and cytotoxic) were normal. Immune responses to conventional antigens were impaired, especially in the early phases of the immune response, but after boosting they were virtually normal, except for IgG3 which remained low. Primary antibody responses to T cell-independent antigens of the class II type (bacterially related antigens) were absent, although precursor frequencies for these antigens were within the expected range. The significance of this finding, as it relates to allelic exclusion of Ig genes, is discussed.

Helper T (Th) cell-dependent activation requirements for 2,4,6-trinitrophenyl (TNP)-specific resting B cells obtained from mice transgenic for Sp-6 mu, kappa genes were analyzed. Carrier-specific T cell help required linked recognition of TNP carrier and was functionally restricted by the B cell major histocompatibility complex. However, histoincompatible T cell-B cell conjugates formed by bridging surface immunoglobulin and Th cell receptor for antigen (TcR) through TNP-conjugated anti-TcR antibodies resulted in the efficient differentiation of TNP-specific B cells. Thus, Th cell-dependent cognate recognition of B cells is not obligatory. Specific conjugate formation could be obviated by using unconjugated fragments of anti-TcR antibodies. If dimeric, these fragments supported the Th cell-dependent differentiation of co-cultured histoincompatible resting B cells. Unconjugated monomeric fragments were ineffective, demonstrating the necessity for TcR cross-linking. Resting B cells from Sp-6+ mice rendered TNP-conjugated monomeric fragments of anti-TcR antibodies effectively multivalent, thereby satisfying conditions for the activation of co-cultured Th cells. The results demonstrate that Th cells do not transduce activation signals through TcR recognition of B cell membrane-associated ligand which limit the induction of B cell differentiation. Cross-linking of TcR on Th cells is required, sufficient and can be induced through interaction with the antigen-specific B cell surface.

The uptake in vitro was studied of 3H-labeled DNA-anti-DNA complexes by neutrophils and monocytes from human blood. Complexes were prepared from 3H-labeled circular double-stranded (dS) DNA of bacteriophage PM2 and anti-dsDNA-containing sera from patients with systemic lupus erythematosus. After phagocytosis, cells and medium were separated. The cells were treated with DNase to remove adherent and noningested complexes before the cell-associated radioactivity was counted. Thus, only complexes inside the cells were measured. The medium was analyzed for acid-precipitable radioactivity. In this way, we found that neutrophils only phagocytose the complexes, whereas monocytes phagocytose the complexes and degrade the antigen. In contrast, both types of phagocyte degraded the antigen in tetanus-anti-tetanus complexes. The degradation took place after phagocytosis, inside the cells. The difference in DNA degradation between neutrophils and monocytes correlated with the difference in acid DNase activity of the lysosomal fractions: monocytes contained DNase activity, neutrophils did not. With complexes made from DNA with 131I-labeled anti-DNA, we found that both cell types degraded the antibody. Uptake of complexes and degradation of antigen increased with incubation time and cell concentration and was saturable with respect to complex concentration. The processes were inhibited by 5 mM mono-iodoacetic acid or by low temperatures.

The influence was studied of the size of DNA-anti-DNA complexes on their capacity to induce phagocytosis and degradation by monocytes and neutrophils from human blood. An estimate of the size of the complexes was obtained by precipitation analysis, sucrose-gradient velocity sedimentation and filtration over Nucleopore filters. The results show that only those complexes are processes that precipitate in 15 min at 3000 x g. These complexes have an S value of more than 25000 and a diameter of more than 0.4 micrometer. Likewise, tetanus-anti-tetanus complexes also had to be precipitable in 15 min at 3000 x g to be processed by the phagocytes.