Nadja Zaborsky

Antigen aggregation decides the fate of the allergic immune response.

Previously, defined naturally occurring isoforms of allergenic proteins were classified as hypoallergens and therefore suggested as an agent for immunotherapy in the future. In this paper, we report for the first time the molecular background of hypoallergenicity by comparing the immunological behavior of hyperallergenic Betula verrucosa major Ag 1a (Bet v 1a) and hypoallergenic Bet v 1d, two isoforms of the major birch pollen allergen Betula verrucosa 1. Despite their cross-reactivity, Bet v 1a and Bet v 1d differ in their capacity to induce protective Ab responses in BALB/c mice. Both isoforms induced similar specific IgE levels, but only Bet v 1d expressed relevant titers of serum IgGs and IgAs. Interestingly, hypoallergenic Bet v 1d activated dendritic cells more efficiently, followed by the production of increased amounts of Th1- as well as Th2-type cytokines. Surprisingly, compared with Bet v 1a, Bet v 1d-immunized mice showed a decreased proliferation of regulatory T cells. Crystallographic studies and dynamic light scattering revealed that Bet v 1d demonstrated a high tendency to form disulfide-linked aggregates due to a serine to cysteine exchange at residue 113. We conclude that aggregation of Bet v 1d triggers the establishment of a protective Ab titer and supports a rationale for Bet v 1d being a promising candidate for specific immunotherapy of birch pollen allergy.

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.

Reshaping the Bet v 1 fold modulates TH polarization

BACKGROUND Several alternative mechanisms have been proposed to explain why some proteins are able to induce a T(H)2-biased and IgE-mediated immune response. These include specific interactions with receptors of the innate immune system, proteolytic activities, allergen-associated carbohydrate structures, and intrinsic structural determinants. OBJECTIVES Available data suggest that a fold-dependent allergy-promoting mechanism could be a driving force for the T(H)2-polarization activity of Bet v 1, the major birch pollen allergen. METHODS Computer-aided sequence and fold analysis of the Bet v 1 family identified a short stretch susceptible for mutations inducing an altered fold of the entire molecule. With this knowledge, 7 consecutive amino acids of Bet v 1 were replaced with the homologous Mal d 1 sequence, creating the derivative BM4. RESULTS The minimal changes of the sequence led to a loss of the Bet v 1-like fold and influenced the immunologic behavior. Compared to wild-type Bet v 1, BM4 induced elevated T-cell proliferation of human PBMCs. In the mouse model, immunization with Bet v 1 absorbed to aluminum hydroxide triggered strong T(H)2 polarization, whereas BM4 immunization additionally recruited T(H)1 cells. Furthermore, the fold variant BM4 showed enhanced uptake by dendritic cells and a decreased susceptibility to endo-/lysosomal proteolysis. CONCLUSION Modifications in the 3-dimensional structure of Bet v 1.0101 resulted in a change of its immunologic properties. We observed that the fold alteration led to a modified crosstalk with dendritic cells and a shift of the immune response polarization toward a mixed T(H)1/T(H)2 cytokine production.

Chemotherapy-induced augmentation of T cells expressing inhibitory receptors is reversed by treatment with lenalidomide in chronic lymphocytic leukemia

While T-cell dysfunction occurring alongside chronic lymphocytic leukemia (CLL) is well documented (reviewed by Pleyer et al. [1][1] and Hamblin et al. [2][2]), it was recently reported that also T-cell exhaustion is associated with CLL, reflected in the presence of PD-1+ CLL T cells and higher

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.

HPK1 Associates with SKAP-HOM to Negatively Regulate Rap1-Mediated B-Lymphocyte Adhesion

Background Hematopoietic progenitor kinase 1 (HPK1) is a Ste20-related serine/threonine kinase activated by a range of environmental stimuli including genotoxic stress, growth factors, inflammatory cytokines and antigen receptor triggering. Being inducibly recruited to membrane-proximal signalling scaffolds to regulate NFAT, AP-1 and NFκB-mediated gene transcription in T-cells, the function of HPK1 in B-cells to date remains rather ill-defined. Methodology/Principal Findings By using two loss of function models, we show that HPK1 displays a novel function in regulating B-cell integrin activity. Wehi 231 lymphoma cells lacking HPK1 after shRNA mediated knockdown exhibit increased basic activation levels of Ras-related protein 1 (Rap1), accompanied by a severe lymphocyte function-associated antigen-1 (LFA-1) dependent homotypic aggregation and increased adhesion to intercellular adhesion molecule 1 (ICAM-1). The observed phenotype of enhanced integrin activity is caused downstream of Src, by a signalling module independent of PI3K and PLC, involving HPK1, SKAP55 homologue (SKAP-HOM) and Rap1-GTP-interacting adaptor molecule (RIAM). This alters actin dynamics and renders focal adhesion kinase (FAK) constitutively phosphorylated. Bone marrow and splenic B-cell development of HPK1−/− mice are largely unaffected, except age-related tendencies for increased splenic cellularity and BCR downregulation. In addition, naïve splenic knockout B-cells appear hyperresponsive to a range of stimuli applied ex vivo as recently demonstrated by others for T-cells. Conclusions/Significance We therefore conclude that HPK1 exhibits a dual function in B-cells by negatively regulating integrin activity and controlling cellular activation, which makes it an interesting candidate to study in pathological settings like autoimmunity and cancer.

Chronic lymphocytic leukaemia induces an exhausted T cell phenotype in the TCL1 transgenic mouse model

Although chronic lymphocytic leukaemia (CLL) is a B cell malignancy, earlier studies have indicated a role of T cells in tumour growth and disease progression. In particular, the functional silencing of antigen‐experienced T cells, called T cell exhaustion, has become implicated in immune evasion in CLL. In this study, we tested whether T cell exhaustion is recapitulated in the TCL1tg mouse model for CLL. We show that T cells express high levels of the inhibitory exhaustion markers programmed cell death 1 (PDCD1, also termed PD‐1) and lymphocyte‐activation gene 3 (LAG3), whereas CLL cells express high levels of CD274 (also termed PD‐ligand 1). In addition, the fraction of exhausted T cells increases with CLL progression. Finally, we demonstrate that exhausted T cells are reinvigorated towards CLL cytotoxicity by inhibition of PDCD1/CD274 interaction in vivo.

AID induces intraclonal diversity and genomic damage in CD86+ chronic lymphocytic leukemia cells

The activation‐induced cytidine deaminase (AID) mediates somatic hypermutation and class switch recombination of the Ig genes by directly deaminating cytosines to uracils. As AID causes a substantial amount of off‐target mutations, its activity has been associated with lymphomagenesis and clonal evolution of B‐cell malignancies. Although it has been shown that AID is expressed in B‐cell chronic lymphocytic leukemia (CLL), a clear analysis of in vivo AID activity in this B‐cell malignancy remained elusive. In this study performed on primary human CLL samples, we report that, despite the presence of a dominant VDJ heavy chain region, a substantial intraclonal diversity was observed at VDJ as well as at IgM switch regions (Sμ), showing ongoing AID activity in vivo during disease progression. This AID‐mediated heterogeneity was higher in CLL subclones expressing CD86, which we identified as the proliferative CLL fraction. Finally, CD86 expression correlated with shortened time to first treatment and increased γ‐H2AX focus formation. Our data demonstrate that AID is active in CLL in vivo and thus, AID likely contributes to clonal evolution of CLL.

Alternative splice variants of AID are not stoichiometrically present at the protein level in chronic lymphocytic leukemia

Activation‐induced deaminase (AID) is a DNA‐mutating enzyme that mediates class‐switch recombination as well as somatic hypermutation of antibody genes in B cells. Due to off‐target activity, AID is implicated in lymphoma development by introducing genome‐wide DNA damage and initiating chromosomal translocations such as c‐myc/IgH. Several alternative splice transcripts of AID have been reported in activated B cells as well as malignant B cells such as chronic lymphocytic leukemia (CLL). As most commercially available antibodies fail to recognize alternative splice variants, their abundance in vivo, and hence their biological significance, has not been determined. In this study, we assessed the protein levels of AID splice isoforms by introducing an AID splice reporter construct into cell lines and primary CLL cells from patients as well as from WT and TCL1tg C57BL/6 mice (where TCL1 is T‐cell leukemia/lymphoma 1). The splice construct is 5′‐fused to a GFP‐tag, which is preserved in all splice isoforms and allows detection of translated protein. Summarizing, we show a thorough quantification of alternatively spliced AID transcripts and demonstrate that the corresponding protein abundances, especially those of splice variants AID‐ivs3 and AID‐ΔE4, are not stoichiometrically equivalent. Our data suggest that enhanced proteasomal degradation of low‐abundance proteins might be causative for this discrepancy.

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.