Maternal immunization with ovalbumin prevents neonatal allergy development and up-regulates inhibitory receptor Fc gamma RIIB expression on B cells

Background: Preconception allergen immunization prevents neonatal allergen sensitization in mice by a complex interaction between regulatory cells/factors and antibodies. The present study assessed the influence of maternal immunization with ovalbumin (OVA) on the immune response of 3 day-old and 3 week-old offspring immunized or non-immunized with OVA and evaluated the effect of IgG treatment during fetal development or neonatal period. Results: Maternal immunization with OVA showed increased levels of Fc gamma RIIb expression in splenic B cells of neonates, which were maintained for up to 3 weeks and not affected by additional postnatal OVA immunization. Maternal immunization also exerted a down-modulatory effect on both IL-4 and IFN-gamma-secreting T cells and IL-4 and IL-12-secreting B cells. Furthermore, immunized neonates from immunized mothers showed a marked inhibition of antigen-specifc IgE Ab production and lowered Th2/Th1 cytokine levels, whereas displaying enhanced Fc gamma RIIb expression on B cells. These offspring also showed reduced antigen-specific proliferative response and lowered B cell responsiveness. Moreover, in vitro evaluation revealed an impairment of B cell activation upon engagement of B cell antigen receptor by IgG from OVA-immunized mice. Finally, in vivo IgG transference during pregnancy or breastfeeding revealed that maternal Ab transference was able to increase regulatory cytokines, such as IL-10, in the prenatal stage; yet only the postnatal treatment prevented neonatal sensitization. None of the IgG treatments induced immunological changes in the offspring, as it was observed for those from OVA-immunized mothers. Conclusion: Maternal immunization upregulates the inhibitory Fc gamma RIIb expression on offspring B cells, avoiding skewed Th2 response and development of allergy. These findings contribute to the advancement of prophylactic strategies to prevent allergic diseases in early life.

RelB nuclear translocation mediated by C-terminal activator reigons of Epstein Barr virus-encoded latent membrane protein 1 and its effect on antigen-presenting function in B cells

Previous studies have shown that Epstein-Barr virus-encoded latent membrane protein 1 (LMP1) is uniquely able to up-regulate the expression of the peptide transporters (referred to as TAP-1 and TAP-2) and major histocompatibility complex (MHC) class I in Burkitt's lymphoma (BL) cell lines. This up-regulation is often accompanied by a restoration of antigen-presenting function as measured by the ability of these cells to present endogenously expressed viral antigen to cytotoxic T lymphocytes. Here we show that the expression of LMP1 resulted in up-regulation and nuclear translocation of RelB that were coincident with increased expression of MHC class I in BL cells. Deletion of the C-terminal activator regions (CTARs) of LMP1 significantly impaired the abilities of LMP1 to translocate RelB into the nucleus and to up-regulate the expression of antigen-processing genes. Further analysis with single-point mutations within the CTARs confirmed that the residues critical for NF-kappaB activation directly contribute to antigen-processing function regulation in BL cells. This LMP1-mediated effect was blocked following expression of either dominant negative IkappaBalpha S32/36A, an NF-kappaB inhibitor, or antisense RelB. These observations indicate that upregulation of antigen-presenting function in B cells mediated by LMP1 is signaled through the NF-kappaB subunit RelB. The data provide a mechanism by which LMP1 modulates immunogenicity of Epstein-Barr virus-infected normal and malignant cells.

Cross-presentation by WASp deficient B cells and dendritic cells

The immune system comprises of different cell types whose role is to protect us against pathogens. This thesis investigates a very important mechanism for our organism protection in a specific disorder: cross-presentation in Wiskott-Aldrich Syndrome (WAS). WAS is caused by loss-of-function mutations in the cytoskeletal regulator WASp and WAS patients suffer from eczema, thrombocytopenia, and immunodeficiency. X-linked neutropenia (XLN) is caused by gain-of-function mutations in WASp and XLN patients suffer from severe congenital neutropenia and immunodeficiency. This thesis was focused on the role of B and T lymphocytes and dendritic cells (DCs). This work will be divided into two main topics: 1) In the first part I studied the capacity of B cells to take up, degrade and present antigen. Moreover I studied the capacity of B cells to induce T cell proliferation. 2) In the second part, I studied T cell proliferation induced by dendritic cells. To increase our understanding about this mechanism, additional experiments were performed, including acidification capacity of CD8+ and CD8- DCs, reactive oxygen species (ROS) production since it is directly connected to acidification. These assays were measured by flow cytometry. Localization of Rac1 and Rac2 GTPases was assessed by confocal microscopy. Proliferation, acidification and ROS production assays were performed also with cells from X-linked neutropenia (XLN) mice. From this study we concluded that B cells cannot induce CD8+ T cell proliferation however they take up and present antigen. Moreover I have shown that increased cross-presentation by WASp KO DCs with ovalbumin is associated with decreased capacity to acidify endosomal compartment; and WASp KO CD8- DCs have increased Rac2 localization to the phagosome. XLN dendritic cells have similar acidification and ROS production capacity than wildtype cells. In conclusion, our data suggests that WASp regulates antigen processing and presentation in DCs.

Regulation of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-[kappa]B) by protein Kinase C theta (PKC-[theta]) and cylindromatosis (CYLD) in murine listeriosis and toxoplasmosis

Magdeburg, Univ., Fak. für Naturwiss., Diss., 2013

Maturation of marginal zone and follicular B cells requires B cell activating factor of the tumor necrosis factor family and is independent of B cell maturation antigen.

B cells undergo a complex series of maturation and selection steps in the bone marrow and spleen during differentiation into mature immune effector cells. The tumor necrosis factor (TNF) family member B cell activating factor of the TNF family (BAFF) (BLyS/TALL-1) plays an important role in B cell homeostasis. BAFF and its close homologue a proliferation-inducing ligand (APRIL) have both been shown to interact with at least two receptors, B cell maturation antigen (BCMA) and transmembrane activator and cyclophilin ligand interactor (TACI), however their relative contribution in transducing BAFF signals in vivo remains unclear. To functionally inactivate both BAFF and APRIL, mice transgenic for a soluble form of TACI were generated. They display a developmental block of B cell maturation in the periphery, leading to a severe depletion of marginal zone and follicular B2 B cells, but not of peritoneal B1 B cells. In contrast, mice transgenic for a soluble form of BCMA, which binds APRIL, have no detectable B cell phenotype. This demonstrates a crucial role for BAFF in B cell maturation and strongly suggests that it signals via a BCMA-independent pathway and in an APRIL-dispensable way.

The changing preference of T and B cells for partners as T-dependent antibody responses develop.

Recirculating virgin CD4+ T cells spend their life migrating between the T zones of secondary lymphoid tissues where they screen the surface of interdigitating dendritic cells. T-cell priming starts when processed peptides or superantigen associated with class II MHC molecules are recognised. Those primed T cells that remain within the lymphoid tissue move to the outer T zone, where they interact with B cells that have taken up and processed antigen. Cognate interaction between these cells initiates immunoglobulin (Ig) class switch-recombination and proliferation of both B and T cells; much of this growth occurs outside the T zones B cells migrate to follicles, where they form germinal centres, and to extrafollicular sites of B-cell growth, where they differentiate into mainly short-lived plasma cells. T cells do not move to the extrafollicular foci, but to the follicles; there they proliferate and are subsequently involved in the selection of B cells that have mutated their Ig variable-region genes. During primary antibody responses T-cell proliferation in follicles produces many times the peak number of T cells found in that site: a substantial proportion of the CD4+ memory T-cell pool may originate from growth in follicles.

Leishmania major-specific B cells are necessary for Th2 cell development and susceptibility to L. major LV39 in BALB/c mice.

B lymphocytes are considered to play a minimal role in host defense against Leishmania major. In this study, the contribution of B cells to susceptibility to infection with different strains of L. major was investigated in BALB/c mice lacking mature B cells due to the disruption of the IgM transmembrane domain (microMT). Whereas BALB/c microMT remained susceptible to infection with L. major IR173 and IR75, they were partially resistant to infection with L. major LV39. Adoptive transfer of naive B cells into BALB/c microMT mice before infection restored susceptibility to infection with L. major LV39, demonstrating a role for B cells in susceptibility to infection with this parasite. In contrast, adoptive transfer of B cells that express an IgM/IgD specific for hen egg lysozyme (HEL), an irrelevant Ag, did not restore disease progression in BALB/c microMT mice infected with L. major LV39. This finding was likely due to the inability of HEL Tg B cells to internalize and present Leishmania Ags to specific T cells. Furthermore, specific Ig did not contribute to disease progression as assessed by transfer of immune serum in BALB/c microMT mice. These data suggest that direct Ag presentation by specific B cells and not Ig effector functions is involved in susceptibility of BALB/c mice to infection with L. major LV39.

Analysis of synaptic-like microvesicle exocytosis of B-cells using a live imaging technique.

Pancreatic β-cells play central roles in blood glucose homeostasis. Beside insulin, these cells release neurotransmitters and other signaling molecules stored in synaptic-like microvesicles (SLMVs). We monitored SLMV exocytosis by transfecting a synaptophysin-pHluorin construct and by visualizing the cells by Total Internal Reflection Fluorescence (TIRF) microscopy. SLMV fusion was elicited by 20 mM glucose and by depolarizing K(+) concentrations with kinetics comparable to insulin secretion. SLMV exocytosis was prevented by Tetanus and Botulinum-C neurotoxins indicating that the fusion machinery of these organelles includes VAMP-2/-3 and Syntaxin-1, respectively. Sequential visualization of SLMVs by TIRF and epifluorescence microscopy showed that after fusion the vesicle components are rapidly internalized and the organelles re-acidified. Analysis of single fusion episodes revealed the existence of two categories of events. While under basal conditions transient fusion events prevailed, long-lasting episodes were more frequent upon secretagogue exposure. Our observations unveiled similarities between the mechanism of exocytosis of insulin granules and SLMVs. Thus, diabetic conditions characterized by defective insulin secretion are most probably associated also with inappropriate release of molecules stored in SLMVs. The assessment of the contribution of SLMV exocytosis to the manifestation of the disease will be facilitated by the use of the imaging approach described in this study.

Heterogeneous secretion of individual B cells in response to D-glucose and to nonglucidic nutrient secretagogues.

We used a hemolytic plaque assay for insulin to determine whether the same pancreatic B cells respond to D-glucose, 2-amino-bicyclo[2,2,1]heptane-2-carboxylic acid (BCH) and the association of this nonmetabolized analogue of L-leucine with either the monomethyl ester of succinic acid (SME) or the dimethyl ester of L-glutamic acid (GME). During a 30-min incubation in the absence of D-glucose, BCH alone (5 mM) had no effect on insulin release. In contrast, the combination of BCH with either SME (10 mM) or GME (3 mM) stimulated insulin release to the same extent observed in the sole presence of 16.7 mM D-glucose. The effects of BCH plus SME and BCH plus GME on both percentage of secreting B cells and total insulin output were little affected in the presence of D-glucose concentrations ranging from 0 to 16.7 mM. Varying the concentration of SME from 2 to 10 mM also did not influence these effects. In other experiments, the very same B cells were first exposed 45 min to 16.7 mM D-glucose, then incubated 45 min in the presence of only BCH and SME. Under these conditions, most (80.3 +/- 2.5%) of the cells contributing to insulin release did so during both incubation periods. Furthermore, virtually all cells responding to BCH and SME during the second incubation corresponded to cells also responsive to D-glucose during the first incubation. Similar observations were made when the sequence of the two incubations was reversed.(ABSTRACT TRUNCATED AT 250 WORDS)

BAFF is a survival and maturation factor for mouse B cells.

Human B cell-activating factor (BAFF) induces mouse surface IgM+ B cells of the immature type from bone marrow and of the immature types 1 and 2 from spleen, as well as of the mature type from spleen to increased longevity in tissue culture. BAFF does so polyclonally and without inducing proliferation in any of these B cell subpopulations. BAFF induces phenotypic and functional maturation of immature to mature B cells so that all immature cells loose C1qRp (AA4.1, 493) expression and type 1 immature cells up-regulate IgD, CD21 and CD23. Immature B cells of types 1 and 2, upon pre-incubation with BAFF, change their reactiveness to Ig-specific antibodies so that they no longer enter apoptosis but now proliferate. However, BAFF does not seem to overcome negative selection of developing immature B cells in vitro.

Efficient generation of human alloantigen-specific CD4+ regulatory T cells from naive precursors by CD40-activated B cells.

CD4(+)CD25(+)Foxp3(+) regulatory T cells (Treg) play an important role in the induction and maintenance of immune tolerance. Although adoptive transfer of bulk populations of Treg can prevent or treat T cell-mediated inflammatory diseases and transplant allograft rejection in animal models, optimal Treg immunotherapy in humans would ideally use antigen-specific rather than polyclonal Treg for greater specificity of regulation and avoidance of general suppression. However, no robust approaches have been reported for the generation of human antigen-specific Treg at a practical scale for clinical use. Here, we report a simple and cost-effective novel method to rapidly induce and expand large numbers of functional human alloantigen-specific Treg from antigenically naive precursors in vitro using allogeneic nontransformed B cells as stimulators. By this approach naive CD4(+)CD25(-) T cells could be expanded 8-fold into alloantigen-specific Treg after 3 weeks of culture without any exogenous cytokines. The induced alloantigen-specific Treg were CD45RO(+)CCR7(-) memory cells, and had a CD4(high), CD25(+), Foxp3(+), and CD62L (L-selectin)(+) phenotype. Although these CD4(high)CD25(+)Foxp3(+) alloantigen-specific Treg had no cytotoxic capacity, their suppressive function was cell-cell contact dependent and partially relied on cytotoxic T lymphocyte antigen-4 expression. This approach may accelerate the clinical application of Treg-based immunotherapy in transplantation and autoimmune diseases.

CpG-ODN-induced sustained expression of BTLA mediating selective inhibition of human B cells.

BTLA (B- and T-lymphocyte attenuator) is a prominent co-receptor that is structurally and functionally related to CTLA-4 and PD-1. In T cells, BTLA inhibits TCR-mediated activation. In B cells, roles and functions of BTLA are still poorly understood and have never been studied in the context of B cells activated by CpG via TLR9. In this study, we evaluated the expression of BTLA depending on activation and differentiation of human B cell subsets in peripheral blood and lymph nodes. Stimulation with CpG upregulated BTLA, but not its ligand: herpes virus entry mediator (HVEM), on B cells in vitro and sustained its expression in vivo in melanoma patients after vaccination. Upon ligation with HVEM, BTLA inhibited CpG-mediated B cell functions (proliferation, cytokine production, and upregulation of co-stimulatory molecules), which was reversed by blocking BTLA/HVEM interactions. Interestingly, chemokine secretion (IL-8 and MIP1β) was not affected by BTLA/HVEM ligation, suggesting that BTLA-mediated inhibition is selective for some but not all B cell functions. We conclude that BTLA is an important immune checkpoint for B cells, as similarly known for T cells.

Antitumour effects of single or combined monoclonal antibodies directed against membrane antigens expressed by human B cells leukaemia.

Background: The increasing availability of different monoclonal antibodies (mAbs) opens the way to more specific biologic therapy of cancer patients. However, despite the significant success of therapy in breast and ovarian carcinomas with anti-HER2 mAbs as well as in non-Hodkin B cell lymphomas with anti-CD20 mAbs, certain B cell malignancies such as B chronic lymphocytic leukaemia (B-CLL) respond poorly to anti-CD20 mAb, due to the low surface expression of this molecule. Thus, new mAbs adapted to each types of tumour will help to develop personalised mAb treatment. To this aim, we analyse the biological and therapeutic properties of three mAbs directed against the CD5, CD71 or HLA-DR molecules highly expressed on B-CLL cells. Results: The three mAbs, after purification and radiolabelling demonstrated high and specific binding capacity to various human leukaemia target cells. Further in vitro analysis showed that mAb anti-CD5 induced neither growth inhibition nor apoptosis, mAb anti-CD71 induced proliferation inhibition with no early sign of cell death and mAb anti-HLA-DR induced specific cell aggregation, but without evidence of apoptosis. All three mAbs induced various degrees of ADCC by NK cells, as well as phagocytosis by macrophages. Only the anti-HLA-DR mAb induced complement mediated lysis. Coincubation of different pairs of mAbs did not significantly modify the in vitro results. In contrast with these discrete and heterogeneous in vitro effects, in vivo the three mAbs demonstrated marked anti-tumour efficacy and prolongation of mice survival in two models of SCID mice, grafted either intraperitoneally or intravenously with the CD5 transfected JOK1-5.3 cells. This cell line was derived from a human hairy cell leukaemia, a type of malignancy known to have very similar biological properties as the B-CLL, whose cells constitutively express CD5. Interestingly, the combined injection of anti-CD5 with anti-HLA-DR or with anti-CD71 led to longer mouse survival, as compared to single mAb injection, up to complete inhibition of tumour growth in 100% mice treated with both anti-HLA-DR and anti-CD5. Conclusions: Altogether these data suggest that the combined use of two mAbs, such as anti-HLA-DR and anti-CD5, may significantly enhance their therapeutic potential.

Anti-tumour effect of monoclonal antibodies against selected antigens expressed by B-cells in Chronic Lymphocytic Leukaemia : strategies to enhance the efficacy of immunotherapy

Résumé : Les anticorps monoclonaux ont une place de plus en plus prépondérante dans le traitement des lymphomes et leucémies. Dans cette étude, trois anticorps monoclonaux murins, dirigés contre les antigènes CDS, CD71 et HLA-DR exprimés à la surface des cellules de leucémies lymphoïdes chroniques (LLC), ont été évalués. In vitro, les anticorps radiomarqués ont montrés des bonnes liaisons spécifiques sur les différentes cellules cibles. L'anti-CD71 inhibait la prolifération de la plupart des lignées cellulaires testées avec une accumulation des cellules en phase S précoce du cycle cellulaire. L'anti-HLA-DR inhibait aussi la prolifération des lignées leucémique JOK1-5.3 et lymphoïde Daudi. Cette inhibition était associée à une agrégation des cellules. Aucune induction d'apoptose n'a pu être clairement observée avec ces anticorps. L'anti-CD5 n'a montré aucun effet d'inhibition de croissance in vitro. In vivo, l'injection des anticorps individuellement augmentait significativement la survie médiane de souris SCID greffées avec des cellules JOK1-5.3 en i.p. De plus, l'anticorps antiCD5 combiné à l'anti-HLA-DR ou l'anti-CD71, sous certaines conditions, inhibait complètement le développement tumoral dans la quasi totalité des souris traitées avec une augmentation significative de l'efficacité comparée aux anticorps seuls. L'augmentation de l'efficacité thérapeutique des anticorps monoclonaux par les cytokines, dont l'IL-2, a déjà été montrée dans la littérature. Au regard du meilleur comportement de l'IL-2 sous la forme complexée à un anticorps anti-IL-2, nous avons évalué l'efficacité de l'IL-2/anti-IL-2 seul ou combinés au rituximab chez différents modèles tumoraux s.c. (BL60.2, Daudi, Ramos) ou i.p. (JOK15.3) de souris SCID. Le complexe IL-2/anti-IL-2 a montré un effet anti-tumoral dans les souris greffées avec BL60.2 et Daudi. Le traitement IL-2/anti-IL-2 combiné au rituximab a montré une efficacité accrue chez des souris avec BL60.2 par rapport au rituximab seul. En revanche, nous n'avons pas observé de différence avec IL-2/anti-IL-2 seul.Aussi, nous avons évalué l'utilisation de l'agent couplant tri-fonctionnel TMEA pour produire des anticorps bispecifiques. Les expériences préliminaires avec les anticorps rituximab et herceptine, ont mis en évidence sur gel SDS-Page la formation de dimers (~100kDa) et de trimers (~150kDa). Les anticorps bispecifiques sont composés d'un fragment Fab' d'une spécificité et de un ou deux fragments Fab' de l'autre spécificité permettant de moduler la capacité de liaison. Nous avons enfin montré qu'une construction anti-CD5/anti-CD20 était capable de se lier indépendamment ou simultanément à ses antigènes cibles. En conclusion, ce travail a montré l'efficacité thérapeutique des trois anticorps monoclonaux étudiés dans un model de LLC in vivo, et plus particulièrement l'intérêt de certaines combinaisons. D'autre part, nous avons montré l'efficacité anti-tumorale du complexe IL-2/anti-IL-2 in vivo. Des études futures devront permettre de définir un régime favorable pour augmenter l'efficacité de la thérapie avec les anticorps monoclonaux. Enfin, nous avons montré la faisabilité d'utiliser l'agent couplant TMEA pour produire des anticorps bispécifiques fonctionnels.Abstract : Monoclonal antibody (mAb) therapy has become an integral part in different treatments of lymphomas and leukaemias. In this study, we describe three murine mAbs directed against the CD5, CD71 and HLA-DR antigens expressed on chronic lymphocytic leukaemia cells (CLL). In vitro, radiolabeled purified mAbs showed good specific binding on live target cells. Anti-CD71 mAb inhibited proliferation of most cell lines with an accumulation of responding cells in early S-phase of the cell cycle, but without induction of apoptosis. Anti-HLA-DR mAb showed proliferation inhibition of leukaemia JOK1-5.3 and lymphoid Daudi cells, associated with cell aggregation, but again no specific sign of apoptosis was observed. Anti-CD5 mAb did not show any growth inhibitory effect in vitro. In vivo, in a model of SCID mice grafted i.p. with JOK1-5.3 cells, injection of individual mAbs induced significant prolongation of median survival, up to complete inhibition of tumour growth in some mice. Antibody combination of anti-CD5 with anti-HLA-DR or anti-CD71, evaluated in an early treatment, completely inhibited tumour growth in most mice, with a significant efficacy enhancement as compared to mAb used as single agents. Previous reports described the improved efficacy of mAb therapy when combined with cytokines such as IL-2. Relying further on the improved efficacy of IL-2 when administered as an immune complex with anti-IL-2 mAb, we evaluated the anti-tumour effect of the IL-2/anti-IL-2 complex alone or combined with rituximab in subcutaneous (BL60.2, Daudi, Ramos) or i.p. (JOK1-5.3) tumour models in SCID mice. The IL-2/anti-IL-2 complex demonstrated an anti-tumour effect in BL60.2 and Daudi grafted SCID mice. Combination of IL-2/anti-IL-2 treatment with rituximab showed increased efficacy as compared to rituximab alone in BL60.2 grafted mice. However, no difference was observed with IL-2/anti-IL-2 complex alone in these experiments. Finally, we evaluated the feasibility of producing bispecific antibodies (bsAbs) using a trifunctional coupling agent, called TMEA. In preliminary experiments coupling rituximab with herceptine Fab' fragments we obtained the formation of dimers (~100kDa) and trimers (~150kDa) as observed on SDS-Page gel. This method allowed us to produce bsAb with one Fab' fragments of one specificity and one or two Fab' fragments of the second specificity. An anti-CD5/anti-CD20 bsAb was shown to bind targeted antigen either independently or simultaneously. In conclusion, these data show that the three mAbs were all able to induce significant growth inhibition of the JOK1-5.3 cell line in vivo, and efficacy was enhanced when used in combination. IL2/anti-IL-2 complex displayed anti-tumour efficacy in vivo. Further evaluation is necessary to define the most favourable combination to improve mAb therapy. BsAb were produced using the tri-functional agent allowing antibody fragments with relatively good binding. The poor yield obtained with such chemical couplings limited the use of these constructs in preclinical experiments.