Maternal T cells of immunized pregnant mice induce immune suppression in their offspring.

The present study focused on the influence of maternal immunity during pregnancy on the responsiveness of the immune system(s) in offspring. Maternal immunization of pregnant mice with T-dependent foreign antigen sheep red blood cells (SRBC) induced suppression of anti-SRBC plaque-forming cell (PFC) responses in their offspring. We attempted to identify the cell species among the maternal lymphoid cells of the immunized pregnant mice that induced this suppression in their offspring, by separating the maternal cells into T cells, B cells and macrophages, or T-cell subsets, and then adoptively transferring them into other normal pregnant mice. The results demonstrated the following: first, maternal CD4+ T cells of immunized pregnant mice induced immune suppression in their offspring. Second, maternal T cells could be activated during pregnancy in the same fashion as in non-pregnant mice. The T-cell factor(s) for the immune suppression in offspring is produced not only by maternal T cells of immunized pregnant mice but also by T cells activated in non-pregnant mice. Third, cellular organization was required for maternal T cells to induce this immune suppression in their offspring.     

Genetic approaches to tyrosine kinase signaling pathways in the immune system

The development of a productive immune response requires the carefully coordinated activation of lymphocytes through their cell-surface antigen receptors, surface immunoglobulin (Ig) on B cells and the T cell receptor (TCR) on T cells. Studies of mutant cell lines, gene-targeted mice and humans with inherited immunodeficiencies have demonstrated that tyrosine kinases are critical components of lymphocyte antigen-receptor-signaling pathways. Our laboratory is interested in the mechanisms by which modulation of signaling pathways involving tyrosine kinases and related signaling molecules can influence cell function and development. We have concentrated our attention on the genetic and biochemical dissection of signaling pathways in the immune system, and how altering these pathways can change responses to infectious disease. As a model system, we are examining the Tec family kinases and their roles in T lymphocyte development and function.     

CD28:B7 interaction is necessary for the protective effect of T cell vaccination in EAE

The mechanisms of T cell vaccination (TCV) are still unclear, especially the molecular interactions for recognition of autoreactive T cells by the immune system. Here we investigated the role of CD28:B7 interaction in TCV-induced protection in the murine EAE model. We demonstrate that there is increased expression of both B7-1 and B7-2 on autoreactive Th1 cells compared to Th2 cells. Blockade of B7 on the vaccinating autoreactive T cell surface or blockade of CD28 in recipient mice reduced the protective effect of TCV. Furthermore, we showed that TCV significantly inhibited Ag-specific CD4 and CD8 T cell proliferation and decreased Ag-specific IFN-gamma production by CD4 T cells in mice undergoing TCV, and blocking of B7 on the surface of vaccinating T cells reduced this inhibition on Ag-specific CD4 and CD8 T cell proliferation, more significantly on Ag-specific CD4 T cell proliferation. These data indicated that B7 expression on autoreactive T cells is necessary for the recognition of autoreactive T cells by the immune system and subsequent protection from EAE in mice undergoing TCV.     

In vivo staphylococcal superantigen-driven polyclonal Ig responses in mice: dependence upon CD4(+) cells and human MHC class II

Staphylococcal enterotoxin (SE) B and seven other staphylococcal superantigens (SAg), despite promoting vigorous Ig production in human peripheral blood mononuclear cell cultures, are exceedingly poor at eliciting Ig responses in cultures of spleen cells from C57BL/10J (B10) or C3H/HeJ mice. In contrast, SEB elicits Ig responses in cultures of spleen cells from human MHC class II-transgenic mice. Whereas i.p. administration of SEB (0.2-20 microg) to non-transgenic B10 mice elicits very weak in vivo Ig responses, identical treatment of CD4(+) cell-intact (but not CD4(+) cell-depleted) human MHC class II-transgenic mice elicits dramatic increases in both splenic Ig-secreting cells and serum Ig levels. Over a 2-week period, the SEB-induced in vivo Ig responses peak and then plateau or fall in association with a preferential increase in splenic CD8(+) cells. Nevertheless, in vivo depletion of CD8(+) cells has no sustained effect on SEB-driven Ig responses. Taken together, these observations demonstrate that the effects of SAg on in vivo humoral immune responses are highly CD4(+) cell dependent, are substantially CD8(+) cell independent and can be successfully investigated using human MHC class II-transgenic mice. This model system may be useful in investigating the polyclonally activating effects of microbial products (prototypic environmental insults) on the development of systemic autoimmunity.     

Mice expressing human CR1/CD35 have an enhanced humoral immune response to T-dependent antigens but fail to correct the effect of premature human CR2 expression

We have previously demonstrated that mice expressing human complement receptor type 2 (CR2/CD21) during the CD43(+)/CD25(-) late pro-B cell stage of B cell development have marked changes in their subsequent B cell ontogeny. Here, we show that the humoral immune response to the T cell dependent antigen, sheep red blood cells (SRBCs) can be moderately enhanced with the addition of human CR1 (driven by the lambda promoter/enhancer transgene) to endogenous mCR1/CR2 expression on the B cell surface but that hCR1 expression alone (on the mouse CR1/2 deficient background) has no effect on the humoral immune response or general B cell development. Furthermore, expression of hCR1 had no recuperative effect on the markedly altered B cell phenotype noted with premature expression of hCR2 (either in the presence or absence of endogenous mCR1/2). We conclude that hCR1 alone cannot replace the role of CR2 in mice and that the effects of premature hCR2 expression during BCR development are not significantly altered by the addition of hCR1 at that developmental stage or beyond; thus hCR2 signaling in the mouse remains dominant over subsequent input from either hCR1 or endogenous receptors.     

B Cell Memory in xid Mice is Long-Lived Despite Reduced Memory B Cell Frequency

Brutons tyrosine kinase (Btk) deficient xid mice have a diminished primary T cell dependent immune response, resulting in a reduced memory B cell frequency. Boosting at 35 days post primary immunization, however, generates a normal secondary immune response, indicating a functional memory B cell compartment. The longevity of B cell memory appears to depend on both the presence of antigen and expression of cell survival genes such as bcl-2 . Since there is a natural decay in the number of memory B cells over time and since xid B cells have been demonstrated to have reduced Bcl-2 levels, we aimed at determining whether B cell memory of xid mice would be long-lasting. This report demonstrates that memory B cell precursors are detectable in xid mice more than 100 days after primary immunization. Furthermore, a secondary immune response of normal magnitude and kinetics can be generated in xid mice at 150 days after primary immunization indicating that B cell memory is long-lived in xid mice. Thus, although survival of B cell memory is presumably dependent on immunoglobulin (Ig)-mediated interaction with antigen, this interaction does not depend solely on signalling through Btk.     

Induction of human B cell proliferation and differentiation by the combination of phorbol ester and ionomycin

Phorbol esters and Ca2+ ionophores are known to mimic intracellular messengers involved in cell activation. We studied the effects of 12-O-tetradecanoylphorbol 13-acetate (TPA) and ionomycin on tonsil and peripheral blood-derived B lymphocytes. We show that TPA and ionomycin are co-mitogenic and induce B lymphocyte differentiation. Although TPA in high concentrations is mitogenic to B lymphocytes by itself, submitogenic concentrations of TPA in combination with ionomycin trigger 50% of B lymphocytes to synthesize DNA. Stimulation of B lymphocytes with TPA plus ionomycin resulted in increased magnitude and a shift in the kinetics of c-fos and c-myc expression compared with either agent used alone. Activation markers such as the transferrin and interleukin 2 (IL2) receptors were markedly increased after 24 h incubation with TPA and ionomycin. In parallel to the rapid proliferative burst, we observed evidence for B lymphocyte differentiation with an increase in the number of cells expressing cytoplasmic immunoglobulin (Ig) and the disappearance of the B1 surface marker. Since the cells remained surface Ig+ and secreted only small quantities of Ig, our results suggest that the combination of TPA and ionomycin is a potent inducer of B cell proliferation and early differentiation; terminal differentiation to an Ig-secreting state, however, is not achieved.     

The absence of immunoglobulin D B cell receptor-mediated signals promotes the production of autoantibodies and exacerbates glomerulonephritis in murine lupus

Immunoglobulin (Ig)D is the major antigen receptor isotype co-expressed with IgM on the surface of most peripheral B cells in mice and humans. However, the biological role of IgD as B cell receptor (BCR) has remained unclear. Previous studies have indicated that IgD may play a role in B cell tolerance. To understand the role of IgD in B cell tolerance and autoimmunity, we have examined the development of autoimmune syndrome in lpr mice deficient for IgD. The present study showed that IgD deficiency did not alter lymphoproliferation and lymphocyte activation in lpr mice. The survival and proliferation of B cells were not affected by the absence of IgD, indicating that IgD BCR-mediated signals do not have an important role in negative selection of autoreactive B cell clones. Interestingly, compared to IgD-competent littermates, lpr mice with IgD deficiency had elevated autoantibody production, increased deposition of immune complex in the kidney and more severe nephritis. Accumulation of abnormal CD4(-) CD8(-) αβ(+) T cells was accelerated in IgD(-/-) lpr mice compared to lpr mice. These results suggest that IgD BCR-mediated signals may be involved in the differentiation of autoreactive B cells into plasma cells and abnormal T cell expansion.     

Maternal allergen immunisation to prevent sensitisation in offspring: Th2-polarising adjuvants are more efficient than a Th1-polarising adjuvant in mice

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 FcγRIIb 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-γ-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 FcγRIIb 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 FcγRIIb 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.     

Allelic exclusion of immunoglobulin genes: models and mechanisms

Summary: The allelic exclusion of immunoglobulin (Ig) genes is one of the most evolutionarily conserved features of the adaptive immune system and underlies the monospecificity of B cells. While much has been learned about how Ig allelic exclusion is established during B-cell development, the relevance of monospecificity to B-cell function remains enigmatic. Here, we review the theoretical models that have been proposed to explain the establishment of Ig allelic exclusion and focus on the molecular mechanisms utilized by developing B cells to ensure the monoallelic expression of Igκ and Igλ light chain genes. We also discuss the physiological consequences of Ig allelic exclusion and speculate on the importance of monospecificity of B cells for immune recognition.     

Estrogen‐inducible sFRP5 inhibits early B‐lymphopoiesis in vivo,but not during pregnancy

Mammals have evolved to protect their offspring during early fetal development. Elaborated mechanisms induce tolerance in the maternal immune system for the fetus. Female hormones, mainly estrogen, play a role in suppressing maternal lymphopoiesis. However, the molecular mechanisms involved in the maternal immune tolerance are largely unknown. Here, we show that estrogen‐induced soluble Frizzled‐related proteins (sFRPs), and particularly sFRP5, suppress B‐lymphopoiesis in vivo in transgenic mice. Mice overexpressing sFRP5 had fewer B‐lymphocytes in the peripheral blood and spleen. High levels of sFRP5 inhibited early B‐cell differentiation in the bone marrow (BM), resulting in the accumulation of cells with a common lymphoid progenitor (CLP) phenotype. Conversely, sFRP5 deficiency reduced the number of hematopoietic stem cells (HSCs) and primitive lymphoid progenitors in the BM, particularly when estrogen was administered. Furthermore, a significant reduction in CLPs and B‐lineage‐committed progenitors was observed in the BM of sfrp5‐null pregnant females. We concluded that, although high sFRP5 expression inhibits B‐lymphopoiesis in vivo, physiologically, it contributes to the preservation of very primitive lymphopoietic progenitors, including HSCs, under high estrogen levels. Thus, sFRP5 regulates early lympho‐hematopoiesis in the maternal BM, but the maternal–fetal immune tolerance still involves other molecular mechanisms that remain to be uncovered.     

Allergic sensitization and allergen exposure during pregnancy favor the development of atopy in the neonate

BACKGROUND: Several studies have considered that the in utero environment plays an important role in the onset of the allergic phenotype. We assessed whether allergic sensitization and allergen exposure during pregnancy favor the postnatal onset of allergy in the neonate. METHODS: BALB/c mice were sensitized to ovalbumin (OVA) before mating followed by allergen aerosol exposure during pregnancy. T and B cell responses in offspring were followed up until day 60 postpartum. At the age of 4 weeks offspring were exposed to a heterologous antigen, beta-lactoglobulin (BLG). RESULTS: Pregnant mice developed immediate hypersensitivity responses and Th-2/ Th-0 immunity following allergen aerosol exposure. At birth, T cells from offspring of nonsensitized BALB/c mice were characterized by an impaired IFN-gamma production, which was lowered even further in offspring of OVA-sensitized BALB/c mice. Offspring of OVA-sensitized BALB/c mice responded with immediate-type cutaneous hypersensitivity reactions to OVA which could be related to the pre- and postnatal transfer of maternal OVA-specific IgG1 antibodies. After exposure to BLG, offspring of OVA-sensitized BALB/c mice developed an accelerated Th-2-driven immune response compared to offspring from nonsensitized BALB/c mice as indicated by enhanced anti-BLG IgG1 antibody production and increased numbers of positive immediate-type cutaneous hypersensitivity reactions to BLG. CONCLUSION: Our data suggest that Th-2/Th-0 immunity present during pregnancy has a decisive impact on shaping the Th-1/Th-2 T cell profile in response to postnatal allergen exposure.     

Preconception maternal immunization to dust mite inhibits the type I hypersensitivity response of offspring

BACKGROUND: The maternal immunologic experience associated with early life exposure to allergens might contribute to the development of allergy during infancy. OBJECTIVES: We sought to analyze the effect of the mother's immunization before conception with the dust mite Dermatophagoides pteronyssinus on the allergen priming and hypersensitivity response in early immunized offspring. The kinetics of D pteronyssinus immunization were observed from newborn to adult age, and the secondary response to D pteronyssinus was followed in offspring immunized in early life. METHODS: Female A/Sn mice were immunized or not with D pteronyssinus and mated with male C57BL/6 mice. The hybrid offspring were immunized to investigate allotypes and subclasses of anti-D pteronyssinus antibody, as well as total IgE levels, by using ELISA and anti-D pteronyssinus IgE antibody by using the passive cutaneous anaphylaxis reaction. Ovalbumin was used for heterologous immunization. Cytokines were measured in the cell-culture supernatant by means of ELISA, and CD4(+)CD25(+) cells were analyzed by means of flow cytometry. RESULTS: Offspring from immune mothers have not shown evidence of prenatal or postnatal allergen priming with respect to humoral level. Immunization with D pteronyssinus of offspring at very early life and in the postweaning period inhibited anti-D pteronyssinus IgE and IgG1 antibody production, along with the expected presence of maternal antibody. Furthermore, offspring antibody responsiveness from immune mothers has remained quiescent on secondary allergenic challenge. This maternal influence on the offspring antibody response was specific to D pteronyssinus because the immunization with a heterologous antigen did not alter IgE response. Maternal D pteronyssinus immunization induced a significant decrease of the IFN-gamma level in the offspring, avoided an exacerbation of T(H)2 cytokine secretion, and, concomitantly, upregulated the number of CD4(+)CD25(+) T cells. CONCLUSION: Maternal immunization to D pteronyssinus seems to protect offspring from the development of allergy.     

Suppression of Allergen‐Specific IgE in Offspring After Preconceptional Immunisation: Maternal,Paternal and Genetic Influences

Immunisation of female mice with the allergen ovalbumin (OVA) during pregnancy reduces the OVA‐specific IgE response in adult offspring. To approach primary prevention strategies for allergy, we investigated to what extent genetic, paternal and maternal factors influence this suppressive effect on allergic sensitisation in offspring and investigated the possibility of pregestational immunisation. Maternal allergen immunisation reduced OVA‐specific IgE levels in immunised offspring, even after maternal immunisation up to 8 weeks before conception without further allergen exposure. Immunisation of immunodeficient BALB/c severe combined immune deficiency (SCID) dams mated with wild type males did not lead to IgE suppression in offspring, indicating the importance of a functional maternal immune system. Immunisation of male mice before the relevant spermatogenesis did not cause antibody suppression in offspring. OVA‐specific IgG1, presumably of maternal origin, was present in naïve offspring only from immunised dams and was associated with suppressed IgE responses after offspring immunisation. The IgE‐suppressive effect of maternal immunisation was demonstrated in all three immunocompetent strains tested (NIH/OlaHsd, BALB/cA and C57BL/6 mice). In conclusion, suppression of allergen‐specific IgE production in offspring could not be induced by paternal immunisation, and genetic factors were of minor importance. In contrast, we demonstrate the necessity of maternal factors, possibly allergen‐specific IgG1, resulting from a functional adaptive immune response, for the IgE‐suppressive effect in offspring. These maternal factors could be induced by immunisation of female mice even before conception.     

Polyclonal activation of the murine immune system by an antibody to IgD. VI. Influence of doses of goat anti-mouse delta chain and normal goat IgG on B lymphocyte proliferation and differentiation

The injection of mice with 800 micrograms of an affinity-purified goat antibody to mouse IgD (GaM delta) induces early, T-independent polyclonal increases in the expression of B cell surface Ia, and B cell size and DNA synthesis, as well as later, T-dependent polyclonal increases in spleen cell number and Ig secretion. We have now studied the effects of varying the doses of injected GaM delta on all phases of B cell activation, as well as the effects of supplementing GaM delta with varying quantities of normal goat IgG (GIgG). We have found that while 12.5 micrograms of GaM delta modulates most of the IgD from the surface of splenic B lymphocytes, it fails to activate these cells. Increases in the expression of B cell surface Ia are first seen when 50 micrograms of GaM delta is injected, while increases in B cell DNA synthesis usually require the injection of 200 micrograms of GaM delta and peak with doses of approximately 800 micrograms. Increases in splenic B cell number and DNA synthesis during the T-dependent phase of GaM delta-induced B cell activation are seen only in those mice that were injected with sufficient quantities of GaM delta to induce DNA synthesis during the T-independent phase. Supplementing the dose of GaM delta injected with additional GIgG has no significant effect on B cell DNA synthesis or B cell number but dramatically increases polyclonal IgG1 secretion. Although mice which have been injected with 50 micrograms of GaM delta or with 800 micrograms of GIgG alone have few polyclonal IgG1-secreting cells, substantial increases in the number of IgG1-secreting cells are seen in mice injected with 50 micrograms of GaM delta plus 750 micrograms of GIgG. GIgG and larger doses of GaM delta similarly act synergistically to increase polyclonal IgG1 secretion. In contrast to the induction of polyclonal IgG1 secretion, the stimulation of polyclonal IgM secretion requires the injection of mitogenic doses of GaM delta and is not enhanced by the injection of additional GIgG. These observations suggest that, in this model system, stimulatory signals that activate B cells through their surface Ig are limiting for the induction of polyclonal proliferation and IgM secretion, while the generation of T helper lymphokines that do not directly interact with B cells through their surface Ig may be more limiting for the stimulation of polyclonal IgG1 secretion.     

Identification of disulfide bonds in the Ig-alpha/Ig-beta component of the B cell antigen receptor using the Drosophila S2 cell reconstitution system

Structural information about immune receptor complexes is important for understanding signal transduction mechanisms. We have used the Drosophila S2 cell reconstitution system for identification of disulfide bonds within and between CD79a (Ig-alpha) and CD79b (Ig-beta), the heterodimeric signal transducing element of the B cell antigen receptor (BCR). Cysteines 113 and 135 of Ig-alpha and Ig-beta, respectively, form the intermolecular disulfide bridge stabilizing the Ig-alpha/Ig-beta heterodimer in both S2 cells and the B cell line J558L. Furthermore, using transfected S2 cells, two putative intramolecular disulfide bonds in the Ig-like domain of Ig-beta were identified. Ig-betaC65 and Ig-betaC120 form the canonical Ig fold disulfide bond. In addition, Ig-betaC43 and Ig-betaC124 also bind covalently. Individual cysteine to serine mutations in Ig-alpha had no influence on membrane-bound Ig (mIg)-M expression on the surface of S2 cells. In contrast, mIgM expression on the surface of B cells expressing Ig-alphaC113S was reduced, indicating that this intermolecular bond is prerequisite for efficient IgM-BCR formation. Our data also suggest that the Ig-alpha/Ig-beta heterodimer can assemble into oligomers.     

Immunoglobulin promotes the diversity and the function of T cells

It is generally accepted in immunology that while T and B cells collaborate for the production of antibodies in response to protein antigens, T cells develop and function in the absence of B cells. However, B cell-deficient subjects and mice have unexplained cellular immune defects. Here, we examined the contribution of B cells/Ig to the generation of diversity and function of T cells. Mice lacking B cells and Ig (JH(-/-)) or having oligoclonal B cells (QM) had a profoundly contracted T cell receptor (TCR) Vbeta repertoire: 0.08 and 1.3% of wild type, respectively. Rejection of H-Y-incompatible skin grafts in QM and JH(-/-) mice was significantly delayed (median, 43 and 22 days, respectively) compared to wild-type mice (median, 16 days). Furthermore, reduction of the TCR Vbeta diversity by thymectomy in wild-type mice significantly increased survival of H-Y-incompatible skin grafts, and reconstitution of the T cell diversity in QM mice with Ig Fab fragments significantly decreased survival of the skin grafts. These results indicate that B cells and/or Ig "help" T cells through the generation and maintenance of T cell diversity, improving T cell function. Our results may have important implications for therapy and immune reconstitution in the context of AIDS, cancer, autoimmunity and post-myeloablative treatments.