Clonal deletion of B lymphocytes in a transgenic mouse bearing anti-MHC class I antibody genes

B lymphocytes can be rendered specifically unresponsive to antigen by experimental manipulation in vivo and in vitro, but it remains unclear whether or not natural tolerance involves B-cell tolerance because B cells are controlled by T lymphocytes, and in their absence respond poorly to antigen (reviewed in ref. 7). In addition, autoantibody-producing cells can be found in normal mice and their formation is enhanced by B-cell mitogens such as lipopolysaccharides. We have studied B-cell tolerance in transgenic mice using genes for IgM anti-H-2k MHC class I antibody. In H-2d transgenic mice about 25-50% of the splenic B cells bear membrane immunoglobulin of this specificity, and abundant serum IgM encoded by the transgenes is produced. In contrast, H-2k x H-2d (H-2-d/k) transgenic mice lack B cells bearing the anti-H-2k idiotype and contain no detectable serum anti-H-2k antibody, suggesting that very large numbers of autospecific B cells can be controlled by clonal deletion.     

Induction of self-tolerance in mature peripheral B lymphocytes

In transgenic mice, mature peripheral B lymphocytes in lymphoid follicles, like immature B cells, are rendered tolerant by encounter with self-antigen, provided receptor occupancy by self-antigen exceeds a critical threshold. The tolerant state of the B cell is closely correlated with down-regulation of membrane IgM but not IgD antigen-receptors. Identical changes in antigen-receptor expression occur in a subset of follicular B cells in nontransgenic mice, suggesting that clonally silenced self-reactive cells are common in the peripheral B-cell repertoire.     

Molecular components of the B-cell antigen receptor complex of the IgM class

The antigen receptors on mature B lymphocytes are membrane-bound immunoglobulins of the IgM and IgD classes whose cross-linking by polyvalent antigens results in B-cell proliferation and differentiation. How these membrane-bound immunoglobulin chains, which lack a cytoplasmic tail, generate a cell activation signal is not at present known. We now show that the IgM molecule is non-covalently associated in the membrane of B cells with two proteins of relative molecular mass 34,000 (Mr 34 K; IgM-alpha) and 39 K (Ig-beta) which form a disulphide-linked heterodimer. Surface expression of IgM seems to require the formation of an appropriate complex between IgM and the heterodimer. A transfection experiment indicates that IgM-alpha is the product of mb-1, a B-cell specific gene encoding a transmembrane protein with sequence homology to proteins of the T-cell antigen receptor-CD3 complex.     

Endogenous antigen tunes the responsiveness of naive B cells but not T cells

In humans, up to 75% of newly generated B cells and about 30% of mature B cells show some degree of autoreactivity. Yet, how B cells establish and maintain tolerance in the face of autoantigen exposure during and after development is not certain. Studies of model B-cell antigen receptor (BCR) transgenic systems have highlighted the critical role of functional unresponsiveness or ‘anergy’. Unlike T cells, evidence suggests that receptor editing and anergy, rather than deletion, account for much of B-cell tolerance. However, it remains unclear whether the mature diverse B-cell repertoire of mice contains anergic autoreactive B cells, and if so, whether antigen was encountered during or after their development. By taking advantage of a reporter mouse in which BCR signalling rapidly and robustly induces green fluorescent protein expression under the control of the Nur77 regulatory region, antigen-dependent and antigen-independent BCR signalling events in vivo during B-cell maturation were visualized. Here we show that B cells encounter antigen during development in the spleen, and that this antigen exposure, in turn, tunes the responsiveness of BCR signalling in B cells at least partly by downmodulating expression of surface IgM but not IgD BCRs, and by modifying basal calcium levels. By contrast, no analogous process occurs in naive mature T cells. Our data demonstrate not only that autoreactive B cells persist in the mature repertoire, but that functional unresponsiveness or anergy exists in the mature B-cell repertoire along a continuum, a fact that has long been suspected, but never yet shown. These results have important implications for understanding how tolerance in T and B cells is differently imposed, and how these processes might go awry in disease.     

A B cell-deficient mouse by targeted disruption of the membrane exon of the immunoglobulin mu chain gene

Of the various classes of antibodies that B lymphocytes can produce, class M (IgM) is the first to be expressed on the membrane of the developing cells. Pre-B cells, the precursors of B-lymphocytes, produce the heavy chain of IgM (mu chain), but not light chains. Recent data suggest that pre-B cells express mu chains on the membrane together with the 'surrogate' light chains lambda 5 and V pre B (refs 2-7). This complex could control pre-B-cell differentiation, in particular the rearrangement of the light-chain genes. We have now assessed the importance of the membrane form of the mu chain in B-cell development by generating mice lacking this chain. We disrupted one of the membrane exons of the gene encoding the mu-chain constant region by gene targeting in mouse embryonic stem cells. From these cells we derived mice heterozygous or homozygous for the mutation. B-cell development in the heterozygous mice seemed to be normal, but in homozygous animals B cells were absent, their development already being arrested at the stage of pre-B-cell maturation.     

Gamma-interferon is one of several direct B cell-maturing lymphokines

Two classes of molecules often released after the interaction of T lymphocytes, macrophages and antigen are B-cell maturation factors (BMF)1-3 and immune (gamma) interferon (IFN-gamma)4-7. BMFs directly induce the maturation of resting B lymphocytes to the state of active immunoglobulin secretion, while IFN-gamma is defined by the reduction of viral infectivity in vitro. However, interferons have been shown to have a variety of effects and they have also been reported both to increase and decrease B-cell differentiation in intact animals and complex cellular mixtures in vitro. Here we show that murine IFN-gamma produced by recombinant DNA technology shows similar biological effects to BMFs from two other sources. All three preparations induce immunoglobulin secretion by both normal resting murine splenic B cells and the comparable B-cell tumour line WEHI-279.1 (refs 1, 3). IFN-gamma and the other two BMFs are not identical, however, as anti-IFN-gamma antibodies block the effects on B cells of IFN-gamma, but not those of the other two lymphokines. IFN-gamma may be one of several molecules with a direct role in driving the maturation of resting B cells to active immunoglobulin secretion.     

Glycosyl-phosphatidylinositol linkage as a mechanism for cell-surface expression of immunoglobulin D.

The B-cell antigen receptor of the IgM and IgD class is a multimeric complex consisting of the membrane-bound form of the immunoglobulin molecule and two other proteins, Ig-alpha and Ig-beta. The Ig-alpha and Ig-beta proteins form a disulphide-linked alpha/beta heterodimer and are encoded by the mb-1 (ref 9, 10) and B29 genes, respectively. Surface expression of the membrane-bound IgM molecule requires assembly with the alpha/beta heterodimer. The IgD molecule, however, can be expressed on the cell surface in an alpha/beta-dependent and -independent form. We show here that in the alpha/beta-independent form the IgD molecule is anchored in the plasma membrane through a glycosyl-phosphatidylinositol linker. In the presence of the alpha/beta heterodimer, most of the otherwise glycosyl-phosphatidylinositol-linked IgD molecule is expressed on the cell surface as transmembrane proteins.     

Altered immunoglobulin expression and functional silencing of self-reactive B lymphocytes in transgenic mice

Immunological tolerance has been demonstrated in double-transgenic mice expressing the genes for a neo-self antigen, hen egg lysozyme, and a high affinity anti-lysozyme antibody. The majority of anti-lysozyme B-cells did not undergo clonal deletion, but were no longer able to secrete anti-lysozyme antibody and displayed markedly reduced levels of surface IgM while continuing to express high levels of surface IgD. These findings indicate that self tolerance may result from mechanisms other than clonal deletion, and are consistent with the hypothesis that IgD may have a unique role in B-cell tolerance.     

Lymphokine-induced IgM secretion by clones of neoplastic B cells

The induction of antibody secretion by B cells requires T-cell-derived factors1-5. Such factors have been described1,2,6-12 but the precise relationship among these various factors is not clear, and it has been difficult to demonstrate that these factors act directly on the B cell and do not exert their effect via T cells or macrophages. In this report we describe the direct induction of IgM synthesis and secretion in cloned lines of long-term tissue culture adapted neoplastic B cells (BCL1) by T-cell supernatants from phorbol-12-myristate 13-acetate (PMA)-induced EL-4 cells or concanavalin A (Con A)-induced 7.1.1a cells5,9. We have termed this activity BCDFmu (B-cell differentiation factor for IgM). The supernatants containing BCDFmu induce activated and neoplastic B cells to secrete IgM5 and the factor responsible is distinct from BCGF13, interleukin-2 (IL-2)5, the classical T-cell replacing factor (TRF) described by Schimpl and Wecker5, and immune interferon (IFN gamma)5.     

Isolation of a cDNA clone corresponding to an X-linked gene family (XLR) closely linked to the murine immunodeficiency disorder xid

The striking number of human and murine immunodeficiency disorders which map to the X chromosome suggests that genes localized on this chromosome must have important roles in lymphocyte development. At least seven distinct disorders in the human and two in the mouse disrupt lymphocyte maturation, particularly that of B cells, at characteristic stages. As functional genes mapping to the X chromosome in one mammal are found on the X chromosome in all other mammals, the same genes regulating lymphocyte development are expected to be found on the X chromosome in mouse and man. Investigations into the possible mechanisms of these X-linked disorders have been hampered by the lack of molecular probes for the genes or gene products affected; because of this, and the possibility of correlating one or more of the several hundred B- or T-cell-specific genes with a specific mutation, we surveyed 15 different B- and T-cell-specific cDNA clones for localization to the X chromosome. We report here the characterization of one of these murine cDNA clones, which hybridizes with a large, X-linked gene family, designated XLR (X-linked, lymphocyte-regulated). We show that the XLR gene family is closely linked to the X-linked immunodeficiency described in the CBA/N mouse strain (xid), by restriction fragment length polymorphism (RFLP) analysis of DNA from mice congeneic for xid. This finding, together with data on the expression of the XLR locus in B cells, indicates that this gene family either includes the locus defined by the xid mutation or is adjacent to it in a gene complex which may be important in lymphocyte differentiation.     

Continued RAG expression in late stages of B cell development and no apparent re-induction after immunization.

Models of B-cell development in the immune system suggest that only those immature B cells in the bone marrow that undergo receptor editing express V(D)J-recombination-activating genes (RAGs). Here we investigate the regulation of RAG expression in transgenic mice carrying a bacterial artificial chromosome that encodes a green fluorescent protein reporter instead of RAG2. We find that the reporter is expressed in all immature B cells in the bone marrow and spleen. Endogenous RAG messenger RNA is expressed in immature B cells in bone marrow and spleen and decreases by two orders of magnitude as they acquire higher levels of surface immunoglobulin M (IgM). Once RAG expression is stopped it is not re-induced during immune responses. Our findings may help to reconcile a series of apparently contradictory observations, and suggest a new model for the mechanisms that regulate allelic exclusion, receptor editing and tolerance.     

T cells with receptors for IgD

The role of IgD in the immune response has been elusive, although its predominance on the cell surface suggests a receptor function. We have shown previously that euthymic but not athymic BALB/c mice, injected with IgD before antigen, exhibit enhanced antibody responses which can be transferred by T cells. Isotype-specific T cells have been reported to have both upward and downward immunoregulatory effects. Here we demonstrate the existence of T cells with receptors for IgD, and show that exposure to IgD in vivo or in vitro significantly increases the number of T delta cells in the spleen and lymph nodes but not in the thymus. The kinetics of T delta-cell appearance in vivo parallels that of the immunoenhancing effect which occurs after injection of IgD. These T delta cells are of the Lyt 1+2- T-cell phenotype.     

尼罗罗非鱼IgM单抗的制备及IgM+B淋巴细胞吞噬能力的研究

采用免疫亲和层析方法纯化尼罗罗非鱼抗TNP特异性IgM，并以此为抗原免疫Babl/c小鼠进行单克隆抗体制备. 免疫的小鼠脾脏细胞与骨髓瘤细胞(SP2/0)融合成杂交瘤细胞，利用酶联免疫吸附法、蛋白质印迹法(Western blot)和流式细胞术筛选获得两株抗IgM重链的单克隆抗体，分别命名为3B3和9D12. 纯化后的单克隆抗体3B3和9D12在1 mg/mL浓度下其抗体效价分别为740,741和359,712 units/mL. 利用制备的单抗结合激光共聚焦显微镜检测发现，膜结合型IgM位于B细胞膜表面(IgM+ B细胞). 流式细胞术检测尼罗罗非鱼IgM+ B细胞的免疫组织分布，发现IgM+ B细胞分布存在组织差异性，其中在外周血(PBL)所占比例最大，约为37.6%，其次是脾脏(SPL)，占百分比33.7%，头肾(AK)占比例约为23.9%，而后肾(PK)占比例约为20%. IgM+ B细胞荧光微球的吞噬能力分析发现，IgM+ B细胞对0.5 m的荧光微球吞噬能力强于1 m. 另外，IgM+ B细胞的吞噬能力存在免疫组织差异性，其中对于0.5 m荧光微球的吞噬，PBL明显高于其他组织，而AK IgM+ B细胞对1 m荧光微球的吞噬能力最强. 以上结果表明，本研究成功制备了鼠抗尼罗罗非鱼IgM单抗工具，并利用其IgM单抗发现IgM+ B细胞具有吞噬能力且其吞噬能力存在组织差异性，表明IgM+ B细胞在先天性免疫中可能发挥重要作用.     

Depletion of the predominant B-cell population in immunoglobulin mu heavy-chain transgenic mice

The transgenic mouse line M54 was generated by introducing a functionally-rearranged immunoglobulin mu heavy-chain gene into the germ line of a C57B1/6 inbred mouse. Previous examination of the antibodies produced by B-cell hybridomas derived from transgenic M54 mice showed that the presence of the mu transgene grossly altered the immunoglobulin repertoire of unimmunized animals, suggesting that these mice suffer from a serious immunoregulatory perturbation. Studies presented here introduce a new perspective on this functional defect. We show that the lymphoid tissues from these transgenic mice lack virtually all conventional bone-marrow-derived B cells, which constitute the predominant B-cell population in normal mice and which typically produce primary and secondary antibody responses to T-cell-dependent antigens. Moreover, the bone marrow from transgenic M54 mice is depleted of pre-B lymphocytes, indicating a serious defect in early B-cell lymphopoiesis. In contrast, CD5 (Ly-1) B cells, a second B-cell population displaying a characteristic set of cell surface markers which are derived from distinct precursors in the peritoneum, are represented at normal frequencies in these transgenic mice. Thus, the presence of the rearranged immunoglobulin heavy-chain transgene in M54 mice results in an unexpected selective developmental defect that impairs the development of bone-marrow-derived pre-B and B cells without affecting Ly-1 B cells.     

A chemokine-driven positive feedback loop organizes lymphoid follicles

Lymphoid follicles are B-cell-rich compartments of lymphoid organs that function as sites of B-cell antigen encounter and differentiation. CXC chemokine receptor-5 (CXCR5) is required for B-cell migration to splenic follicles, but the requirements for homing to B-cell areas in lymph nodes remain to be defined. Here we show that lymph nodes contain two types of B-cell-rich compartment: follicles containing follicular dendritic cells, and areas lacking such cells. Using gene-targeted mice, we establish that B-lymphocyte chemoattractant (BLC/BCA1) and its receptor, CXCR5, are needed for B-cell homing to follicles in lymph nodes as well as in spleen. We also find that BLC is required for the development of most lymph nodes and Peyer's patches. In addition to mediating chemoattraction, BLC induces B cells to up-regulate membrane lymphotoxin alpha1beta2, a cytokine that promotes follicular dendritic cell development and BLC expression, establishing a positive feedback loop that is likely to be important in follicle development and homeostasis. In germinal centres the feedback loop is overridden, with B-cell lymphotoxin alpha1beta2 expression being induced by a mechanism independent of BLC.     

Resistance of mice suppressed for IgM production to Babesia microti infection.

The immunological mechanisms responsible for overcoming infections with Babesia, an intra-erythrocytic protozoan, are not fully understood. Although high titres of specific anti-babesial antibodies have been observed in several species of animals, and protection has been obtained by transfer of large volumes of recovery serum, the role of antibody in the immune response to an infection is uncertain. The present study investigates the nature of B-cell participation during Babesia microti infections by observing the course of the disease in mice in which IgM production has been suppressed from birth and which contain no B cells. The results show that, in contrast to control mice, which develop and subsequently clear circulating parasitaemias, suppressed mice show an unexpected resistance to infection as reflected by a virtual absence of parasites in the peripheral circulation.     

Isotype exclusion and transgene down-regulation in immunoglobulin-lambda transgenic mice

A given B lymphocyte makes an antibody containing either kappa- or lambda-light chains, but not both. This isotype exclusion is effected at the level of the rearrangement of the immunoglobulin gene segments, although by an unknown mechanism. An attractive possibility is that, following productive rearrangement of one of the light-chain loci, the newly synthesized light-chain polypeptide inhibits DNA rearrangement for the other isotype. To test such feedback regulation, we have created transgenic mice carrying a rearranged lambda 1-gene. By contrast with the B cells in normal newborn mice which are mainly kappa+lambda-, the B cells in the newborn transgenic mice express lambda- but not kappa-chains. We propose that the synthesis of any light chain, be it kappa or lambda, that allows expression of IgM on the cell surface results in a cessation of all V-J joining. Interestingly, the limited light-chain repertoire of the transgenic mice does not persist and most adult B cells express endogenous kappa-rearrangements and down-regulate the transgene.     

TACI and BCMA are receptors for a TNF homologue implicated in B-cell autoimmune disease

B cells are important in the development of autoimmune disorders by mechanisms involving dysregulated polyclonal B-cell activation, production of pathogenic antibodies, and co-stimulation of autoreactive T cells. zTNF4 (BLyS, BAFF, TALL-1, THANK) is a member of the tumour necrosis factor (TNF) ligand family that is a potent co-activator of B cells in vitro and in vivo. Here we identify two receptors for zTNF4 and demonstrate a relationship between zTNF4 and autoimmune disease. Transgenic animals overexpressing zTNF4 in lymphoid cells develop symptoms characteristic of systemic lupus erythaematosus (SLE) and expand a rare population of splenic B-Ia lymphocytes. In addition, circulating zTNF4 is more abundant in NZBWF1 and MRL-lpr/lpr mice during the onset and progression of SLE. We have identified two TNF receptor family members, TACI and BCMA, that bind zTNF4. Treatment of NZBWF1 mice with soluble TACI-Ig fusion protein inhibits the development of proteinuria and prolongs survival of the animals. These findings demonstrate the involvement of zTNF4 and its receptors in the development of SLE and identify TACI-Ig as a promising treatment of autoimmune disease in humans.     

Molecular characterization of single memory B cells

Primary antigenic exposure results in an initial antibody response and the T cell-dependent induction of B-cell memory. Memory B-cell differentiation is characterized by somatic hypermutation in antibody variable region genes (V) and selection of B cells expressing high-affinity variants of this antigen receptor. Despite our current understanding of B-cell memory, the origin of memory B cells and the regulation of their differentiation remain elusive. This is largely due to the difficulties in observing and purifying this minor component of the immunized spleen. Further, molecular characterization of memory B cells requires hybridoma formation which restricts analyses to only those clones capable of fusion and does not allow isolation of cells in a normal physiological state. We have therefore developed a unique system which allows isolation and unambiguous enumeration of IgG1+ memory B cells, based on six-parameter flow cytometry, secretion of antibody in clonal cultures and analysis of clonally expressed V genes using the polymerase chain reaction. Here we report that single IgG1+ antigen-binding B cells from an early secondary immune response proliferate in lipopolysaccharide-driven microcultures and produce antigen-specific IgG1 antibodies. Individual B-cell clones in these cultures express somatically mutated heavy chain V genes, confirming their designation as memory B cells. Although isolated memory B cells undergo extensive proliferation in vitro, V gene sequence analysis of their individual progeny shows that further hypermutation does not occur.