Mature murine B lymphocytes immortalized by Kirsten sarcoma virus

Clonal, antigen-specific, functionally responsive cell populations have proved critical for the analysis of the activation and regulation of lymphocytes. Such studies with B lymphocytes, the precursors of antibody-secreting cells, are hampered by the difficulty in generating phenotypically mature, antigen-reactive lines from defined cell populations. One method is to use acutely transforming retroviruses, which can transform B-lineage lymphocytes in vitro. However, Abelson murine leukaemia virus (A-MuLV) infection of murine bone marrow cells in vitro yields mostly immature B-cell lines, and infection of murine bone marrow cells with murine sarcoma viruses carrying ras related genes produces only immature lymphoid cell lines. Retroviruses which contain ras can immortalize nonlymphoid cells without causing loss of mature phenotypic characteristics. We used ras-containing Kirsten sarcoma virus (KiSV) pseudotyped with an amphotropic MuLV helper virus, to infect a purified population of mature, hapten-binding murine splenic B lymphocytes, aiming to generate mature B-cell lines to use as models for the study of B-cell growth and differentiation physiology. Immortalized B-cell lines which retain the same mature phenotype as the starting population, including hapten-specific binding, were produced. This is the first demonstration of a method for immortalizing selected antigen-binding B lymphocytes, and the first example of immortalization of mature B cells in vitro with an acutely transforming ras-containing retrovirus.     

Bipotential precursors of B cells and macrophages in murine fetal liver.

LYMPHOCYTES (B and T cells) derive continuously from the same multipotential stem cells that produce myeloid cells, including erythrocytes, granulocytes and macrophages. Tri- and bipotential myeloid intermediates between the multipotential stem cells and later unipotential cells have been identified using clonal methods in culture. Although similar methods have detected committed pre-B cells in mouse fetal liver, earlier progenitors with additional non-B lineage options have not been demonstrated in normal tissues. We report the characterization and purification of fetal liver cells that generate clones containing both macrophages and B cells, identified biochemically and morphologically. The common origin of the two cell types was shown by culture of single precursor cells. Their dual potential and unrearranged immunoglobulin loci place the precursors before exclusive B-lineage commitment in the haematopoietic hierarchy. The availability of such cells in purified form will allow direct study of lineage choice in cells having both lymphoid and non-lymphoid options.     

Diversity of immunoglobulin expression in leukaemic cells resembling B-lymphocyte precursors

Approximately 20% of patients with acute lymphocytic leukaemia (ALL) have leukaemic blasts with features of pre-B cells which are the recently characterized precursors of B lymphocytes in normal development (for a review, see ref. 2). Pre-B cells isolated from normal bone marrow or fetal liver, and malignant cells from patients with pre-B cell leukaemia, are rapidly dividing lymphoid cells that contain cytoplasmic immunoglobulin mu heavy chains, but have no detectable surface immunoglobulin. The resemblance of immunoglobulin-containing ALL cells to normal precursors of B lymphocytes and their availability in relatively pure preparations allowed us to explore them as models of early stages in the differentiation of the B-lymphocyte line. We report here observations on the occurrence of intermediate pre-B/B-cell phenotypes, immunoglobulin isotype switching and the asynchrony of immunoglobulin heavy and light chain expression in 30 cases of ALL and 3 cases of chronic myelogenous leukaemia in lymphoblastic crisis (CML-BC).     

Interleukin-3 supports growth of mouse pre-B-cell clones in vitro

Study of the differentiation of immunoglobulin-producing B lymphocytes has been hampered by the inability to maintain homogeneous populations of precursor cells in vitro. We describe here that interleukin-3 supports the growth of freshly isolated fetal liver pre-B cells and the long-term culture of interleukin-3 dependent pre-B-cell clones that can be induced to mature into antibody secreting cells in vitro.     

Characterization of murine interleukin B by a monoclonal antibody

Interleukin B (IL-B), formerly termed BEF (B-cell-derived enhancing factor) or IL-B4, was originally described as a non-immunoglobulin regulatory factor spontaneously produced by B lymphocytes and B-cell lines that enhances the in vitro antigen-driven antibody response of unfractionated spleen cells stimulated by thymus-dependent antigens. Since then we have examined the function of interleukin B in a number of immune reactions, both in vitro and in vivo, and found that it inhibits the activation of suppressor T lymphocytes. We report here the production of two monoclonal antibodies (mAb) that specifically inhibit interleukin B activity. The use of these mAb in the purification and characterization of IL-B is described. IL-B from both normal and transformed B cells consists of two subunits of similar size and amino-acid composition. The structure of interleukin B and its specific behaviour in biological assay distinguish it from many other known lymphokines.     

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.     

Functional V region formation during in vitro culture of a murine immature B precursor cell line

B lymphocytes originate from pluripotential haematopoietic stem cells and differentiate into immunoglobulin (Ig)-producing cells. B-cell lineage differentiation is accompanied by two types of immunoglobulin gene rearrangements--rearrangement of V, D and J gene segments to create a functional V gene and rearrangement of CH genes for heavy-chain switching. These results, however, have been obtained mainly by analysis of immunoglobulin gene organization of myeloma cells. Baltimore and his colleagues have established Abelson murine leukaemia virus (A-MuLV)-transformed cell lines and found a few lines capable of carrying out kappa-gene rearrangement or undergoing isotype switching during in vitro culture. To study early B-cell lineage differentiation events, we have now also established A-MuLV-transformed cell lines which are capable of differentiating from mu- to mu+ and of undergoing continuing rearrangement of heavy-chain genes in culture. Analysis of immunoglobulin gene organization of these transformed cells revealed that mu- cells have already undergone DNA rearrangements involving JH segments but an additional rearrangement of JH segments is required for initiation of mu-chain synthesis. Southern blot analysis of the DNA and two-dimensional gel electrophoresis of intracytoplasmic mu-chain show that mu-chain diversity with respect to antigen specificity may be generated during this second rearrangement process. As no rearrangement of light-chain genes takes place in these cells, this implies that light-chain gene rearrangement requires some further change, or a different enzyme.     

Molecular characterization and expression of the gene encoding human erythroid-potentiating activity

Erythropoietin is the primary physiological regulator of erythropoiesis; however, in vitro studies have identified another class of mediators which appear to be important in stimulating erythroid progenitors. These factors have generally been referred to as burst-promoting activities (BPA), because they stimulate the growth of early erythroid progenitors referred to as burst-forming units-erythroid (BFU-E) which give rise to colonies of up to thousands of haemoglobinized cells. We recently reported purification of a burst-promoting activity from medium conditioned by the Mo T-lymphoblast cell line infected with human T-cell lymphotropic virus type II (HTLV-II). This purified glycoprotein of relative molecular mass (Mr) 28,000 also stimulates colony formation by more mature erythroid precursors (CFU-E) and is therefore referred to as erythroid-potentiating activity (EPA). Purified EPA specifically stimulates human and murine cells of the erythroid lineage, unlike murine interleukin-3 (IL-3) which stimulates precursor cells from all haematopoietic lineages. We report here the isolation of a complementary DNA molecular clone encoding EPA and its use in producing EPA in COS (monkey) cells and CHO (Chinese hamster ovary) cells. We also define the organization of the EPA gene in human DNA.     

Regulated progression of a cultured pre-B-cell line to the B-cell stage

The variable (V) regions of heavy and light immunoglobulin chains are encoded by multiple germline DNA elements which are assembled into complete variable-region genes in precursor(pre-) B lymphocytes. The heavy-chain V region (VH) is assembled from three separate germline DNA elements, the variable (VH), diversity (D) and joining (JH) segments; whereas light-chain variable regions of either the kappa or lambda type are assembled from two elements, the VL and JL. Analysis of tumour cell lines or sorted cell populations which represent early and late pre-B cells has suggested that heavy-chain assembly and expression generally precedes that of light chains; but, primarily because of the lack of appropriate model systems to study the phenomenon, the mechanism and significance of this apparently orderly differentiation process are much debated. Here we describe for the first time a transformed cell line, 300-19, which sequentially undergoes all of the immunoglobulin gene rearrangement and expression events associated with the differentiation of pre-B cells to surface immunoglobulin-positive B lymphocytes. Analysis of the in vitro differentiation of 300-19 cells provides direct evidence for distinct differentiation phases of first VH and subsequently VL assembly during B-cell differentiation. Furthermore, these analyses suggest that the mu heavy chain, resulting from a productive VHDJH rearrangement, has both a positive and a negative regulatory role in mediating this ordered differentiation process, that is, signalling the cessation of VH gene assembly and simultaneously signalling the onset of VL assembly.     

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.     

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.     

Protein kinase Cdelta controls self-antigen-induced B-cell tolerance

Interaction of a B cell expressing self-specific B-cell antigen receptor (BCR) with an auto-antigen results in either clonal deletion or functional inactivation. Both of these processes lead to B-cell tolerance and are essential for the prevention of auto-immune diseases. Whereas clonal deletion results in the death of developing autoreactive B cells, functional inactivation of self-reactive B lymphocytes leads to complex changes in the phenotype of peripheral B cells, described collectively as anergy. Here we demonstrate that deficiency in protein kinase Cdelta (PKC-delta) prevents B-cell tolerance, and allows maturation and terminal differentiation of self-reactive B cells in the presence of the tolerizing antigen. The importance of PKC-delta in B-cell tolerance is further underscored by the appearance of autoreactive anti-DNA and anti-nuclear antibodies in the serum of PKC-delta-deficient mice. As deficiency of PKC-delta does not affect BCR-mediated B-cell activation in vitro and in vivo, our data suggest a selective and essential role of PKC-delta in tolerogenic, but not immunogenic, B-cell responses.     

绒白乳菇中提取的乳菇菌素-D免疫抑制作用的研究

研究从绒白乳菇菌中分离出的一种新型倍半萜类化合物-乳菇菌素D的免疫抑制作用及机制。运用体外培养法取BALB/C小鼠脾细胞,研究乳菇菌素D对静止脾细胞、Con A(刀豆蛋白A)和LPS(脂多糖)刺激后的脾细胞(含B淋巴细胞和T淋巴细胞)增殖的抑制作用;运用MTT法经酶标仪检测吸光度,并计算刺激指数(stimulating index,SI);体外培养小鼠脾细胞,运用ELISA检测法观察乳菇菌素D对脾脏中T淋巴细胞分泌IL-2的抑制作用。结果显示该化合物对静止的脾细胞无抑制作用;对Con A和LPS刺激后的脾细胞增殖有显著抑制作用(P0.05),最大浓度药物组刺激指数SI低至0.28;高浓度的乳菇菌素D对Con A刺激的小鼠脾细胞产生的IL-2,具有显著抑制作用(P0.05)。结果显示乳菇菌素D通过抑制抗原刺激后的T淋巴细胞和B淋巴细胞的增殖、抑制其分泌细胞因子,从而发挥免疫抑制作用。研究结果将为开发以乳菇菌素D为母体、经结构修饰、安全低毒的免疫抑制剂提供理论基础。     

Adult T-cell progenitors retain myeloid potential

During haematopoiesis, pluripotent haematopoietic stem cells are sequentially restricted to give rise to a variety of lineage-committed progenitors. The classical model of haematopoiesis postulates that, in the first step of differentiation, the stem cell generates common myelo-erythroid progenitors and common lymphoid progenitors (CLPs). However, our previous studies in fetal mice showed that myeloid potential persists even as the lineage branches segregate towards T and B cells. We therefore proposed the 'myeloid-based' model of haematopoiesis, in which the stem cell initially generates common myelo-erythroid progenitors and common myelo-lymphoid progenitors. T-cell and B-cell progenitors subsequently arise from common myelo-lymphoid progenitors through myeloid-T and myeloid-B stages, respectively. However, it has been unclear whether this myeloid-based model is also valid for adult haematopoiesis. Here we provide clonal evidence that the early cell populations in the adult thymus contain progenitors that have lost the potential to generate B cells but retain substantial macrophage potential as well as T-cell, natural killer (NK)-cell and dendritic-cell potential. We also show that such T-cell progenitors can give rise to macrophages in the thymic environment in vivo. Our findings argue against the classical dichotomy model in which T cells are derived from CLPs; instead, they support the validity of the myeloid-based model for both adult and fetal haematopoiesis.     

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.     

Bone marrow cells give rise to distinct cell clones within the thymus

The thymus is the major, if not the sole site of maturation of T lymphocytes from their haematopoietic precursors. During embryonic life (at a few well-defined intervals, at least in birds) the thymus receives thymus-homing haematopoietic precursors that give rise to antigen-specific functional T lymphocytes. Although the number and thymic location of distinct T-cell lineages destined to form the peripheral T-cell pool are not yet well defined, at least two independent pathways have been proposed. First, thymic subcapsular lymphoblasts divide and differentiate to give rise to small deep cortical thymic lymphocytes, medullary lymphocytes and thymus emigrants (I.W., unpublished data) and second, the medulla contains an independent self-renewing population that contains the precursors of the peripheral T-cell pool. Following irradiation the thymus may be repopulated by injected haematopoietic cells presumably related to the thymus-homing haematopoietic cells of the embryo. Here we have reconstituted irradiated mice with limiting numbers of bone marrow cells from Thy-1 congeneic donors and have found distinct clones of cells within the thymus. The pattern of reconstitution by the precursor cells indicates that two independent thymus lineages exist: cortex plus medulla, and medulla alone.     

Epstein-Barr virus infection and replication in a human epithelial cell system.

Epstein-Barr virus, a human herpesvirus with oncogenic potential, infects two target tissues in vivo: B lymphocytes, where the infection is largely non-productive, and stratified squamous epithelium in which virus replication occurs. The interaction with B cells, initiated through virus binding to the B-cell surface molecule CR2 (ref. 4), has been studied in vitro and the virus 'latent' genes associated with B-cell growth transformation defined. By comparison, viral infection of epithelium remains poorly understood, reflecting the lack of an appropriate cell-culture model. Here we describe the development of such a model using as targets CR2-expressing transfected cells of two independent human epithelial lines. A high proportion of these cells bind virus and become actively infected, expressing the small EBER RNAs (small non-polyadenylated virus-coded RNAs) and the Epstein-Barr nuclear antigen 1 but not other latent proteins; thereafter, under conditions favouring epithelial differentiation, up to 30% of the cells can be induced to enter virus productive cycle with some progressing to full virus replication. We find significant differences between laboratory virus strains in their ability to infect epithelium that do not correlate with their B-cell growth-transforming activity.     

Interleukin 21 and its receptor are involved in NK cell expansion and regulation of lymphocyte function

Cytokines are important in the regulation of haematopoiesis and immune responses, and can influence lymphocyte development. Here we have identified a class I cytokine receptor that is selectively expressed in lymphoid tissues and is capable of signal transduction. The full-length receptor was expressed in BaF3 cells, which created a functional assay for ligand detection and cloning. Conditioned media from activated human CD3+ T cells supported proliferation of the assay cell line. We constructed a complementary DNA expression library from activated human CD3+ T cells, and identified a cytokine with a four-helix-bundle structure using functional cloning. This cytokine is most closely related to IL2 and IL15, and has been designated IL21 with the receptor designated IL21 R. In vitro assays suggest that IL21 has a role in the proliferation and maturation of natural killer (NK) cell populations from bone marrow, in the proliferation of mature B-cell populations co-stimulated with anti-CD40, and in the proliferation of T cells co-stimulated with anti-CD3.     

Precursor and effector phenotypes of activated human T lymphocytes

In mice, thymus-derived lymphocytes are differentiated into functional subclasses by their cell surface antigens. The Ly 1 determinants are present on T cells with a helper function, whereas Ly 2 and Ly 3 antigens are expressed on the surface of lymphocytes with suppressor or cytotoxic functions. In man also, T-cell subsets have been identified using allo- and heteroimmune sera and, more recently, using monoclonal antibodies, which seem to identify helper and suppressor or cytotoxic subpopulations. The major histocompatibility system (MHS)-encoded Ia antigens belong to several polymorphic families of membrane associated glycoproteins originally found on B lymphocytes; however, they have also been shown to be markers for suppressor T cells in mice. Recent studies have shown that in both mouse and man, T cells activated by a mixed lymphocyte reaction or by mitogens become Ia+. Furthermore, some human T lymphoid cells, either freshly isolated from peripheral blood or after in vitro activation by lectins or alloantigens, possess suppressor properties. We report here the phenotype of a T suppressor-cell subpopulation which was induced in long-term culture of lymphoid cells after activation with phytohaemagglutinin (PHA). Our results suggest that a subset of T cells was progressively expanded over a period of 8 days in culture and that, with the expression on the surface of these cells of 'Ia-like' antigens, they acquired the capacity to suppress the proliferative response of syngeneic or allogeneic lymphocytes to alloantigens or mitogens.