Induction of light chain expression in a pre-B cell line by fusion to myeloma cells

Pre-B cells, the first cells in the B-lymphocyte differentiation pathway which express immunoglobulin, have recently been shown to express cytoplasmic mu heavy chain (H) but not light chain (L). If, as is believed, pre-B cells are the precursors of immature B lymphocytes, which express surface IgM, the differentiation of pre-B cells to immature B lymphocytes must be accompanied by the expression of light chains. In this case, it should be possible for the progeny of a single pre-B cell to express a variety of light chains in association with the same heavy chain. We have tested this hypothesis by hybridizing a pre-B cell line 18-81 expressing only cytoplasmic mu chains with variant myeloma cells which do not express light chains. Hybridization of B-lymphoma cells with myeloma cells usually produces a hybrid with the phenotype of the more differentiated parent. In this case, the fusion resulted in the induction of light chain expression from the 18-81 genes and we have been able to demonstrate that independent hybrids express different light chains, in accordance with the hypothesis that a pre-B cell committed to expression of a single mu heavy chain can generate progeny expressing different slight chains.     

Pre-B cells in kappa-transgenic mice

Recent experiments have shown that the microinjected kappa-chain gene of transgenic mice is expressed in a tissue-specific fashion only in B lymphocytes. The next step was to determine whether, within the B-lymphocyte lineage, the kappa-chain gene was expressed in a normal developmental fashion. Normally, only mu heavy(H)-chain genes, and not kappa-chain genes, are expressed in pre-B cells. To obtain cloned cell lines derived from early cells of the B-cell lineage, we transformed bone marrow cells from kappa-transgenic mice with Abelson murine leukaemia virus (A-MuLV) and tested the resultant cell lines for the retention of the kappa transgene and its expression in RNA and protein. We found that cells with the pre-B phenotype exist in kappa-transgenic mice. We further observed that in A-MuLV-transformed cell lines from a kappa-transgenic mouse with a high copy number of the transgene, the proportion of cell lines expressing kappa (transgenic kappa) was higher than in cell lines from normal or low copy number transgenic mice.     

Lambda 5, a new light-chain-related locus selectively expressed in pre-B lymphocytes

The development from stem cells to pre-B cells, B lymphocytes and, finally, plasma cells and memory cells proceeds through various stages which have been defined by the genomic context in which immunoglobulin (Ig) heavy (H) and light (L) chain gene segments are found, as well as by their state of expression. They have also been identified by surface marker analysis and susceptibility to various stimuli regulating growth and differentiation. We have searched for genes that are expressed at given stages in the B-lymphocyte development pathway and which might function to control this development at various stages. A complementary DNA sequence called pZ183 was found in a library constructed from messenger RNA of the murine pre-B lymphoma cell line 70Z/3 which is selectively expressed in pre-B cells. Here we report the nucleotide sequence of a cDNA clone (pZ183-1) containing 0.7 kilobases (kb) of the pZ183 gene. Part of this sequence shows strong homology to constant (C) and joining (J) region sequences of lambda 1 L chains. Our findings define a new immunoglobulin L-chain-related locus, which we call lambda 5, that is selectively transcribed in pre-B lymphocytes.     

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.     

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.     

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.     

The t(8; 14) chromosomal translocation occurring in B-cell malignancies results from mistakes in V-D-J joining

The reciprocal chromosome translocation, t(8;14), involving the heavy chain locus on chromosome 14 and the c-myc oncogene on chromosome 8 is a characteristic of the B-cell malignancies Burkitt's lymphoma and acute lymphoblastic leukaemia (ALL). We have cloned and sequenced the t(8; 14) breakpoints of an African Burkitt's lymphoma cell line, P3HR-1, and a pre-B cell ALL cell line, 380. In each case the region of chromosome 8 involved has recombined with a JH region on chromosome 14. The two sites of breakage on chromosome 8 lie within 70 base pairs (bp) of one another. At each joining site, sequences homologous to the signal sequences thought to be recognized by the V-D-J recombinase were identified, as were N regions. In B-cell chronic lymphocytic leukaemias (B-CLL) carrying the t(11; 14) chromosome translocation and in follicular lymphomas carrying the t(14; 18) translocation, the V-D-J recombinase is implicated in the mechanism of chromosomal translocations. We speculate that the same enzymatic mechanism is responsible for the t(8; 14) translocations in African Burkitt's lymphoma and pre-B cell ALL.     

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.     

Genome-wide analysis of genetic alterations in acute lymphoblastic leukaemia

Chromosomal aberrations are a hallmark of acute lymphoblastic leukaemia (ALL) but alone fail to induce leukaemia. To identify cooperating oncogenic lesions, we performed a genome-wide analysis of leukaemic cells from 242 paediatric ALL patients using high-resolution, single-nucleotide polymorphism arrays and genomic DNA sequencing. Our analyses revealed deletion, amplification, point mutation and structural rearrangement in genes encoding principal regulators of B lymphocyte development and differentiation in 40% of B-progenitor ALL cases. The PAX5 gene was the most frequent target of somatic mutation, being altered in 31.7% of cases. The identified PAX5 mutations resulted in reduced levels of PAX5 protein or the generation of hypomorphic alleles. Deletions were also detected in TCF3 (also known as E2A), EBF1, LEF1, IKZF1 (IKAROS) and IKZF3 (AIOLOS). These findings suggest that direct disruption of pathways controlling B-cell development and differentiation contributes to B-progenitor ALL pathogenesis. Moreover, these data demonstrate the power of high-resolution, genome-wide approaches to identify new molecular lesions in cancer.     

A novel cell surface molecule on early B-lineage cells

B cells and their antibody-secreting progeny represent one of several differentiation pathways that haematopoietic stem cells (HSC) may enter. Cells representing intermediate stages between HSC and B cells have been identified in mammalian haematopoietic tissues and studied intensively over the past decade. This population of early B-lineage cells, termed pre-B, is characterized by cellular proliferation and an orderly cascade of immunoglobulin gene rearrangements, a combination of events leading to the generation of clonally diverse B cells which then migrate to peripheral lymphoid tissues. It remains to be determined what elements determine the polyclonal growth of pre-B cells, how immunoglobulin gene rearrangements are regulated, and what happens to pre-B cells undergoing 'non-productive' immunoglobulin gene rearrangements. These issues could be resolved more easily if early B-lineage cells could be identified precisely and isolated. Here, we describe a cell surface glycoprotein that is selectively expressed by pre-B and newly formed B cells in murine haematopoietic tissues. The molecule, a homodimer formed by disulphide-linked chains of relative molecular mass (Mr) 140,000, is identified by a mouse monoclonal alloantibody called BP-1.     

Isolation of HTLV-transformed B-lymphocyte clone from a patient with HTLV-associated adult T-cell leukaemia

The human T-cell leukaemia/lymphoma virus (HTLV) is an exogenous retrovirus which has been associated with adult T-cell leukaemia/lymphoma (ATL). This malignancy of T lymphocytes is endemic to southern Japan, the West Indies, and to a lesser extent, the Middle East, Central Africa and the southeastern United States. ATL cells from patients of diverse geographical origins have been found to be infected with HTLV-1 (ref.6). HTLV is normally tropic for mature T lymphocytes, especially those expressing the helper-inducer surface antigen phenotype (OKT4 or Leu-3-positive), and the neoplastic T cells infected with HTLV generally express receptors for T-cell growth factor (detected by reactivity with anti-Tac antibody). However, we report here the isolation of a HTLV-infected B-lymphocyte clone from the peripheral blood of a patient with ATL. This clone is cytogenetically normal and is not infected with Epstein-Barr virus (EBV). Co-culture of cells from this clone with cord blood lymphocytes resulted in transmission of HTLV and the immortalization of either T or B lymphocytes. These results suggest that HTLV may be associated with a broader range of host cells than previously recognized.     

Immunoglobulin heavy chain binding protein

Pre-B lymphocytes, and hybridomas derived from them, synthesize immunoglobulin heavy (IgH) chain in the absence of light (L) chain. In the Abelson virus transformed line 18-81, which is representative of the pre-B cell stage, we observed that at least some of the H-chains are bound to a protein other than L-chain. Here we show that the protein (which we term immunoglobulin heavy-chain binding protein, BiP) binds non-covalently to free IgH, but not to IgH associated with IgL.     

T-cell control of Epstein-Barr virus-infected B cells is lost during P. falciparum malaria

Endemic Burkitt's lymphoma, a tumour of children in which B lymphocytes are infected with Epstein-Barr virus (EBV), is common in areas of Africa where malaria is holoendemic. The tumour is characterized by chromosome translocations; usually the terminal portion of chromosome 8 containing the c-myc gene is translocated to chromosome 14, near the enhancer of the immunoglobulin heavy-chain locus. Less frequent are translocations of chromosome 8 to the kappa light-chain locus of chromosome 2 or to the lambda light-chain locus of chromosome 22. In vitro, EBV induces B cells to proliferate and secrete immunoglobulin and antibody. However, in vivo the infected B lymphocytes are under immunological control, so that abnormal proliferation is found only in immunosuppressed patients. Such patients are subsequently liable to develop lymphomas. Burkitt believed that the tumour he had described resulted from interaction between a virus(es) and a "reticuloendothelial system altered by chronic and heavy infection by malarial or other parasites". We report here that during an attack of Plasmodium falciparum malaria, T-cell subpopulations are radically altered so that, in vitro, B lymphocytes infected with EBV proliferate abnormally to secrete large amounts of immunoglobulin and antibody. This phenomenon offers some explanation for the increased incidence of Burkitt's tumour and the high levels of immunoglobulin found in people living in areas where P. falciparum malaria is common.     

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.     

The immunoglobulin mu constant region gene is expressed in mouse thymocytes

It has been a matter of controversy whether the functional capacity of T cells to discriminate between antigens is mediated via immunoglobulin, an immunoglobulin-like molecule, or by the product(s) of unrelated genes. The progenitors of immunoglobulin-secreting cells, B cells, express membrane-bound immunoglobulin as the antigen-specific receptor on their surface. For T cells, although products of immunoglobulin heavy chain variable region genes are implicated as receptor components, there has been no compelling immunochemical evidence for participation of either immunoglobulin light chains or heavy chain constant regions (see refs 2-6 for the disparate views). Recently, using cloned immunoglobulin DNA sequences as hybridization probes, we have demonstrated that the immunoglobulin Cmu gene, but not the Cmu gene, is expressed as polyadenylated RNA in some T cell tumour (T lymphoma) cell lines. Individual T lymphoma lines yielded up to three discrete mu RNA species of different sizes (1.9, 2.2 and 3.0 kilobases), each species being different in size from the major mu RNA species present in B lymphoma cells (2.4 and 2.7 kilobases). We show here that cells from the normal mouse thymus contain mu RNA species, indistinguishable in size from those in T lymphoma cells, but contain little if any kappa RNA.     

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.     

A new human immunoglobulin VH family preferentially rearranged in immature B-cell tumours

The prevalent forms of adult and childhood B-cell neoplasia are chronic lymphocytic (CLL) and acute lymphocytic (ALL) leukaemia, and are typified by a nearly monoclonal accumulation of cells expressing a single heavy (H) and light (L) chain variable (V) region. V gene selection could be random, or quite biased if the disease or the developmental status of the transformed cell somehow influenced DNA rearrangement. We have cloned and sequenced three germ-line VH gene segments that constitute a new human VH family (subgroup V) linked within 160 kilobase pairs of the DH-JH complex. One VH(V) member is rearranged in about 30% of patients with CLL and ALL, but not in IgM-expressing B-cell lines from peripheral blood. In some tumours, we detect a truncated (VH(V) RNA devoid of constant regions that originates from unrearranged VH(V) genes. In other tumours and in resting splenocytes, we detect large amounts of normally sized VH(V)-associated mRNA, although stimulation by mitogen of splenic B cells results in loss of VH(V)-hybridizing RNA. These features suggest that biased rearrangement of subgroup V may be under developmental selection.