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.     

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.     

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.     

DNase I hypersensitive sites in the chromatin of human mu immunoglobulin heavy-chain genes

An immunoglobulin polypeptide chain is encoded by multiple gene segments that lie far apart in germ-line DNA and must be brought together to allow expression of an immunoglobulin gene active in B lymphocytes. For the immunoglobulin heavy chain genes, one of many variable (V) region genes becomes joined to one of several diversity (D) segments which are fused to one of several joining (J) segments lying 5' of the constant region (C) genes. Here we show that the rearranged mu genes of an IgM-producing human B-lymphocyte cell line exhibit pancreatic deoxyribonuclease (DNase I) hypersensitive sites in the JH-C mu intron that are absent in naked DNA or the chromatin of other differentiated cell types. DNA sequence analysis reveals that the major hypersensitive site maps to a conserved region of the JH-C mu intron recently shown to function as a tissue-specific enhancer of heavy-chain gene expression. A similar association of an enhancer-like element with a DNase I hypersensitive site has been reported for the mouse immunoglobulin light-chain J kappa-C kappa intron. These results implicate disruption of local chromatin structure in the mechanism of immunoglobulin enhancer function.     

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.     

Recombination between an expressed immunoglobulin heavy-chain gene and a germline variable gene segment in a Ly 1+ B-cell lymphoma

The early stages of murine B-cell differentiation are characterized by a series of immunoglobulin gene rearrangements which are required for the assembly of heavy(H) and light(L)-chain variable regions from germline gene segments. Rearrangement at the heavy-chain locus is initiated first and consists of the joining of a diversity (DH) gene segment to a joining (JH) gene segment. This forms a DJH intermediate to which a variable (VH) gene segment is subsequently added. Light-chain gene rearrangement follows and consists of the joining of a VL gene segment to a JL gene segment: once a productive light-chain gene has been formed the cell initiates synthesis of surface immunoglobulin M (sIgM) receptors (reviewed in ref. 1). These receptors are clonally distributed and may undergo further diversification either by somatic mutation or possibly by continued recombinational events. Such recombinational events have been detected in the Ly 1+ B-cell lymphoma NFS-5, which has been shown to rearrange both lambda and H-chain genes subsequent to the formation of sIgM (mu kappa) molecules. Here we have analysed a rearrangement of the productive allele of NFS-5 and found that it is due to a novel recombination event between VH genes which results in the replacement of most or all of the coding sequence of the initial VHQ52 rearrangement by a germline VH7183 gene. Embedded in the VH coding sequence close to the site of the cross-over is the sequence 5' TACTGTG 3', which is identical to the signal heptamer found 5' of many DH gene segments. This embedded heptamer is conserved in over 70% of known VH genes. We suggest that this heptamer mediates VH gene replacement and may play an important part in the development of the antibody repertoire.     

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.     

Immunoglobulin heavy-chain class switching in a pre-B cell line is accompanied by DNA rearrangement

Switching of an Abelson virus-transformed mouse lymphoid cell line from immunoglobulin mu to gamma 2b heavy-chain synthesis in vitro is accompanied by loss of DNA sequences between the JH and C gamma 2b gene segments, and thus cannot be explained by differential RNA processing. The light-chain loci of both mu- and gamma 2b-producing cell clones are in the embryonic configuration, which indicates that class switching can occur in pre-B cells.     

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).     

Preferential utilization of the most JH-proximal VH gene segments in pre-B-cell lines

The most JH-proximal VH gene segments are used highly preferentially to form VHDJH rearrangements in pre-B-cell lines. This result demonstrates that the rate at which immunoglobulin VH gene segments recombine is influenced by their chromosomal organization, and that the initial repertoire of VH genes expressed in pre-B cells is strikingly different from that seen in mature populations.     

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.     

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.     

Critical test of a sister chromatid exchange model for the immunoglobulin heavy-chain class switch

B lymphocytes may switch from producing an immunoglobulin heavy chain of the mu class to that of the gamma, epsilon or alpha class. To maintain the specificity, the new heavy chain must keep the original variable (V) region; this is achieved by deleting DNA sequences so that the V (consisting of joined VH, diversity (DH) and joining (JH) gene segments) and C (constant) gene segments coding for the new heavy chain are brought into close proximity (reviewed in ref. 5; we do not consider here the mu-delta situation). There are, in principle, three types of chromosomal rearrangements that yield a deletion: rearrangement within a chromatid; unequal sister chromatid exchange (as suggested by Obata et al.); and unequal recombination between chromosomal homologues. We have analysed the arrangement of C mu DNA in clones of the pre-B-cell line 18-81 that switches in vitro from mu to gamma 2b. The clones examined produce either mu, gamma 2b or no immunoglobulin chain. We report here that all the gamma 2b clones had lost at least one copy of C mu and no clones contained three copies of C mu. These findings formally exclude both unequal sister chromatid exchange and recombination between homologues as mechanisms for creating a gene encoding the gamma 2b chain.     

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.     

Major reorganization of immunoglobulin VH segmental elements during vertebrate evolution

In mammals, the immunoglobulin heavy-chain variable region (VH) locus is organized in a linear fashion; individual VH, diversity (DH), joining (JH) and constant (CH) region segments are linked in separate regions. During somatic development, coding segments flanked by characteristic short recombination signal sequences, separated by intervening sequence regions that may exceed 2,000 kilobases (kb), are recombined. Combinatorial joining of different segments as well as imprecision in this process contribute to the diversity of the primary antibody response; subsequent mutation further alters functionally rearranged genes. This basic somatic reorganization mechanism is shared by six major families of genes encoding antigen receptors. Previously, we have shown that multiple germline genes and mammalian-like recombination signal sequences are associated with the VH gene family of Heterodontus francisci (horned shark), a primitive elasmobranch. Studies presented here demonstrate that segmental reorganization involving mammalian-like DH and JH segments occurs in the lymphoid tissues of this species. In marked contrast to the mammalian system, we find multiple instances of close linkage (approximately 10 kb) between individual VH, DH, JH, and CH segments. This unique organization may limit combinatorial joining and be a factor in the restricted antibody response of this lower vertebrate.