Association of Ig L chain-like protein lambda 5 with a 16-kilodalton protein in mouse pre-B cell lines is not dependent on the presence of Ig H chain protein

Using a polyclonal rabbit antiserum against recombinant mouse lambda 5 protein, we determined that the pre-B cell specific mouse lambda 5 gene encodes a 22-kDa protein. The lambda 5 protein, which is related to conventional Ig lambda L chain proteins forms a complex with Ig mu H chain protein and an as yet unidentified 16-kDa protein (p16) in mu+ pre-B cell lines carrying a functionally rearranged VH-DH-JH allele. In pre-B cell lines which carry DH-JH rearrangements and do not express mu H chain protein, lambda 5 protein is associated with p16. Thus the expression of lambda 5 protein precedes the expression of intact mu H chain protein. This suggests the existence of developmentally regulated protein complexes involving the Ig L chain-like protein lambda 5 and p16 in mu- pre-B cells; lambda 5, p16, and Ig H chain protein in mu+ pre-B cells and Ig H chain and conventional Ig L chain proteins in B cells and plasma cells.     

Activation of V kappa gene rearrangement in pre-B cells follows the expression of membrane-bound immunoglobulin heavy chains.

During B cell development V kappa gene rearrangement seems to occur only in mu-positive pre-B cells. To study the role of the mu chain in the activation of the Ig kappa locus, we introduced expression vectors carrying different forms of the mu gene into null pre-B cells. The activation of the Ig kappa locus followed the expression of the membrane form (micron) of the mu chain. The expression of the secreted form (microS) did not result in the activation of the Ig kappa locus. We further show that both forms of the mu chain differ in their intracellular transport in pre-B cells.     

Russell bodies: a general response of secretory cells to synthesis of a mutant immunoglobulin which can neither exit from, nor be degraded in, the endoplasmic reticulum

Dilated cisternae of the ER resembling Russell Bodies (RBs) are induced in light (L) chain producing myeloma cell lines by transfection of a mu heavy (H) chain gene lacking the first constant domain (mu delta CH1). RBs do not appear to be tissue specific, since they are also induced in a rat glioma cell line transfected with mu delta CH1 and L chain genes. Efficient RB biogenesis requires H-L assembly and polymerization. The mutant Ig is partially degraded in a pre-Golgi compartment. The remnant, however, becomes an insoluble lattice when intersubunit disulphide bonds are formed. The resulting insoluble aggregate accumulates in RBs. Replacing the COOH-terminal cysteine of mu delta CH1 chains with alanine reverses the RB-phenotype: the double mutant mu ala delta CH1 chains assemble noncovalently with L and are secreted as H2L2 complexes. Similarly, secretion of mu delta CH1 chains can be induced by culturing transfectant cells in the presence of reducing agents. The presence of RBs does not alter transport of other secretory or membrane molecules, nor does it affect cell division. Resident proteins of the ER and other secretory proteins are not concentrated in RBs, implying sorting at the ER level. Sorting could be the result of the specific molecular structure of the insoluble lattice. We propose that RBs represent a general response of the cell to the accumulation of abundant, nondegradable protein(s) that fail to exit from the ER.     

A transgenic immunoglobulin mu gene prevents rearrangement of endogenous genes

Transgenic mice containing a microinjected rearranged immunoglobulin (Ig) mu heavy chain gene were examined for the effects on DNA rearrangement of the endogenous Ig genes. Abelson murine leukemia virus (A-MuLV) cell lines were isolated from pre-B cells of transgenic mice and of normal littermates. Microinjected mu gene RNA and a mu heavy chain protein were synthesized in every transgenic A-MuLV cell line. Only 10% of normal mouse A-MuLV transformants synthesized mu protein. A germ-line JH allele was observed in 40% of the transgenic lines, demonstrating that the block to endogenous Ig DNA rearrangement occurred at the first step of heavy chain DNA joining. All alleles were rearranged in normal mouse A-MuLV lines. Germline JH alleles were also detected in 10% of the transgenic hybridomas derived from proliferating B cells. Our results support a model of active prevention of rearrangement by the product of successfully rearranged mu genes.     

VPREB3: cDNA characterization and expression in human and chromosome mapping in human and mouse

The pre-B cell receptor (pre-BCR) regulates pre-B cell expansion and allelic exclusion at the immunoglobulin (Ig) heavy chain locus and mediates the selection of Ig heavy chain variable gene segments. During the early phase of pre-BCR assembly in the mouse, the membrane Ig mu heavy chain transiently associates with the VPREB3 protein in the endoplasmic reticulum. Here, we present the human VPREB3 cDNA sequence and its B cell-specific expression in hematopoietic cell lines. We have localized this gene to chromosome 22q11 close to IGLL genes in human and to chromosome 10C in mouse.     

Isolation of scid pre-B cells that rearrange kappa light chain genes: formation of normal signal and abnormal coding joins.

Consistent with an ordered immunoglobulin (Ig) gene assembly process during precursor (pre-) B cell differentiation, we find that most Abelson murine leukemia virus (A-MuLV)-transformed pre-B cells derived from scid (severe combined immune deficient) mice actively form aberrant rearrangements of their Ig heavy chain locus but do not rearrange endogenous kappa light chain variable region gene segments. However, we have identified several scid A-MuLV transformants that transcribe the germline Ig kappa light chain constant region and actively rearrange the kappa variable region gene locus. In one case progression to the stage of kappa light chain gene rearrangement did not require expression of Ig mu heavy chains; furthermore, this progression could not be efficiently induced following expression of mu heavy chains from an introduced vector. As observed in pre-B cell lines from normal mice, attempted V kappa-to-J kappa rearrangements in scid transformants occur by inversion at least as frequently as by deletion. The inverted rearrangements result in retention of both products of the recombination event in the chromosome, thus allowing their examination. scid kappa coding sequence joins are aberrant and analogous in structure to previously described scid heavy chain coding joins. In contrast, the recognition signals that flank involved coding segments frequently are joined precisely back-to-back in normal fashion. The scid VDJ recombinase defect therefore does not significantly impair recognition of, site-specific cutting at, or juxtaposition and appropriate ligation of signal sequences. Our finding that the scid defect prevents formation of correct coding but not signal joins distinguishes these events mechanistically.     

Chromatin remodeling at the Ig loci prior to V(D)J recombination.

Rearrangement of Ig H and L chain genes is highly regulated and takes place sequentially during B cell development. Several lines of evidence indicate that chromatin may modulate accessibility of the Ig loci for V(D)J recombination. In this study, we show that remodeling of V and J segment chromatin occurs before V(D)J recombination at the endogenous H and kappa L chain loci. In recombination-activating gene-deficient pro-B cells, there is a reorganization of nucleosomal structure over the H chain J(H) cluster and increased DNase I sensitivity of V(H) and J(H) segments. The pro-B/pre-B cell transition is marked by a decrease in the DNase I sensitivity of V(H) segments and a reciprocal increase in the nuclease sensitivity of Vkappa and Jkappa segments. In contrast, J(H) segments remain DNase I sensitive, and their nucleosomal organization is maintained in mu(+) recombination-activating gene-deficient pre-B cells. These results indicate that initiation of rearrangement is associated with changes in the chromatin structure of both V and J segments, whereas stopping recombination involves changes in only V segment chromatin. We further find an increase in histone H4 acetylation at both the H and kappa L chain loci at the pro-B cell stage. Although histone H4 acetylation appears to be an early change associated with B cell commitment, acetylation alone is not sufficient to promote subsequent modifications in Ig chromatin.     

DNase I sensitivity of immunoglobulin light chain genes in Abelson murine leukemia virus transformed pre-B cell lines.

We have used Abelson murine leukemia virus (A-MuLV) transformed pre-B cell lines to test the hypothesis that the rearrangement potential of a developing B-lymphocyte is dependent on an "opening" of the chromatin structure surrounding immunoglobulin (Ig) genes, thus allowing accessibility to an Ig gene recombinase. The chromatin structures surrounding heavy (H), kappa (kappa), and lambda (lambda) chain constant-region genes were assessed by DNase I sensitivity in A-MuLV transformed cell lines capable of H, kappa or lambda gene rearrangement. Our results indicate that DNase I-sensitive chromatin structures of these Ig constant-region genes correlate closely with the ability of the genes to undergo recombination. We also find that the chromatin structure of an Ig constant-region locus becomes DNase I sensitive before any DNA rearrangement events occur.     

Surface mu heavy chain expressed on pre-B lymphomas transduces Ca2+ signals but fails to cause growth arrest of pre-B lymphomas.

Little is known about the role of signals transduced by cell surface IgM (sIgM) expressed during early B cell development. A subclone (1.6) of the late pre-B cell lymphoma 70Z/3.12 was used to study signal transduction by surface mu heavy (H) chain before and after transition to the early immature B cell stage, and the functional consequences thereof. Although kappa L chain expression can be induced on 1.6 cells by LPS or cytokines, immunoprecipitations indicated that the non-induced 1.6 cells expressed mu H chain with an alternative protein(s) which may be a surrogate light chain(s). Consistent with this, anti-mu but not anti-kappa or anti-lambda antibodies caused transient Ca2+ mobilization in noninduced 1.6 cells. The Ca2+ signal was derived from both intracellular stores and Ca2+ influx in either noninduced cells or in cells that had been preinduced to express kappa L chain. Thus, the ability of mu H chain to mobilize Ca2+ as a second messenger does not depend upon the expression of mature L chains. The immature B lymphomas, WEHI-231 and CH1, express mature forms of IgM and undergo growth arrest when stimulated by anti-mu antibody. In contrast, signals generated by mu H chain on either noninduced or preinduced 1.6 cells or in the sIgM+ pre-B cell transfectant 300-19 mu lambda 36/8 did not cause growth arrest. These results suggest that mu H chain expressed on pre-B cells is capable of mobilizing Ca2+, but that this signal alone is insufficient to induce growth arrest in the pre-B cell.     

Deletion of the immunoglobulin kappa chain intron enhancer abolishes kappa chain gene rearrangement in cis but not lambda chain gene rearrangement in trans.

Immunoglobulins (Ig) secreted from a plasma cell contain either kappa or lambda light chains, but not both. This phenomenon is termed isotypic kappa-lambda exclusion. While kappa-producing cells have their lambda chain genes in germline configuration, in most lambda-producing cells the kappa chain genes are either non-productively rearranged or deleted. To investigate the molecular mechanism for isotypic kappa-lambda exclusion, in particular the role of the Ig kappa intron enhancer, we replaced this enhancer by a neomycin resistance (neoR) gene in embryonic stem (ES) cells. B cells heterozygous for the mutation undergo V kappa-J kappa recombination exclusively in the intact Ig kappa locus but not in the mutated Ig kappa locus. Homozygous mutant mice exhibited no rearrangements in their Ig kappa loci. However, splenic B cell numbers were only slightly reduced as compared with the wild-type, and all B cells expressed lambda chain bearing surface Ig. These findings demonstrate that rearrangement in the Ig kappa locus is not essential for lambda gene rearrangement. We also generated homozygous mutant mice in which the neoR gene was inserted at the 3' end of the Ig kappa intron enhancer. Unexpectedly, mere insertion of the neoR gene showed some suppressive effect on V kappa-J kappa recombination. However, the much more pronounced inhibition of V kappa-J kappa recombination by the replacement of the Ig kappa intron enhancer suggests that this enhancer is essential for V kappa-J kappa recombination.     

Immunoglobulin mu heavy chains do not mediate tyrosine phosphorylation of Ig alpha from the ER-cis-Golgi

Signals delivered by Ig receptors guide the development of functional B lymphocytes. For example, clonal expansion of early mu heavy chain ( mu HC)-positive pre-B cells requires the assembly of a signal-competent pre-B cell receptor complex (pre-BCR) consisting of a mu HC, a surrogate L chain, and the signal dimer Ig alpha beta. However, only a small fraction of the pre-BCR is transported to the cell surface, suggesting that pre-BCR signaling initiates already from an intracellular compartment, e.g., the endoplasmic reticulum (ER). The finding that differentiation of pre-B cells and allelic exclusion at the IgH locus take place in surrogate L chain-deficient mice further supports the presence of a mu HC-mediated intracellular signal pathway. To determine whether a signal-competent Ig complex can already be assembled in the ER, we analyzed the consequence of pervanadate on tyrosine phosphorylation of Ig alpha in J558L plasmacytoma and 38B9 pre-B cells transfected with either a transport-competent IgL chain-pairing or an ER-retained nonpairing micro HC. Flow cytometry, combined Western blot-immunoprecipitation-kinase assays, and confocal microscopy revealed that both the nonpairing and pairing mu HC assembled with the Ig alpha beta dimer; however, in contrast to a pairing mu HC, the nonpairing mu HC was retained in the ER-cis-Golgi compartment, and neither colocalized with the src kinase lyn nor induced tyrosine phosphorylation of Ig alpha after pervanadate treatment of cells. On the basis of these findings, we propose that a signal-competent Ig complex consisting of mu HC, Ig alpha beta, and associated kinases is assembled in a post-ER compartment, thereby supporting the idea that a pre-BCR must be transported to the cell surface to initiate pre-BCR signaling.     

Establishment of a leukemic cell model for studying human pre-B to B cell differentiation

Reproducible models for examining early stages of human B cell differentiation are poorly developed. We now describe the establishment and characterization of a novel human leukemic cell line that recapitulates the pre-B to B cell stage of differentiation. This cell line, designated BLIN-1, was initially established in tissue culture medium containing low m.w. B cell growth factor, and consistently shows a dependency on this cytokine for optimal growth at low density. BLIN-1 cells have a 9p chromosomal abnormality, identical to the abnormality present in the leukemic blasts from the patient's original bone marrow aspirate. The immunologic phenotype of BLIN-1 is consistent with a cell arrested at the pre-B cell stage of development. Analysis of Ig gene rearrangement and Ig expression in a series of BLIN-1 subclones show that the cells spontaneously rearrange kappa light chain genes, leading to the differentiation of surface kappa-negative pre-B cells into surface kappa-positive B cells. The BLIN-1 cell line is, to our knowledge, the first defined human model for examining this critical developmental stage in human B cell ontogeny. As such, it offers a unique resource for examining variables influencing onset of kappa L chain gene rearrangement and expression.     

Enhanced transcription in a pre-B-like cell line transformed with cloned antibody genes after antigen-specific stimulation

The transducing vector, pSV2-neo, carrying the rearranged immunoglobulin (Ig) heavy (mu) and light (kappa) chain genes specific for the hapten 2,4,6-trinitrophenyl (TNP) was introduced into a pre-B cell line. The transformants expressed the TNP-specific IgM receptor on the surface. Furthermore, the addition of TNP-bovine serum albumin (hapten-carrier conjugate) to the culture media activated the expression of the transferred Ig genes and several endogenous genes such as v-abl and beta-tubulin. However, expression of the beta2-microglobulin gene was not affected. The results presented in this paper show that transfection of cloned Ig genes into B cells is a useful system for establishing monoclonal B cell lines expressing functional Ig receptor molecules with defined hapten specificity.     

Control of IgM synthesis in the murine pre-B cell line, 70Z/3'

The murine 70Z/3 tumor resembles a pre-B cell in synthesizing only intracellular mu-chains and no detectable light chain. However, one kappa gene is already rearranged, and after overnight incubation with lipopolysaccharide (LPS), most of the cells are induced to synthesize light chain. The induced cells display IgM on their surface, but do not secrete IgM. Thus, 70Z/3 cells resemble cells poised at the pre-B cell/B lymphocyte border. We have examined synthesis and post-translational modification of mu-chains in uninduced and induced 70Z/3 cells. Isolation of mu-chains and peptide maps demonstrated that both populations synthesize intracellular forms that correspond to membrane-specific mum and secretion-specific mus chains. These intracellular forms have completed only the first of the two glycosylation steps characteristic of eukaryotic cells. After induction by LPS, L chain synthesis commences, mum and mus synthesis are both increased twofold to threefold (due to an increased rate of synthesis rather than decreased degradation), and both complex with L chain to form mu2L2 tetramers. Furthermore, the glycosylation of a subset of the mum chains is completed, and these are placed on the membrane. However, unglycosylated mu2L2 tetramers can be placed on the membrane, so glycosylation is not a requirement. These data suggest that L chain may not be sufficient for externalization of mum and mus chains. These data support the idea that the controls of membrane placement and secretion of mu chains are post-translational and that different mechanisms operate for mum and mus chains.