The scid defect affects the final step of the immunoglobulin VDJ recombinase mechanism

Abelson murine leukemia virus-transformed precursor B lymphocytes from scid (severe combined immunodeficient) mice, like A-MuLV transformants from normal mice, actively rearrange segments of their Ig heavy chain variable region gene locus during growth in culture. Targeting of recombination to appropriate segments appears normal in these lines as evidenced by initial rearrangement of sequences from within the D and JH locus to form aberrant "DJH" rearrangements and secondary rearrangement of sequences from within the VH locus to the aberrant "DJH" intermediates. A detailed analysis of the joints in these rearrangements indicates that the VDJ recombinase in scid pre-B cells can correctly recognize heptamernonamer signal sequences and perform precise endonucleolytic scissions at these sequences. We propose that the scid defect involves the inability of scid precursor lymphocytes to join correctly the cleaved ends of the coding strands of variable region gene segments.     

Wild-type V(D)J recombination in scid pre-B cells.

Homozygous mutation at the scid locus in the mouse results in the aberrant rearrangement of immunoglobulin and T-cell receptor gene segments. We introduced a retroviral vector containing an inversional immunoglobulin rearrangement cassette into scid pre-B cells. Most rearrangements were accompanied by large deletions, consistent with previously characterized effects of the scid mutation. However, two cell clones were identified which contained perfect reciprocal fragments and wild-type coding joints, documenting, on a molecular level, the ability of scid pre-B cells to generate functional protein-coding domains. Subsequent rearrangement of the DGR cassette in one of these clones was accompanied by a deletion, suggesting that this cell clone had not reverted the scid mutation. Indeed, induced rearrangement of the endogenous kappa loci in these two cell clones resulted in a mixture of scid and wild-type V-J kappa joints, as assayed by a polymerase chain reaction and DNA sequencing. In addition, three immunoglobulin mu- scid pre-B cell lines showed both scid and wild-type V-J kappa joins. These experiments strongly suggest that the V(D)J recombinase activity in scid lymphoid cells is diminished but not absent, consistent with the known leakiness of the scid mutation.     

Differentiation of cloned populations of immature B cells after transformation with Abelson murine leukemia virus

The nature of the target cell for Abelson virus transformation and the effect of transformation on B cell differentiation were studied with six cloned lines of nontransformed immature B lymphocytes. Three clones were at the pre-B cell stage of maturation prior to A-MuLV infection; two were at the B cell stage, and one appeared to represent a stage prior to rearrangement of the mu heavy chain gene. All six cloned lines could be transformed by Abelson virus. Many of the transformants of the pre-B cell clones underwent kappa light chain gene rearrangement and expression following viral infection. Distinct light chain gene rearrangements were segregated by further subcloning these transformed lines. Abelson virus infection of one cloned cell line believed to represent a stage of maturation prior to the pre-B cell stage produced pre-B cell transformants with a variety of heavy chain gene rearrangements. Thus B lymphoid target cells for Abelson virus are not restricted to a single developmental stage, and some transformed subclones can undergo extensive immunoglobulin gene rearrangements shortly after viral infection.     

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.     

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.     

Abnormal recombination of Igh D and J gene segments in transformed pre-B cells of scid mice

Studies of Ig and TCR genes in transformed lymphocytes of scid mice have revealed aberrant DNA rearrangements. Here we present a more detailed analysis of the Igh gene recombination in nine scid pre-B cell lines transformed by Abelson murine leukemia virus. We found 85% of the rearranged Igh alleles to contain abnormal Dh-Jh deletions of varying size. All of these deletions encompassed Jh elements and extended into the Igh enhancer region, occasionally involving the switch (S) region of the C mu gene. Some of these rearrangements removed most of the Dh elements, but none appeared to extend to the Vh genes. DNA sequence analysis of the two abnormally rearranged Igh alleles in one pre-B cell line showed that no Dh or Jh coding sequences were retained at the recombination sites though heptamer-like (CACTGTG) recognition signal sequences were present in the absence of nonamer (GGTTTTTGT) recognition signal sequences. These results imply that a deregulated recombinase activity may be responsible for the abnormal Dh-Jh deletions and the absence of Vh-Dh joining in established lines of Abelson murine leukemia virus-transformed scid pre-B cells.     

VHDJH formation and DJH replacement during pre-B differentiation: non-random usage of gene segments.

The Abelson murine leukemia virus (A-MuLV) transformed cell line 300-19 was derived from the bone marrow of an adult NIH/Swiss outbred mouse. The original 300-19 clonal isolate carried DHH rearrangements of both JH alleles, a molecular genotype characteristic of early pre-B cells. During propagation in culture, the 300-19 line frequently generates secondary rearrangements of its JH alleles including rearrangements which append VH segments to the pre-existing DJH complexes to form complete VHDJH variable region genes and secondary D to JH rearrangements which replace the pre-existing DJH rearrangement by joining an upstream D to a downstream JH. The two types of secondary rearrangement events occur at approximately equal frequency. Approximately 30% of the VH to DJH joins lead to the production of mu heavy chains providing support for a regulated model of allelic exclusion. Like pre-B cell lines from other origins, the 300-19 line preferentially utilized VH gene segments from the more JH-proximal (3') families to form VHDJH rearrangements. However, the VH segments preferentially employed by 300-19 were from a different family than those previously demonstrated to be utilized by pre-B lines of BALB/c origin; we relate these different utilization patterns to differences in the organization of the more 3' VH families between the two strains. The initial DJH rearrangements of the 300-19 line employed more 3' (JH-proximal) D segments; however, the DJH replacements preferentially employed the most 5' D segment. We discuss this phenomenon in the context of a mechanism which may target recombinase to regions of the chromosome more 5' to the D locus (VH-containing regions) once an initial DJH complex is formed.     

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.     

Murine lambda gene rearrangements: the stochastic model prevails over the ordered model.

The ontogeny of the immunoglobulin (Ig) gene rearrangement in mammalian B cells seems to be ordered. Heavy chain gene segments rearrange first, followed by light chain gene segments, kappa before lambda. The genomic organization of murine lambda locus does not preclude the simultaneous expression of two subtypes from the same chromosome. In order to distinguish between an ordered and a stochastic model of rearrangement, a panel of 67 B cell hybridomas secreting either lambda 1, lambda 2, lambda 3 or lambda x (recently described) were analysed for V lambda J lambda rearrangements. The results show that in 97% of cases, a single rearrangement occurred, favouring the stochastic model over the ordered one. Strikingly, the possibility of having a productive rearrangement if the first try results in an aberrant one is rare. We propose therefore, that the lambda Ig is not necessarily required to ensure allelic and subtypic exclusion mechanisms. Moreover, in 97% of the cases, at least one kappa allele is rearranged. Furthermore, the RS recombination has been detected in 77% of the cases. This suggests that, although the stimulation of kappa precedes that of lambda locus, the RS recombination acts as a transacting albeit dispensable lambda activator.     

Induced kappa receptor editing shows no allelic preference in a mouse pre-B cell line

B cell Ag receptor editing is a process that can change kappa antigen recognition specificity of a B cell receptor through secondary gene rearrangements on the same allele. In this study we used a model mouse pre-B cell line (38B9) to examine factors that might affect allelic targeting of secondary rearrangements of the kappa locus. We isolated clones that showed both productive and nonproductive rearrangements of one kappa allele, while retaining the other kappa allele in the germline configuration (kappa(+)/kappa degrees or kappa(-)/kappa degrees ). In the absence of any selective pressures, subsequent rearrangement of the germline alleles occurred at the same frequency as secondary rearrangement of the productive or nonproductive rearranged alleles. Because 38B9 cells lack Ig heavy chains, we stably expressed mu heavy chain protein in 38B9 cells to determine whether heavy-light pairing might affect allelic targeting of secondary kappa rearrangements. Although the expression of heavy chain was found to both pair with and stabilize kappa protein in these cells, it had no effect on preferential targeting Vkappa-Jkappa receptor editing compared with rearrangement of a germline allele. These studies suggest that in the absence of selection to eliminate autoreactive Vkappa-Jkappa genes, there is no allelic preference for secondary rearrangement events in 38B9 cells.     

Molecular requirements for the mu-induced light chain gene rearrangement in pre-B cells.

During B cell differentiation rearrangement of immunoglobulin (Ig) genes is partially regulated by the Ig proteins. Rearrangement of heavy (H) chain genes is inhibited, whilst that of light (L) chain genes is induced by the membrane form of the mu H chain. In order to analyse additional structural requirements of mu induced L chain gene rearrangement we transfected wild-type mu and mutant mu constructs lacking functional exons encoding the first or second constant domains into Abelson murine leukemia virus (AMuLV) transformed pre-B cells. All mu chains are expressed on the surface of the pre-B cell and all associate with omega and iota, two proteins forming a surrogate light chain, necessary for mu membrane expression. Nevertheless, only wild-type mu and not the mutant mu proteins promote L gene rearrangement. A heterodimer of proteins with Mr of 33 kd and 36 kd was found associated with wild-type but not with the mutant mu proteins. Continuous presence of mu is required for L chain gene recombination since loss of mu stopped and readdition of mu started L gene rearrangement. We propose that the protein complex composed of mu and the 33 kd/36 kd protein heterodimer is responsible for the activation of the L chain gene locus and its rearrangement.     

Immunoglobulin synthesis and gene rearrangements in lymphoid cells transformed by replication-competent Rauscher murine leukemia virus: transformation of B cells at various stages of differentiation

Lymphoid cells transformed by Rauscher murine leukemia virus (R-MuLV) belonged to the B cell lineages. One group of cells exhibited Fc receptors but completely lacked immunoglobulin mu heavy and kappa light chains. The majority of the cells resemble pre-B type. They displayed mu chains but kappa chains were completely absent. Very rarely certain cells synthesized both mu and kappa chains. Based on the presence of Fc receptors and IgM synthesis the cells transformed by R-MuLV belonged to three B cell developmental stages. These cells were tested for immunoglobulin gene rearrangements using JH and CK probes. DNA from cell lines without any detectable levels of IgM mu exhibited embryonic as well as rearranged JH genes, whereas cells expressing IgM possess, in addition, productive and non-productive light chain gene rearrangements. The most terminally differentiated cell possesses JH and CK rearrangement associated with the synthesis of mu and kappa chains. Presumably the cells with rearranged JH and CK genes without immunoglobulin synthesis represent a developmental transition. We conclude that cells transformed by R-MuLV belonged to five step-wise compartments of B cell development. Our findings implicate definite sequential events of immunoglobulin gene rearrangement and expression during B cell development.     

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.     

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.     

Cloning and sequencing of immunoglobulin variable-region genes using degenerate oligodeoxyribonucleotides and polymerase chain reaction

A procedure is described for using the polymerase chain reaction (PCR) to amplify and clone the cDNA from mouse immunoglobulin (Ig) variable (V) regions. This method uses a set of universal 5'-oligodeoxyribonucleotide primers that are degenerate and allow for the amplification of Ig V-region sequences from gamma and mu heavy chains and from kappa light chains. Selective first-strand cDNA synthesis is performed using Ig constant region primers and then a PCR is achieved by using the appropriate universal 5'-primer. The universal Ig heavy-chain primer was used to amplify the V-region cDNA from gamma and mu isotypes and the universal light-chain primer was used to amplify three separate kappa light V-region sequences. This procedure was used to obtain Ig V-region gene sequences from hybridomas secreting IgG1/kappa, IgG2b/kappa and IgM/kappa isotypes.     

Characterization of B lymphocyte lineage progenitor cells from mice with severe combined immune deficiency disease (SCID) made possible by long term culture

A single gene mutation results in near absence of B and T lymphocytes and their immediate progenitors in mice with severe combined immunodeficiency disease (SCID). However, long term culture conditions allowed rapid outgrowth of lymphocytes from SCID bone marrow suspensions, and this permitted their detailed analysis. The cells were judged to be committed to the B lymphocyte lineage on the basis of expression of the BP-1 antigen, as well as by the density and pattern of expression of other markers. Cultured SCID lymphocytes were indistinguishable from control BALB/c cells in terms of morphology, typing for 13 cell surface markers, and changes in cell surface antigen expression with time in culture. In contrast to cultures of normal cells, which always included IgM synthesizing cells, SCID lymphocytes rarely expressed mu heavy chains. Southern blot analysis demonstrated that at least the first Ig gene rearrangement step had occurred in most of the cultured cells. The patterns of JH gene rearrangements suggested that relatively limited population diversity existed in individual cultures of SCID and normal BALB/c marrow. In addition, there was evidence that abnormal Ig heavy chain gene rearrangements had taken place in lymphocytes from approximately 25% of the SCID cultures. These cells were distinguished by the absence of detectable JH gene segments. kappa light chain genes appeared to be unrearranged in SCID cultured lymphocytes. We conclude that the lymphopoietic microenvironments of SCID mice are probably normal, and the animals have infrequent progenitors of B cells. Aberrant or nonproductive IgH gene rearrangements may account for the absence of pre-B and B cells in SCID mice. This study demonstrates the usefulness of long term culture methodology for isolating rare subsets of non-transformed lymphoid cells from normal and genetically defective hemopoietic tissues.     

A second gene, VpreB in the lambda 5 locus of the mouse, which appears to be selectively expressed in pre-B lymphocytes.

The murine gene lambda 5 is selectively expressed in pre-B lymphocytes. Of the three exons encoding lambda 5, exons II and III show strong homologies to immunoglobulin lambda light (L) chain gene segments, i.e. to J lambda intron and exon, and C lambda exon sequences respectively. We have now found, 4.6 kb upstream of lambda 5, another gene composed of two exons which is selectively expressed in pre-B cell lines as a 0.85 kb mRNA potentially coding for a protein of 142 amino acids including a 19 amino acid-long signal peptide. The 5' sequences of this gene show homologies to sequences encoding the variable regions of kappa and lambda L chains and of heavy (H) chains. The deduced amino acid sequence contains the consensus cysteine residues as well as other consensus amino acids at positions which characterize immunoglobulin (Ig) domains. We call the second gene VpreB. The 3' end of VpreB encoding the 26 carboxyl terminal amino acids shows no homology to any known nucleotide sequence. The putative protein encoded by VpreB is a potential candidate for association with the putative protein encoded by lambda 5, and thereby a candidate for association with H chains in pre-B cells. Southern blot analysis of DNA from liver (germ line) and 70Z/3 pre-B cell lines reveals two genes which hybridize to the VpreB gene. We call VpreB1 the gene which is found 5' of lambda 5. The other gene, called VpreB2, which has not yet been located within the genome, shows 97% nucleotide sequence homology to VpreB1 in an area of 1 kb which covers the coding region of the gene.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.