Lambda5 is required for rearrangement of the Ig kappa light chain gene in pro-B cell lines.

Lambda5 associates with V(pre-B) to form the surrogate light (L) chain. The phenotype of lambda5 knockout mice showed severe impairment of B cell development from pro-B to immature B cell stages. To investigate the function of the surrogate L chain at this stage, we restored expression of lambda5 to lambda5-deficient pro-B cell lines which were established from bone marrow cells of lambda5 knockout mice in the presence of IL-7 and a stromal cell line. Some of these lines are severely impaired in B cell development from pro-B to immature B cell stages as is seen in vivo in lambda5 knockout mice. Restoration of lambda5 protein by retroviral-mediated gene transfer into established lambda5-deficient pro-B cell lines induced rearrangement of the Ig kappa L chain genes after removal of IL-7 from the culture. Immunoprecipitation revealed that the restored lambda5 in the cell line is coupled with V(pre-B) to form the surrogate L chain. The results demonstrate that formation of a complete surrogate L chain, consisting of both lambda5 and V(pre-B), stimulates efficient rearrangement of the kappa L chain genes.     

Independent rearrangement of Ig lambda genes in tissue culture-derived murine B cell lines

Rearrangement of the lambda light chain locus is considered a late event in pre-B cell differentiation which occurs after successful heavy chain and unsuccessful kappa light chain rearrangement. However, this view has recently been challenged by the observation of apparently independent lambda rearrangement in certain B cell lines and Ig transgenic populations. In this study we have examined the pattern of Ig rearrangements and expression in several tissue culture-derived murine B cell lines. One pre-B cell line (BDL-1) displayed germline heavy and kappa light chain genes despite the presence of a productive lambda 1 light chain rearrangement. Two other cell lines (DAC-2, BDL-2) had multiple lambda rearrangements despite the presence of productive kappa chain rearrangements. These cell lines provide new precedents for rearrangement of the lambda locus independent of the kappa locus. Their phenotype suggests that accessibility at the different Ig loci may be controlled by a non-sequential mechanism.     

Partial block in B lymphocyte development at the transition into the pre-B cell receptor stage in Vpre-B1-deficient mice

The surrogate light chain (SL) is composed of two polypeptides, Vpre-B and lambda5. In large pre-BII cells the SL chain associates with Ig mu heavy chain (muH) to form the pre-B cell receptor (pre-BCR). In mice there are two Vpre-B genes which are 98% identical within the coding regions. The two genes are co-expressed at the RNA level and encode functional proteins that can assemble with lambda5. However, it is not known whether both gene products serve the same function in vivo. Here we have established mice that lack the Vpre-B1 gene (VpreB1(-/-)), but still express the Vpre-B2 gene, both as RNA and protein. In Vpre-B1(-/-) mice, the bone marrow cellularity and the percentage of B220+ cells is normal. However, among the B220+ cells, the percentage of pre-BI cells is increased, and the percentage of pre-BII and immature B cells is slightly decreased, suggesting that the lack of Vpre-B1 causes a partial block at the transition from pre-BI to pre-BII cells, i.e. into the pre-BCR stage. The number of cells that produce a functional pre-BCR is thus lower, but the cells that reach this stage are normal as they can be expanded by proliferation and then differentiate into more mature cells. The spleens of Vpre-B1 homozygous mutant mice show normal numbers of B and T lymphocytes. Moreover, the Ig loci are allelicly excluded and the homozygous mutant mice respond with normal levels of antigen-specific antibodies to T-dependent antigens. These results demonstrate that VpreB2 alone is capable of supporting B lymphocyte development in the bone marrow and can give rise to immuno-competent cells in the periphery.     

The pre-B cell receptor and its function during B cell development

The process of B cell development in the bone marrow occurs by the stepwise rearrangements of the V, D, and J segments of the Ig H and L chain gene loci. During early B cell genesis, productive Ig H chain gene rearrangement leads to assembly of the pre-B cell receptor (pre-BCR), which acts as an important checkpoint at the pro-B/preB transitional stage. The pre-BCR, transiently expressed by developing precursor B cells, comprises the Ig muH chain, surrogate light (SL) chains VpreB and lambda5, as well as the signal-transducing heterodimer Ig alpha/Ig beta. Signaling through the pre-BCR regulates allelic exclusion at the Ig H locus, stimulates cell proliferation, and induces differentiation to small post-mitotic pre-B cells that further undergo the rearrangement of the Ig L chain genes. Recent advances in elucidating the key roles of pre-BCR in B cell development have provided a better understanding of normal B lymphopoiesis and its dysregulated state leading to B cell neoplasia.     

Mu heavy chains can associate with a pseudo-light chain complex (psi L) in human pre-B cell lines

In pre-B cells, the earliest identifiable stage of B cell differentiation, there is an asynchrony of immunoglobulin chain expression in that mu heavy chains are synthesized in the absence of light chain synthesis. These mu chains largely remain intracellular and are degraded. Here we demonstrate that a fraction of mu chains in human pre-B cell lines can reach the surface in association with three pre-B-specific proteins with relative molecular masses of 22, 18, and 16 kd, which we term collectively the pseudo-light chain complex, psi L. This association generates a multimeric complex, mu 2-psi L. Two of the psi L proteins (22 and 16 kd) are lambda-immunoreactive and form disulfide bonds with mu chains, suggesting that they are closely related to conventional lambda light chains. The 18 kd psi L species is a non-covalently-associated member of the complex. The expression of mu-psi L complexes on the surface of pre-B cells could have a functional role in the control of pre-B growth and differentiation by the hematopoietic microenvironment.     

Transformation of B and non-B cell lines with the 2,4,6,-trinitrophenyl (TNP)-specific immunoglobulin genes

The rearranged mu and kappa genes from the 2,4,6-trinitrophenyl (TNP)-specific hybridoma Sp6 have been introduced into B cells from three different stages of differentiation as well as 5 non-B cell lines to determine the levels and modes of immunoglobulin (Ig) gene expression. In pre-B cells transformed with the mu and kappa genes, low levels of Sp6-specific mu RNA were produced and approximately 210-fold less mu and 800-fold less kappa proteins were produced than in the hybridoma Sp6. The Ig proteins were present intracellularly, but were not detected on the cell membrane. In mature surface sIg+ B cell transformants, higher levels of mu Sp6 and kappa Sp6 proteins and RNA were produced than in the pre-B cell transformants (12 X mu, 70 X kappa). These transformants displayed the mu Sp6 and kappa Sp6 proteins on the cell membrane and also secreted the transfected Ig product. Plasma cell transformants produced the highest amounts of mu Sp6 and kappa Sp6 proteins. These transformants secreted pentameric IgM but did not display detectable amounts of these proteins on the cell membrane. T cell and one fibroblast transformant produced Ig as normal sized mu Sp6 and kappa Sp6 proteins. All other mu Sp6 and kappa Sp6 non-B cell transformants (melanoma, teratoma and macrophage) failed to produce enough Ig to determine whether the Ig proteins were of the correct molecular weights. The T cell and fibroblast transformants that produced Ig proteins did not secrete or display detectable Ig on the cell membrane. The expression of Ig did not inhibit the expression of the T cell antigen Thy-1 in the T cell transformants.     

Re-evaluation of the probabilities for productive rearrangements on the {kappa} and{lambda}loci

V-J rearrangements at Ig light chain (IgL) genes occur in restingsmall pre-B cells. In the absence of cell division, the probabilityof productive and rearrangements is proportional to the outputof ⁺ B and ⁺ B cells in bone marrow. The kinetics and probabilityof productive or rearrangements was assessed in three groupsof mice carrying two (wild-type), one or no intact Ig gene,and the following conclusion are drawn, and rearrangementsoccur independently at different kinetics, and rearrangementsare initiated at a time when rearrangements are stopping. Theprobability of productive and rearrangements per chromosomeis calculated to be –60 and –20% respectively. Thus,a gene can attempt rearrangements up to three times per chromosomeduring B cell development. These findings explain that the observedratio of ⁺ B/⁺ B cell production in wild-type mice is 95/5.     

The immunoglobulin light chain related protein lambda 5 is expressed on the surface of mouse pre-B cell lines and can function as a signal transducing molecule.

Abelson Leukemia Virus-transformed mouse cell lines with an early pre-B phenotype carry partially rearranged or unrearranged Ig-H genes and consequently do not express intact IgM-H protein (mu protein). Such early mu- pre-B cells express an intracellular protein complex of the pre-B cell specific 22 kDa protein lambda 5 and a 16 kDa protein designated p16. Late pre-B cell lines which carry a rearranged IgM-H chain gene in which a continuous translational reading frame has been established in the fused V-D-J element express intact mu-protein, which forms an intracellular complex with lambda 5 and p16. We show here that both the lambda 5/p16 or the mu/lambda 5/p16 complexes can be immunoprecipitated from lysates of cells surface labeled with 125I. Thus early pre-B cells express the lambda 5/p16 complex on the cell surface in the absence of mu protein, while mu+ late pre-B cells express a surface mu/lambda 5/p16 complex. To investigate a possible signal transduction function of the lambda 5/p16 and mu/lambda 5/p16 complexes on the surface of pre-B cell lines we measured the changes in intracellular free Ca2+ after treatment of cells with anti-lambda 5 or anti-mu antibodies. Two mu- early pre-B cell lines showed a rapid and transient increase in intracellular free Ca2+ when incubated with anti-lambda 5 antibodies but not when incubated with anti-mu, while the mu+ late pre-B cell line CB32 showed a rapid and transient increase in intracellular Ca2+ after incubation with anti-lambda 5 or anti-mu. These results show that both the lambda 5/p16 and the mu/lambda 5/p16 cell surface protein complexes can transduce an external signal to the inside of the cell, which implicates these complexes in the regulation of pre-B cell physiology.     

Only VpreB1, but not VpreB2, is expressed at levels which allow normal development of B cells

The surrogate light chain (SLC) consists of the polypeptides lambda5 and, in the mouse, either VpreB1 or VpreB2. SLC associates with BILL-Cadherin and other glycoproteins to form the pro-B cell receptor (pro-BCR) at the pre-BI cell stage, and with the immunoglobulin mu heavy chain to form the pre-BCR at the pre-BII cell stage. The function of the pro-BCR, if any, is unknown, whereas the pre-BCR is crucial for proliferative expansion of pre-BII cells. To shed light on the functional properties of VpreB1 and VpreB2 in vivo, mice with either one or two VpreB1, or one or two VpreB2, alleles have been investigated. We show that B cell development in mice with two VpreB1 alleles is indistinguishable from that of normal mice. In contrast, mice with two VpreB2 alleles show an approximately 1.6-fold increase in pre-BI and a 35% decrease in pre-BII cell numbers, while mice with only one VpreB2 allele show a reduction in B cell development manifested in a 2-fold enrichment in pre-BI cells and a 75% reduction in pre-BII cells. However, such a gene dosage effect is not observed for VpreB1. Our results suggest that the difference between VpreB1- and VpreB2-deficient mice is due to lower VpreB2 protein expression, thus limiting the formation of pre-BCRs and thereby the number of large, cycling pre-BII cells.     

Regulation of immunoglobulin light chain gene rearrangements during early B cell development in the human.

Southern blot analyses of immunoglobulin light chain gene rearrangements in human leukemias and myelomas indicated that lambda loci in kappa-producing cells are largely unrearranged while kappa loci in lambda producers are often rearranged and inactivated by rearrangements of the kappa-deleting element (KDE). For a systematic analysis of the regulation of light chain rearrangements during early B cell development in normal human B cells also considering functionality of the rearrangements, we used FACS-sorted single naive kappa- and lambda-expressing B cells from peripheral blood of healthy humans. V(kappa)J(kappa) and V(lambda)J(lambda) joints and rearrangements involving the KDE were amplified simultaneously from single cells and sequenced. Whereas only 2 - 3 % of kappa-expressing cells carry V(lambda)J(lambda) joints, nearly all lambda-expressing cells have rearranged kappa loci and indeed carry V(kappa)J(kappa) joints. The V(kappa)J(kappa) joints in lambda-expressing cells exhibit preferential J(kappa)4 and J(kappa)5 over J(kappa)1 and J(kappa)2 usage compared to kappa-expressing cells. Thirty percent of the V(kappa)J(kappa) joints in lambda producers are rearranged in-frame. These data indicate extensive sequential V(kappa)-J(kappa) rearrangements and inactivation of functional V(kappa)J(kappa) joints in lambda-expressing cells, presumably before V(lambda)J(lambda) joining.     

Endogenous immunoglobulin expression in mu transgenic mice

Transgenic mice (M54) containing a functional mu heavy chain were examined to determine the effects of the transgene on rearrangement and expression of endogenous immunoglobulin genes. Two major novel findings are presented. (i) In transgenic mice, the expressed endogenous VH repertoire in LPS-generated B cell blasts and hybridomas is skewed toward expression of JH-proximal VH families (VH7183 and Q52). (ii) There is an increase in the frequency of B cells expressing lambda light chain genes in transgenic mice. Furthermore, in Abelson-MuLV transformed pre-B cells, VH to DJH is inhibited more than the D to JH rearrangement. The results presented indicate that the transgene skews the expressed VH repertoire by inhibiting the VH to DJH rearrangement while permitting an expansion of B cells expressing limited VH and lambda light chain genes.     

Heparin alters the expression of different forms of immunoglobulin mu heavy chains and their associated proteins by pre-B cell lines and normal Ly-1 (CD5+) B cells.

Studies presented here show that heparin alters immunoglobulin expression by murine pre-B cell lines and normal Ly-1 (CD5+) B cells. Previous studies have shown that pre-B cell lines 70Z/3 and NFS-5.3 express mu heavy chains in the cytoplasm and a small amount on the cell surface. Both these cytoplasmic and surface mu are disulfide-linked to omega (lambda 5) surrogate light chains and are noncovalently associated with iota (Vpre-B) variable region-like proteins. We show that culturing 70Z/3 with heparin reduces the amount of the membrane-form mu (micron) on the cell surface. Culturing NFS-5.3 with heparin similarly decreases the membrane-form mu; however, it increases the surface level of a pentameric mu molecule containing secreted-form mu (microS) heavy chains, disulfide-linked omega (lambda 5) chains, and noncovalently associated proteins. Culturing peritoneal B cells with heparin also increases the production of the secreted-form microS, detectable in this case by the secretion of classical pentameric IgM. Similarly, injecting heparin intraperitoneally increases IgM secretion by peritoneal Ly-1 B cells. Thus heparin could influence pre-B cell and B cell differentiation and function.     

Predominant immunoglobulin gene rearrangements in two patients with immunodeficiency: restricted use of V gene segments and DNA hypermethylation

The majority of common variable immunodeficiencies (CVID) is caused by intrinsic B cell defects which impede distinct stages of B cell differentiation. B cell differentiation is accompanied by the rearrangement of immunoglobulin (Ig) genes. The first step in the rearrangement process is the assembly of IgH genes, and subsequently, IgL genes are rearranged. During B cell maturation, Ig genes are demethylated in a stepwise, locus-specific manner. Here, we examined the Ig gene rearrangements of four patients with classical CVID and of one child suffering from an unusual immunodeficiency associated with CD5+ B cell lymphocytosis. In one of the four adult patients with CVID, we observed a predominant type of VHDJH-gene rearrangement. In the child, different polyclonal VHDJH-gene rearrangements were found together with a predominant type of kappa light chain gene rearrangement. The rearranged kappa chain genes were methylated (as in the pre-B cell stage). These findings together with the cell phenotype analysis and the clinical course of the disease in the child suggests that in some patients with primary immunodeficiency a maturation arrest may occur in B cells leading to a predominant Ig V gene rearrangement.     

The kappa/lambda ratio in surface immunoglobulin molecules on B lymphocytes differentiating from DHJH-rearranged murine pre-B cell clones in vitro

The expression of kappa and lambda light chains in surface immunoglobulin (sIg) molecules on B lymphocytes differentiating from murine pre-B cell clones in vitro was analyzed. The four pre-B cell clones used represent a very early pre-B cell stage. They have their heavy chain loci DJ rearranged and their light chain loci in germ-line configuration. In order to grow in vitro, these clones require contact with stromal cells and the stimulatory activity of interleukin (IL) 7. Upon removal of IL 7 from the cultures, these clones differentiate within 3 days into sIg+ B cells. Between 7% and 12% of IgM+ B cells could be detected in these cultures. The majority (78%-92%) of the IgM+ B cells co-expressed kappa light chains. The percentage of lambda light chain expressing B cells was below detectable level. Upon lipopolysaccharide (LPS) stimulation, the percentages of IgM+ B cells increased dramatically (from 32%-64%). The majority (91%-97%) of the IgM+ B cells express kappa chains, but a very small percentage (3.1%-5.0%) express lambda. A similarly high kappa/lambda ratio was found in 418 hybridomas prepared from these LPS-stimulated B cells (388 kappa+ and 30 lambda+). Thus, the high kappa/lambda ratio characteristic of the mouse peripheral B cell repertoire is already evident in the antigen-independent transition from pre-B to B cells.