Secondary V(D)J rearrangements and B cell receptor-mediated down-regulation of recombination activating gene-2 expression in a murine B cell line

It has recently become clear that recombination of Ig genes is not restricted to B cell precursors but that secondary rearrangements can also occur under certain conditions in phenotypically immature bone marrow and peripheral B cells. However, the nature of these cells and the regulation of secondary V(D)J recombination in response to B cell receptor (BCR) stimulation remain controversial. In the present study, we have analyzed secondary light chain gene rearrangements and recombination activating gene (RAG) expression in the surface IgM+, IgD- murine B cell line, 38C-13, which has previously been found to undergo kappa light chain replacement. We find that 38C-13 cells undergo spontaneous secondary Vkappa-Jkappa and RS rearrangements in culture, with recombination occurring on both productive and nonproductive alleles. Both 38C-13 cells and the Id-negative variants express the RAG genes, indicating that the presence of RAG does not depend on activation via the 38C-13 BCR. Moreover, BCR cross-linking in 38C-13 cells leads to a rapid and reversible down-regulation of RAG2 mRNA. Therefore, 38C-13 cells resemble peripheral IgM+, IgD- B cells undergoing light chain gene rearrangement and provide a possible in vitro model for studying peripheral V(D)J recombination.     

Models for antigen receptor gene rearrangement. I. Biased receptor editing in B cells: implications for allelic exclusion.

Recent evidence suggests that lymphocyte Ag receptor gene rearrangement does not always stop after the expression of the first productively rearranged receptor. Light chain gene rearrangement in B cells, and alpha-chain rearrangement in T cells can continue, which raises the question: how is allelic exclusion maintained, if at all, in the face of continued rearrangement? In this and the accompanying paper, we present comprehensive models of Ag receptor gene rearrangement and the interaction of this process with clonal selection. Our B cell model enables us to reconcile observations on the kappa:lambda ratio and on kappa allele usage, showing that B cell receptor gene rearrangement must be a highly ordered, rather than a random, process. We show that order is exhibited on three levels: a preference for rearranging kappa rather than lambda light chain genes; a preference to make secondary rearrangements on the allele that has already been rearranged, rather than choosing the location of the next rearrangement at random; and a sequentiality of J segment choice within each kappa allele. This order, combined with the stringency of negative selection, is shown to lead to effective allelic exclusion.     

Models for antigen receptor gene rearrangement. II. Multiple rearrangement in the TCR: allelic exclusion or inclusion?

This series of papers addresses the effects of continuous Ag receptor gene rearrangement in lymphocytes on allelic exclusion. The previous paper discussed light chain gene rearrangement and receptor editing in B cells, and showed that these processes are ordered on three different levels. This order, combined with the constraints imposed by a strong negative selection, was shown to lead to effective allelic exclusion. In the present paper, we discuss rearrangement of TCR genes. In the TCR alpha-chain, allelic inclusion may be the rule rather than the exception. Several previous models, which attempted to explain experimental observations, such as the fractions of cells containing two productive TCRalpha rearrangements, did not sufficiently account for TCR gene organization, which limits secondary rearrangement, and for the effects of subsequent thymic selection. We present here a detailed, comprehensive computer simulation of TCR gene rearrangement, incorporating the interaction of this process with other aspects of lymphocyte development, including cell division, selection, cell death, and maturation. Our model shows how the observed fraction of T cells containing productive TCRalpha rearrangements on both alleles can be explained by the parameters of thymic selection imposed over a random rearrangement process.     

Models for antigen receptor gene rearrangement. III. Heavy and light chain allelic exclusion

The extent of allelic exclusion in Ig genes is very high, although not absolute. Thus far, it has not been clearly established whether rapid selection of the developing B cell as soon as it has achieved the first productively rearranged, functional heavy chain is the only mechanism responsible for allelic exclusion. Our computational models of Ag receptor gene rearrangement in B lymphocytes are hereby extended to calculate the expected fractions of heavy chain allelically included newly generated B cells as a function of the probability of heavy chain pairing with the surrogate light chain, and the probability that the cell would test this pairing immediately after the first rearrangement. The expected fractions for most values of these probabilities significantly exceed the levels of allelic inclusion in peripheral B cells, implying that in most cases productive rearrangement and subsequent cell surface expression of one allele of the heavy chain gene probably leads to prevention of rearrangement completion on the other allele, and that additional mechanisms, such as peripheral selection disfavoring cells with two productively rearranged heavy chain genes, may also play a role. Furthermore, we revisit light chain allelic exclusion by utilizing the first (to our knowledge) computational model which addresses and enumerates B cells maturing with two productively rearranged kappa light chain genes. We show that, assuming that there are no selection mechanisms responsible for abolishing cells expressing two light chains, the repertoire of newly generated B lymphocytes exiting the bone marrow must contain a significant fraction of such kappa double-productive B cells.     

Corrective recombination of mouse immunoglobulin kappa alleles in Abelson murine leukemia virus-transformed pre-B cells.

Previous characterization of mouse immunoglobulin kappa gene rearrangement products cloned from murine plasmacytomas has indicated that two recombination events can take place on a single kappa allele (R. M. Feddersen and B. G. Van Ness, Proc. Natl. Acad. Sci. USA 82:4792-4797, 1985; M. A. Shapiro and M. Weigert, J. Immunol. 139:3834-3839, 1987). To determine whether multiple recombinations on a single kappa allele can contribute to the formation of productive V-J genes through corrective recombinations, we have examined several Abelson murine leukemia virus-transformed pre-B-cell clones which rearrange the kappa locus during cell culture. Clonal cell lines which had rearranged one kappa allele nonproductively while maintaining the other allele in the germ line configuration were grown, and secondary subclones, which subsequently expressed kappa protein, were isolated and examined for further kappa rearrangement. A full spectrum of rearrangement patterns was observed in this sequential cloning, including productive and nonproductive recombinations of the germ line allele and secondary recombinations of the nonproductive allele. The results show that corrective V-J recombinations, with displacement of the nonproductive kappa gene, occur with a significant frequency (6 of 17 kappa-producing subclones). Both deletion and maintenance of the primary (nonfunctional) V-J join, as a reciprocal product, were observed.     

A shared kappa reciprocal fragment and a high frequency of secondary Jk5 rearrangements among influenza hemagglutinin specific B cell hybridomas

Ig kappa-chain gene rearrangement results in the displacement or loss of the DNA immediately 5' of Jk. This retained DNA is found on a different size fragment than in the germline (a reciprocal fragment), and contains the reciprocal joint of rearranged Vk and Jk genes, the back-to-back fusion of the heptamer/nonamer recombination signals. B cells of independent origin rarely have reciprocal fragments of the same size. However, we report that 9 of 15 B cell hybridomas of independent origin have reciprocal fragments of the same size (8-kb BamHI fragments) unrelated to their productive rearrangements. An 8-kb reciprocal fragment has also occurred on about 25% of the kappa alleles of normal splenic B cells. We find that the reciprocal fragments in two of these hybridomas contain the reciprocal joints of Jk1 genes and different Vk8 genes. In addition, we find that at least 8 of the 12 Jk4 or Jk5 expressing hybridomas have undergone double recombinations on their productive kappa alleles. The implications of these findings on the high frequency of 8-kb reciprocal rearrangements and on Vk rearrangement are discussed.     

VH replacement rescues progenitor B cells with two nonproductive VDJ alleles

Inaccurate VDJ rearrangements generate a large number of progenitor (pro)-B cells with two nonproductive IgH alleles. Such cells lack essential survival signals mediated by surface IgM heavy chain (muH chain) expression and are normally eliminated. However, secondary rearrangements of upstream VH gene segments into assembled VDJ exons have been described in mice transgenic for productive muH chains, a process known as VH replacement. If VH replacement was independent of muH chain signals, it could also modify nonproductive VDJ exons and thus rescue pro-B cells with unsuccessful rearrangements on both alleles. To test this hypothesis, we homologously replaced the JH cluster of a mouse with a nonproductive VDJ exon. Surprisingly, B cell development in IgHVDJ-/VDJ- mice was only slightly impaired and significant numbers of IgM-positive B cells were produced. DNA sequencing confirmed that all VDJ sequences from muH chain-positive B lymphoid cells were generated by VH replacement in a RAG-dependent manner. Another unique feature of our transgenic mice was the presence of IgH chains with unusually long CDR3-H regions. Such IgH chains were functional and only modestly counter-selected, arguing against a strict length constraint for CDR3-H regions. In conclusion, VH replacement can occur in the absence of a muH chain signal and provides a potential rescue mechanism for pro-B cells with two nonproductive IgH alleles.     

Ig gene rearrangement and expression in the progeny of B-cell progenitors in the course of clonal expansion in bone marrow cultures.

In cultures of murine bone marrow cells colonies of 10(3)-10(4) cells were identified which consisted to a large part of pre-B and B cells. Cell mixing experiments with genetically marked cells indicated that each colony is derived from a single progenitor cell not yet committed to the expression of either IgH locus. A concanavalin-A-mediated electrofusion method allowed us to rescue and amplify individual cells from a given colony by hybridization with X63.Ag8.653 cells. The molecular analysis of 12 such hybridomas revealed that all IgH loci were rearranged into DJH or productive or nonproductive VHDJH complexes. Most kappa and all lambda light chain loci were in germline configuration. Kappa chain expression was only seen in heavy (mu) chain expressing hybridomas. Hybridomas from a given colony were heterogeneous in terms of DJH and VHDJH rearrangements and in no cell was more than one productive VHDJH complex detected. None of the productive VHDJH complexes contained a VH gene of group 1 (J558), the largest VH gene family with about half of the VH genes. This is in marked contrast to VH gene usage in splenic B cells.     

Ig lambda and heavy chain gene usage in early untreated systemic lupus erythematosus suggests intensive B cell stimulation.

To determine the distribution of Vlambda and Jlambda as well as VH and JH gene usage in a patient with systemic lupus erythematosus (SLE), productive and nonproductive VJ and V(D)J rearrangements were amplified from individual peripheral CD19+ B cells and were analyzed. No differences in the Vlambda and Jlambda or the VH and JH gene usage in the nonproductive gene repertoire of this SLE patient were found compared with the distribution of genes found in normal adults, whereas marked skewing of both Vlambda and VH was noted among the productive rearrangements. The distribution of productive Vlambda rearrangements was skewed, with significantly greater representation of the Jlambda distal cluster C Vlambda genes and the Vlambda distal Jlambda7 element, consistent with the possibility that there was receptor editing of the Vlambda locus in this patient. Significant bias in VH gene usage was also noted with VH3 family members dominating the peripheral B cell repertoire of the SLE patient (83%) compared with that found in normal subjects (55%; p < 0.001). Notably, a clone of B cells employing the VH3-11 gene for the heavy chain and the Vlambda1G segment for the light chain was detected. These data are most consistent with the conclusion that extreme B cell overactivity drives the initial stages of SLE leading to remarkable changes in the peripheral V gene usage that may underlie on fail to prevent the emergence of autoimmunity.     

The V lambda J lambda repertoire in human fetal spleen: evidence for positive selection and extensive receptor editing

VlambdaJlambda rearrangements obtained from genomic DNA of individual IgM(+) B cells from human fetal spleen were analyzed. A nonrandom pattern of lambda gene rearrangements that differed from the adult Vlambda repertoire was found. The Vlambda distal genes 8A and 4B were absent from the nonproductive fetal repertoire, whereas 2E and 3L were overrepresented and 1B was underrepresented in the productive fetal repertoire. Positive selection of the Vlambda gene, 2E, along with Vlambda rearrangements employing homologous VlambdaJlambda joins were observed in the fetal, but not in the adult Vlambda repertoire. Overrepresentation of Jlambda distal cluster C genes rearranging to the Vlambda distal J segment, Jlambda7, in both productive and nonproductive fetal repertoires suggested that receptor editing/replacement was more active in the fetus than in adults. Numerous identical VlambdaJlambda junctions were observed in both the productive and nonproductive repertoire of the fetus and adult, but were significantly more frequent in the productive repertoire of the fetus, suggesting expansion of B cells expressing particular lambda-light chains in both stages of development, with more profound expansion in the fetal repertoire. Notably, B cells expressing identical lambda-light chains expressed diverse heavy chains. These data demonstrate that three mechanisms strongly influence the shaping of the human fetal lambda-chain repertoire that are less evident in the adult: positive selection, receptor editing, and expansion of B cells expressing specific lambda-light chains. These events imply that the expressed fetal repertoire is shaped by exposure to self Ags.     

Predominance of nonproductive rearrangements of VH81X gene segments evidences a dependence of B cell clonal maturation on the structure of nascent H chains

Ig H chain V regions using the VH81X gene segment were PCR amplified from genomic DNA obtained from either splenic B cells or surface (s)Ig- bone marrow cells of BALB/c mice. Sequence analysis demonstrated that 93% of VH81X containing H chain V region genes in splenic B cells were rearranged nonproductively. Furthermore, 74% of rearrangements of VH81X among sIg- bone marrow cells were nonproductive. This contrasts with previous results obtained for rearrangements of members of the VH36-60 gene segment family among sIg- cells wherein, as a consequence of extensive clonal expansion after productive H chain V gene rearrangement, 80% of rearrangements were productive. The low proportion of productive rearrangements of VH81X is interpreted as indicating that most productive rearrangements of VH81X cannot facilitate clonal expansion, which would support the hypothesis that selection for clonal expansion and maturation is dependent on the amino acid sequence of nascent H chains. Additionally, because most productive rearrangements of VH81X cannot facilitate clonal maturation but do appear to mediate allelic exclusion, these processes are likely to be regulated independently.     

Kappa editing rescues autoreactive B cells destined for deletion in mice transgenic for a dual specific anti-laminin Ig

We explored mechanisms involved in B cell self-tolerance in a double- and triple-transgenic mouse model bearing the LamH-C mu Ig H chain conventional transgene and a gene-targeted replacement for a functional V kappa 8J kappa 5 L chain gene. Whereas the H chain is known to generate anti-laminin Ig in combination with multiple L chains, the H + L Ig binds ssDNA in addition to laminin. Immune phenotyping indicates that H + L transgenic B cells are regulated by clonal deletion, receptor editing via secondary rearrangements at the nontargeted kappa allele, and anergy. Collectively, the data suggest that multiple receptor-tolerogen interactions regulate autoreactive cells in the H + L double-transgenic mice. Generation of H + LL triple-transgenic mice homozygous for the targeted L chain to exclude secondary kappa rearrangements resulted in profound B cell depletion with absence of mature B cells in the bone marrow. We propose that the primary tolerogen of dual reactive B cells in this model is not ssDNA, but a strongly cross-linking tolerogen, presumably basement membrane laminin, that triggers recombination-activating gene activity, L chain editing, and deletion.     

Simultaneously arising Ly-1(CD5) B cell lymphomas have identical expressed IgH and kappa-genes but different nonproductive heavy chain rearrangements.

A group of CD5(Ly-1) B cell lymphomas are described. They were derived from mice which received a common pool of syngeneic mouse spleen cells. Southern blot analysis revealed that the lymphomas exhibited an unusual set of Ig gene rearrangements. Six lymphomas analyzed had either of two rearrangement patterns. EcoRI restriction digests of tumor DNA probed for rearrangements in the JH region, resulted in restriction fragments of 4.7 and 5.6 kb or of 4.7 and 8.5 kb. Each had an identical HindIII restriction fragment identified when probed for kappa gene rearrangements. Inasmuch as several B cell lymphomas from mice receiving a common pool of spleen cells had identical kappa-rearrangements and one identical IgH rearrangement, it was important to determine the DNA sequence of expressed IgH and kappa-genes. Each tumor was found to have identical nucleotide sequences of VH-DH-JH and VK-JK. The nonproductive IgH rearrangements each consisted of incomplete DH-JH rearrangements. The 8.5-kb EcoRI fragment was generated from a DFL16 gene segment rearranged into JH3, and the 5.6-kb fragment was generated from DQ52 rearranged into JH)1. We conclude that these Ly-1 B tumors are most likely derived from a single clone of cells which underwent a secondary rearrangement on the nonproductive allele after kappa-rearrangement had occurred. The alternate possibility of independently arising lymphomas with identical expressed VH and VK sequences is discussed.     

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.     

Gene targeting in the Ig kappa locus: efficient generation of lambda chain-expressing B cells, independent of gene rearrangements in Ig kappa.

The production of lambda chain-expressing B cells was studied in mice in which either the gene encoding the constant region of the kappa chain (C kappa) or the intron enhancer in the Ig kappa locus was inactivated by insertion of a neomycin resistance gene. The two mutants have similar phenotypes: in heterozygous mutant mice the fraction of lambda chain-bearing B cells is twice that in the wildtype. Homozygous mutants produce approximately 7 times more lambda-expressing B cells (and about 2.3 times fewer total B cells) in the bone marrow than their normal counterparts, suggesting that B cell progenitors can differentiate into either kappa- or lambda-producing cells and do the latter in the mutants. Whereas gene rearrangements in the Ig kappa locus are blocked in the case of enhancer inactivation, they still occur in that of the C kappa mutant, although in this mutant RS rearrangement is lower than in the wildtype. This indicates that gene rearrangements in the Ig lambda locus can occur in the absence of a putative positive signal resulting from gene rearrangements in Ig kappa, including RS recombination. Complementing these results, we also present data indicating that in normal B cell development kappa chain rearrangement can be preceded by lambda chain rearrangement and that the frequency of kappa/lambda double producers is small and insufficient to explain the massive production of lambda chain-expressing B cells in the mutants.     

Productive coupling of accessible Vbeta14 segments and DJbeta complexes determines the frequency of Vbeta14 rearrangement

To elucidate mechanisms that regulate Vbeta rearrangement, we generated and analyzed mice with a V(D)J recombination reporter cassette of germline Dbeta-Jbeta segments inserted into the endogenous Vbeta14 locus (Vbeta14(Rep)). As a control, we first generated and analyzed mice with the same Dbeta-Jbeta cassette targeted into the generally expressed c-myc locus (c-myc(Rep)). Substantial c-myc(Rep) recombination occurred in both T and B cells and initiated concurrently with endogenous Dbeta to Jbeta rearrangements in thymocytes. In contrast, Vbeta14(Rep) recombination was restricted to T cells and initiated after endogenous Dbeta to Jbeta rearrangements, but concurrently with endogenous Vbeta14 rearrangements. Thus, the local chromatin environment imparts lineage and developmental stage-specific accessibility upon the inserted reporter. Although Vbeta14 rearrangements occur on only 5% of endogenous TCRbeta alleles, the Vbeta14(Rep) cassette underwent rearrangement on 80-90% of alleles, supporting the suggestion that productive coupling of accessible Vbeta14 segments and DJbeta complexes influence the frequency of Vbeta14 rearrangements. Strikingly, Vbeta14(Rep) recombination also occurs on TCRbeta alleles lacking endogenous Vbeta to DJbeta rearrangements, indicating that Vbeta14 accessibility per se is not subject to allelic exclusion.