Ongoing diversification of the rearranged immunoglobulin light-chain gene in a bursal lymphoma cell line.

The chicken immunoglobulin light-chain gene (IgL) encodes only a single variable gene segment capable of recombination. To generate an immune repertoire, chickens diversify this unique rearranged VL gene segment during B-cell development in the bursa of Fabricius. Sequence analysis of IgL cDNAs suggests that both gene conversion events derived from VL segment pseudogene templates (psi VL) and non-template-derived single-base-pair substitutions contribute to this diversity. To facilitate the study of postrecombinational mechanisms of immunoglobulin gene diversification, avian B-cell lines were examined for the ability to diversify their rearranged IgL gene during in vitro passage. One line that retains this ability, the avian leukosis virus-induced bursal lymphoma cell line DT40, has been identified. After passage for 1 year in culture, 39 of 51 randomly sequenced rearranged V-J segments from a DT40 population defined novel subclones of the parental tumor. All cloned V-J segments displayed the same V-J joint, confirming that the observed diversity arose after V-J rearrangement. Most sequence variations that we observed (203 of 220 base pairs) appeared to result from psi VL-derived gene conversion events; 16 of the 17 novel single nucleotide substitutions were transitions. Based on these data, it appears that immunoglobulin diversification during in vitro passage of DT40 cells is representative of the diversification that occurs during normal B-cell development in the bursa of Fabricius.     

Somatic diversification of the chicken immunoglobulin light chain gene is limited to the rearranged variable gene segment

Previous studies have shown that the chicken lambda immunoglobulin light chain gene undergoes a single rearrangement that results in functional VJ joining of the unique variable (V lambda 1) and joining (J lambda) coding regions. The immunologic repertoire of lambda genes is created through extensive sequence diversification within the rearranged locus during B cell development in the bursa of Fabricius. This sequence diversification was detected only at the rearranged V lambda 1 segment and not within the 5' leader sequence, the J lambda segment, or the unrearranged V lambda 1 segment. The selective diversification of the rearranged V lambda 1 segment was associated with unique DNAase I-hypersensitive sites on the rearranged allele. While probes for V lambda 1 sequences detect multiple homologous V lambda segments, probes for both the 5' leader and J lambda segments fail to detect homologous sequences. Taken together, these results suggest that a highly selective process, possibly gene conversion, operates during B cell ontogeny to generate diversity within the lambda gene.     

Expression of cloned immunoglobulin genes introduced into mouse L cells.

Functionally rearranged immunoglobulin heavy-chain (gamma 2b) and light-chain (lambda 1 and kappa) genes were introduced into mouse L tk- cells by co-transformation with the Herpes virus tk gene. Cloned cell lines were selected in HAT medium and tested for the presence of transfected immunoglobulin gene sequences by Southern blotting analysis. It was found that the gamma 2b gene was accurately transcribed at a low level in transfected mouse L cells and cytoplasmic gamma 2b, heavy-chain protein was detected by immunoprecipitation of cell extracts. Light-chain genes, on the other hand, were not accurately transcribed. Instead, lambda 1 or kappa RNA species were detected which were approximately 200 to 300 bases longer than the authentic mRNAs. These results suggest that the expression of rearranged heavy-chain and light-chain genes are controlled differently and that these differences can be seen in transfected, non-lymphoid cells.     

Involvement of immunoglobulin heavy- and light-chain (kappa) gene clusters in a human B-cell translocation, t(2;14)

The breakpoints of a translocation, t(2;14)(p11;q32), detected in an Epstein-Barr virus-transformed lymphoid B-cell line were mapped by Southern analysis, field-inversion gel electrophoresis, and in situ hybridisation. The translocation involved the immunoglobulin light-chain (kappa) locus on chromosome 2 and the heavy-chain locus on chromosome 14. The breakpoint on chromosome 2 was between VK and CK, most likely within JK. The chromosome 14 break was located within the VH cluster, no more than 220 kb 5' of the productively rearranged JH locus. The translocation probably resulted from an aberrant rearrangement of the kappa light-chain genes.     

Relationship between the rearrangement of immunoglobulin genes, the appearance of a B lymphocyte antigen, and immunoglobulin synthesis in murine pre-B cell lines

Eighteen Abelson virus-transformed immature B cell lines were established and immunoglobulin biosynthesis, expression of a B lymphocyte antigen detected by a monoclonal antibody, and rearrangement of immunoglobulin genes in these cell lines were studied. Only one cell line (A1) synthesized micro-chains but no light chains, and the other cell lines synthesized no detectable immunoglobulins. None of the cell lines established had detectable membrane-associated IgM. Fifteen cell lines expressed a B lymphocyte antigen on their cell surfaces. In three cell lines, however, the majority (greater than 99%) of cells did not express this antigen. Heavy chain genes were rearranged on both chromosomes in all the cell lines, although one heavy chain gene was deleted in three cell lines. In 12 of 18 cell lines, one or both kappa-chain genes were rearranged. In six cell lines, however, both kappa-chain genes remained in embryonic form; lambda-chain genes were in embryonic form in all the cell lines. These results suggested the hierarchy of Ig gene rearrangements, beginning with mu and proceeding to kappa and then to lambda. JH rearrangement was also shown to precede the appearance of a B lymphocyte antigen. In three cell lines (A1-A3), which were considered subclones derived from a single common precursor, it was suggested that one rearranged JH gene was functional, and the other was nonfunctional, indicating that allelic exclusion already operated in pre-B cells.     

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.     

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.     

Increased frequency of N-region insertion in a murine pre-B-cell line infected with a terminal deoxynucleotidyl transferase retroviral expression vector.

The role of terminal deoxynucleotidyl transferase (TdT) in the insertion of N regions into the junctional sites of immunoglobulin genes was investigated. Pre-B-cell lines capable of continuous rearrangement of immunoglobulin light-chain genes and differing only in the presence or apparent absence of TdT were derived by infecting cells with a TdT retroviral expression vector or a control vector. The cell lines were then superinfected with a retrovirus-based artificial immunoglobulin gene rearrangement substrate. The substrate was allowed to rearrange in the cell lines and the rearranged proviruses were rescued from the cell lines. Nucleotide sequence analysis of the V-J junctions of the proviral rearranged genes showed a fivefold greater frequency of N-region insertion in proviruses rescued from the TdT+ cell lines than in those rescued from the TdT- cell lines, so that at least 50% of the rearrangements that occurred in the presence of TdT had N regions. It is thus evident that TdT can stimulate N-region insertion, and the enzyme is presumably directly responsible for adding nucleotides at V-J and other immunoglobulin and T-cell receptor gene junctions.     

RAG-1 and RAG-2 are not sufficient to direct all phases of immunoglobulin gene rearrangement in pre-B-cell lines.

To probe the factors controlling immunoglobulin heavy-chain gene rearrangement, we analyzed Abelson virus-transformed pre-B-cell lines that fail to undergo VH-to-DJH joining at an appreciable frequency. Despite this feature, some of these cell lines (rechi) rearrange an extrachromosomal recombination substrate at levels normal for transformed pre-B cells. Others (reclo) rearrange these substrates at levels characteristic of nonlymphoid hematopoietic cells. The DJH rearrangements from a representative rechi cell line were aberrant, suggesting that these cells probably fail to complete heavy-chain gene assembly because some of the necessary cis-acting signals are missing. In contrast, both DJH rearrangements from a reclo cell line appeared normal in structure, indicating that trans-acting factors necessary for recombination might be missing. Introduction of the RAG-1 and RAG-2 genes, genes encoding two such factors, failed to confer a rechi phenotype to these cells. However, fusion of the reclo cells to a rechi cell line generated a high frequency of rechi hybrids. In addition, most of the hybrids rearranged the endogenous kappa light-chain locus. Neither the rechi phenotype nor kappa-chain rearrangement correlated with levels of RAG-1 and RAG-2 expression in all of the hybrids. Thus, both gene transfer and cell fusion experiments indicate that RAG-1 and RAG-2 are not sufficient to activate immunoglobulin gene recombination in at least some pre-B-cell lines. In addition, the fusion experiments suggest that two gene products in addition to RAG-1 and RAG-2 may be required for kappa-gene rearrangement.     

Avian reticuloendotheliosis virus-transformed lymphoid cells contain multiple pp59v-rel complexes.

The v-rel oncogene of avian reticuloendotheliosis virus type T (REV-T) encodes a 59-kilodalton (kDa) phosphoprotein located principally in the cytosol of transformed lymphoid cells. All of the detectable pp59v-rel was present in high-molecular-weight complexes containing at least five cellular proteins (p124, p115, p75c-rel, p70hsc, and pp40). Antiserum was developed against the 40-kDa protein, the most abundant cellular protein associated with the complex. The 40-kDa phosphoprotein was complexed with pp59v-rel in REV-T-transformed lymphoid cell lines arrested at different stages of B-cell development as well as in lymphoid tumor cells and in fibrosarcomas. The half-life (8 h) of pp40 in REV-T-transformed lymphoid cells was the same as that of pp59v-rel. Antiserum against pp40 permitted the identification of two pp59v-rel complexes. The most abundant cytoplasmic complex contained approximately 75% of the pp59v-rel and all of the detectable pp40 in REV-T-transformed lymphoid cells. Twenty-five percent of the pp59v-rel was present in a minor complex that contained the majority of p75c-rel along with p115 and p124. In nuclear extracts of REV-T-transformed lymphoid cells, pp59v-rel was complexed with pp40. The two high-molecular-weight proteins (p115 and p124) and p75c-rel were not detected in the nuclear complex. In the cytosolic complexes, pp40 was heavily phosphorylated, whereas the nuclear form was much less extensively phosphorylated.     

Activation of the c-myb locus is insufficient for the rapid induction of disseminated avian B-cell lymphoma.

We have previously reported that infection of 9- to 13-day-old chicken embryos with RAV-1 results in rapid development of a novel B-cell lymphoma in which proviral insertion has activated expression of the c-myb gene (E. Pizer and E. H. Humphries, J. Virol. 63:1630-1640, 1989). The biological properties of these B-cell lymphomas are distinct from those associated with the B-cell lymphomas that develop following avian leukosis virus proviral insertion within the c-myc locus. In an extension of this study, more than 200 chickens, infected as 10- to 11-day-old embryos, were examined for development of lymphomas that possess disrupted c-myb loci. Fourteen percent developed disseminated B-cell lymphoma. In the majority of these tumors, the RAV-1 provirus had inserted between the first and second exons that code for p75c-myb. However, insertions between the second and third exons and between the third and fourth exons were also detected. In situ analysis of myb protein expression in tumor tissue revealed morphological features suggesting that the tumor originates in the bursa. Within the bursa, the lymphoma appeared to spread from follicle to follicle without compromising the structural integrity of the organ. Tumor masses in liver demonstrated heterogeneous levels of myb protein suggestive of biologically distinct subpopulations. In contrast to the morbidity data, immunohistological analysis of bursae from 4- to 6-week-old chickens at risk of developing lymphomas bearing altered c-myb loci revealed lesions expressing elevated levels of myb in 16 of 19 birds. The activated myb lymphoma displayed very poor capacity to proliferate outside its original host. Only 1 of 33 in vivo transfers of tumor to recipient hosts established a transplantable tumor. None of the primary tumor tissue nor the transplantable tumor exhibited the capacity for in vitro proliferation. Similar experimental manipulation has yielded in vitro lines established from avian B-cell lymphomas expressing elevated levels of c-myc or v-rel. The dependence on embryonic infection for development of activated-myb lymphoma suggests a requirement for a specific target cell in which c-myb is activated by proviral insertion. It is likely, moreover, that continued tumor development requires elevated expression of myb proteins within a specific cell population in a restricted stage of differentiation.