Anti-CD40 plus interleukin-4-activated human naive B cell lines express unmutated immunoglobulin genes with intraclonal heavy chain isotype variability

1995 ◽  
Vol 25 (3) ◽  
pp. 733-737 ◽  
Author(s):  
Laurent Galibert ◽  
Joelle van Dooren ◽  
Isabelle Durand ◽  
Françoise Rousset ◽  
Royston Jefferis ◽  
...  
Keyword(s):  
B Cell ◽  
Cell Lines ◽  
Heavy Chain ◽  
Interleukin 4 ◽  
Immunoglobulin Genes

scholarly journals Rate of replication of the murine immunoglobulin heavy-chain locus: evidence that the region is part of a single replicon.

1987 ◽  
Vol 7 (1) ◽  
pp. 450-457 ◽  
Author(s):  
E H Brown ◽  
M A Iqbal ◽  
S Stuart ◽  
K S Hatton ◽  
J Valinsky ◽  
...  
Keyword(s):  
B Cell ◽  
Cell Line ◽  
Gene Cluster ◽  
Cell Lines ◽  
Heavy Chain ◽  
Dna Content ◽  
Immunoglobulin Heavy Chain ◽  
S Phase ◽  
C Value ◽  
Murine Erythroleukemia

We measured the temporal order of replication of EcoRI segments from the murine immunoglobulin heavy-chain constant region (IgCH) gene cluster, including the joining (J) and diversity (D) loci and encompassing approximately 300 kilobases. The relative concentrations of EcoRI segments in bromouracil-labeled DNA that replicated during selected intervals of the S phase in Friend virus-transformed murine erythroleukemia (MEL) cells were measured. From these results, we calculated the nuclear DNA content (C value; the haploid DNA content of a cell in the G1 phase of the cell cycle) at the time each segment replicated during the S phase. We observed that IgCH genes replicate in the following order: alpha, epsilon, gamma 2a, gamma 2b, gamma 1, gamma 3, delta, and mu, followed by the J and D segments. The C value at which each segment replicates increased as a linear function of its distance from C alpha. The average rate of DNA replication in the IgCH gene cluster was determined from these data to be 1.7 to 1.9 kilobases/min, similar to the rate measured for mammalian replicons by autoradiography and electron microscopy (for a review, see H. J. Edenberg and J. A. Huberman, Annu. Rev. Genet. 9:245-284, 1975, and R. G. Martin, Adv. Cancer Res. 34:1-55, 1981). Similar results were obtained with other murine non-B cell lines, including a fibroblast cell line (L60T) and a hepatoma cell line (Hepa 1.6). In contrast, we observed that IgCh segments in a B-cell plasmacytoma (MPC11) and two Abelson murine leukemia virus-transformed pre-B cell lines (22D6 and 300-19O) replicated as early as (300-19P) or earlier than (MPC11 and 22D6) C alpha in MEL cells. Unlike MEL cells, however, all of the IgCH segments in a given B cell line replicated at very similar times during the S phase, so that a temporal directionality in the replication of the IgCH gene cluster was not apparent from these data. These results provide evidence that in murine non-B cells the IgCH, J, and D loci are part of a single replicon.

Immunoglobulin heavy-chain enhancer is required to maintain transfected gamma 2A gene expression in a pre-B-cell line

1990 ◽  
Vol 10 (3) ◽  
pp. 1076-1083
Author(s):  
B Porton ◽  
D M Zaller ◽  
R Lieberson ◽  
L A Eckhardt
Keyword(s):  
B Cell ◽  
Cell Lines ◽  
Heavy Chain ◽  
Gene Activation ◽  
Immunoglobulin Heavy Chain ◽  
Transcription Unit ◽  
Enhancer Activity ◽  
Igh Genes ◽  
High Level

The immunoglobulin heavy-chain (IgH) enhancer serves to activate efficient and accurate transcription of cloned IgH genes when introduced into B lymphomas or myelomas. The role of this enhancer after gene activation, however, is unclear. The endogenous IgH genes in several cell lines, for example, have lost the IgH enhancer by deletion and yet continue to be expressed. This might be explained if the role of the enhancer were to establish high-level gene transcription but not to maintain it. Alternatively, other enhancers might lie adjacent to endogenous IgH genes, substituting their activity for that of the lost IgH enhancer. To address both of these alternatives, we searched for enhancer activity within the flanking regions of one of these IgH enhancer-independent genes and designed an experiment that allowed us to consider separately the establishment and maintenance of expression of a transfected gene. For the latter experiment we generated numerous pre-B cell lines stably transformed with a gamma 2a gene. In this gene, the IgH enhancer lay at a site outside the heavy-chain transcription unit, between DH and JH gene segments. After expression of the transfected gene was established, selective conditions were chosen for the outgrowth of subclones that had undergone D-J joining and thus IgH enhancer deletion. Measurements of gamma 2a expression before and after enhancer deletion revealed that the enhancer was required for maintenance of expression of the transfected gene. The implication of this finding for models of enhancer function in endogenous genes is discussed.

scholarly journals Interleukin 4 causes isotype switching to IgE in T cell-stimulated clonal B cell cultures.

1988 ◽  
Vol 168 (3) ◽  
pp. 853-862 ◽  
Author(s):  
D A Lebman ◽  
R L Coffman
Keyword(s):  
B Cells ◽  
B Cell ◽  
Cell Cultures ◽  
Heavy Chain ◽  
Clonal Expansion ◽  
Precursor Cells ◽  
Interleukin 4 ◽  
Isotype Switching ◽  
Th Cell ◽  
Clonal Cultures

Although it has been established that IL-4 enhances both IgG1 and IgE secretion in LPS-stimulated B cell cultures, these studies failed to determine whether IL-4 preferentially induces isotype switching or preferentially allows for the maturation of precommitted precursor cells. To distinguish between these possibilities, it is necessary to ascertain the effect of IL-4 on the isotypes secreted by individual precursor cells during clonal expansion. Therefore, clonal cultures of B cells stimulated with a Th2 helper cell line specific for rabbit Ig and rabbit anti-mouse IgM were established. The majority of B cells are capable of undergoing clonal expansion under these conditions. To vary the level of IL-4 present, either IL-4 or anti-IL-4 was added to cultures. In the presence of IL-4 there was an increase in the proportion of clones that secreted IgE and a decrease in the proportion of clones that secreted IgM. The addition of IL-4 to cultures also increased the amount of IgE secreted by individual clones. Thus, these experiments definitively prove that IL-4 causes specific heavy chain class switching to IgE in Th2-stimulated B cell cultures. In contrast, IL-4 does not affect the proportion of clones secreting IgG1, suggesting that other consequences of Th cell-B cell interactions play a role in the generation of an IgG1 response.

Stimulation of kappa light-chain gene rearrangement by the immunoglobulin mu heavy chain in a pre-B-cell line

1993 ◽  
Vol 13 (9) ◽  
pp. 5679-5690
Author(s):  
A M Shapiro ◽  
M S Schlissel ◽  
D Baltimore ◽  
A L DeFranco
Keyword(s):  
B Cell ◽  
Cell Lines ◽  
Heavy Chain ◽  
Gene Rearrangement ◽  
Germ Line ◽  
Gene Rearrangements ◽  
Matched Pairs ◽  
Immunoglobulin Gene ◽  
Kappa Light Chain ◽  
Immunoglobulin Mu

B-lymphocyte development exhibits a characteristic order of immunoglobulin gene rearrangements. Previous work has led to the hypothesis that expression of the immunoglobulin mu heavy chain induces rearrangement activity at the kappa light-chain locus. To examine this issue in more detail, we isolated five matched pairs of mu- and endogenously rearranged mu+ cell lines from the Abelson murine leukemia virus-transformed pro-B-cell line K.40. In four of the five mu+ cell lines, substantial expression of mu protein on the cell surface was observed, and this correlated with an enhanced frequency of kappa immunoglobulin gene rearrangement compared with that in the matched mu- cell lines. This increased kappa gene rearrangement frequency was not due to a general increase in the amount of V(D)J recombinase activity in the mu+ cells. Consistently, introduction of a functionally rearranged mu gene into one of the mu- pre-B-cell lines resulted in a fivefold increase in kappa gene rearrangements. In three of the four clonally matched pairs with increased kappa gene rearrangements, the increase in rearrangement frequency was not accompanied by a significant increase in germ line transcripts from the C kappa locus. However, in the fourth pair, K.40D, we observed an increase in germ line transcription of the kappa locus after expression of mu protein encoded by either an endogenously rearranged or a transfected functional heavy-chain allele. In these cells, the amount of the germ line C kappa transcript correlated with the measured frequency of rearranged kappa genes. These results support a regulated model of B-cell development in which mu protein expression in some way targets the V(D)J recombinase to the kappa gene locus.

Decoupling of Normal CD40 / Interleukin-4 Immunoglobulin Heavy Chain Switch Signal Leads to Genomic Instability in RPMI 8226 and SGH-MM5 Multiple Myeloma Cell Lines.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1424-1424
Author(s):  
William Hwang ◽  
Charles Gullo ◽  
Gerrard Teoh
Keyword(s):  
Dna Repair ◽  
Cell Line ◽  
Genomic Instability ◽  
Cell Lines ◽  
Heavy Chain ◽  
Clonal Evolution ◽  
Chromosomal Translocations ◽  
Interleukin 4 ◽  
Dna Dsbs ◽  
Rpmi 8226

Abstract Certain genetic events accompany transformation of MM to more aggressive disease. For example, there is a predominance of new and promiscuous chromosomal translocations into the switch region of the immunoglobulin heavy chain (IgH) gene on chromosome 14 (i.e. at 14q32). Development of chromosomal translocations involves the process of DNA double strand break repair (DSBR) by non-homologous end joining (NHEJ). DNA protein kinase (DNA-PK) is the principal DNA repair enzyme mediating DNA DSBR. It is made up of a catalytic subunit (DNA-PKcs) and a regulatory subunit (the Ku70/Ku86 heterodimer). Interestingly, the majority (86% to 100%) of freshly isolated patient MM cells express a variant form of Ku86 protein (Ku86v), which has been associated with abnormalities in DNA repair. Since, the combined effects of CD40 plus interleukin-4 (IL-4) are required for normal IgH isotype class switch recombination (CSR), and this process involves DNA DSBR, NHEJ and DNA-PK; we hypothesized that CD40 and/or IL-4 activation of MM cells could induce abnormalities in DNA DSBR, which could lead eventually to genomic instability and clonal evolution. In this study, we first showed that RPMI 8226 and SGH-MM5 MM cell lines (but not the CESS Epstein-Barr virus (EBV) immortalized normal B cell line) that are optimally triggered via CD40 and/or IL-4 demonstrate abnormal decoupling of IL-4 signal transduction from CD40. Specifically, CD40 alone was sufficient to trigger growth of tumor cell lines, suggesting that biological sequelae mediated by CD40 could be dysregulated in MM cells. Whether this process involves Ku86v is presently being investigated. We further demonstrate that CD40 triggering induced both DNA DSBs as well as new (acquired) karyotypic abnormalities in the SGH-MM5 MM cell line. These complex karyotypic changes included at least 5 new and clonal chromosomal translocations and deletions. Since, normal IgH isotype CSR is accompanied by induction of activation induced cytidine deaminase (AID) expression, we next demonstrated that CD40 triggering of MM cell lines (without IL-4) was sufficient to upregulate AID expression. These data suggest that DNA DSBs induced by CD40 were part of IgH isotype CSR rather than CD40 induced apoptosis of tumor cells. This is an important distinction to make because CD40 has been demonstrated to induce apoptosis by both p53 -dependent and -independent pathways. In order to confirm that CD40-triggered MM cells did not undergo apoptosis, we performed annexin V/propidium iodide (PI) staining on CD40-triggered MM cells. We showed that MM cell lines not only remained viable after CD40 triggering, but also demonstrated G1 cell cycle exit. In conclusion, our present study shows that CD40 alone could act as an inducer of genomic instability in MM cell lines, and lead to clonal evolution. Since CD40 ligand (CD40L) is naturally expressed during inflammation and T cell activation, it is tempting to speculate that the normal inflammatory process could potentially participate in clonal evolution and even myelomagenesis in vivo.

Heavy chain immunoglobulin gene rearrangement in acute nonlymphocytic leukemia

Blood ◽  
1984 ◽  
Vol 63 (5) ◽  
pp. 1023-1027
Author(s):  
U Rovigatti ◽  
J Mirro ◽  
G Kitchingman ◽  
G Dahl ◽  
J Ochs ◽  
...  
Keyword(s):  
B Cell ◽  
Cell Lines ◽  
Heavy Chain ◽  
Gene Rearrangement ◽  
Leukemia Cell ◽  
Leukemic Cell ◽  
Immunoglobulin Gene ◽  
Acute Nonlymphocytic Leukemia ◽  
Lymphoid Leukemia ◽  
Immunoglobulin Gene Rearrangement

Samples of leukemic cell DNA from 14 children with acute nonlymphocytic leukemia (ANLL) and 4 human myeloid leukemia cell lines were analyzed for rearrangement in the heavy chain region of the immunoglobulin gene. The diagnosis of ANLL was confirmed in all patients by morphological, cytochemical, and immunologic studies. By restriction endonuclease digestion and hybridization with cloned heavy chain immunoglobulin gene probes for the constant (Cmu) and joining (JH) regions, the DNA of 2 patients and 1 cell line (ML-1) was found to contain rearrangements. The DNA from the remaining 12 patients and 3 cell lines was not rearranged (germline configuration). Both patients with apparent immunoglobulin gene rearrangement achieved complete remission on therapy for ANLL. Immunoglobulin gene rearrangement in phenotypically defined ANLL suggests (1) that such changes may not be limited to lymphoid leukemia of B cell lineage, or (2) that, in some patients, the leukemic transforming event may involve stem cells capable of both B cell and myeloid differentiation.

scholarly journals The immunoglobulin heavy chain locus contains another B-cell-specific 3' enhancer close to the alpha constant region.

1993 ◽  
Vol 13 (3) ◽  
pp. 1547-1553 ◽  
Author(s):  
P Matthias ◽  
D Baltimore
Keyword(s):  
Transcription Factors ◽  
B Cell ◽  
Heavy Chain ◽  
Variable Region ◽  
Immunoglobulin Heavy Chain ◽  
Immunoglobulin Genes ◽  
Constant Region ◽  
Transcriptional Enhancer ◽  
Additional Control ◽  
Complex Regulation

The transcription of immunoglobulin genes is controlled by variable region promoters and by enhancers, both of which are lymphoid specific. Because immunoglobulin genes are subject to an extremely complex regulation, we anticipated that there might be additional control elements for these genes. We therefore sought additional enhancers and demonstrate here that there is indeed another weak transcriptional enhancer just 3' to the mouse alpha constant region. This novel immunoglobulin enhancer is lymphoid specific and at two positions can bind members of the Oct family of transcription factors.

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