Germline Immunoglobulin Heavy Chain Research Articles

Involvement of the E2A basic helix-loop-helix protein in immunoglobulin heavy chain class switching

The basic helix-loop-helix (bHLH) transcriptional factor E2A has previously been shown to play a critical role in early B cell development, with E2A knockout mice and Id1 transgenic mice showing an arrest at the pro-B cell stage of development. More recent data suggest that E2A, through an interaction with the immunoglobulin heavy chain 3′ enhancer, might also regulate later events in B cell development such as heavy chain class switching. The patterns of E2A protein expression in secondary lymphoid tissues support a role in later stages of B cell maturation. In particular, immunostaining reveals upregulation of E2A protein in cells of the dark zone of the germinal center, the site of immunoglobulin heavy chain class switching. To examine the role of E2A in class switching, the inhibitory HLH protein Id1 was expressed in B cell lines which normally undergo spontaneous and inducible switching from IgM to IgA. The forced expression of Id1 in these cell lines effectively blocked class switching. This Id1 blockade of class switching did not occur via downregulation of immunoglobulin heavy chain germline transcription or through inhibition of cell cycling. Further-more, Id1 inhibited spontaneous and, to a lesser degree, cytokine inducible class switching. From these data, we conclude that E2A plays an important role in the class switching process.

Analysis and Comparison of the Mouse and Human Immunoglobulin Heavy Chain JH–Cμ–Cδ Locus

We report here 23,686 bases of contiguous DNA sequences from the mouse germline immunoglobulin heavy chain (H) constant (C) μδ region. The sequence spans the joining (JH) regions, the μ constant region (Cμ), the δ constant region (Cδ) coding regions, a domain relic, the μ switch region (Sμ), seven blocks of simple sequence repeats, a large unique sequence inverted repeat, a large unique sequence forward repeat, and all of the intervening material. A comparison of this 23.7-kb region with the corresponding human Cμ/Cδ region reveals clear homology in the coding and introns of Cμ but not in the 5′ flanking J gene segments nor in the intergenic and Cδ regions. This mixed pattern of similarity between the human and the mouse sequences contrasts with high levels of similarity found in the T-cell receptor Cα/Cδ region and α and β myosin genes and the very low levels found in the γ-crystallin, XRCC1, and β-globin gene clusters. The human and mouse comparison further suggests the incorporation of novel sequences into expressed genes of IgD.

Chromosomal translocations cause deregulated BCL6 expression by promoter substitution in B cell lymphoma.

The BCL6 gene codes for a zinc-finger transcription factor and is involved in chromosomal rearrangements in 30-40% of diffuse large-cell lymphoma (DLCL). These rearrangements cluster within the 5' regulatory region of BCL6 spanning its first non-coding exon. To determine the functional consequences of these alterations, we have analyzed the structure of the rearranged BCL6 alleles and their corresponding RNA and protein species in two DLCL biopsies and one tumor cell line which carried the t(3;14)(q27;q32) translocation involving the BCL6 and immunoglobulin heavy-chain (IgH) loci. In all three cases, the breakpoints were mapped within the IgH switch region and the BCL6 first intron, leading to the juxtaposition of part of the IgH locus upstream and in the same transcriptional orientation to the BCL6 coding exons. An analysis of cDNA clones showed that these recombinations generate chimeric IgH-BCL6 transcripts which initiated from IgH germline transcript promoters (I mu or I gamma 3), but retain a normal BCL6 coding domain. In the tumor cell line, the chimeric I gamma 3-BCL6 allele, but not the germline BCL6 gene, was transcriptionally active and produced a normal BCL6 protein. These findings indicate that t(3;14) translocations alter BCL6 expression by promoter substitution and imply that the consequence of these alterations is the deregulated expression of a normal BCL6 protein.

Characterization of an interleukin 4 (IL-4) responsive region in the immunoglobulin heavy chain germline epsilon promoter: regulation by NF-IL-4, a C/EBP family member and NF-kappa B/p50.

A large body of data indicate that antibody class switching is directed by cytokines by inducing or repressing transcription from unrearranged, or germline, CH genes. Interleukin 4 (IL-4) induces transcription of the germline C epsilon genes in activated B cells and subsequently, cells in this population will undergo switch recombination to immunoglobulin E. Furthermore, the data suggest that transcription of germline C epsilon genes is required for class switching. In this paper we define DNA elements required for induction of transcription of the germline C epsilon genes by IL-4. To do this, segments of DNA from the 5' flank of the initiation sites for germline epsilon RNA were ligated to a luciferase reporter gene and transfected into two mouse B cell lines, one of which can be induced to switch to IgE. By analysis of a series of 5' deletion constructs and linker-scanning mutations, we demonstrate that a 46-bp segment (residing at -126/-79 relative to the first RNA initiation site) contains an IL-4 responsive region. By electrophoretic mobility shift assays, we find that this segment binds three transcription factors: the recently described NF-IL4, one or more members of the C/EBP family of transcription factors, and NF-kappa B/p50. Mutation of any of the binding sites for these three factors abolishes or reduces IL-4 inducibility of the epsilon promoter. A 27-bp segment within this IL-4 response region containing binding sites for NF-IL4 and a C/EBP factor is sufficient to transfer IL-4 inducibility to a minimal c-fos promoter.

The IL-4 induced increase in the frequency of resting murine splenic B cells expressing germline Ig heavy chain gamma 1 transcripts correlates with subsequent switching to IgG1.

Cytokine induced germline immunoglobulin heavy chain gene transcription appears to signal commitment to an isotype switch and may be the mechanism by which specific switch regions are targeted as the sites for recombination. In this study, the structure and expression of mouse germline gamma 1 RNAs are described. The 5'-ends of these transcripts are derived from an exon denoted I gamma 1, located upstream of the gamma 1 switch region and initiate at multiple sites over a 200 nucleotide region. Sequence analysis of cDNA and genomic clones reveals that these RNAs, unlike other germline CH transcripts, may encode a novel I gamma 1/C gamma 1 heavy chain protein, of which the N-terminal 27 residues are encoded by I gamma 1. In vitro culture of resting or pre-activated splenic B cells in the presence of lipopolysaccharides and interleukin-4 (IL-4) generates clones that secrete both IgM and IgG1 or either isotype alone. IL-4 increases the frequency of clones secreting both IgM and IgG1 and IgG1 alone, suggesting that commitment to IgG1 secretion may be independent of, or associated with, IgM secretion. PCR analysis of gamma 1 germline transcript expression in clonal B cell cultures or single pre-activated B cells, shows that the IL-4 induced increase in the frequency of cells expressing gamma 1 germline transcripts directly correlates with the increased frequency of cells switching from IgM to IgG1 production. This finding statistically confirms at a clonal level the relationship between cytokine induced germline transcription and isotype switching.

Radiation sensitivity of human B-lineage lymphoid precursor cells

We studied the radiation sensitivity of eight immunophenotypically distinct B-lineage lymphoid precursor cell (LPC) lines of acute lymphoblastic leukemia (ALL) or fetal liver origin corresponding to discrete developmental stages of human B-cell ontogeny. The radiation sensitivity of B-lineage LPC showed a temporal association with the distinct stages of development. FL112 and FL114 fetal liver pro-B cells (Stage 0 B-lineage LPC) with germline immunoglobulin heavy chain (IgH) genes but rearranged T-cell receptor gamma (T γ) genes (D O of FL112 = 80.3 cGy, D O of FL114 = 50.2 cGy), REH ALL pre-pre-B cells (Stage I B-lineage LPC) with rearranged IgH and T γ genes (D O = 66.1 cGy), and NALM-6 ALL pre-pre-B/pre-B cells (Stage II B-lineage LPC) (D O = 50.5 cGy) corresponding to the earliest three stages of human B-lymphocyte development were the most radiation sensitive B-lineage LPC populations. By comparison, KM-3 ALL pre-B (Stage III B-lineage LPC) (D O = 194.7 cGy), HPB-NULL ALL pre-B (Stage IV B-lineage LPC) (D O = 134.6 cGy), and sIgM + RAJI/NAMALWA early B (Stage Va/b B-lineage LPC) cell lines (D O of RAJI = 144.0 cGy, D O of NAMALWA = 165.5 cGy) corresponding to the later stages of human B-lymphocyte development were much more radiation resistant. These results indicate that the radiation sensitivity of B-lineage LPC decreases during maturation within the B-lineage lymphoid precursor pathway. By comparison, the S-phase index (% of S-phase cells as determined by DNA flow cytometry) or proliferation index (% S+G 2M), cellular protein content, intracellular glutathione (GSH) level, glutathione-S-transferase (GST) activity, intracellular pH, or free cytoplasmic calcium concentration did not correlate with the radiation sensitivity of the B-lineage LPC.

Structure and expression of mouse germline immunoglobulin γ3 heavy chain transcripts induced by the mitogen lipopolysaccharide

Germline immunoglobulin heavy chain gene transcription is though to direct isotype switching by modulating the accessibility of specific switch regions to a recombinase. In this study, cloned cDNA copies of mouse germline Igh-8 RNAs have been used to characterize the Igh-8 transcription unit. The 5' end of these transcripts are derived from an exon denoted Ig3, located 1 kilobase 5' of the Igh-8 switch region. Sequence analysis of cDNA and genomic clones reveals that these RNAs are noncoding. In splenic B cell cultures treated with lipopolysaccharide (LPS), germline Igh-8 transcript levels are upregulated after 8 h due to increased transcription. This induction is consistent with the identification of a putative binding site for the LPS inducible transcription factor NF-kappa B approximately 150 nucleotides upstream of the sites of transcript initiation. Furthermore, nucleotide sequence comparisons reveal that the region encompassing the site of germline Igh-8 transcription initiation is highly homologous to part of the Ig2b exon, and is also conserved upstream of the Igh-1 switch region. The implications of these findings for the control of germline Igh-8 transcription is discussed.

Stage-specific transcription of germline lgH Cγ and Cα regions during human B cell differentiation

Previous studies have suggested that transcription of germline heavy chain constant region (CH) genes in murine B cells may determine the potential of their different CH regions to undergo isotype switch recombination. We have examined the transcriptional activity across the immunoglobulin heavy chain (IgH) locus in human B lineage cells. Transcription of germline C gamma and C alpha was observed in every surface IgM+ or surface IgM+/IgD+ B cell stage cell line and malignancy. In contrast, such transcription could not be detected in pre-B cells and only low levels of C alpha but not C gamma transcription were evident in IgM-secreting plasmablast cells. Transcriptional activity of germline IgH C epsilon was singularly absent at all stages of B cell development. Our results suggest that germline transcription of the C gamma and C alpha regions may be a constitutive feature of the human B cell differentiation program. Because this transcriptional activity is limited primarily to the B cell stage and occurs prior to the actual isotype switch, the induction of C gamma and C alpha transcription may represent preparation of the downstream IgH chromatin for potential switch recombination.

Structure and expression of murine germ-line immunoglobulin epsilon heavy chain transcripts induced by interleukin 4.

The murine lymphokine, interleukin 4 (IL-4) is able to specifically promote isotype switching to IgG1 and IgE in cultures of mitogen-stimulated B cells. Emerging evidence suggests that germ-line immunoglobulin heavy chain gene transcription may direct switching by modulating switch-region accessibility to a recombinase. In this study, cloned cDNA copies of the germ-line epsilon heavy chain transcript have been used to determine the genomic organization of this transcription unit. The 5' end of these transcripts are derived from an exon, denoted I epsilon, located 2 kilobases 5' of the C epsilon switch region [C epsilon = epsilon heavy chain constant (C) region gene]. Nucleotide sequence analysis reveals that this RNA does not encode a protein, as the I epsilon exon contains termination codons in all reading frames. Germ-line epsilon chain transcripts can be detected in cultures of normal splenic B cells treated with IL-4 within 24 hr, and this expression correlates with subsequent switching to C epsilon. Consistent with the IL-4 inducibility of this RNA is the identification of a motif upstream from the site of transcription initiation that closely resembles a transcription element implicated in the IL-4 regulation of the gene encoding the murine class II histocompatibility antigen, A alpha k. These data lend support to the accessibility model of isotype switching and implicate IL-4 in the transcriptional activation of the C epsilon locus.

RNA splicing generates alternate forms of germline immunoglobulin α heavy chain transcripts

Emerging evidence implicates germline immunoglobulin heavy chain gene transcription in the targeting of heavy chain genes for switch recombination. In this study, cloned cDNA copies of the major germline alpha heavy chain transcript expressed in the murine B cell lymphoma 1.29 mu, a cell line that switches to IgA in culture, have been used to characterize the germline alpha transcription unit. The 5' end of these transcripts are heterogeneous, being derived from an exon denoted I alpha located approximately 2.2 kb 5' of the alpha switch region. Sequence analysis of cDNA and genomic clones reveals that alternate splice donor sites generate I alpha exons of varying length. While the two smaller spliced forms of I alpha contain stop codons in the open reading frame of the C alpha gene, transcripts utilizing the 3' most splice donor signal may encode a protein in which amino acids derived from the 3' end of the I alpha exon are fused to the C alpha domain.

Terminal Deoxynucleotidyl Transferase Expression Can Precede T Cell Receptor β Chain and γ Chain Rearrangement in T Cell Acute Lymphoblastic Leukemia

The nuclear enzyme terminal deoxynucleotidyl transferase (TdT) is thought to contribute to the diversity of certain immunoglobulin and T cell receptor gene rearrangements through the addition of random nucleotides at their variable (V)-joining (J) region junctions. An acute lymphoblastic leukemia (ALL) with an immature T cell phenotype (CD7+, CD5+, CD1+/-, CD2+/-, CD3-, CD4-, CD8-) was found to be TdT+ with germline immunoglobulin heavy chain, T cell receptor beta chain, and T cell gamma chain genes. The data indicate that TdT expression can precede T gamma and T beta rearrangement during T lymphoid ontogeny consistent with its proposed association with the T cell receptor rearrangement process. Southern analysis of certain cases of T-ALL may not result in the detection of a monoclonal population of cells.

Terminal deoxynucleotidyl transferase expression can precede T cell receptor beta chain and gamma chain rearrangement in T cell acute lymphoblastic leukemia

The nuclear enzyme terminal deoxynucleotidyl transferase (TdT) is thought to contribute to the diversity of certain immunoglobulin and T cell receptor gene rearrangements through the addition of random nucleotides at their variable (V)-joining (J) region junctions. An acute lymphoblastic leukemia (ALL) with an immature T cell phenotype (CD7+, CD5+, CD1+/-, CD2+/-, CD3-, CD4-, CD8-) was found to be TdT+ with germline immunoglobulin heavy chain, T cell receptor beta chain, and T cell gamma chain genes. The data indicate that TdT expression can precede T gamma and T beta rearrangement during T lymphoid ontogeny consistent with its proposed association with the T cell receptor rearrangement process. Southern analysis of certain cases of T-ALL may not result in the detection of a monoclonal population of cells.

Human bone marrow cells positive for terminal deoxynucleotidyl transferase (TdT), HLA-DR, and a T cell marker may represent prothymocytes.

Recent evidence suggests that prothymocytes, which occur in a low frequency in murine bone marrow (BM), are already committed to thymocyte differentiation and discrete from precursor B cells as well as pluripotent hematopoietic stem cells. Furthermore, it was suggested that, in rodents, prothymocytes are positive for the nuclear enzyme terminal deoxynucleotidyl transferase (TdT) and a T cell surface antigen. The human prothymocyte has not been identified as yet. We analyzed human BM cells by double immunofluorescence staining for TdT and the T cell surface markers Tp41 (recognized by the monoclonal antibodies WT1 and 3A1), T11, T1, and T6. In the BM samples tested, neither T1+/TdT+ nor T6+/TdT+ cells were detected, but Tp41+/TdT+ and T11+/TdT+ cells were present in low frequencies. In childhood BM, the frequency was about two to five in 10,000, whereas in adult BM and regenerating BM, these cells were not always detectable, but if detected, their frequency was five- to 10-fold lower. In a triple staining, using fluorescein, rhodamine, and colloidal gold particles as labels, it appeared that all Tp41+/TdT+ cells were also positive for HLA-DR. These Tp41+/HLA-DR+/TdT+ cells were also detectable in low frequencies in the thymus, and occasionally Tp41+/TdT+ and T11+/TdT+ cells were detected in the peripheral blood (PB), suggesting a migration from the BM to the thymus via the PB. The malignant counterpart of the Tp41+/HLA-DR+/TdT+ cell was detected in a patient with acute lymphoblastic leukemia with the Tp41+/T11+/HLA-DR+/TdT+/T1-/T6- phenotype and germ-line immunoglobulin heavy chain genes. We postulate that the Tp41+/T11+/HLA-DR+/TdT+/T1-/T6- cell represents a human prothymocyte.

Allelic immunoglobulin VH genes in two mouse strains: possible germline gene recombination.

The nucleotide sequence of two germline immunoglobulin heavy chain variable region (VH) genes of mouse BALB/c origin was determined. These two genes are highly homologous to each other. They both have the unusual codon CCT for proline at position 7, which so far has been found only in a specific set of VH genes, called the NPb family. We show that the two VH genes belong to this set. One of our BALB/c genes, VH124, is more homologous to a C57BL/6 NPb VH gene than to any BALB/c VH gene, and we propose that these two genes are alleles. A comparison of the substitutions between these two genes with published sequences of all other BALB/c and C57BL/6 NPb VH genes reveals evidence for past homologous recombination events between related germline VH genes Homologous recombination may play an important role in the diversification of germline immunoglobulin VH genes.