Constant Region Gene Segments Research Articles

Transgenic mice whose γ 1 and κ genes have been replaced by human ε and κ genes (HYP3P.402)

Abstract IgE is a primary mediator in most allergic diseases. Human IgE specific for various allergens are very important reagents in diagnostic assays for the management of allergic diseases. However, allergen-specific human IgEs are present in patients’ blood at very low levels and hence extremely difficult to prepare. We have now constructed a transgenic homozygous mouse strain, referred to as HεκKI strain, whose γ1 constant region gene segment has been replaced by human ε gene constant region segment and whose κ constant gene replaced by human κ constant gene. In those HεκKI mice, the human IgE levels are about 10 times of mouse IgE. Upon immunization with an allergen papain, both human IgE and mouse IgE were increased, with human IgE increased to about 30 folds of mouse IgE. We have prepared a number of hybridomas secreting monoclonal human IgE (the variable regions being mouse) specific for papain. Cells of a basophilic leukemic line pulsed with such human IgE could be activated by papain to undergo degranulation process. These results indicate that the transgenic mouse strain HεκKI could be employed to prepare polyclonal and monoclonal human IgE specific for various allergens.

Tracking germinal center B cells expressing germ-line immunoglobulin γ1 transcripts by conditional gene targeting

Germinal centers (GCs) represent the main sites for the generation of high-affinity, class-switched antibodies during T cell-dependent antibody responses. To study gene function specifically in GC B cells, we generated Cgamma1-cre mice in which the expression of Cre recombinase is induced by transcription of the Ig gamma1 constant region gene segment (Cgamma1). In these mice, Cre-mediated recombination at the fas, Igbeta, IgH, and Rosa26 loci occurred in GC B cells as early as 4 days after immunization with T cell-dependent antigens and involved >85% of GC B cells at the peak of the GC reaction. Less than 2% of IgM(+) B cells showed Cre-mediated recombination. These cells carried few Ig somatic mutations, expressed germ-line Cgamma1- and activation-induced cytidine deaminase-specific transcripts and likely include GC B cell founders and/or plasma cell precursors. Cre-mediated recombination involved most IgG1, but also a fraction of IgG3-, IgG2a-, IgG2b-, and IgA-expressing GC and post-GC B cells. This result indicates that a GC B cell can transcribe more than one downstream C(H) gene before undergoing class switch recombination. The efficient induction of Cre expression in GC B cells makes the Cgamma1-cre allele a powerful tool for the genetic analysis of these cells, as well as, in combination with a suitable marker for Cre-mediated recombination, the tracking of class-switched memory B and plasma cells in vivo. To expedite the genetic analysis of GC B cells, we have established Cgamma1-cre F(1) embryonic stem cells, allowing further rounds of gene targeting and the cloning of compound mutants by tetraploid embryo complementation.

Absence of Immunoglobulin Class Switch in Primary Lymphomas of the Central Nervous System

Primary lymphomas of the central nervous system (PCNSLs) were investigated for their capacity to perform further maturation steps. We studied a series of 11 PCNSLs derived from immunocompetent patients for immunoglobulin (Ig) class switch recombination (CSR) by performing reverse transcriptase-polymerase chain reaction (RT-PCR) for transcripts of Ig constant region gene segments (IGHC). This analysis revealed exclusive transcription of IgM and IgD mRNA in the absence of IgG, IgA, or IgE transcription. This finding was corroborated at the protein level by the immunohistochemical demonstration of IgM on the surface of the tumor cells. The unexpected lack of CSR may be due to internal switch mu region deletions, which were detected in 7 of 11 cases. We also found that expression of activation-induced cytidine deaminase (AID), which is required for CSR and somatic hypermutation, was detectable by RT-PCR in 4 of 10 cases and by immunohistochemistry in one of three cases analyzed. This may indicate that ongoing somatic mutation, which is often observed in PCNSL, could be due to sustained AID expression in a fraction of cases and that intraclonal V gene diversity may occur in other cases at an earlier phase of tumor clone expansion, when AID may have been expressed.

Identification of a novel substitution in the constant region of a gene coding for an amyloidogenic κ 1 light chain

Current concepts regarding the association between immunoglobulin (Ig) light chain structure and AL amyloidosis (AL) emphasize Ig variable region amino acid substitutions because the majority of light chain amyloid fibrils that have been sequenced contain amino termini of the variable region with only small amounts of the constant region. In this report, we describe a patient with rapidly progressive AL whose amyloid deposits contained primarily monoclonal κ light chain constant region fragments. We sequenced and analyzed this AL protein, determining that it was an O18-O8 κ 1 variant and that the constant region possessed an unusual Ser→Asn substitution at position 177. Using pre-mortem bone marrow cells, we cloned and sequenced the cDNA for this AL protein (HCAK1) and, using DNA from post-mortem somatic tissue, we cloned and sequenced the patient’s κ germline O18-O8 donor and κ constant region ( Cκ) gene segments. The cDNA that coded for HCAK1 contained a variable region that was derived from O18-O8, showing 96.1% homology to germline, and a Cκ that had a nucleotide substitution (A G C to A A C), resulting in the 177 Ser→Asn replacement. Two Cκ genes were cloned from somatic tissue DNA, one identical to a known Cκ sequence and another containing this substitution which likely is a new Cκ allotype. Our findings indicate that further investigation is warranted into the contributions genetic polymorphisms and light chain constant regions may make to amyloidogenesis.

An extrachromosomal switch recombination substrate reveals kinetics and substrate requirements of switch recombination in primary murine B cells.

Ig class switch recombination occurs in B lymphocytes upon activation, and is targeted to distinct switch (S) regions by cytokine-mediated induction of switch transcripts spanning the entire S region and the adjacent constant region gene segments. Using a novel type of switch recombination substrate, constructed according to the intron-exon structure of the IgH locus, but with heterologous elements, we here have tested the structural requirements for targeting and the kinetics of switch recombination in activated primary murine B cells. When transfected at various times after activation, up to 10% of the transfected B cells perform recombination of the substrate within 12 h. Switch recombination in primary B cells is restricted to the first 72 h after onset of activation, then rapidly decreases to background levels, as obtained in plasmacytoma cells or with substrates carrying no S region sequences. In terms of structural requirements, switch recombination is targeted to any transcription unit that contains an intronic S region and depends on processing of the primary transcript by splicing.

Quantitative assessment of the human TCRBV repertoire by competitive PCR

A novel quantitative protocol was developed for the measurement of the relative expression levels of the human TCRBV genes based on reverse transcription (RT) and subsequent competitive polymerase chain reaction (cPCR). Competitor DNA templates for the analysis of 24 different TCRBV families were generated by a simple and rapid one-step PCR procedure with a special PCR primer, which introduces a deletion in the constant region gene segment. A defined amount of TCRBV family-specific competitor DNA and the reverse transcribed cDNA of interest were amplified in the same tube with the same primer pair in a competitive way. The resulting fragments were separated on agarose gels and the densitometrical values were evaluated directly without the requirement for additional hybridization steps. For all of the 24 different TCRBV family-specific cPCRs equal amplification efficiencies were demonstrated by titration experiments for wild-type and competitor templates. For TCRBV repertoire studies, a short form of the cPCR assay was performed, requiring only one cPCR for quantitation of each TCRBV family. The exact initial amount of wild-type template in each cPCR was interpolated from TCRBV family-specific reference calibration curves. The RT-cPCR assay was applied for the quantitative assessment of the TCRBV repertoire of CD4+ and CD8+ T cell subsets in unstimulated PBMC and compared to flow cytometric analyses with a panel of monoclonal antibodies specific for TCRBV determinants. The RT-cPCR experiments revealed a differential expression of several BV families in either the CD4+ or the CD8+ fraction. The TCRBV family-specific cPCR assay presented here combines the simplicity and speed of conventional TCRBV family-specific PCR with the quantitative features of competitive PCR. TCRBV family-specific RT-cPCR has a broad application for all kinds of quantitative T cell repertoire studies and could be easily adapted for the usage with different BV-specific primer sets.

Class switching in human immunoglobulin transgenic mice.

We have introduced human germline-configuration heavy and kappa light chain minilocus transgenes into mice that have been engineered so that their endogenous heavy and kappa light chain loci are inactive. The two human transgenes are inserted by pronuclear microinjection, while the two endogenous mouse genes are disrupted by homologous recombination in embryonic stem cells. The resulting animals contain four unlinked genetic modifications and must rely on the introduced transgenes for the development of the B-cell lineage and for the generation of an antibody repertoire. The heavy chain transgene includes both the human mu and the human gamma 1 constant region gene segments, as well as upstream switch region sequences. Although mouse B cells and human B cells exhibit species-specific differences in the induction of gamma isotype expression, the transgenic mouse B cells appear to undergo regulated switching to human gamma 1 both in vivo and in vitro. This observation defines a subset of the heavy chain constant region that is sufficient for class switching, and implies that the human gamma 1 switch region includes a core of sequence that is functionally homologous to those cis-acting regulatory elements that direct mouse gamma switching.

The organization of the human immunoglobulin lambda gene locus.

To elucidate the complex structure of the human immunoglobulin lambda gene locus, a 1020-kb contig was constructed using 184 cosmid clones and one bacterial artificial chromosome (BAC) clone. A high-resolution physical map of this contig revealed that the entire lambda gene locus is 911 kb in length. It contains seven constant region (C lambda) gene segments and 69 unique EcoRI-HindIII segments that hybridize to variable region gene (V lambda) probes. The VpreB gene, BCRL4, and gamma-glutamyl transpeptidase gene (GGT)-like sequences are also located within the lambda gene locus. Hybridization analysis suggested that the lambda gene locus has undergone extensive amplification events in evolution.

B Lymphocytes Are Not Required for Murine Resistance to the Human Filarial Parasite, Brugia malayi

Immunocompetent mice are resistant to the growth and development of human lymphatic filarial parasites, including the aperiodic strain of Brugia malayi. We have recently established that mice homozygous for the severe combined immunodeficiency (scid) mutation, and therefore deficient in both T and B lymphocytes, are permissive for infection. This observation suggests that components of the adaptive (antigen-specific) immune system are obligate requirements for murine resistance to B. malayi. In order to determine more precisely the component of the immune system that mediates murine resistance to B. malayi, we have used mouse strains in which individual genes involved in the maturation of specific components of the immune system have been disrupted by homologous recombination. In previous studies, we demonstrated that mice that lack either major histocompatibility (MHC) class I restricted, CD8+ T lymphocytes (beta 2-microglobulin knockout mice; beta 2M-/-) or CD4+ T lymphocytes (CD4 knockout mice; CD4-/-) are as resistant to B. malayi as intact mice. In the current study, we have used mice in which the membrane exon of the immunoglobulin (Ig) mu (heavy chain) constant region gene segment has been disrupted by homologous recombination. These mice cannot develop mature B lymphocytes and lack serum Ig. We demonstrate that such "B-less" mice are completely resistant to B. malayi. These data, taken in combination with the observation that T-cell-deficient athymic mice homozygous for the nu (nude) mutation are fully permissive for infection, suggest that B lymphocytes and their products are neither required nor sufficient to mediate resistance to B. malayi.

Relative size and evolution of the germline repertoire of T-cell receptor β-chain gene segments in nonhuman primates

The mammalian T-cell receptor (TCR) gene complexes exist as multiple tandemly arrayed gene segments that have apparently arisen by gene duplication mechanisms. A study of the number of TCR germline gene segments in several primate species might provide insight into the relative rate and patterns of gene duplication and deletion within these gene complexes. DNA probes from the TCR β-chain variable (TCRBV) region gene segment subfamilies 1 through 25 and the constant region gene segment were sequentially hybridized under low stringency to Southern blots containing genomic DNA of human, gorilla, orangutan, and pig-tailed macaque. The number of gene members in each subfamily was estimated from the number of hybridizing DNA fragments. The results show apparent examples of both TCRB V gene duplication and deletion since speciation of the Hominoids from Cercopithecoid (Old World) primates. For one putative duplication/deletion event involving six TCRBV gene segments, derivation and comparison of germline DNA sequence from macaque and human as well as Southern blot analysis of additional primates demonstrated that this event was a duplication that occurred after the divergence of the family Pongidae (Greater Apes) from Hylobatidae (Lesser Apes). Southern blot analysis of multiple pig-tailed macaques and their offspring suggests a degree of DNA sequence variability in these gene segments similar to that observed in humans. An appreciation of the size and variability of each TCRBV subfamily will be useful when considering the DNA primers and probes necessary to measure the relative usage of these TCRBV genes as part of the immune response in these nonhuman primates.

Switch recombination in normal IgA1+ B lymphocytes.

Most B lymphocytes in normal individuals express two classes of cell-surface immunoglobulins, IgM and IgD. The specificity of the two antigen receptors is identical since they are produced by transcription and differential splicing of the same variable region gene segment to the heavy-chain constant region gene segments for both mu and delta heavy chains. B lymphocytes expressing other immunoglobulin isotypes, IgG, IgA, or IgE, are rare and not well characterized. Particularly controversial is the molecular mechanism of their isotype switch. Here we use high-gradient magnetic cell sorting and fluorescence-activated cell sorting to purify surface IgA1-bearing B lymphocytes from human blood for cellular and molecular analysis. These cells express no immunoglobulin class other than IgA1 and are a relatively uniform population with regard to expression of other cell-surface molecules. They are resting cells in terms of cell cycle and activation marker analysis. The molecular basis for class switching in the IgA1+ cells is not differential transcription or splicing. Rather, switch recombination involving deletion of DNA has occurred on both immunoglobulin heavy-chain gene loci, including the allelically excluded one, and appears to have been directed to IgA1 under normal physiological conditions.

Identification of a secondary Fc gamma RI binding site within a genetically engineered human IgG antibody.

Although human IgG2 is not cytophilic, we have shown previously that an IgG2 antibody expressing the sequence PLLGG (underline = substitution) spanning CH2 domain residues 233-237 (Eu numbering) displayed IgG1-like Fc gamma RI binding activity. In contrast, IgG1 PLLGG exhibited 3-fold less affinity, whereas IgG2 ELLGG was 3-fold more active than native IgG1. These results suggested that additional site(s) conferred enhanced binding properties to the engineered, cytophilic IgG2 variant. These sites were shown to reside in the IgG2 CH2 domain, since the IgG1 CH2 module did not have enhanced activity in a panel of hybrid IgG1/IgG2 antibodies. To map these sites further, human IgG1 and IgG2 constant region gene segments were modified to allow reciprocal COOH-terminal half segment exchanges of CH2 exons. These were cloned into a pSV2neo expression vector bearing a rearranged MOPC 315 heavy chain variable region gene and transfected into a MOPC 315 heavy chain deletion mutant. The dinitrophenol affinity-purified IgGs were radiolabeled and assessed for Fc gamma RI binding activity in direct binding assays using U937 cells. The COOH terminus of the IgG2 CH2 domain was found to contain accessory site(s) since it enhanced the binding properties of both IgG1 PLLGG and native IgG1. In contrast, grafting of the COOH terminus of the IgG1 CH2 domain onto IgG2 PLLGG and IgG2 ELLGG diminished their cytophilic activity. The amino acid responsible for the enhancing properties of the COOH terminus of the IgG2 CH2 domain was shown to be threonine 339, since IgG1 PLLGG/Thr339 displayed increased Fc gamma RI binding affinity. Kinetics studies revealed that this is accomplished through an increase in the forward rate constant of the IgG-Fc gamma RI interaction.

Divergent evolution of T cell repertoires: extensive diversity and developmentally regulated expression of the sheep gamma delta T cell receptor.

Sheep gamma delta T cells express an unprecedented repertoire of antigen receptors contributed by increased diversity in both variable and constant region gene segments. Variable region diversity results mainly from the utilization of a large family of duplicated V delta genes that have retained two distinct hypervariable segments comparable with the complementarity determining regions present in other antigen receptor V genes. This implies that sheep V delta chains have been intensely selected during evolution, probably at sites involved in ligand recognition. The sheep gamma delta heterodimer occurs in at least five isotypic variants formed by the association of a single C delta segment with one of five functional C gamma segments, each with distinctive hinge regions. Our analysis also shows that the establishment of a normal peripheral repertoire is both developmentally regulated and dependent on the continual presence of a functional thymus during ontogeny. The existence of an expanded V gene repertoire and multiple receptor isotypes together with the prominence of gamma delta T cells in the sheep immune system argues that this lineage of T cells has a more elaborate functional role in this evolutionary pathway.

Involvement of both HLA and Ig heavy chain haplotypes in human IgA deficiency.

Immunoglobulin-A deficiency (IgA-D) is the most common human Ig class deficiency with an estimated frequency of approximately 1 in 500 in the Swedish population. We investigated the immunoglobulin heavy chain constant region gene segments (IGHC) in 103 individuals with IgA-D and the immunoglobulin heavy chain variable region gene segments (IGHV) in 20 of these, in order to identify a possible molecular basis of the defect. No deletions of IGHV gene segments of the VH2, VH5, and VH6 families or the IGHG genes were observed. In the IGHC, there were, however, differences in the restriction fragment length polymorphism frequencies of IGHG genes where the Bam HI haplotype "H2" [IGHGP, 10 kilobases (kb), IGHG2, 25 kb; and IGHG4, 9.0 kb] was overrepresented. The mean serum levels of IgG4 and IgE were significantly lower in individuals (both IgA-D subjects and healthy controls) homozygous for the H2 haplotype than in individuals homozygous for the H1 haplotype (IGHGP, 8.8 kb, IGHG2, 13.5 kb, and IGHG4, 9.4 kb). IgA-D subjects homozygous for HLA DQB1*0201 (DQw2), a marker that has previously been reported to show a strong association with IgA deficiency, showed a similar reduction of serum levels of IgG4 and IgE as compared with DQB1*0201 negative IgA-D subjects. These findings suggest that the two loci found to be associated with IgA deficiency may act via a common pathway.

An enhancer at the 3′ end of the mouse immunoglobulin heavy chain locus

A tissue-specific enhancer (E mu) lies between the joining (JH) and mu constant region (C mu) gene segments of the immunoglobulin heavy chain (IgH) locus. Since mouse endogenous IgH genes are efficiently transcribed in its absence, the normal function of this enhancer remains ill-defined. Recently, another lymphoid-specific enhancer of equal strength has been identified 3' of the rat IgH locus. We have isolated an analogous sequence from mouse and have mapped it 12.5 kb 3' of the 3'-most constant region gene (C alpha-membrane) of the BALB/c mouse locus. The mouse and rat sequences are 82% homologous and share with other enhancers several DNA sequence motifs capable of binding protein. However, in transient transfection assays, the mouse sequence behaves as a weaker enhancer. The role of this distant element in the expression of endogenous IgH genes, both in E mu-deficient, Ig-producing cell lines and during normal B cell development, is discussed.

The T alpha 2 nuclear protein binding site from the human T cell receptor alpha enhancer functions as both a T cell-specific transcriptional activator and repressor.

T cell-specific expression of the human T cell receptor alpha (TCR-alpha) gene is regulated by the interaction of variable region promoter elements with a transcriptional enhancer that is located 4.5 kb 3' of the TCR-alpha constant region (C alpha) gene segment. The minimal TCR-alpha enhancer is composed of two nuclear protein binding sites, T alpha 1 and T alpha 2, that are both required for the T cell-specific activity of the enhancer. The T alpha 1 binding site contains a consensus cAMP response element (CRE), and binds a set of ubiquitous nuclear proteins. The T alpha 2 binding site does not contain known transcriptional enhancer motifs. However, it binds at least two nuclear protein complexes, one of which is T cell specific. We now report that although the T alpha 2 nuclear protein binding site displays transcriptional activator activity in the context of the TCR-alpha enhancer, this site alone can function as a potent, T cell-specific transcriptional repressor when positioned either upstream, or downstream of several heterologous promoter and enhancer elements. These results demonstrate that a single nuclear protein binding site can function as a T cell-specific transcriptional activator or repressor element, depending upon the context in which it is located.

Patterns and extent of isotype-specificity in the murine H chain switch DNA rearrangement.

We have analyzed the configuration of the H chain locus of 41 hybridomas by Southern blot analysis. Each H chain switch region was determined to be germ line, rearranged, or deleted. Including 13 previously analyzed hybridomas, 60% of those with rearrangements on both alleles showed a correlation of the two alleles, i.e., both the expressed and the nonexpressed alleles have rearranged to the same H chain constant region gene segment. When the two H chain alleles did not rearrange to the same gene, they often rearranged to neighboring H chain genes. These results support a role for isotype-specific factors in H chain switch recombination. The action of these isotype-specific factors may be propagated to some extent along the chromosome, which would lead to rearrangements to neighboring genes.

Inheritance of a Large Deletion within the Human Immunoglobulin Heavy Chain Constant Region Gene Complex and Immunological Implications

A deletion of the immunoglobulin heavy chain (IgH) pseudo-gamma, gamma-2, gamma-4, epsilon, and alpha-2 constant region gene segments was found to segregate unchanged in three generations of a family. The IgG1 locus on the IgH allele carrying the deletion was expressed to the same extent as its normal counterpart. One individual who was heterozygous for the deletion had an IgG2 deficiency, whereas the four other heterozygous individuals had serum levels of IgG2 and IgG4 within the normal ranges. IgA2 levels were low or below the normal range in all heterozygous individuals. The data indicate that the expression of some Ig isotypes can be decreased by hemizygous deletions, possibly due to a lower probability for switching.

Translation affects immunoglobulin mRNA stability

When termination codons were introduced into exons of the gene for Ig mu chain, steady-state levels of mu mRNA were reduced, both at the pre-B cell stage and at the plasma cell stage. A termination codon in the variable region gene segment and a termination codon in the second exon of the constant region gene segment had effects of similar magnitude. When the termination codon was deleted, the original level of mRNA was restored. The rate of mu gene transcription was the same whether or not a termination codon was present. Therefore, the termination codons must reduce the amount of the mRNA by reducing its stability. Since the introduced termination codons prematurely terminate translation and, in so doing, change the ribosome load on the mRNA, we conclude that mu mRNA stability is conferred in part by ribosomal protection from enzymatic degradation. We propose that the differences in mu mRNA stability during B lymphocyte differentiation are due to different amounts of ribosomes available for translation.

Diverse organization of immunoglobulin VH gene loci in a primitive vertebrate.

The immunoglobulin (Ig) heavy chain variable (VH) gene family of Heterodontus francisci (horned shark), a phylogenetically distant vertebrate, is unique in that VH, diversity (DH), joining (JH) and constant region (CH) gene segments are linked closely, in multiple individual clusters. The V regions of 12 genomic (liver and gonad) DNA clones have been sequenced completely and three organization patterns are evident: (i) VH-D1-D2-JH-CH with unique 12/22 and 12/12 spacers in the respective D recombination signal sequences (RSSs); VH and JH segments have 23 nucleotide (nt) spacers, (ii) VHDH-JH-CH, an unusual germline configuration with joined VH and DH segments and (iii) VHDHJH-CH, with all segmental elements being joined. The latter two configurations do not appear to be pseudogenes. Another VH-D1-D2-JH-CH gene possesses a D1 segment that is flanked by RSSs with 12 nt spacers and a D2 segment with 22/12 spacers. Based on the comparison of spleen, VH+ cDNA sequences to a germline consensus, it is evident that both DH segments as well as junctional and N-type diversity account for Ig variability. In this early vertebrate, the Ig genes share unique properties with higher vertebrate T-cell receptor as well as with Ig and may reflect the structure of a common ancestral antigen binding receptor gene.