Nucleic acid structures and enzymes in the immunoglobulin class switch recombination mechanism

Class switch recombination is the gene rearrangement process by which our B lymphocytes change from IgM production to IgG, IgA, or IgE. Unlike the well-characterized V(D)J recombination, the mechanism of class switch recombination has been largely enigmatic until very recent progress has begun to shed light on this gene rearrangement process. Progress has been made on the enzymes involved in leading to the DNA cleavage events and on identifying the unusual DNA structures that those enzymes recognize.     

IL-4 induces the specific rearrangement of gamma 1 genes on the expressed and unexpressed chromosomes of lipopolysaccharide-activated normal murine B cells

Small, resting, surface IgM+/surface IgD+ murine B cells undergo an Ig class switch to IgG1 or IgE after stimulation with LPS and T cell supernatants containing IL-4. To firmly establish the role of IL-4 in the directed switch recombination observed in IgG1-secreting cells, we have 1) used highly purified native IL-4 instead of T cell supernatants, 2) used resting B cells from F1 mice in which the active IgH allele was determined before culture, 3) taken advantage of the allelic differences in the restriction fragment lengths of mu, gamma 1, gamma 2b, and gamma 3 loci to determine the status of the CH genes on both the expressed and unexpressed chromosomes, and 4) used different restriction enzymes to distinguish between deletion and rearrangement of a given CH gene. Our results indicate that LPS alone induces rearrangement of the gamma 3 genes on both chromosomes whereas stimulation with LPS plus IL-4 results in deletion of gamma 3 genes and rearrangement of gamma 1 genes on both chromosomes. The studies definitively establish the role of IL-4 in directed switch recombination to the gamma 1 locus in LPS-stimulated murine B cells.     

Kinetics of interleukin-4 induction and interferon-gamma inhibition of IgE secretion by Epstein-Barr virus-infected human peripheral blood B cells.

Interleukin-4 (IL-4) acts directly on purified human peripheral blood B cells cultured in the presence of Epstein-Barr virus (EBV) to induce IgE secretion and to enhance the secretion of IgG and IgM. Interferon-gamma (IFN-gamma) inhibits IgE secretion in this system, without affecting the secretion of the other Ig isotypes. To identify the time period during which EBV-infected B cells can be induced by IL-4 to secrete IgE, we have studied the effects of delayed addition of IL-4, or the termination of IL-4 stimulation by wash out or by neutralization with anti-IL-4 antibodies, on the induction of an IgE response. To induce a maximal IgE response, IL-4 had to be added to cultures of B cells plus EBV no later than 2 days after the initiation of culture, and had to remain present through the tenth day of culture. These two time points correspond to the initiation of detectable DNA synthesis (Days 3 to 4) and the earliest detectable Ig secretion (Days 10 to 12) by EBV-stimulated B cells. No IgE response was induced if the period during which EBV-stimulated B cells were cultured with IL-4 was less than 4 days, or if IL-4 were added later than the tenth day of culture, regardless of how long the culture was continued beyond that time. In contrast, IL-4 considerably enhanced IgG and IgM secretion and B cell CD23 expression, even if it was added after the tenth day of culture. IFN-gamma strongly inhibited the IgE response of B cells cultured with IL-4 plus EBV if added within 6 days of the initiation of culture, but had little effect on the generation of IgM or IgG responses made by these cells, regardless of the time of addition. Neither IL-4 nor IFN-gamma affected ongoing IgE secretion by an established, IgE-secreting, EBV-transformed cell line. These observations suggest that: (i) IL-4 first becomes able to induce EBV-activated B cells to secrete IgE as these cells begin to synthesize DNA, must stimulate B cells for at least 4 days to induce IgE secretion, and loses its ability to induce IgE secretion as these cells differentiate into Ig-secreting cells; (ii) the ability of IFN-gamma to suppress an IgE response is limited to this same time period; and (iii) IL-4 enhancement of CD23 expression and IgM and IgG secretion are independent of IL-4 induction of an IgE response.     

Unique sequences are interspersed among tandemly repeated elements in the murine gamma 1 switch segment.

Early in its differentiative pathway, a given B lymphocyte expresses immunoglobulin of the mu heavy chain class (IgM). Subsequent differentiative processes may involve rearrangement within the immunoglobulin heavy chain chromosomal locus to enable cells of the same lineage to synthesize immunoglobulins of other heavy chain classes (e. g. IgG, IgE or IgA), but with specificity for the same antigen as the original IgM molecule. Switch recombination, the molecular event which facilitates this chromosomal rearrangement, has been shown to occur between segments of DNA consisting of tandemly repeated unit sequences. These DNA segments have been functionally defined as switch regions. We have cloned the gamma 1 switch region from the BALB/c germline, and have demonstrated that significantly divergent sequence elements are interspersed among the tandemly repeated units characteristic of this switch region. We show that these unique elements exist in at least three copies within the switch segment, and discuss the implications of this novel and previously unreported primary structure.     

The role of rearrangement at the second Ig heavy chain locus in maintaining B cell tolerance to DNA

In recently generated B6.56R anti-DNA autoantibody-transgenic mice, it was noted that a substantial fraction of the B cells that had avoided DNA reactivity had done so through the rearrangement and usage of the endogenous, nontargeted H chain (HC) allele. This suggested that rearrangement at the second HC locus might be an important mechanism through which self-reactive B cells might successfully revise their initial Ag specificity. To test the importance of this mechanism in B cell tolerance, we generated B6.56R/56R mice that possessed the 56R anti-DNA H chain transgene inserted into both HC loci. These transgenic homozygotes developed higher titers of anti-DNA Abs, with an expanded population of B220(low)MHC class II(low) B cells, enriched for CD21(low)CD23(low) preplasmablasts. The analysis of hybridomas from these mice revealed that the only avenue by which these B cells could avoid DNA reactivity was through the use of the editor L chains, V(k)20 or V(k)21. Hence, in addition to LC editing, rearrangement and usage of the second HC locus/allele constitutes an important safety valve for B cells the primary BCR of which confers DNA reactivity. In contrast to these tolerance mechanisms, editing the first rearranged HC locus (through HC replacement) and somatic mutations appear to be less frequently used to edit/revise self-reactive B cells.     

Rapid loss of IgM expression by normal murine B cells undergoing IgG1 and IgE class switching after in vivo immunization

Injection of mice with polyclonal goat anti-mouse IgD antibody (G alpha M delta) stimulates a potent T cell-dependent immune response characterized by large increases in serum IgG1 and IgE concentrations and by the generation of substantial numbers of membrane (m)IgG1+ B cells. The onset of this response occurs 6 days after G alpha M delta injection and peaks by day 7 to 8. Utilizing two color fluorescence analysis and cell sorting we demonstrate that most mIgG1-expressing B cells lack mIgM during the period of onset of Ig isotype switching (day 6). Both IgG1 and IgE are produced predominantly by mIgM- cells. On day 6, IgG1 and IgE are secreted predominantly by cells expressing mIgG1 and mIgE, respectively. By day 8, a majority of the IgG1 secretion occurs among the mIgG1- cells but virtually all IgE secretion continues to come from the mIgE+ population. B cells that strongly express mIgG1 secrete little IgM or IgE. Freshly harvested B cells expressing mIgG1, 6 days after G alpha M delta injection, have undergone substantial deletion of CH mu-specific DNA in contrast to their mIgG1- counterparts. Hence, the great majority of B cells that switch to the IgG1 or IgE isotypes in vivo rapidly lose their expression of IgM.     

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.     

Induced kappa receptor editing shows no allelic preference in a mouse pre-B cell line

B cell Ag receptor editing is a process that can change kappa antigen recognition specificity of a B cell receptor through secondary gene rearrangements on the same allele. In this study we used a model mouse pre-B cell line (38B9) to examine factors that might affect allelic targeting of secondary rearrangements of the kappa locus. We isolated clones that showed both productive and nonproductive rearrangements of one kappa allele, while retaining the other kappa allele in the germline configuration (kappa(+)/kappa degrees or kappa(-)/kappa degrees ). In the absence of any selective pressures, subsequent rearrangement of the germline alleles occurred at the same frequency as secondary rearrangement of the productive or nonproductive rearranged alleles. Because 38B9 cells lack Ig heavy chains, we stably expressed mu heavy chain protein in 38B9 cells to determine whether heavy-light pairing might affect allelic targeting of secondary kappa rearrangements. Although the expression of heavy chain was found to both pair with and stabilize kappa protein in these cells, it had no effect on preferential targeting Vkappa-Jkappa receptor editing compared with rearrangement of a germline allele. These studies suggest that in the absence of selection to eliminate autoreactive Vkappa-Jkappa genes, there is no allelic preference for secondary rearrangement events in 38B9 cells.     

Partial IgA-deficiency with increased Th2-type cytokines in TGF-beta 1 knockout mice.

Though it has been shown that TGF-beta 1 directs B cells to switch to IgA in vitro, no studies have assessed TGF-beta 1 effects on mucosal vs systemic immunity in vivo. When the B cell functions of TGF-beta 1 gene-disrupted (TGF-beta 1-/-) mice were analyzed, significantly decreased IgA levels and increased IgG and IgM levels in serum and external secretions were observed. Further, analysis of Ab forming cells (AFC) isolated from both mucosal and systemic lymphoid tissue showed elevated IgM, IgG, and IgE, with decreased IgA AFC. A lack of IgA-committed B cells was seen in TGF-beta 1-/- mice, especially in the gastrointestinal (GI) tract. Splenic T cells triggered via the TCR expressed elevated Th2-type cytokines and, consistent with this observation, a 31-fold increase in serum IgE was seen in TGF-beta 1-/- mice. Thus, uncontrolled B cell responses, which include elevated IgE levels, a lack of antiinflammatory IgA, and an excess of complement-binding IgG and IgM Abs, will promote inflammation at mucosal surfaces in TGF-beta 1-/- mice and likely contribute to pulmonary and GI tract lesions, ultimately leading to the early death of these mice.     

Tolerance to DNA in (NZB x NZW)F1 mice that inherit an anti-DNA V(H) as a conventional micro H chain transgene but not as a V(H) knock-in transgene

Lupus-prone (NZB x NZW)F(1) (BWF(1)) mice were made transgenic (Tg) for an anti-DNA Ab inherited either as a conventional V(H)3H9- micro H chain Tg (3H9- micro ) with or without a conventional V(kappa)8-kappa Tg, or a V(H)3H9 V(H) knock-in Tg allele (3H9R) with or without a V(kappa)4 V(kappa) knock-in Tg allele (V(kappa)4R). V(H)3H9 yields an anti-DNA Ab with most L chains including an anti-ssDNA with the V(kappa)8 Tg and an anti-dsDNA with the V(kappa)4 Tg. BWF(1) mice that inherited the conventional 3H9- micro had normal serum IgM, little to none of which was encoded by 3H9- micro, and only a small percentage of those mice had serum anti-DNA, none of which was transgene encoded. B cells expressing the conventional 3H9- micro Tg were anergic. BWF(1) mice that inherited the knock-in 3H9R Tg allele also had normal serum IgM, one-half of which was encoded by 3H9R, and produced anti-DNA encoded by the Tg allele. Most B cells expressing the knock-in 3H9R Tg also had an anergic phenotype. The results indicate that autoimmune-prone BWF(1) mice initially develop effective B cell tolerance to DNA through anergy, and anergy was sustained in 3H9- micro Tg peripheral B cells but not in 3H9R Tg B cells. B cells expressing the 3H9R knock-in Tg allele were able to achieve an activation threshold that B cells expressing the 3H9- micro conventional Tg could not. The maintenance of B cell tolerance to DNA in autoimmune-prone BWF(1) mice appears to differ from both normal mice and autoimmune-prone MRL(lpr/lpr) mice.     

Expression of v-rel induces mature B-cell lines that reflect the diversity of avian immunoglobulin heavy- and light-chain rearrangements.

The infection of newly hatched chickens with reticuloendotheliosis virus strain T (REV-T) and a nonimmunosuppressive helper virus, chicken syncytial virus, induces rapidly metastatic B-cell lymphomas. In vivo analysis of these tumors with monoclonal antibodies detected the expression of the B-cell surface markers immunoglobulin M (IgM), CIa, Bu2, and CLA-1, but not IgG, Bu1, or a T-cell surface marker, CT-1. Cell lines derived from tumors exhibited the same pattern of staining, suggesting that expression of cell surface markers does not change during in vitro cell line development. All cell lines examined synthesized IgM in varying amounts. Northern (RNA blot) analysis confirmed abundant expression of v-rel mRNA, and Southern analysis revealed rearrangement of both heavy- and light-chain immunoglobulin loci. Analysis of the light-chain locus demonstrated that 20 of 22 lines contained a single rearranged allele. With respect to specific restriction enzyme sites within the V lambda 1 gene, the active allele in any given clone was either diversified or nondiversified. In contrast, examination of the heavy-chain loci within these lines demonstrated that 16 of the 22 had both alleles rearranged. Further diversification of the V lambda 1 locus did not occur after prolonged in vitro passage of the cell lines. We propose that v-rel expression arrests diversification of the light-chain locus in these lymphoid cells, allowing the production of stable, clonal B-cell populations. The development of these and similar cell lines will make it possible to identify specific stages of avian lymphoid ontogeny and to study the mechanism of rearrangement and diversification in the avian B lymphocyte.     

Frequency and characterization of phenotypic Ig heavy chain allelically included IgM-expressing B cells in mice

Ig H chain (IgH) allelic exclusion remains a puzzling topic. Here, we address the following question: Do phenotypic IgH allelically included cells exist in normal mice and, if so, at what frequency? Sorted cells from heterozygous mice were evaluated for the expression of both IgM allotypes by double intracytoplasmic stainings. Dual expressors were found at a frequency of 1 in 104 splenic B cells. These data were confirmed by direct sequencing of IgH-rearranged alleles obtained after single cell (or clone) PCR on dual expressors. Typically, these cells have one rearranged J558 VH whereas, in the other allele, a D-proximal VH gene is used. Interestingly, dual expressors have rearranged IgH alleles with similar CDR3 lengths. These results show that, in contrast to the kappa L chain and the TCR beta-chain, IgH allelic exclusion is the result of an extremely stringent mechanism. We discuss two non-mutually exclusive scenarios for the origin of IgH dual expressors: 1) IgH allelically included cells arise when the first allele to rearrange productively is unable to form a pre-BCR; dual expressors could be a subset of this population in which, upon conventional L chain rearrangement, both IgH are expressed at the surface; and 2) synchronous rearrangement of the IgH alleles.     

Detection of True IgE-expressing Mouse B Lineage Cells

B lymphocyte immunoglobulin heavy chain (IgH) class switch recombination (CSR) is a process wherein initially expressed IgM switches to other IgH isotypes, such as IgA, IgE and IgG. Measurement of IgH CSR in vitro is a key method for the study of a number of biologic processes ranging from DNA recombination and repair to aspects of molecular and cellular immunology. In vitro CSR assay involves the flow cytometric measurement surface Ig expression on activated B cells. While measurement of IgA and IgG subclasses is straightforward, measurement of IgE by this method is problematic due to soluble IgE binding to FcεRII/CD23 expressed on the surface of activated B cells. Here we describe a unique procedure for accurate measurement of IgE-producing mouse B cells that have undergone CSR in culture. The method is based on trypsin-mediated cleavage of IgE-CD23 complexes on cell surfaces, allowing for detection of IgE-producing B lineage cells by cytoplasmic staining. This procedure offers a convenient solution for flow cytometric analysis of CSR to IgE.     

Antigen receptor editing in anti-DNA transitional B cells deficient for surface IgM

In response to encounter with self-Ag, autoreactive B cells may undergo secondary L chain gene rearrangement (receptor editing) and change the specificity of their Ag receptor. Knowing at what differentiative stage(s) developing B cells undergo receptor editing is important for understanding how self-reactive B cells are regulated. In this study, in mice with Ig transgenes coding for anti-self (DNA) Ab, we report dsDNA breaks indicative of ongoing secondary L chain rearrangement not only in bone marrow cells with a pre-B/B cell phenotype but also in immature/transitional splenic B cells with little or no surface IgM (sIgM(-/low)). L chain-edited transgenic B cells were detectable in spleen but not bone marrow and were still found to produce Ab specific for DNA (and apoptotic cells), albeit with lower affinity for DNA than the unedited transgenic Ab. We conclude that L chain editing in anti-DNA-transgenic B cells is not only ongoing in bone marrow but also in spleen. Indeed, transfer of sIgM(-/low) anti-DNA splenic B cells into SCID mice resulted in the appearance of a L chain editor (Vlambdax) in the serum of engrafted recipients. Finally, we also report evidence for ongoing L chain editing in sIgM(low) transitional splenic B cells of wild-type mice.     

Both naive and memory T cells can provide help for human IgE production, but with different cytokine requirements.

Most in vitro systems for the induction of IgE production by human B cells require both IL-4 and the presence of T cells. Little is known about the mechanism of T cell help or the ability of different T cell subsets to provide this helper activity. In the present study we demonstrate that, in the presence of exogenous IL-4, anti-CD3 stimulated naive T cells (CD4+CD45RA+) are potent helper cells for human IgE production. In their presence, as little as 750 autologous B cells can produce up to 100 ng/ml IgE. This response was found over a broad range of anti-CD3 concentrations. IgE helper activity by naive T cells was inhibited by IL-2. Under all conditions tested, naive T cells were unable to provide help for IgM production. This is in contrast to activated memory T cells (CD4+CD45RO+), which are very efficient helper cells for IgM or IgE production, provided that IL-2 or IL-2 plus IL-4 are present respectively.