Reversible switching of immunoglobulin hypermutation machinery in a chicken B cell line

A chicken B lymphoma line, DT40, hypermutates immunoglobulin (Ig) genes spontaneously during culture. Thus, cultured DT 40 cells constitute a useful Ig library for screening antibodies (Abs) in vitro. To fix desirable Ig mutants by stopping hypermutation or to resume mutation for further improvement of Ab affinity, activation-induced cytidine deaminase (AID), a key enzyme responsible for the Ig mutation machinery, must be switched on or off. To this end, we generated a DT40 line whose one AID allele was disrupted, and the other allele was replaced by the loxP-flanked AID construct. In this engineered cell line designated as DT40-SW, AID expression could be switched reversibly by tamoxifen-regulated Cre recombinase. Devices were also introduced to discriminate between the "AID-ON" and the "AID-OFF" cells by GFP expression and puromycin resistance, respectively. Starting from a single DT40-SW cell, Ig gene repertoire was efficiently diversified during culture only when AID expression was on.     

Induction of isotype switching and Ig production by CD5+ and CD10+ human fetal B cells.

In the present study the capacity of early fetal B cells to produce Ig was investigated. It is shown that B cells from fetal liver, spleen, and bone marrow (BM) can be induced to produce IgM, IgG, IgG4, and IgE, but not IgA, in response to IL-4 in the presence of anti-CD40 mAb or cloned CD4+ T cells. Even splenic B cells from a human fetus of only 12 wk of gestation produced these Ig isotypes. IFN-alpha, IFN-gamma, and transforming growth factor-beta inhibited IL-4-induced IgE production in fetal B cells, as described for mature B cells. The majority of B cells in fetal spleen expressed CD5 and CD10 and greater than 99% of B cells in fetal BM were CD10+. Highly purified CD10+, CD19+ immature B cells and CD5+, CD19+ B cells could be induced to produce Ig, including IgG4 and IgE, in similar amounts as unseparated CD19+ B cells. Virtually all CD19+ cells still expressed CD10 after 12 days of culture. However, the IgE-producing cells at the end of the culture period were found in the CD19-,CD10- cell population, suggesting differentiation of CD19+,CD10+ B cells into CD19-,CD10- plasma cells. Pre-B cells are characterized by their lack of expression of surface IgM (sIgM). Only 30 to 40% of BM B cells expressed sIgM. However, in contrast to sIgM+,CD10+,CD19+ immature B cells, sorted sIgM-,CD10+,CD19+ pre-B cells failed to differentiate into Ig-secreting cells under the present culture conditions. Addition of IL-6 to these cultures was ineffective. Taken together, these results indicate that fetal CD5+ and CD10+ B cells are mature in their capacity to be induced to Ig isotype switching in vitro as soon as they express sIgM.     

Induction of secretion of IgM from cells of the B cell line 38c-13 by somatic cell hybridization.

Cells of the murine B cell line 38C-13 possess immunoglobulins of the IgM class on their surface but do not secrete them. Upon hybridization of 38C-13 cells with murine myeloma cells, hybridoma clones were obtained that secreted both pentameric IgM of 38C-13 origin and the myeloma protein. All hybridoma clones synthesized and secreted large amounts of homogeneous IgM with a half disappearance time of about 2 hr, typical of mature plasma cells. Concomitantly with the induction of IgM secretion, the hybridoma cells lost their surface IgM. The possibility of separate pathways for the synthesis of membrane and secreted IgM is discussed.     

B cell development leads off with a base hit: dU:dG mismatches in class switching and hypermutation

The mechanisms underlying somatic hypermutation (SHM) and class switch recombination (CSR) have been the subject of much debate. Recent studies from the Neuberger and Honjo labs have lent insight into these distinct processes, and we discuss a new, comprehensive model for how AID, uracil DNA glycosylase (UNG) and the mismatch repair system function in both SHM and CSR.     

Lack of detectable somatic hypermutation in the V region of the Ig H chain gene of a human chronic B lymphocytic leukemia

Monoclonal human B cell tumors are a model system for the study of somatic hypermutation of the Ig genes of humans. It was previously shown that a number of B cell lymphomas exhibited striking V region point mutation, hypothesized to result from the somatic hypermutation mechanism. In this study we have extended the analysis to chronic lymphocytic leukemia. We have cloned and sequenced the productive Vh representing five different cells from a monoclonal chronic lymphocytic leukemia. All five Vh sequences were identical. Therefore, the Vh region in this leukemia was not the subject of detectable somatic mutation. These data suggest that chronic lymphocytic leukemia might lack the mechanism for somatic hypermutation and represent a stage of normal B lymphocyte differentiation in which the somatic hypermutation mechanism is not active.     

Differential endocytosis of IgM and IgD in murine B cell lines

The internalization of surface immunoglobulin (Ig) by B lymphocytes is the first step in the antigen-presenting function performed by these cells. Mature B cells coexpress on their surface IgM and IgD. At this time, there is controversy over whether these two isotypes serve different functions in the antigen-presenting process. The results presented here show that the intracellular pattern of distribution of IgM and IgD after internalization is strikingly different in the B cell lines studied. These findings support the hypothesis that the role of the two Ig classes in the antigen-presenting function may be different.     

B cell clonal expansion and somatic hypermutation of Ig variable heavy chain genes in the synovial membrane of patients with osteoarthritis

Inflammatory mediators have been explored as possible factors in the initiation and/or progression of osteoarthritis (OA). This study shows that synovial infiltration by B lymphocytes is present in almost half of the knee OA cases. The degree of B lymphocyte infiltration is associated with more pronounced synovial inflammation and with the presence of plasma cells and lymphoid follicles in more severe cases. To examine whether these B cells are merely bystanders or could be involved in the pathogenesis of OA, we analyzed the Ig H chain variable region (V(H)) genes of B cells recovered from the synovial membrane of five OA patients with marked B cell infiltration. Sequence analysis of CDR3 regions of rearranged VDJ genes revealed clonal or oligoclonal B cell expansions in all cases. Expanded B cell clones in four of five OA patients showed clustered somatic mutations, occurring mainly in the CDRs and with a high replacement-to-silent ratio (>2.9), indicating that these cells are postgerminal center B cells that had been positively selected through their Ag receptor. These data demonstrate the presence in inflamed knee OA synovium of clonally expanded, Ag-driven B cells that may contribute to the development or progression of the disease.     

Molecular components of the B cell antigen receptor complex of class IgD differ partly from those of IgM

Two classes of immunoglobulin, IgM and IgD, are present as antigen receptors on the surface of mature B lymphocytes. We show here that IgD molecules are noncovalently associated in the B cell membrane with a heterodimer consisting of two proteins of 35 kd (IgD-alpha) and 39 kd (Ig-beta), respectively. The two novel proteins are not found in the IgD-expressing myeloma J558L delta m, which fails to bring IgD antigen receptor onto the cell surface. In a surface IgD positive variant line of this myeloma, however, membrane-bound IgD molecules are associated with the heterodimer, suggesting that the formation of an antigen receptor complex is required for surface IgD expression. We further demonstrate that the IgD-associated heterodimer differs partly from that of the IgM antigen receptor and that its binding to the heavy chain only requires the presence of the last constant domain and the transmembrane part of the delta m chain.     

Cross-linking surface Ig delays CD40 ligand- and IL-4-induced B cell Ig class switching and reveals evidence for independent regulation of B cell proliferation and differentiation

T cells stimulate B cells to divide and differentiate by providing activating signals in the form of inducible membrane-bound molecules and secreted cytokines. Provision of these signals in vitro reproduces many of the consequences of T-B collaboration in the absence of any form of Ag stimulation. Although clearly not obligatory, Ag signals appear to play an important regulatory role in numerous aspects of the B cell response. To examine directly the effect of an Ag signal, naive B cells were stimulated in the presence of rCD40 ligand, with or without IL-4 in the presence or absence of different anti-Ig mAbs. Anti-Ig mAbs exerted variable effects on the B cell division rate, from enhancement to no effect to inhibition. In contrast, all anti-Ig mAbs tested inhibited division-linked isotype switching to IgG1 and IgE. Thus, B cell Ag receptor ligands could modify the rates of B cell expansion and class switching independently. The ability of anti-Ig reagents to modify class switching suggests the B cell Ag receptor may play an important role in the selection of Ig isotypes during T cell-dependent humoral immune responses to Ags of different physical structure.     

Overexpression of human DNA polymerase mu (Pol mu) in a Burkitt's lymphoma cell line affects the somatic hypermutation rate

DNA polymerase μ (Pol μ) is a DNA-dependent DNA polymerase closely related to terminal deoxynucleotidyl transferase (TdT), and prone to induce template/primer misalignments and misincorporation. In addition to a proposed general role in non-homologous end joining of double-strand breaks, its mutagenic potential and preferential expression in secondary lymphoid tissues support a role in somatic hypermutation (SHM) of immunoglobulin genes. Here, we show that human Pol μ protein is expressed in the nucleus of centroblasts obtained from human tonsils, forming a characteristic foci pattern resembling that of other DNA repair proteins in response to DNA damage. Overexpression of human Pol μ in Ramos cells, in which the SHM process is constitutive, augmented the somatic mutations specifically at the variable (V) region of the immunoglobulin genes. The nature of the mutations introduced, mostly base substitutions, supports the contribution of Pol μ to mutation of G and C residues during SHM. In vitro analysis of Pol μ misincorporation on specific templates, that mimic DNA repair intermediates and correspond to mutational hotspots, indicated that many of the mutations observed in vivo can be explained by the capacity of Pol μ to induce transient template/primer misalignments.     

Ig isotype switching in B lymphocytes. Isolation and characterization of clonal variants of the murine Ly-1+ B cell lymphoma, CH12, expressing isotypes other than IgM

We have selected and cloned variant cells from the murine B cell lymphoma, CH12, that produce a variety of other Ig isotypes in addition to or in place of the original IgM and IgD. Variants were selected by flow cytometry and automated cloning and isotype production was analyzed by membrane immunofluorescence and ELISA of culture fluids. Variants have been isolated that produce the single isotypes IgA, IgG2b, and IgG3, as well as variants that produce more than one isotype simultaneously, i.e., IgM, IgD, and IgA; IgG2b and IgA; IgG3 and IgA. All isotypes have been seen as cell surface proteins and all except IgD have been found in culture supernatants. All isotypes display the same idiotype and Ag-binding specificity for phosphatidyl choline as the original IgM and all are translated from the same VDJH and VJ kappa gene assemblies. Production of more than one isotype by a variant clone is due to simultaneous production of all the isotypes by each cell within the clone. The finding that the variants producing more than one isotype are all tetraploid suggests the interesting possibility that each isotype is derived from an independently switching chromosome. All isotype variants can be stimulated by LPS to secrete the appropriate Ig isotype at an increased rate similar to the IgM expressing parent. The variants differ in stability; some have remained stable for more than 9 months in culture, whereas other have undergone further isotype switching. The facts that some isotypes have not been seen, that multistep switching has occurred, and that many variants produce IgA in addition to another isotype are discussed in relation to current notions of isotype switching mechanisms.     

Immunoglobulin secretion by human splenic lymphocytes in vitro: the effects of antibodies to IgM and IgD.

The effects of F(ab')2 fragments of affinity-purified rabbit anti-human mu chain antibody (RaHmu) and rabbit anti-human delta chain antibody (RaHdelta) on spontaneous and mitogen-stimulated immunoglobulin (Ig) secretion by normal human spleen cells were studied. IgM and IgG secretion by human spleen cells cultured in vitro was measured by incubating the cells with 3H-amino acids precipitating the secreted labeled Ig with anti-Ig, and analyzing the precipitates by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Both RaHmu and RaHdelta suppressed spontaneous and LPS-induced IgM and IgG secretion as well as PWM-stimulated IgG secretion. In different experiments, RaHmu and RaHdelta either suppressed or augmented PWM-induced IgM secretion. The anti-Ig induced augmentation of PWM-triggered IgM secretion was most apparent when spleen cells were cultured at lower cell densities or when lower concentrations of anti-Ig were employed. These date indicate that perturbation of B cell surface immunoglobulin receptors with specific anti-Ig antibody can alter markedly the ability of these cells to differentiate into antibody-secreting cells.     

Protein evolution by hypermutation and selection in the B cell line DT40

Genome-wide mutations and selection within a population are the basis of natural evolution. A similar process occurs during antibody affinity maturation when immunoglobulin genes are hypermutated and only those B cells which express antibodies of improved antigen-binding specificity are expanded. Protein evolution might be simulated in cell culture, if transgene-specific hypermutation can be combined with the selection of cells carrying beneficial mutations. Here, we describe the optimization of a GFP transgene in the B cell line DT40 by hypermutation and iterative fluorescence activated cell sorting. Artificial evolution in DT40 offers unique advantages and may be easily adapted to other transgenes, if the selection for desirable mutations is feasible.     

Modulation of a human lymphoblastoid B cell line by cyclic AMP. Ig secretion and phosphatidylcholine metabolism

A transformed human B cell line, LA350, was found to be sensitive to cAMP-elevating agents by responding with rapid (0 to 2 h) severalfold elevations of intracellular cAMP to treatment with cholera toxin, isobutylmethylxanthine (IBMX), forskolin, and dibutyryl cAMP (all p less than 0.001). These cAMP-elevating agents also produced significant inhibitions of subsequent (48 to 72 h) Ig secretion by the same B cells as measured by a reverse hemolytic plaque assay and an enzyme-linked immunoadsorbent assay for IgM (both p less than 0.001). PMA- and IBMX-treated cells were particularly responsive to the effects of cholera toxin, showing a doubling of cAMP content and profound decrease in Ig production (p less than 0.001). Because our previous studies had correlated activation of the metabolic turnover of the phosphatidylcholine (PC) fraction of membrane phospholipids with enhanced Ig secretion, we examined the sensitivity of PC metabolism to cAMP in control and PMA-stimulated cells. Formation of PC was found to be inhibited by forskolin and IBMX (both p less than 0.002) but breakdown of PC was stimulated (p less than 0.001). These findings imply that as the enzymatic products of PC, choline phosphate and diacylglycerol, are depleted due to the combined effects of cAMP upon synthesis and turnover of PC, there is a decrease in Ig secretion. Since diacylglycerol activates protein kinase C, it appears reasonable that Ig secretion is at least partially regulated by cAMP-responsive alterations in PC metabolism produced by protein kinase C-induced phosphorylation. We conclude that the early cAMP-sensitive changes in PC metabolism in this activated B cell line may signal for subsequent alterations in Ig secretion.     

Induction of IgG2a class switching in B cells by IL-27

IL-27 is a novel IL-12 family member that plays a role in the early regulation of Th1 initiation. However, its role in B cells remains unexplored. We here show a role for IL-27 in the induction of T-bet expression and regulation of Ig class switching in B cells. Expression of WSX-1, one subunit of IL-27R, was detected at the mRNA level in primary mouse spleen B cells, and stimulation of these B cells by IL-27 rapidly activated STAT1. IL-27 then induced T-bet expression and IgG2a, but not IgG1, class switching in B cells activated with anti-CD40 or LPS. In contrast, IL-27 inhibited IgG1 class switching induced by IL-4 in activated B cells. Similar induction of STAT1 activation, T-bet expression and IgG2a class switching was observed in IFN-gamma-deficient B cells, but not in STAT1-deficient ones. The induction of IgG2a class switching was abolished in T-bet-deficient B cells activated with LPS. These results suggest that primary spleen B cells express functional IL-27R and that the stimulation of these B cells by IL-27 induces T-bet expression and IgG2a, but not IgG1, class switching in a STAT1-dependent but IFN-gamma-independent manner. The IL-27-induced IgG2a class switching is highly dependent on T-bet in response to T-independent stimuli such as LPS. Thus, IL-27 may be a novel attractive candidate as a therapeutic agent against diseases such as allergic disorders by not only regulating Th1 differentiation but also directly acting on B cells and inducing IgG2a class switching.     

Induction of proliferation and Ig production in human B leukemic cells by anti-immunoglobulins and T cell factors

The proliferation and differentiation of human leukemic B cells (B-CLL cells) with anti-Ig and T cell-derived helper factors are described. Stimulation of B-CLL cells with anti-Ig and T helper factors could induce proliferation as well as differentiation into IgM- and IgG-producing cells. Neither anti-Ig nor T helper factors alone could induce any proliferation and/or differentiation of B-CLL cells. Not only whole molecules of anti-Ig but also F(ab')2 fragments could induce proliferation and differentiation of B-CLL cells in the presence of T helper factors, but monovalent Fab' fragments were not effective. Induction of both IgM and IgG with the same idiotype was confirmed by immunofluorescent and SDS-PAGE analysis. By employing an IL 2-dependent cytotoxic T cell line and a TRF-responsive B cell line, T cell factors were separated into a fraction with IL2 activity but no TRF activity and a fraction with TRF activity but no IL 2 activity by chromatofocusing. Anti-Ig and IL 2 fraction could induce proliferation of B-CLL cells, but TRF fraction was not effective for the induction of proliferation in anti-IG-stimulated cells. For IgM and IgG production, anti-Ig and both IL 2 and TRF fractions were required. Depletion of IL 2 fraction in the first 2 days' culture inhibited Ig production, whereas the absence of TRF fraction in the first 2 days did not show any inhibitory effect on Ig production.     

Induction of a germinal center phenotype in B cells in vitro by a Th2 cell line

We have investigated the contribution of various stimuli for generating in vitro the changes in surface phenotype characteristic of B cells responding to a T-dependent antigen in a germinal center (GC). We show that, unlike many other stimuli such as B cell mitogens, cytokines, and surrogate antigen, alone or in combination, an alloreactive Th2 clonal line induces splenic B cells to become cell surface peanut agglutinin (PNA)(hi), Ig(lo), CD62L(lo), and CD44(hi) to produce mRNA for M17 and to express a GC-specific transgene even without B cell receptor ligation. Neither proliferation nor prior activation of responding B cells is needed, but B cells from CD45-null mice show reduced efficiency of this induction. These findings open up possibilities for separation and dissection of the various components of the GC response.