The Eµ enhancer region influences H chain expression and B cell fate without impacting IgVH repertoire and immune response in vivo.

The IgH intronic enhancer region Eµ is a combination of both a 220-bp core enhancer element and two 310-350-bp flanking scaffold/matrix attachment regions named MARsEµ. In the mouse, deletion of the core-enhancer Eµ element mainly affects VDJ recombination with minor effects on class switch recombination. We carried out endogenous deletion of the full-length Eµ region (core plus MARsEµ) in the mouse genome to study VH gene repertoire and IgH expression in developing B-lineage cells. Despite a severe defect in VDJ recombination with partial blockade at the pro-B cell stage, Eµ deletion (core or full length) did not affect VH gene usage. Deletion of this regulatory region induced both a decrease of pre-B cell and newly formed B cell compartments and a strong orientation toward the marginal zone B cell subset. Because Igµ H chain expression was decreased in Eµ-deficient pre-B cells, we propose that modification of B cell homeostasis in deficient animals was caused by "weak" pre-B cell and BCR expression. Besides imbalances in B cell compartments, Ag-specific Ab responses were not impaired in animals carrying the Eµ deletion. In addition to its role in VDJ recombination, our study points out that the full-length Eµ region does not influence VH segment usage but ensures efficient Igµ-chain expression required for strong signaling through pre-B cells and newly formed BCRs and thus participates in B cell inflow and fate.

Antigen-specific human monoclonal antibodies from mice engineered with human Ig heavy and light chain YACs.

We describe a strategy for producing human monoclonal antibodies in mice by introducing large segments of the human heavy and kappa light chain loci contained on yeast artificial chromosomes into the mouse germline. Such mice produce a diverse repertoire of human heavy and light chains, and upon immunization with tetanus toxin have been used to derive antigen-specific, fully human monoclonal antibodies. Breeding such animals with mice engineered by gene targeting to be deficient in mouse immunoglobulin (Ig) production has led to a mouse strain in which high levels of antibodies are produced, mostly comprised of both human heavy and light chains. These strains should provide insight into the adoptive human antibody response and permit the development of fully human monoclonal antibodies with therapeutic potential.

XBP-1 specifically promotes IgM synthesis and secretion, but is dispensable for degradation of glycoproteins in primary B cells.

Differentiation of B cells into plasma cells requires X-box binding protein-1 (XBP-1). In the absence of XBP-1, B cells develop normally, but very little immunoglobulin is secreted. XBP-1 controls the expression of a large set of genes whose products participate in expansion of the endoplasmic reticulum (ER) and in protein trafficking. We define a new role for XBP-1 in exerting selective translational control over high and sustained levels of immunoglobulin M (IgM) synthesis. XBP-1(-/-) and XBP-1(+/+) primary B cells synthesize IgM at comparable levels at the onset of stimulation with lipopolysaccharide or CpG. However, later there is a profound depression in synthesis of IgM in XBP-1(-/-) B cells, notwithstanding similar levels of micromRNA. In marked contrast, lack of XBP-1 does not affect synthesis and trafficking of other glycoproteins, or of immunoglobulin light chains. Contrary to expectation, degradation of proteins from the ER, using TCRalpha or US11-mediated degradation of class I major histocompatibility complex molecules as substrates, is normal in XBP-1(-/-) B cells. Furthermore, degradation of membrane mu was unaffected by enforced expression of XBP-1. We conclude that in primary B cells, the XBP-1 pathway promotes synthesis and secretion of IgM, but does not seem to be involved in the degradation of ER proteins, including that of mu chains themselves.

Two distinct populations of H chain-edited B cells show differential surrogate L chain dependence.

Developing autoreactive B cells may edit (change) their specificity by secondary H or L chain gene rearrangement. Recently, using mice hemizygous for a site-directed VDJH and VJkappa transgene (tg) encoding an autoreactive Ab, we reported ongoing L chain editing not only in bone marrow cells with a pre-B/immature B cell phenotype but also in immature/transitional splenic B cells. Using the same transgenic model, we report here that editing at the H chain locus appears to occur exclusively in bone marrow cells with a pro-B phenotype. H chain editing is shown to involve VH replacement at the tg allele or VH rearrangement at the wild-type (wt) allele when the tg is inactivated by nonproductive VH replacement. VH replacement/rearrangement at the tg/wt alleles was found to entail diverse usage of VH genes. Whereas the development of edited B cells expressing the wt allele was dependent on the lambda5 component of the surrogate L chain, the development of B cells expressing the tg allele, including those with VH replacement, appeared to be lambda5 independent. We suggest that the unique CDR3 region of the tg-encoded muH chain is responsible for the lambda5 independence of tg-expressing B cells.

Differential regulation of membrane and secretory mu chain synthesis in human beta cell lines. Regulation of membrane mu or secreted mu.

Regulation of membrane and secretory mu synthesis was examined in human lymphoblastoid cell lines representing various stages of differentiation. Immunoglobulin phenotype was determined by surface and cytoplasmic staining with fluorochrome-conjugated antibodies and by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of anti-mu precipitable cellular products. The thymidine analogue, 5-bromo-2'-deoxyuridine (BUdR), which inhibits differentiation-specific proteins in a variety of systems, was used to examine regulation of immunoglobulin synthesis. We found that BUdR had a differential effect on membrane (mum) and secretory (mus) type mu heavy chains. Ig production in pre-B and plasma cell-like lines, which make mus, was unaffected by BUdR. However, surface expression of IgM (mum) in B cell lines was drastically inhibited at similar doses of BUdR without diminishing total Ig or protein synthesis. Examination of labeled mu chains from control and BUdR-treated B cell lines by SDS-PAGE revealed the production of two sizes of mu (mum and mus) in control cells and only the smaller size (mus) in BUdR-treated cells. This size difference could not be attributed to alterations in glycosylation of the molecules. These data show that BUdR inhibits the production of membrane mu chains without diminishing secretory mu chain synthesis in the same cell. Our findings suggest that thymidine-rich regions of the genome are involved in the regulation of mum vs. mus during B cell differentiation.

Counterselection against D mu is mediated through immunoglobulin (Ig)alpha-Igbeta.

The pre-B cell receptor is a key checkpoint regulator in developing B cells. Early events that are controlled by the pre-B cell receptor include positive selection for cells express membrane immunoglobulin heavy chains and negative selection against cells expressing truncated immunoglobulins that lack a complete variable region (D mu). Positive selection is known to be mediated by membrane immunoglobulin heavy chains through Ig alpha-Ig beta, whereas the mechanism for counterselection against D mu has not been determined. We have examined the role of the Ig alpha-Ig beta signal transducers in counterselection against D mu using mice that lack Ig beta. We found that D mu expression is not selected against in developing B cells in Ig beta mutant mice. Thus, the molecular mechanism for counterselection against D mu in pre-B cells resembles positive selection in that it requires interaction between mD mu and Ig alpha-Ig beta.

Double isotype production by a neoplastic B cell line. II. Allelically excluded production of mu and gamma 1 heavy chains without CH gene rearrangement.

In our accompanying paper, we described a switch variant (BCL1.2.58) that expresses membrane and secreted forms of IgM and IgG1. Both IgM and IgG1 share the same idiotype and use the same VDJ rearrangement. Here, a detailed Southern blot analysis of the entire constant region of the Ig heavy chain (Ig CH) locus of parental (BCL1.B1) and variants (BCL1.B2) DNA showed no detectable rearrangement. Similar analysis of the JH-C mu region led to the conclusion that two heavy chain alleles present in the IgM/IgG1-producing variants carried the same VDJ rearrangement but differed in their 3' flanking regions. One chromosome 12 did not carry any Ig CH genes, whereas, the other chromosome 12 carried one copy of CH genes. In BCL1.B1, however, each of the chromosome 12 alleles carried a full copy of CH genes. Karyotypic analysis confirmed the presence of two translocated t(12;16) chromosomes in both BCL1.2.58 and BCL1.B1 cells, with a break 5' to the VH locus at the distal region (12F2) of chromosome 12, and at the proximal region below the centromere (16B3) of chromosome 16. We conclude that double production of IgM and IgG1 in BCL1.B2 is accomplished by transcription of the corresponding CH genes in germline configuration using a single VDJ on the same chromosome 12.

Truncated mu (mu') chains in murine IgM. Evidence that mu' chains lack variable regions.

Secreted IgM was shown to contain truncated mu (mu') chains with an apparent molecular mass of approximately 55 kD. The estimated percentage of IgM heavy (H) chains in the mu' form ranged from less than or equal to 1% in the case of one tumor IgM protein (104E) to greater than or equal to 30% in normal serum IgM. Serum mu' chains lacked antigenic determinants characteristic of immunoglobulin variable regions and showed a restricted isoelectric focusing pattern compared with that of conventional mu chains. Intracellular mu' chains were readily detected in bone marrow cells but not in spleen or lymph node cells; mu' chains were also detected in IgM-producing tumor cells and in a hybridoma cell line that deleted its productive mu allele. These results predict irregularities in IgM structure and recall an old controversy concerning the valence of IgM molecules.

A novel mechanism for B cell repertoire maturation based on response by B cell precursors to pre-B receptor assembly.

The expression of different sets of immunoglobulin specificities by fetal and adult B lymphocytes is a long-standing puzzle in immunology. Recently it has become clear that production of immunoglobulin mu heavy chain and subsequent assembly with a surrogate light chain to form the pre-B cell receptor complex is critical for development of B cells. Here we show that instead of promoting pre-B cell progression as in adult bone marrow, this complex inhibits pre-B cell growth in fetal liver. Curiously, we identify a fetal-associated VH11 mu heavy chain that allows continued pre-B proliferation in fetal liver. Interestingly, this heavy chain does not associate efficiently with a surrogate light chain, providing a previously unrecognized mechanism for skewing the expression of distinctive VH genes toward fetal through early neonatal life.

Regulation of an early developmental checkpoint in the B cell pathway by Ig beta.

Many of the cell fate decisions in precursor B cells and more mature B cells are controlled by membrane immunoglobulin (Ig)M heavy chain (mu) and the Ig alpha-Ig beta signal transducers. The role of Ig beta in regulating early B cell development was examined in mice that lack Ig beta (Ig beta-/-). These mice had a complete block in B cell development at the immature CD43+B220+ stage. Immunoglobulin heavy chain diversity (DH) and joining (JH) segments rearranged, but variable (VH) to DJH recombination and immunoglobulin messenger RNA expression were compromised. These experiments define an unexpected, early requirement for Ig(beta) to produce B cells that can complete VDJH recombination.

A new isotype of immunoglobulin heavy chain in the urodele amphibian Pleurodeles waltl predominantly expressed in larvae.

Up to now, it was thought that urodele amphibians possessed only two IgH isotypes, IgM (mu) and IgY (upsilon). By screening a Pleurodeles waltl Ig cDNA mini-library, we identified three isotypes: IgM, IgY and a previously unknown class. IgM are multimeric molecules and represent the most abundant isotype throughout the life of P. waltl. IgY are likely the counterpart of mammalian IgA. The new isotype has typical Ig H-chain characteristics and is expressed as both secretory and membrane forms. Our analyses indicate that this isotype is restricted to Pleurodeles. Consequently, we named it "IgP" (pi) for Pleurodeles. This isotype is mainly expressed after hatching. Its expression decreases after metamorphosis. Our data indicate that IgP-expressing B cells present some similarities with mammalian B1-cells.

Extensive immunoglobulin production sensitizes myeloma cells for proteasome inhibition.

Multiple myeloma is an incurable plasma cell neoplasia characterized by the production of large amounts of monoclonal immunoglobulins. The proteasome inhibitor bortezomib (PS-341, Velcade) induces apoptosis in various malignant cells and has been approved for treatment of refractory multiple myeloma. Inhibition of the antiapoptotic transcription factor nuclear factor-kappaB (NF-kappaB) apparently contributes to the antitumor effects of bortezomib; however, this mechanism cannot fully explain the exceptional sensitivity of myeloma cells. Extensive protein synthesis as in myeloma cells is inherently accompanied by unfolded proteins, including defective ribosomal products (DRiPs), which need to be degraded by the ubiquitin-proteasome system. Therefore, we hypothesized that the proapoptotic effect of bortezomib in multiple myeloma is mainly due to the accumulation of unfolded proteins in cells with high protein biosynthesis. Using the IgG-secreting human myeloma cell line JK-6L and murine muH-chain-transfected Ag8.H myeloma cells, apoptosis induction upon proteasome inhibition was clearly correlated with the amount of immunoglobulin production. Preferentially in immunoglobulin-high myeloma cells, bortezomib triggered activation of caspases and induction of proapoptotic CHOP, a component of the terminal unfolded protein response induced by endoplasmic reticulum (ER) stress. In immunoglobulin-high cells, bortezomib increased the levels of proapoptotic Bax while reducing antiapoptotic Bcl-2. Finally, IgG-DRiPs were detected in proteasome inhibitor-treated cells. Hence, proteasome inhibitors induce apoptosis preferentially in cells with high synthesis rate of immunoglobulin associated with accumulation of unfolded proteins/DRiPs inducing ER stress. These findings further elucidate the antitumor activities of proteasome inhibitors and have important implications for optimizing clinical applications.

Effects of intron length on differential processing of mouse mu heavy-chain mRNA.

Production of membrane-bound and secreted forms of mouse mu heavy-chain mRNA is controlled by differential processing in a developmental-stage-specific manner. We have analyzed the effects of various deletions and insertions in the C4-M1 intron of the mouse mu gene on the differential processing of mu mRNA. We show that there is a correlation between the length of the C4-M1 intron and the molar ratio of membrane-bound to secreted mu mRNAs, i.e., the shorter the C4-M1 intron, the higher the ratio. Since the poly(A) addition signal in the C4-M1 intron seems to be intact in the mutant mu genes, it is likely that the efficiency of splicing of the C4-M1 intron is affected by changes in the intron length.

Identification of an activity in B-cell extracts that selectively impairs the formation of an immunoglobulin mu s poly(A) site processing complex.

The immunoglobulin mu heavy-chain transcription unit is differentially expressed during B-cell development, producing mRNAs that encode secreted (mu s) and membrane-bound (mu m) forms of the heavy-chain polypeptide. Whereas the mu s mRNA and the mu m mRNA are produced in approximately equal abundance in B cells, an increase in the utilization of the mu s poly(A) site contributes to the production of the mu s mRNA as the predominant form in a plasma cell. Previous experiments have demonstrated a correlation between the formation of a stable complex on a poly(A) site and the relative function of the poly(A) site. We have thus investigated the parameters determining the interaction of these factors with the immunoglobulin poly(A) sites. Assays of complex formation involving the two immunoglobulin poly(A) sites by using HeLa cell activities revealed the formation of stable complexes with no apparent difference between the mu s site and the mu m site. In contrast, the mu s-specific complex was markedly less stable when a B-cell extract was used. Fractionation of B-cell extracts has revealed an activity that specifically destabilizes the mu s polyadenylation complex, suggesting that the function of this poly(A) site may be regulated by both positive- and negative-acting factors.

An RNA polymerase pause site is associated with the immunoglobulin mus poly(A) site.

Immunoglobulin mu alternative RNA processing is regulated during B-cell maturation and requires balanced efficiencies of the competing splice (mum) and cleavage-polyadenylation (mus) reactions. When we deleted sequences 50 to 200 nucleotides beyond the mus poly(A) site, the mus/mum mRNA ratio decreased three- to eightfold in B, plasma, and nonlymphoid cells. The activity could not be localized to a smaller fragment but did function in heterologous contexts. Our data suggest that this region contains an RNA polymerase II pause site that enhances the use of the mus poly(A) site. First, known pause sites replaced the activity of the deleted fragment. Second, the mu fragment, when placed between tandem poly(A) sites, enhanced the use of the upstream poly(A) site. Finally, nuclear run-ons detected an increase in RNA polymerase loading just downstream from the mus poly(A) site, even when the poly(A) site was inactivated. When this mu fragment and another pause site were inserted 1 kb downstream from the mus poly(A) site, they no longer affected the mRNA expression ratio, suggesting that pause sites affect poly(A) site use over a limited distance. Fragments from the immunoglobulin A gene were also found to have RNA polymerase pause site activity.

Light chain loss and reexpression leads to idiotype switch. Surrogate light chains are probably responsible for this process.

The murine B-lymphocyte cell line 38C-13 is characterized by several cell surface markers typical for an early stage of B-cell differentiation. Cells of this cell line possess cell surface membrane IgM molecules composed of mu and kappa polypeptide chains. They also produce "surrogate" or "pseudo" light chains (psi L) coded by the lambda 5 and VpreB genes. Variants of the 38C-13 cell line which do not synthesize kappa chains can be isolated from the 38C-13 population by the use of anti-idiotype antibodies in vivo and in vitro. In some kappa chain-deficient variant cell lines, cells which have regained surface IgM expression but have lost the original idiotype specificity, can be isolated. This idiotype switch is probably due to a secondary rearrangement of the kappa gene. In the kappa chain-deficient variant cells, the mu chains assemble with the surrogate light chains but the assembled IgM-like molecules are not expressed on the cell surface. It is suggested that surrogate light chains play an important role in the induction of kappa gene rearrangement but that surface expression of mu-psi L complexes is not required for this process.

TPA-induced differentiation of chronic lymphocytic leukemia cells: studies on mu-chain expression.

The regulation of IgM synthesis and secretion was studied in chronic lymphocytic leukemia cells, with a phenotype roughly similar to peripheral resting B cells, during phorbol ester (12-O-tetradecanoylphorbol-13-acetate)-induced differentiation. TPA treatment caused a 20 times increase in total RNA synthesis and 20 to 50 times increase in the protein synthesis as compared to control cells. Morphologically, 70-90% of the cells reached the lympho- or plasmablast stage of differentiation. In control culture cells, approximately equal amounts of mRNA coding for secretory (s) and membrane (m) mu-chains were found. The micron message was translated as surface IgM expression was detected. A posttranscriptional regulation of microsecond synthesis appears to exist, since only low amounts of cytoplasmic mu-chains were detected by immunoprecipitation and SDS-PAGE, and no secretion of pentameric IgM was detected as measured by an RIA. TPA induction caused a relative increase in the microseconds to microns mRNA ratio, demonstrating differentiation associated control mechanisms operating at the level of mRNA processing. The high levels of cytoplasmic microsecond-chain precursor and the efficient secretion of pentameric IgM in TPA-induced chronic lymphocytic leukemia cells indicated the presence also of posttranscriptional controls.

The control of membrane and secreted heavy chain biosynthesis varies in different immunoglobulin isotypes produced by a monoclonal B cell lymphoma.

The control of production of the membrane (m) vs secreted (s) forms of immunoglobulin heavy chains was investigated in a panel of cell lines expressing different heavy chain classes but identical light chains (lambda) and variable regions. These cell lines could be induced towards Ig secretion by mitogen treatment. During this process a shift from m to s heavy chain production takes place. Here we show that, similarly to IgA- and IgE-producing B cells, in IgG2a-producing I.29 cells the gamma m-gamma s shift was accompanied by a shift in the corresponding mRNAs, with a decrease of gamma m mRNA and an increase of the gamma s mRNA in LPS-stimulated cells. By contrast, the micron mRNA was increased in LPS-stimulated IgM-producing cells, albeit these cells synthesized reduced amounts of micron polypeptides. The utilization of the translational level in the early steps of B lymphocyte maturation thus distinguishes the mode of regulation of mu chains from those of the other isotypes. In addition, in B cells a post-translational event blocks the secretion of IgM but not of IgG or IgE.

Transient expression of pentameric IgM on the surface of NZB B cells.

In contrast to normal B lymphocytes, splenic B cells from autoimmune NZB mice express on their surface greater amounts of IgM than IgD. We have found that the predominant mu chain species present on these cells is derived from pentameric secretory IgM which can be radioiodinated during transient association with the cell membrane. If the contribution from 19S IgM is discounted, then the surface IgM:IgD ratio of NZB mice is only moderately higher than that of B cells from normal strains.

Effects of various forms of monoclonal anti-Fc gamma R II (2.4G2) on B lymphocyte responses.

The effects of monoclonal anti-Fc gamma R II (2.4G2) in various forms on B lymphocyte proliferation and antibody (mu) secretion in vitro were evaluated. Soluble native 2.4G2 did not stimulate or inhibit the responses of B lymphocytes, either unstimulated or stimulated with rabbit F(ab')2 anti-mouse mu (anti-mu) plus lymphokines (the supernatant of Concanavalin A stimulated rat spleen cells). The failure of native 2.4G2 to affect responses was observed over a broad range of concentrations, and even when the B lymphocytes were incubated with the 2.4G2 for 24 hr prior to stimulation with anti-mu and lymphokines. Similarly, soluble chemically cross-linked 2.4G2 failed to affect B lymphocyte responses. Binding studies indicated that this failure was not due to a lack of binding and suggested that the polymerized 2.4G2 was cross-linking at least four Fc gamma R II. Larger multimers of 2.4G2 could not be evaluated due to a loss of binding activity. In contrast to the above results, 2.4G2 which was capable of extensively cross-linking Fc gamma R II (2.4G2 bound to Sepharose) or of cross-linking Fc gamma R II to surface IgM (2.4G2 hetero-cross-linked with anti-mu) specifically inhibited B lymphocyte responses to anti-mu and lymphokines. Antibody secretion was affected more than proliferation. These results provide additional evidence that Fc gamma R II regulate the responses of B lymphocytes, and suggest that cross-linking of more than four Fc gamma R II is necessary to generate the inhibitory signal. Further, the results indicate that the ligand does not have to be internalized in order to generate the regulatory signal. Finally, the results with the heterodimer suggest that it may be possible to regulate a particular antibody response using anti-Fc gamma R II cross-linked to antigen or to anti-receptor antibody (e.g. an anti-idiotype).