Control of IgM synthesis in the murine pre-B cell line, 70Z/3'

The murine 70Z/3 tumor resembles a pre-B cell in synthesizing only intracellular mu-chains and no detectable light chain. However, one kappa gene is already rearranged, and after overnight incubation with lipopolysaccharide (LPS), most of the cells are induced to synthesize light chain. The induced cells display IgM on their surface, but do not secrete IgM. Thus, 70Z/3 cells resemble cells poised at the pre-B cell/B lymphocyte border. We have examined synthesis and post-translational modification of mu-chains in uninduced and induced 70Z/3 cells. Isolation of mu-chains and peptide maps demonstrated that both populations synthesize intracellular forms that correspond to membrane-specific mum and secretion-specific mus chains. These intracellular forms have completed only the first of the two glycosylation steps characteristic of eukaryotic cells. After induction by LPS, L chain synthesis commences, mum and mus synthesis are both increased twofold to threefold (due to an increased rate of synthesis rather than decreased degradation), and both complex with L chain to form mu2L2 tetramers. Furthermore, the glycosylation of a subset of the mum chains is completed, and these are placed on the membrane. However, unglycosylated mu2L2 tetramers can be placed on the membrane, so glycosylation is not a requirement. These data suggest that L chain may not be sufficient for externalization of mum and mus chains. These data support the idea that the controls of membrane placement and secretion of mu chains are post-translational and that different mechanisms operate for mum and mus chains.     

Mouse pre-B cells synthesize and secrete mu heavy chains but not light chains

The immunoglobulins produced by the earliest recognizable B cell precursors (pre-B cells) were characterized in the mouse and human. Immunofluorescent analysis revealed no evidence of surface IgM components, and only mu heavy chains could be detected intracytoplasmically in pre-B cells. Surface IgM components could not be isolated from intact fetal liver cells that lacked sIgM+ B lymphocytes but possessed pre-B cells. Pre-B cells were shown to synthesize and secrete mu heavy chains but not light chains by immunochemical analysis. These mu chains constituted less than 0.01% of TCA precipitable protein synthesized and secreted by fetal liver cells during an 8 hr labelling period. Migration of both intracellular and secreted mu chains on SDS-PAGE suggested that they were smaller than mu chains secreted by mouse and human plasmacytomas. These data indicate that mu chain synthesis precedes light chain expression during B cell ontogeny and suggest a new role for pre-B cells in the generation and expression of a diverse immunoglobulin repertoire.     

Mutations of the mouse mu H chain which prevent polymer assembly

Earlier work has shown that truncated mu-chains lacking the carboxy-terminal C mu 4-tail region are secreted as monomeric rather than polymeric IgM and that the monomer phenotype is not due to the lack of a disulfide bond at Cys-575 in the tail. In order to define with greater precision, the molecular requirements for IgM polymer assembly, we have isolated several mutant hybridomas which produce monomeric IgM. For three such mutants, we synthesized cDNA clones of their mu mRNA and identified a mutation in the mu-chain which was responsible for the failure to assemble polymers. Mutant 205 has a 2-bp deletion which results in a termination codon after amino acid 556, effectively deleting the last 20 amino acids of the mu-chain. In conjunction with earlier reports, this result shows that the tail plays some role in assembly other than providing Cys-575, the penultimate amino acid, for disulfide bond formation. Both mutant 21 and mutant 201 have an A to G transition, which results in Tyr-455 in the fourth constant domain being replaced by a cysteine. We conclude that the integrity of both the C mu 4 domain and the 19 amino acid tail are required for the mu H chain to be assembled into polymeric IgM.     

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.     

ERp44 and ERGIC‐53 Synergize in Coupling Efficiency and Fidelity of IgM Polymerization and Secretion

Owing to the quality control mechanisms operating in the early secretory compartment, only native proteins are secreted. Despite the difficulties in assembling planar immunoglobulin M (IgM) polymers, antibody‐secreting cells can release up to thousands of IgM per second. The finding that secretory μ (μ_s) chains bind to ERGIC‐53, a lectin transporter that cycles in the early secretory compartment, suggested that ERGIC‐53 hexamers could provide a polymerization platform. Here, we show that ERGIC‐53 binds to the conserved Asn563 glycan in the C‐terminal μ_s tailpiece (μ_stp). Removal of this glycan inhibits ERGIC‐53 binding and results in the rapid formation of larger polymeric assemblies. In contrast, removal of the Asn402 oligosaccharides prevents both polymerization and secretion. ERp44, a chaperone that interacts with ERGIC‐53, binds to Cys575 in the μ_stp, providing a fail‐safe mechanism that retrieves unpolymerized IgM subunits and promotes polymerization. The coordinated action of ERGIC‐53 and ERp44 provides a way to improve the efficiency of IgM secretion without perturbing its fidelity.     

Growth inhibition of myeloma cells by anti-idiotype antibodies in the absence of membrane-bound immunoglobulin

Immunoglobulins are expressed as membrane-bound or secreted forms. Plasma cells produce little or no membrane immunoglobulin but secrete immunoglobulin molecules in large amounts. Immunoglobulin idiotypes of malignant B cells are tumor-specific antigens that may be targeted for immunotherapy. Thus, idiotype vaccination is being evaluated in clinical trials to control residual disease in multiple myeloma and non-Hodgkin's lymphoma. It is traditionally considered that anti-idiotype antibodies are not effective against plasma cell tumors, because the large amounts of immunoglobulin molecules secreted by the tumors block anti-idiotype antibodies, and because the absence of membrane immunoglobulin on the surface of these tumor cells renders them resistant to the effect of anti-idiotype antibodies. While the obstacle of abundant circulating idiotype may be obviated by reducing tumor burden to minimal residual disease, the absence of membrane immunoglobulin has been considered as a limiting factor that prevents tumor eradication by anti-idiotype antibodies. We demonstrate here that murine plasmacytoma cells can produce small amounts of membrane immunoglobulin M (IgM) heavy chains. However, the latter are precursor molecules that do not reach the cell surface. Although membrane-bound IgM is absent, the cells stain positively for surface IgM, reflecting molecules of the secreted form in the process of secretion. In spite of the relatively low levels of secreted immunoglobulin on the cell surface, anti-idiotype antibodies are effective in retardation of tumor growth in vivo. Thus, while there is no doubt that idiotype-specific cell-mediated responses are very important, myeloma patients in complete remission may additionally benefit from idiotype-specific humoral responses.     

Patterns of isotype commitment in human B cells: limiting dilution analysis of Epstein Barr virus-infected cells

The frequency of Epstein Barr virus- (EBV) inducible IgM, IgG, and IgA-secreting cells in human peripheral blood and tonsil was determined by performing limiting dilution experiments in suspension culture. We devised a method of propagating small numbers of EBV-infected B cells with irradiated umbilical cord blood cells as a feeder layer. Precursor cell frequencies can be derived from these experiments; we have shown by statistical analysis that they conform to the single hit model of the Poisson distribution. By using this technique, a significant percentage of surface immunoglobulin-bearing lymphocytes are activated to secrete immunoglobulin in vitro. On the average, 8 to 38% of B cells in peripheral blood and tonsil can be propagated and the secreted immunoglobulin from the clonal progeny can be analyzed. Neither the EBV immune status of the donor nor the source of the umbilical cord blood feeder layer could account for the variations in cloning efficiency observed among donors. A surprisingly high frequency of B cells secreted IgA in vitro and we have shown that a small proportion of B cell clones in tonsil and peripheral blood secrete both IgM and IgA during the 4-wk culture period. Other B cells, including all those that produce IgG, appear to be committed to the secretion of a single isotype. Thus, these studies establish methodology for the analysis of the secreted products of human B cells at the single cell level and demonstrate that the progeny of at least some clones can secrete more than one isotype in vitro.     

Direct evidence that J chain regulates the polymeric structure of IgM in antibody-secreting B cells.

IgM is secreted in two functional polymeric forms. Secreted IgM was originally thought to be exclusively a pentameric molecule containing J (joining) chain, but many B cells also secrete hexameric IgM lacking J chain. Hexameric IgM may play an important role in the immune system, since it is up to 20 times more active than pentameric IgM in initiating the complement cascade. The predominant polymeric form of IgM secreted by B cell lines, either pentameric or hexameric, correlates with the concentration of J chain present during polymerization, and cells that express high levels of J chain secrete mostly IgM pentamers. The B cell lymphoma WEHI-231 does not express J chain, and the majority of its secreted IgM is polymerized as hexamers. When a J chain-encoding cDNA was expressed in these cells, the secreted IgM was found to be almost exclusively pentameric. However, although the expression of J chain dramatically altered the phenotype of the IgM secreted by these cells, it had little effect on their secretory rate. We conclude that J chain regulates the structure and function of the IgM polymers secreted by B cells, but it is not necessary for either IgM polymerization or secretion.     

Characterization of a spontaneous murine B cell leukemia (BCL1). II. Tumor cell proliferation and IgM secretion after stimulation by LPS.

A spontaneous BALB/c B lymphocyte leukemia could be stimulated in vitro by the polyclonal B cell activator lipopolysaccharide (LPS) and the conditions for activation were studied. Spleen cells or peripheral blood lymphocytes from tumor-bearing animals responded by increased DNA synthesis and the peak of activation occurred earlier than with normal mouse spleen cells. Tumor cells harvested from the spleen, but not from the peripheral blood, could be induced by LPS to secrete IgM. Direct demonstration that the response was due to tumor cell activation and not that of contaminating normal B lymphocytes was provided by karyotype analysis and by immunoprecipitation, which showed the restriction of light chains on secreted IgM molecules to the lambda isotype.     

AID-/-mus-/- mice are agammaglobulinemic and fail to maintain B220-CD138+ plasma cells

The terminal stage of B cell differentiation culminates in the formation of plasma cells (PC), which secrete large quantities of Igs. Despite recent progress in understanding the molecular aspect of PC differentiation and maintenance, the requirement for the synthesis of secretory Igs as a contributing factor has not been explored. To address this issue, we generated activation-induced cytidine deaminase (AID)/secretory mu-chain (mus) double-knockout mice, in which a normally diverse repertoire of B cell receptors is retained, yet B cells are unable to synthesize secretory Igs. These mice possess polyclonal B cells but have no serum Igs. Following immunization in vivo, PCs, identified by CD138 expression and loss of the B220 marker, were starkly reduced in number in spleen and bone marrow of AID(-/-)mus(-/-) agammaglobulinemic mice compared with wild-type mice. Upon mitogenic stimulation in vitro, AID(-/-)mus(-/-) B cells differentiated into plasmablasts to some extent, but showed reduced survival compared with wild-type B cells. We found no evidence that this reduced survival was attributable to accumulation of membrane IgM. Our results indicate that the synthesis of secretory Igs is a requirement for maintenance of B220(-)CD138(+) PCs.     

B-cell and plasma-cell splicing differences: a potential role in regulated immunoglobulin RNA processing

The immunoglobulin micro pre-mRNA is alternatively processed at its 3' end by competing splice and cleavage-polyadenylation reactions to generate mRNAs encoding the membrane-associated or secreted forms of the IgM protein, respectively. The relative use of the competing processing pathways varies during B-lymphocyte development, and it has been established previously that cleavage-polyadenylation activity is higher in plasma cells, which secrete IgM, than in B cells, which produce membrane-associated IgM. To determine whether RNA-splicing activity varies during B-lymphocyte development to contribute to micro RNA-processing regulation, we first demonstrate that micro pre-mRNA processing is sensitive to artificial changes in the splice environment by coexpressing SR proteins with the micro gene. To explore differences between the splice environments of B cells and plasma cells, we analyzed the splicing patterns from two different chimeric non-Ig genes that can be alternatively spliced but have no competing cleavage-polyadenylation reaction. The ratio of intact exon splicing to cryptic splice site use from one chimeric gene differs between several B-cell and several plasma-cell lines. Also, the amount of spliced RNA is higher in B-cell than plasma-cell lines from a set of genes whose splicing is dependent on a functional exonic splice enhancer. Thus, there is clear difference between the B-cell and plasma-cell splicing environments. We propose that both general cleavage-polyadenylation and general splice activities are modulated during B-lymphocyte development to ensure proper regulation of the alternative micro RNA processing pathways.     

A human lymphoid cell line secreting immunoglobulin G and retaining immunoglobulin M in the plasma membrane

A selected clone, LA 85.2, of a human lymphoid cell line produces μ, γ, and light chains. The cells secrete IgG but not IgM. Assembly of μ chains and light chains produces 8S IgM which is retained in the plasma membrane. IgM is produced at a slow rate and in lesser amounts than IgG. LA 85.2 cells produce a plasma membrane protein which can bind to antibody-antigen precipitates. It is suggested that this protein plays a role in holding the surface IgM in the plasma membrane.     

The immunoglobulin mu chains of membrane-bound and secreted IgM molecules differ in their C-terminal segments

The B lymphocytes synthesizes two forms of IgM molecules during its development from a stem cell to a mature antibody-secreting plasma cell. The monomeric receptor IgM molecule is affixed to the plasma membrane and triggers the later stages of B cell differentiation, whereas the pentameric secreted IgM molecule is an effector of humoral immunity. The structural differences between membrane-bound and secreted IgM molecules are reflected in the differences between their heavy or mu chains. We have previously determined the complete amino acid sequence of a murine secreted mu (microsecond) chain. In this study, we have compared the structures of the secreted and membrane-bound mu (micron) heavy chains by peptide mapping, micro-sequence and carboxypeptidase analyses. These studies demonstrate that the micron and microsecond chains are very similar throughout their VH, C mu 1, C mu 2, C mu 3 and C mu 4 domains. The micron and microsecond chains differ in the amino acid sequence of their C-terminal segments. These studies in conjunction with those carried out on the micron and microsecond mRNAs and the C mu gene suggest that the micron and microsecond chains from a given B cell are identical except for their 41 and 20 residue C-terminal segments, respectively. The amino acid sequence of the 41 residue C membrane terminal segment predicted from the corresponding micron mRNA is in agreement with all the protein studies reported in this paper.     

In vitro regulation of IgA subclass production. III. Selective transformation of IgA1 producing cells by Epstein-Barr virus

In past experiments, using limited dilution analysis, we have demonstrated that a high percentage of immunoglobulin-secreting clones derived from Epstein-Barr virus- (EBV) stimulated lymphocytes secrete IgA. To further characterize the IgA produced by these clones, the IgA subclass of supernatants from clones stimulated 4 to 6 wk previously with EBV was determined by radioimmunoassay. All of 17 IgA-producing clones secreted IgA1; none secreted IgA2. Because we have shown that surface IgM+ (sIgM+) B cells are an enriched source of IgA2 plasma cell precursors, panning techniques were used to purify sIgM+ B cells from tonsils. Of 103 clones derived from these sIgM+ B cells, 102 secreted IgA1 and only one secreted IgA2. The relative absence of IgA2-producing clones could not be attributed to an absence of EBV receptors on IgA2 cells. A mean of 84 +/- 4% of freshly isolated IgA2 B cells and 78 +/- 6% of IgA1 B cells could be stained with a monoclonal antibody binding the EBV receptor; and there was no failure of EBV to infect IgA2 plasma cells precursors. Of IgA2 plasma cells derived from peripheral blood lymphocytes stimulated 7 days previously with EBV, 54 +/- 7% were positive for the EBV nuclear antigen, compared with 54 +/- 18% of IgA1 plasma cells from the same cultures. Seven days after EBV stimulation, a mean of 25% of the total IgA plasma cells were positive for cytoplasmic IgA2, whereas by 21 days after stimulation only 7% were positive for IgA2. This shift in the proportions of IgA1 and IgA2 plasma cells could be attributed to a failure of the IgA2 plasma cell number to increase after 10 days in culture. There was no evidence for selective suppression of IgA2 production by T cells or selective lysis of IgA2 plasma cells by infectious EBV particles. These results demonstrate that although precursors for both IgA1- and IgA2-producing cells can be stimulated to differentiate in response to EBV, there is preferential transformation of IgA1-producing cells.     

Biosynthesis and structure of membrane and secretory immunoglobulins

Summary Almost all of the body's extracellular immunoglobulin (Ig) is derived from Ig-secreting plasma cells of lymphoid tissues. The secreted material is a heterogeneous mixture of different classes and specificities. Lymphoid tissues also contain a large number of essentially non-secretory cells — B lymphocytes — which bear Ig firmly associated with their plasma membranes. Ig molecules thus exist in two functionally different forms, as membrane-bound antigen receptors on the surface of B lymphocytes on the one hand, and as humoral secreted Ig antibodies on the other. On B cells, membrane-bound heavy chains have an apparent mol. wt. slightly larger than that of secreted heavy chains from plasma cells. Membrane-bound but not secreted heavy chains bind detergents, thus suggesting the presence of a hydrophobic region in membrane-bound heavy chains, which is absent in secreted heavy chains. Most investigations have dealt with immunoglobulin M. The two types of IgM heavy chains differ at their carboxy termini. Recent investigations at the nucleic acid level demonstrate that membrane-associated µ chains contain a 41-residue hydrophobic tail adjacent to the last constant domain, whereas secretory µ chains contain a 20-residue hydrophilic tail. At the present time, evidence is accumulating that all membrane-bound Ig heavy chain classes may contain similar hydrophobic structures necessary for anchorage of the molecules into the lipid bilayer.     

The intracellular ratio of the membrane to the secreted form of immunoglobulin M heavy chain is a measure of the developmental stage of B cell lymphomas

Tumors of B lymphocyte origin have been used as models for normal B cells “frozen” at particular stages of their development. Surface properties, amount, and intracellular location of immunoglobulin and the synthesis of J chain have all been used as indicators of developmental stages. Each requires special techniques or yields data that are difficult to compare from one experiment to the next. For these reasons, we have developed a metric for B cell development that is simple to perform and allows quick quantitative comparisons of cell lines. It has recently been established that the membrane (μ_m) and secreted (μ_s) forms of the IgM heavy chain differ at their extreme carboxy termini. The two proteins differ slightly in size and are easily distinguished when they are compared without their carbohydrate on sodium dodecyl sulfate (SDS) polyacrylamide gels. We have examined four mouse tumors derived from the B lymphocyte lineage whose phenotypes resemble late pre-B cells (internal μ only; uninduced 70Z/3), small B lymphocytes (high levels of surface IgM; LPS-induced 70Z/3, WEHI 231), lymphoblasts (both membrane and secreted IgM; WEHI 279.1), and plasma cells (copious IgM secretion; MOPC 104E). Despite the fact the 70Z/3 and WEHI 231 secrete no detectable IgM, all of the tumors synthesize at least intracellular forms of both μ_m and μ_s. The proportion of μ_m is stable and is characteristic of each tumor. The 70Z/3 cells and WEHI 231 cells synthesize about 75% of their total μ as μ_m; WEHI 279.1 cells synthesize about 30% and MOPC 104E cells about 5% of their total μ as μ_m. The population of LPS-stimulated B lymphocytes shows a similar progression during its differentiation. The proportion of μ_m correlates with other developmentally regulated parameters (Fc receptor, Ia and plasma cell antigen levels, and J chain) and can be used as a simple metric for comparison with developing B lymphocytes and determination of the developmental stage of a B cell tumor.     

Quality control of ER synthesized proteins: an exposed thiol group as a three-way switch mediating assembly, retention and degradation.

Plasma cells secrete IgM only in the polymeric form: the C-terminal cysteine of the mu heavy chain (Cys575) is responsible for both intracellular retention and assembly of IgM subunits. Polymerization is not quantitative, and part of IgM is degraded intracellularly. Neither chloroquine nor brefeldin A (BFA) inhibits degradation, suggesting that this process occurs in a pre-Golgi compartment. Degradation of IgM assembly intermediates requires Cys575: the monomeric IgMala575 mutant is stable also when endoplasmic reticulum (ER) to Golgi transport is blocked by BFA. Addition of the 20 C-terminal residues of mu to the lysosomal protease cathepsin D is sufficient to induce pre-Golgi retention and degradation of the chimeric protein: the small amounts of molecules which exit from the ER are mostly covalent dimers. By contrast, when retained by the KDEL sequence, cathepsin D is stable in the ER, indicating that retention is not sufficient to cause degradation. Replacing the C-terminal cysteine with serine restores transport through the Golgi. As all chimeric cathepsin D constructs display comparable protease activity in vitro, their different fates are not determined by gross alterations in folding. Thus, also out of its normal context, the mu chain Cys575 plays a crucial role in quality control, mediating assembly, retention and degradation.