Gene transfer of immunoglobulin light chain restores heavy chain secretion

Several lines of evidence suggest that immunoglobulin (Ig) light (L) chain plays a role in the secretion of heavy (H) chain. For example, myeloma variant lines, which synthesize the Ig H chain but not the L chain, fail to secrete H chain protein. Here we have tested directly the role of chain assembly in the control of Ig secretion by the transfer of functional L chain genes into two such L chain-defective myeloma mutants. A lambda 2 or kappa L chain gene was introduced into variant lines of the mouse myelomas MOPC 315 (IgA, lambda 2) or PC7 (IgM, kappa), respectively. Although the two mutant lines are unable to secrete the H chain they produce, rescue of secretion of complete Ig protein molecules (IgA or IgM) was observed after transfection. These results imply that the secretory apparatus of these cells is intact and that the failure to secrete free H chain reflects a structural feature intrinsic to that protein. The implications of these results with respect to control of secretion of multi-subunit proteins are discussed.     

Nucleotide sequences of immunoglobulin mu heavy chain deletion mutants.

Mutants of an IgM producing hybridoma cell line were isolated which produce mu heavy chain fragments. Two such mutants were found to have internal deletions in the mu gene and the nucleotide sequence of the deletion endpoints was determined. No evidence was found for a role of the heavy chain switch region in the formation of these deletions. The implications of these mutants in defining the requirements of immunoglobulin gene expression are discussed.     

Polymeric immunoglobulin M is secreted by transfectants of non-lymphoid cells in the absence of immunoglobulin J chain.

Plasmids were constructed in which expression of genes encoding the heavy and light chains of a hapten-specific IgM antibody is under control of a heat shock promoter. Glioma, phaeochromocytoma and other non-lymphoid cell lines transfected with the plasmids were able to process and secrete immunoglobulin following heat induction. The glioma transfectants were studied in detail and were shown to secrete polymeric IgM in a yield similar to that obtained with a plasmacytoma. However, the glioma IgM was not associated with J chain and was largely composed of pentamers and hexamers. Thus, neither J chain nor other lymphoid-specific proteins are required for assembly and secretion of polymeric IgM although the absence of J chain may encourage hexamer formation.     

T-cell regulation of B-lymphoblastoid cell line function

Seventeen B-lymphoblastoid cell lines (B-LCLs) have been studied for their ability to intereact with normal T cells and produce IgG and IgM in culture. All B-LCLs were HLA homozygous, having been derived from consanguineous donors by in vitro transformation with Epstein-Barr virus, 1 × 10⁴ B-LCLs were cultured with 0, 5, 10, or 20 times as many normal peripheral blood T cells in a 0.2-ml culture. Culture supernatants were removed after 3 and 6 days and assayed for IgG and IgM by a radioimmunoassay. Thirteen of the cell lines were able to secrete immunoglobulin (50–6000 ng/ml), primarily IgG, when cultured without T cells. Addition of T cells (sheep erythrocyte rosette-forming cells) modulated immunoglobulin production, causing either marked enhancement or suppression depending upon the B-cell line. T cells cultured without the B-LCLs did not secrete immunoglobulin above the background level of the immunoassays (6.25 ng/ml). Cell lines which did not secrete IgM when cultured alone could frequently be induced to do so when T cells from select donors were added. Under these conditions, IgM was generally found only in the supernatant fluid removed after 6 days. Taken together, these results suggest that B-LCLs contain cells of at least two stages, those that secrete IgG and resting cells capable of secreting IgM. Furthermore, cells at both stages can be regulated by normal T cells.     

Isolation and characterization of a variant of mouse plasmacytoma J558 synthesizing a 110,000-dalton immunoglobulin heavy chain and of secondary variants synthesizing either a 55,000-dalton or an 80,000-dalton immunoglobulin heavy chain: possible implications.

A mutant has been isolated from the J558 (immunoglobulin A, lambda, anti-alpha 1 leads to 3 dextran) cell line which synthesizes a heavy-chain immunoglobulin twice the size of normal heavy chain. Secondary variants that synthesized heavy chains either 1.5 times as large as wild type or the same size as wild type were identified. All mutants were serologically immunoglobulin continued to bind antigen, and retained the individual idiotype of the parent. Northern blot analysis and in vitro synthesis studies showed that the large heavy chains were primary synthetic products and not the consequence of abnormal covalent bonds. Cleavage of genomic DNA with restriction endonucleases and molecular hybridization studies showed new fragments in the 2 X and 1.5 X mutants which disappeared in the 1 X revertant. These data cannot easily be reconciled with the mutants arising either by unequal recombination or gene conversion. Further molecular characterization of these mutants should give additional insight into immunoglobulin gene evolution.     

Pre-pre-B cells containing only cytoplasmic B220 can be induced by dendritic cells and T cells to differentiate in vitro

We have derived from spleens of nude mice early B lineage cells that were phenotypically compatible with a pre-pre-B cell stage of differentiation. Although these cells containing large basophilic granules had the B lymphocyte antigen B220, in the cytoplasm, they had no surface B220, no cytoplasmic or surface immunoglobulin heavy or light chains, no surface Thy-1, and no surface Ia. In addition, they appeared to have little or no heavy chain gene rearrangements, including the D to J that occurs on both chromosomes prior to the VH rearrangement that forms the code for the C mu heavy chain polypeptide. Cells at even this early stage of differentiation could be induced by DC-T to express B220 on the surface and to synthesize and then to secrete immunoglobulins. These phenotypic changes were associated with a morphologic change in the cells to a lymphoblastoid appearance. Different patterns of immunoglobulin secretion resulted when pre-pre-B cells were cocultivated with DC-T from different tissues; SP DC-T induced the secretion of only IgM, PP DC-T induced the secretion of IgM as well as IgG and IgA. The early inductive event(s) appeared to occur during cell-cell contact in aggregates of the inducing DC-T and the pre-pre-B cells.     

The effect of peptide deletions on the glycosylation of murine immunoglobulin M heavy chains

The glycosylation and processing of the asparagine-linked oligosaccharides at individual glycosylation sites on the mu-chain of murine immunoglobulin M were investigated using variant cell lines that synthesize and secrete IgM heavy chains with known peptide deletions. Normal murine IgM has five N-linked oligosaccharides in the constant region of each heavy or mu-chain. Each mu-chain has four complex-type oligosaccharides as well as a single high mannose-type oligosaccharide near the carboxyl terminus of the molecule. The peptide deletion of the C mu 1 constant region domain in the heavy chains synthesized by one variant cell line did not prevent subsequent glycosylation at more distal glycosylation sites. In fact, the presence of this deletion resulted in more complete glycosylation at the C-terminal glycosylation site. Evaluation of glycopeptides containing individual glycosylation sites by Concanavalin A-Sepharose indicated that this deletion had no significant effect on the processing of structures from high mannose-type to complex-type oligosaccharide chains. In contrast, a deletion of the C-terminal peptide region of the heavy chain of IgM synthesized by a second variant cell line resulted in intracellular processing to more highly branched oligosaccharide structures at several of the glycosylation sites not involved in the deletion.     

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.     

Secretion of either of a pair of immunoglobulins,IgM or IgX,in somatic hybrid cells derived by fusion of a B-cell lymphoma cell line carrying both immunoglobulin isotypes

The I.29 cell line is a nonsecreting B-cell leukemia which bears two different immunoglobulin isotypes on its surface, IgM and IgX. The I.29 cells were hybridized with nonsecreting myeloma cells giving rise to dozens of immunoglobulin secreting hybridomas. These fall into three groups differing in the class of immunoglobulin they secrete. Cells of the first group secrete pentameric IgM (, ), those of the second group secrete an unknown immunoglobulin, IgX, which may constitute an allotype of IgA, and those of the third group produce light chains only. The two complete immunoglobulins, IgM and IgX, have the same idiotype, as revealed by serological cross-reactivity of an exhaustively absorbed rabbit anti-idiotype serum.The molecular sizes of the heavy chains of the secreted IgM and IgX are slightly smaller than the and chains, respectively, which are derived from the surface of normal B cells as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.Abbreviations used in this paper Ig immunoglobulin - NMS normal mouse serum - NaDodSO₄ sodium dodecyl sulfate - PAGE polyacrylamide gel electrophoresis - 2ME 2-mercaptoethanol - BPB bromophenol blue - NP40 nonidet P40 This particular immunoglobulin heavy chain has not been fully characterized. It is neither , , nor but is related to, although not identical with, . Because this immunoglobulin has unique properties, it is referred to as IgX.     

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.     

Immunological phenotype of lymphomas induced by avian leukosis viruses.

The production of immunoglobulin by six cell lines derived from bursal tumors induced by avian leukosis virus follows two general patterns: (i) three cell lines that have been extensively passaged in culture synthesize and secrete light chains only; (ii) three cell lines that are recently isolated produce and secrete monomeric immunoglobulin M in addition to free light chains. All six cell lines synthesize and secrete both glycosylated and unglycosylated forms of light chain. We conclude that the cell lines established from lymphomas induced by avian leukosis virus represent relatively mature, but possibly abnormal, stages in the development of chicken B-lymphocytes. The immunoglobulin M produced by the cell lines failed to form detectable immune complexes with avian leukosis virus. It therefore appears that the immunoglobulin M is not directed against viral antigens and that autogenous antigenic stimulus cannot account for the sustained growth of the neoplastic B-lymphocytes.     

Regulation of mucosal B cell immunoglobulin secretion by intestinal epithelial cell-derived cytokines

Intestinal epithelial cells (IEC) secrete a variety of cytokines and, because of their close proximity to B cells in the lamina propria, may affect local antibody production via these cytokines. However, studies have not yet addressed which and to what extent these IEC-derived cytokines may affect B cell antibody production. In this study, rat mesenteric lymph node B cells were cultured with culture supernatants from the rat IEC-6 intestinal epithelial cell line to determine their effect on immunoglobulin (Ig) secretion. Unstimulated IEC-6 cells were found to secrete sufficient levels of IL-6 to enhance IgA, IgG and IgM secretion by unstimulated B cells. However, culture of lipopolysaccharide (LPS)-stimulated B cells with the unstimulated IEC-6 supernatant resulted in an enhancement of IgA secretion while IgM secretion was significantly suppressed. Depletion of the IEC-6 supernatant using cytokine specific antibodies revealed that both interleukin 6 (IL-6) and transforming growth factor beta (TGF-beta) were responsible for the enhanced IgA secretion while TGF-beta suppressed IgM secretion. More importantly, culture supernatants from LPS stimulated IEC-6 cells contained enhanced levels of IL-6 which enhanced both IgG and IgA production and partially overcame the suppressive effect of TGF-beta on IgM secretion. These results suggest that intestinal epithelial cells may secrete IL-6 and TGF-beta to regulate local B cell antibody secretion and their effect may be highly dependent upon the activation state of the epithelial cells.     

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.     

Synthesis of abnormal immunoglobulins by hybridomas from autoimmune "viable motheaten" mutant mice.

Secretory defects in abnormal plasma cells, called Mott cells, that appear in lymphoid tissues of spontaneously autoimmune, "viable motheaten" (mev/mev) mice lead to deposition of immunoglobulin in RER-bound vesicles. Such vesicles have been termed Russel bodies. Cells with Russel bodies can also be observed rarely in normal animals, usually as a result of extreme antigenic loads or pathologic states. To understand why these abnormal cells appear commonly in mev/mev mice, we have established a panel of hybridomas that contain Russell bodies. Using immunochemical analysis and immunoelectron microscopy, we have characterized the secretory defects. Although these hybridoma cells synthesize a normal size heavy chain and it associates with light chain, the Russell bodies have many characteristics of inclusion bodies, which commonly appear in cells synthesizing mutant proteins and often are associated with incompletely or abnormally folded proteins. Pulse-chase experiments showed that immunoglobulins synthesized by these hybridomas accumulate rapidly into insoluble complexes and have an intracellular half life approximately 10 time greater than normal immunoglobulins. The defect affected only the immunoglobulin derived from the mev/mev mice and did not affect the secretion of normal immunoglobulin produced by an IgG1-secreting fusion partner. In addition to accumulating intracellular immunoglobulins, many mutant cell lines also secreted immunoglobulin. Endoglycosidase H digestion was used to determine the state of processing of the N-linked carbohydrates on the immunoglobulin molecules. This analysis demonstrated that the N-linked carbohydrates on the secreted immunoglobulin were resistant to endoglycosidase H digestion, indicating that they were processed normally. The insoluble IgM molecules were sensitive to endoglycosidase H, which is consistent with their localization to the RER. We propose several models by which these abnormal immunoglobulin-secreting cells commonly appear in this autoimmune mutant mouse.     

Fish immunoglobulins and the genes that encode them

The nature of fish antibodies (concentrating primarily on the most studied species of bony and cartilaginous fishes) is discussed in terms of their immunoglobulin biochemistry and immunobiology. The major serum immunoglobulin (IgM) is described in detail, and structural variants of IgM are discussed in terms of their distribution in different fish species, and different anatomical sites within a fish (e.g. blood, mucus, bile). Structural variation in IgM includes the size of the constituent heavy and light polypeptide chains, and the extent to which they are covalently associated with one another. The intramolecular heterogeneity of binding sites for antigen on IgM is reviewed and possible mechanisms for the phenomenon are presented. The evidence suggests that some, but not all, species of fish possess a detectable J chain in their IgM. The general nature of the fish immune response is that IgM antibody of moderate affinity is produced and prolonged or repeated immunization: (a) fails to produce a switch to production of a non-IgM class of antibody, and (b) fails to induce substantial increases in the affinity of the specific antibodies. Evidence supports a conclusion that fish lack the typical secondary antibody response seen in mammals, and possess antibodies of limited heterogeneity. Our current understanding of the genetic basis for fish antibodies is presented. Fish appear to utilize the same basic genetic elements as mammals to encode and regulate the expression of their immunoglobulins. The teleost heavy chain (IgH) locus resembles that of mammals and amphibia in its organization. The IgH locus of elasmobranchs is arranged in a unique multicluster organization. The light chain loci of elasmobranchs are organized analogously to the heavy chain locus (in multiclusters). The structure of the light chain locus of teleosts is presently unknown. Teleost fish utilize a unique pattern of RNA processing to generate the secreted and membrane receptor forms of the IgM heavy chain. The genes encoding the unique low molecular weight Ig heavy chain found in skates and rays have been cloned and sequenced, and also display the multicluster pattern of organization. Teleost fish appear to have normal numbers of variable regions: it is hypothesized (but as yet unproven) that the failure of their IgM to increase in affinity is due to a deficiency of somatic hypermutational mechanisms in their Ig gene variable regions during B lymphocyte differentiation.     

Developmental regulation of IgM secretion: the role of the carboxy-terminal cysteine

B lymphocytes do not secrete IgM, and plasma cells only secrete IgM polymers. Here we show that both events are attributable to the tailpiece found at the carboxyl terminus of mus chains, and we specifically implicate Cys-575. Thus, if Cys-575 was mutated, IgM was secreted by B cells. Similarly, a mutant IgG containing a mus tailpiece became largely retained within the cell; secretion was restored upon mutation of the tailpiece cysteine. Removal of Cys-575 also allowed hypersecretion of monomeric IgM by plasmacytoma cells. Following further removal of Cmu1, heavy chains were secreted in the absence of light chains. Thus, in B and plasma cells, Cys-575 is involved both in the polymerization of IgM and in intracellular retention of unpolymerized intermediates.     

Abnormalities in the glycosylation of immunoglobulin heavy chain and an h-2 transplantation antigen in a mouse myeloma mutant.

Two mutant cell lines derived from the MPC-11 mouse myeloma synthesize immunoglobulin with abnormal heavy chains and normal light chains. The defective heavy chains have molecular weights of 38,000-42,000 (M3.11) and 50,000 daltons (ICR 11.19) as compared to 55,000 daltons of the wild-type. The glycosylation of the defective heavy chains demostrated several unusual features: first, 30-50% of the M3.11 heavy chain contained no carbonydrate, while 100% of the wildtype and ICR 11.19 heavy chains were glycosylated; second, the glycopeptides of the M3.11 heavy chains revealed an altered gel filtration pattern when compared with the wild-type; and third, digestion with an endoglycosidase indicated that the heterogeneity of the wild-type and M3.11 glycopeptides involved structural changes in the core region of the oligosaccharide. Examination of two other glycoproteins (the major histocompatibility complex antigens) in these cell lines showed that in M3.11, the H-2D but not the H-2K product was abnormally glycosylated and contained a smaller glycopeptide. However, in a subclone of M3.11 that had lost the ability to produce immunoglobulin heavy chains, the H-2D glycopeptide had returned to wild-type size. We concluded from these studies that the defective M3.11 immunoglobulin heavy chain interfered both with its own glycosylation and the glycosylation of another protein, H-2D.     

Duplicated variable region genes account for double isotype expression in a human leukemic B-cell line that gives rise to single isotype-expressing cells.

The SSK41 cell is an immunoglobulin IgM+ human neoplastic B-cell line that switches to IgG+ cells (SSK gamma) spontaneously. We isolated a derivative of SSK41 (SSKWB) that expressed both IgM and IgG. Studies on the Ig heavy chain gene organization have shown that the SSKWB cell had two identical VDJ genes on the same chromosome; one linked to the C mu gene and the other to the C gamma 1 gene, both of which are transcribed to produce mu- and gamma-mRNAs with the same VDJ sequence. We also isolated the two switch variants derived from the SSKWB cell by cell sorter: 1G (IgG+) and 11M (IgM+). The 1G cells contained two populations; one had a similar genetic organization as SSK gamma and expressed only IgG1, and the other carried the same genetic organization as the SSKWB cell but produced aberrantly spliced mu-chain mRNA, in which the hydrophobic signal sequence exon is directly joined to the C mu exon. Te 11M cell deleted the VDJ-C gamma 1 segment of the SSKWB cell and expressed IgM. These results raise the interesting possibility of another mechanism for class switching.