Somatic cell hybrids of mouse myeloma cells and B lymphocytes from a patient with agammaglobulinemia: failure to secrete human immunoglobulin.

Somatic cell hybrid clones were isolated from the fusion of RPC5.4 mouse myeloma cells and B lymphocytes from a patient with common varied agammaglobuinemia. The patient has B lymphocytes that synthesize immunoglobulin but fail to secrete immunoglobulin. The hybrid character of the six clones was established by examination of metaphase chromosome spreads. Most of the hybrid clones expressed mouse and human surface immunoglobulin. All of the clones synthesized immunoglobulin of mouse and human parental origin. Mouse parental immunoglobulin was secreted, whereas the human parental immunoglobulin was not secreted. Human light chain molecules were secreted as part of hybrid H2L2 molecules formed with mouse heavy chains. Human heavy chains had a reduced m.w. in comparison to the mouse heavy chains. Kinetic experiments indicated that human Ig was synthesized in amounts that were comparable to the mouse Ig. Pulse-chase experiments showed that that the intracellular human Ig was removed from the cytoplasm, probably by degradation. These experiments demonstrate that the hybrid cells are an in vitro model of naturally occurring failure of immunoglobulin secretion from agammaglobulinemia. The failure of fusion with mouse myeloma cells to complement the secretion defect suggests that these B cells produce an altered immunoglobulin molecule that is not programmed for secretion.     

Two mRNAs with different 3′ ends encode membrane-bound and secreted forms of immunoglobulin μ chain

During differentiation, B lymphocytes undergo a shift from expression of membrane-bound IgM to IgM secretion. The μ chains of membrane and secreted IgM, μ_m and μ_s, respectively, differ in the amino acid sequence of their carboxy terminal regions. In this paper, we demonstrate that μ_m and μ_s heavy chains are encoded by separate mRNAs of 2.7 and 2.4 kb, respectively. Restriction mapping and sequence analysis of μ cDNA clones from a myeloma tumor that produces both types of μ chain indicate that the μ_m and μ_s mRNAs are identical throughout the coding region up to the 3′ end of the fourth constant region (C_μ4) domain, but differ in their C terminal coding and 3′ untranslated segments. From the nucleotide sequence of the μ_m cDNA clone, we predict the amino acid sequence of the 41-residue μ_m C terminal segment or “M” (membrane) segment. This sequence has characteristics consistent with its being a transmembrane peptide. Thus the μ_s chain has a 20-residue hydrophilic C terminal segment after the C_μ4 domain, and the μ_m chain has a 41-residue C terminal segment containing a hydrophobic sequence. We propose that comparable C terminal segments also will be found in other membrane-bound immunoglobulin heavy chains.     

Amplification of IgG VH and VL (Fab) from single human plasma cells and B cells

Amplification of human immunoglobulin has many potential applications such as analysis of clonality, isolation of immunogenic antigens and antigen-specific immunotherapy. Here we describe a method for amplification of human immunoglobulin heavy and light chains from single B lymphocytes or plasma cells. Cells are isolated by FACS, and Ig is amplified by semi-nested RT–PCR. The method is versatile, sensitive and reliable: it provides appropriately paired heavy and light chains, requiring as little as 2 days to produce amplified Fab DNA from human tissues.     

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.     

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.     

Posttranslational association of immunoglobulin heavy chain binding protein with nascent heavy chains in nonsecreting and secreting hybridomas

A rat monoclonal antibody specific for immunoglobulin (Ig) heavy chain binding protein (BiP) has allowed the examination of the association of BiP with assembling Ig precursors in mouse B lymphocyte-derived cell lines. The anti-BiP monoclonal antibody immunoprecipitates BiP along with noncovalently associated Ig heavy chains. BiP is a component of the endoplasmic reticulum and binds free intracellular heavy chains in nonsecreting pre-B (mu+, L-) cell lines or incompletely assembled Ig precursors in (H+, L+) secreting hybridomas and myelomas. In the absence of light chain synthesis, heavy chains remain associated with BiP and are not secreted. The association of BiP with assembling Ig molecules in secreting hybridomas is transient and is restricted to the incompletely assembled molecules which are found in the endoplasmic reticulum. BiP loses affinity and disassociates with Ig molecules when polymerization with light chain is complete. We propose that the association of BiP with Ig heavy chain precursors is a novel posttranslational processing event occurring in the endoplasmic reticulum. The Ig heavy chains associated with BiP are not efficiently transported from the endoplasmic reticulum to the Golgi apparatus. Therefore, BiP may prevent the premature escape and eventual secretion of incompletely assembled Ig molecules.     

Impaired lymphocyte functions in heterozygous rabbits recovering from neonatal allotype suppression

Lymphocytes from heterozygous rabbits suppressed for an allotypic determinant on kappa light chains by exposure to maternally derived antibodies specific for the paternal gene product were analyzed for their capacity to express membrane-bound and secreted immunoglobulin (Ig). Individual cells displaying allotypic membrane Ig (mIg) were enumerated by a rosette test, while Ig-secreting cells were assessed by means of a hemolytic plaque assay. In a group of suppressed rabbits varying in age from 3 to 19 months, the proportion of cells with mIg of the paternal type was markedly higher than that of cells secreting that type of Ig. The same high proportion of lymphocytes displaying mIg of the suppressed type was observed whether lymphocytes from blood, spleen, or lymph nodes of suppressed rabbits were examined. In contrast, similar analyses performed with cells of normal heterozygous rabbits showed no discrepancy between mIg expression and secretion of either allotype. Lymphocytes synthesizing Ig of the paternal type were also defective in responses to lipopolysaccharide (LPS), which stimulates differentiation to Ig secretion in normal B lymphocytes. These results support the idea that B lymphocytes capable of synthesizing the suppressed type of Ig have functional impairments affecting secretion and responses to environmental stimuli.     

Evidence for an IgD homologue on chicken lymphocytes

Chicken lymphocyte membrane immunoglobulins (Ig), were precipitated with mouse monoclonal antibodies specific for heavy and light chain isotypes and analyzed by polyacrylamide gel electrophoresis. Very little or no membrane-bound IgG and IgA was detected. After sequential precipitation and removal of IgM reactive with any of three monoclonal anti-mu antibodies, anti-light chain antibody precipitated residual Ig with a relative electrophoretic mobility similar to that of IgM. Under reducing conditions, these surface Ig molecules had a heavy chain that appeared slightly larger (approximately 81,000 daltons) than mu-chain (approximately 79,000 daltons), and light chains of approximately 25,000 daltons. Complete clearance of membrane-bound IgM reactive with an anti-mu allotype antiserum left similar molecules precipitate by monoclonal anti-light chain antibody. These non-IgM molecules could be detected on the surface of lymphocytes from blood, spleen, bursa and the B cell line RAV-1, but not from thymus or blood from an agammaglobulinemic chicken. After capping of B cell surface IgM with anti-mu, immunofluorescent staining with anti-light chain antibody revealed residual Ig molecules disturbed across the surface of more than 90% of the IgM-bearing cells. The data suggest the existence of an avian homologue of mammalian IgD. Affinity-purified goat anti-mu antibodies and a fourth monoclonal anti-mu antibody reacted with both IgM and the putative IgD molecules, which suggests that the IgD homologue shares at least one common determinant with chicken IgM.     

Cellular basis of B cell clonal populations in old mice.

Previous studies from this laboratory have shown that >85% of old mice have stable B cell clonal populations detectable by Ig heavy chain complementary-determining region 3 mRNA size analysis and confirmed by sequence analysis. B cells from the same clone are frequently detected in several lymphoid compartments of the same mouse. We now report the phenotype of all ten stable B cell clonal populations detected in five 20-month-old C57BL/6 mice. These clonal B cells appear to develop in the periphery and nine of the ten B cell clonal populations expressed the CD5 cell surface marker. Stable B cell expansions may be dominated by cells at two stages of differentiation. Some B cell populations were detected with DNA as well as RNA and represent large clonal populations of B cells, detectable in several lymphoid compartments. These populations are found predominantly in B cell populations expressing CD45R/B220 and the mRNA coding for the membrane-bound form of the mu Ig heavy chain, which suggests a predominance of B lymphocytes in these populations. In other cases, smaller clonal populations were detected only in splenic RNA samples. These clonal populations were found predominantly among CD45R/B220- B cells and did not express the membrane-bound form of the micro Ig heavy chain. We offer the hypothesis that the B cell clonal populations present in old mice may be precursors of the two types of B cell neoplasms which are dominated by CD5+ B cells (B cell chronic lymphocytic leukemia) or plasma cells (multiple myeloma).     

Assembly and secretion of heavy chains that do not associate posttranslationally with immunoglobulin heavy chain-binding protein

Heavy chain-binding protein (BiP) associates posttranslationally with nascent Ig heavy chains in the endoplasmic reticulum (ER) and remains associated with these heavy chains until they assemble with light chains. The heavy chain-BiP complex can be precipitated by antibody reagents against either component. To identify sites on heavy chain molecules that are important for association with BiP, we have examined 30 mouse myelomas and hybridomas that synthesize Ig heavy chains with well characterized deletions. Mutant Ig heavy chains that lack the CH1 domain could not be demonstrated to associate with BiP, whereas mutant Ig heavy chains with deletions of the CH2 or CH3 domain were still able to associate with BiP. In two light chain negative cell lines that produced heavy chains with deletions of the CH1 domain, free heavy chains were secreted. When Ig assembly and secretion were examined in mutants that did not associate with BiP, and were compared with normal parental lines, it was found that the rate of Ig secretion was increased in the mutant lines and that the Ig molecules were secreted in various stages of assembly. In one mutant line (CH1-) approximately one-third of the secreted Ig molecules were incompletely assembled, whereas the Ig molecules secreted by the parental line were completely assembled. Our data show the CH1 domain to be important for association with BiP and that when this association does not occur, incompletely assembled heavy chains can be secreted. This implies a role for BiP in preventing the transport of unassembled Ig molecules from the ER.     

Mitogen activation of human chronic lymphatic leukemia cells. I. Synthesis and secretion of immunoglobulin.

The response of CLL (chronic lymphatic leukemia) lymphocytes to PHA, PWM, and Con A with respect to changes in surface markers and synthesis and secretion of immunoglobulin were examined. After PHA stimulation the percentage of cells bearing readily detectable surface immunoglobulin (SmIg) diminished rapidly whereas cells forming rosettes with sheep erythrocytes (E-rosettes) increased from less than 1% to 30 to 50%. The great majority of blast-transformed cells were E-rosette-positive cells with a small population of SmIg-positive blast cells also observed. Ig production in four of seven CLL lymphocyte populations was increased 2.5 to greater than 40-fold after 4 to 6 days of culture in the presence of PHA. In contrast, pokeweed mitogen did not affect Ig synthesis. Furthermore, the Ig secreted into the culture supernatant fluids from seven of eight CLL cases examined consisted almost entirely of free light chain molecules. In contrast, the cell lysates contained a significant proportion of intact Ig molecules. These results indicate that CLL cells can, under certain circumstances, be stimulated to synthesize and secrete increased amounts of Ig, but that there is a basic defect in the biosynthetic mechanism of these cells which result in the secretion of free light chains rather than intact immunoglobulin molecules.     

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.     

An Hsp70-like protein in the ER: identity with the 78 kd glucose-regulated protein and immunoglobulin heavy chain binding protein

We have characterized a cDNA clone that encodes a protein related to the 70 kd heat shock protein, but is expressed in normal rat liver. This protein has a hydrophobic leader and is secreted into the endoplasmic reticulum. We show that it is identical with two previously described proteins: GRP78, whose synthesis is induced by glucose starvation, and BiP, which is found bound to immunoglobulin heavy chains in pre-B cells. This protein, which is abundant in antibody-secreting cells, can be released from heavy chains by ATP, a reaction analogous to the release of hsp70 from heat shocked nuclear structures. We propose a specific role for this protein in the assembly of secreted and membrane-bound proteins.     

B cells

B cells are an important component of adaptive immunity. They produce and secrete millions of different antibody molecules, each of which recognizes a different (foreign) antigen. The fact that humans express a very large repertoire of antibodies is due to the complex mechanism of V(D)J recombination of immunoglobulin (Ig) genes as well as other processes including somatic hypermutation, gene conversion and class switching. The B cell receptor (BCR) is an integral membrane protein complex that is composed of two Ig heavy chains, two Ig light chains and two heterodimers of Igalpha and Igbeta. To eliminate foreign antigens, B cells cooperate with other cells of the immune system including macrophages, dendritic cells and T cells. B cell development is a tightly controlled process in which over 75% of the developing cells become apoptotic because of inappropriate immunoglobulin gene rearrangements or recognition of self antigens by Igs. Hence, the majority of B cell-associated disorders are caused by the incorrect function of genes/proteins involved in B cell development.     

Human hybrid immunoglobulin M containing immunoglobulin A

Summary Some hybridoma clones made by fusion of a human lymphoblastoid cell line, HO323 with human B lymphocytes, secreted not only IgA but also IgM-like immunoglobulin molecules. The IgM-like immunoglobulin had a molecular size of 900 K which corresponded to that of IgM. Immunochemical analyses revealed that the IgM-like immunoglobulin contained two monomeric IgA and three monomeric IgM molecules. In the IgA moieties, half of original light chains were replaced withx chains derived from the IgM, and vice versa.     

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.     

Assembly, secretion, and vacuolar delivery of a hybrid immunoglobulin in plants

Secretory immunoglobulin (Ig) A is a decameric Ig composed of four alpha-heavy chains, four light chains, a joining (J) chain, and a secretory component (SC). The heavy and light chains form two tetrameric Ig molecules that are joined by the J chain and associate with the SC. Expression of a secretory monoclonal antibody in tobacco (Nicotiana tabacum) has been described: this molecule (secretory IgA/G [SIgA/G]) was modified by having a hybrid heavy chain sequence consisting of IgG gamma-chain domains linked to constant region domains of an IgA alpha-chain. In tobacco, about 70% of the protein assembles to its final, decameric structure. We show here that SIgA/G assembly and secretion are slow, with only approximately 10% of the newly synthesized molecules being secreted after 24 h and the bulk probably remaining in the endoplasmic reticulum. In addition, a proportion of SIgA/G is delivered to the vacuole as at least partially assembled molecules by a process that is blocked by the membrane traffic inhibitor brefeldin A. Neither the SC nor the J chain are responsible for vacuolar delivery, because IgA/G tetramers have the same fate. The parent IgG tetrameric molecule, containing wild-type gamma-heavy chains, is instead secreted rapidly and efficiently. This strongly suggests that intracellular retention and vacuolar delivery of IgA/G is due to the alpha-domains present in the hybrid alpha/gamma-heavy chains and indicates that the plant secretory system may partially deliver to the vacuole recombinant proteins expected to be secreted.     

Regulation of immunoglobulin production in human peripheral bood leukocytes: cellular interactions.

The intercellular influences regulating immunoglobulin (Ig) synthesis by normal human peripheral blood leukocytes (PBL) were investigated in cells stimulated by pokeweed mitogen (PWM). This system was shown to be totally T lymphocyte dependent as purified B lymphocytes (less than or equal to 1% T lymphocytes) failed to make significant amounts of Ig. No evidence was obtained for an Ig class switch as all classes of Ig (IgM, IgG, IgA) were shown to be produced in increasing amounts over a 6-day time period. T lymphocytes demonstrated maximum helper effect when mixed with equal numbers of B cells. This helper effect was mediated through the dual mechanisms of increasing the number of B lymphocytes containing cytoplasmic Ig and by increasing the maturity of these B lymphocytes as demonstrated by an increasing Ig production per B lymphocyte. When present in higher numbers, T lymphocytes were also capable of suppressing Ig production. This T-mediated suppression was first evident as a decrease in the Ig produced per B lymphocyte (decreased maturity). With maximum T suppression Ig-containing B lymphocyte numbers were also diminished. T lymphocyte help was relatively independent of macrophages (phagocytic cells) and did not require DNA synthesis for expression. Both T help and suppression were shown to cross allogeneic barriers. Immature T lymphocytes (thymocytes) were incapable of mediating either activity. Normal human PBL contain T lymphocytes campable of mediating both T help and suppression and the Ig produced by PBL was shown to be the balance of these activities. This balance probably represent the participation of distinct T lymphocyte subpopulations analogous to the T helper (Ly 1+) and T suppressor (Ly 2+, 3+) populations in the mouse.     

The T cell differentiation antigen Leu-2/T8 is homologous to immunoglobulin and T cell receptor variable regions

Leu-2/T8 is a cell surface glycoprotein expressed by most cytotoxic and suppressor T lymphocytes. Its expression on T cells correlates best with recognition of class I major histocompatibility complex antigens, and it has been postulated to be a receptor for these proteins. We have determined the complete primary structure of Leu-2/T8 from the nucleotide sequence of its cDNA. The protein contains a classical signal peptide, two external domains, a hydrophobic transmembrane region, and a cytoplasmic tail. The N-terminal domain of the protein has striking homology to variable regions of immunoglobulins and the T cell receptor. The membrane-proximal domain appears to be a hinge-like region similar to that of immunoglobulin heavy chains. The superfamily of immunologically important surface molecules can now be extended to include Leu-2/T8.     

Emergence of a surface immunoglobulin recycling process during B lymphocyte differentiation

Surface immunoglobulin (Ig)-mediated endocytosis has been investigated in rat B lymphocytes and plasma cells, using horseradish peroxidase (HRP)-labeled sheep anti-rat Ig Fab' fragment of antibody and HRP as monomeric ligands, respectively. Quantitative estimates of HRP activity associated either with plasma membrane or with endomembrane compartments were made in several experimental conditions. Binding of HRP-conjugate on B lymphocytes was followed by its endocytosis in combination with surface Ig, as shown by the progressive disappearance of plasma membrane-associated HRP activity. Between 1 and 6 h at 37 degrees C in presence of conjugate the total amount of cell-associated activity was constant. These results indicate that during this time no reappearance of surface Ig occurred by neosynthesis, by the expression of an intracellular pool or by the recycling in a free form of the previously internalized molecules. On the contrary, at saturating doses, internalization of HRP by anti-HRP plasma cells increased linearly with time at 37 degrees C in presence of antigen, when, during the same time, the plasma membrane HRP-binding capacity remained constant. Cycloheximide did not affect continuous HRP uptake. The existence of a large intracellular pool of receptors has been ruled out by experiments of removal of binding sites with pronase. In addition, monensin caused a progressive decrease in the number of surface receptors on plasma cells but not on B lymphocytes. Our data then indicate that, unlike B lymphocytes, plasma cells were able to recycle their surface Ig.