Different Responses Elicited in Vitro by Antibodies to IgM and IgD in Cells from a Surface IgM + D-Positive Human Follicular Lymphoma

The effect of anti-Igs in combination with the tumour promotor TPA on DNA and Ig synthesis in a human follicular germinal centre cell lymphoma carrying sIgM and sIgD was investigated. The lymphoma cells responded to both anti-mu and anti-delta chains and to anti-lambda light chains by DNA synthesis, as measured by methyl-(3H)-thymidine incorporation. Flow cytofluorometric measurements, however, showed that only anti-mu chains induced marked increase in cytoplasmic Ig content. This result suggests that in certain B-cell subsets the signals elicited via sIgM and sIgD are different.     

Free immunoglobulin light chain synthesis by human foetal liver and cord blood lymphocytes.

Immunoglobulin (Ig) synthesis by immature B lymphocytes from human foetal liver and cord blood has been investigated. Seven out of fifteen preparations of foetal liver cells and eight out of eleven cord bloods synthesized Ig detectable by biosynthetic labelling. All cultures of foetal lymphocytes with detectable Ig synthesis secreted free light chain. Two of these cases also synthesized free mu heavy chain. Cord blood lymphocyte synthesis patterns were variable ranging from free light chain as the only secreted Ig product to balanced synthesis of heavy and light chains. No cord blood cultures synthesized detectable free mu chains. Free Ig-light-chain synthesis appears to be associated with normal immature B lymphocytes and the results are discussed in relation to the B-cell differentiation pathway.     

Immunoglobulin diversification in embryonic chicken bursae and in individual bursal follicles

Previous studies have shown that the same immunoglobulin (Ig) V lambda gene (V lambda 1) is rearranged in all chicken B cells, and that extensive sequence diversification of this gene occurs during B cell development in the bursa of Fabricius. We used two-dimensional gel electrophoresis to compare the heterogeneity of Ig lambda light chains produced by B cells at different stages of bursal development. Somatically diversified light chains were observed in Ig molecules produced by bursal cells as early as 15 days of embryonic incubation. The two principal species of light chain observed probably represent glycosylated and nonglycosylated forms of lambda chain encoded by alleles of a single lambda gene. Extensive diversification was observed during late embryogenesis. We also studied lambda light chain diversity in cyclophosphamide-treated birds repopulated with normal bursal cells. In these birds, individual bursal follicles are repopulated by single B cell precursors. Follicular cells derived from single B cell precursors were able to produce a spectrum of light chains almost as diverse as that of the total bursal cell population. We used two monoclonal anti-idiotype antibodies to study idiotype expression in individual normal or reconstituted follicles. About 30% of follicles contained 0.1% to 5% of lymphocytes which reacted with one or both of the antibodies. The results indicate that within individual bursal follicles bursa stem cells undergo Ig hyperdiversification.     

Interferon-gamma induces light chain synthesis in interleukin 2 stimulated human B cells

Human B cells were cultured without added lymphokines, with interleukin 2 (IL2) or interferon-gamma (IFN-gamma) alone, or with a combination of IL2 and IFN-gamma. Treatment with IL2 alone induced differentiation of the B cells, as shown by an increase in the number of immunoglobulin (Ig)-containing cells and plaque-forming cells, and by a larger amount of Ig secreted into the medium. In most of the induced Ig-containing cells, heavy chains but not light chains were detectable by cytoplasmic staining. Treatment with IFN-gamma alone did not stimulate B cell differentiation. However, a combination of IFN-gamma and IL2 was more effective than IL2 alone in inducing B cell differentiation, as measured by further increases in the number of plaque-forming cells and in total Ig secretion. Furthermore, after treatment with both IL2 and IFN-gamma, most cells that contained cytoplasmic heavy chains also contained cytoplasmic light chains. We conclude that IFN-gamma acts in synergy with IL2 in B cell differentiation by enhancing light chain synthesis, leading to secretion of Ig molecules.     

Re-organization of the immunoglobulin kappa gene on both alleles is not an obligatory prerequisite for Ig lambda gene expression in human cells.

Re-arrangement and expression of the immunoglobulin (Ig) genes in B cells occurs under an ordered developmental control. The sequential model of Ig light chain exclusion predicts that only after non-productive re-organization or deletion of both Ig kappa alleles would re-arrangement of Ig lambda gene segments occur. To prove this model, we asked whether expression of Ig lambda light chains is always associated with rearrangement and/or deletion of both Ig kappa alleles in human cells. Therefore, we established human diploid B-cell clones in vitro that produce Ig lambda light chains. Southern blot analysis of the Ig kappa alleles revealed that three Ig lambda expressing cell lines (out of six Ig lambda+ cell lines tested) harbour one Ig kappa allele in germline configuration. Furthermore, a 1.5 kb RNA derived from the germline Ig kappa locus was detected by Northern blot hybridizations. The results implicate that the mechanism of Ig light chain exclusion is not precisely sequential and that it does not necessarily need re-arrangement or deletion of both Ig kappa alleles as a prerequisite for Ig lambda light chain expression in human cells.     

Phorbol ester-induced differentiation of chronic B lymphocytic leukaemia cells--regulatory impact of autologous and allogeneic accessory cells.

Phorbol ester (TPA) induction of chronic lymphocytic leukaemia (CLL) cells can be used as a model system for the study of human B cell differentiation. We have analysed the role of accessory cells and the correlation to target cell surface Ig phenotype in TPA-induced morphological and functional differentiation of 12 CLL populations. A more than three-fold increase in secreted IgM was seen in 10 of 12 cases, with the strongest responses in patients having monoclonal serum IgM. CLL populations negative for or having only weak surface mu chain expression were less inducible. The impact of autologous and allogeneic accessory cells on TPA induction was studied in cell enrichment/depletion experiments using both physical and cytotoxic antibody techniques. CLL cells physically depleted of autologous E+ and monocytic (light density fraction) cells still responded to TPA. This response could be enhanced by allogeneic E- light fraction cells. Further depletion of autologous accessory cells by treatment of the E- high density fraction CLL cells with a panel of monoclonal antibodies plus complement demonstrated the permissive role of one or two populations of autologous cells expressing low avidity E receptors and the T3, T4 and T8 antigens. Augmenting T cells of similar phenotype were found among allogeneic cells from normal individuals. Thus, TPA-induced IgM secretion in biopsy B-CLL cells is regulated by minute numbers of autologous helper T cells. Furthermore, the Ig secretory response of CLL populations seems to be correlated with the surface Ig the surface immunoglobulin phenotype of the leukaemic cells.     

Confirmation of the assignment of genes of human immunoglobulin heavy chains to chromosome 14 by analysis of Ig synthesis by man-mouse hybridomas

Hybridomas were produced by fusing the NS1 mouse myeloma line, which does not produce mouse heavy chain Ig, with human peripheral B lymphocytes from a normal individual. Two vigorously growing colonies from this fusion were found to secrete human Ig heavy chains and were recloned. Two secondary clones, which secreted human chains, were again recloned. Among the tertiary clones, two were identified which produced intracellular human Ig chains, but did not secrete immunoglobulin. These tertiary clones were recloned, generating 6 quaternary clones which failed to produce human Ig heavy chains, and 15 quaternary clones which produced intracellular Ig chains. Hybrid clones from each successive subcloning were examined for their human chromosomal content and only those clones which were found to be individually chromosomally distinct, a total of 56 clones in all, were used to analyze the segregation of human chromosomes and human Ig heavy chain synthesis. Results of this study indicate concordant segregation of human Ig heavy chain synthesis and chromosome 14. These studies therefore confirm the previous assignment by C. M. Croce et al. (Proc. Natl. Acad. Sci. USA 1979. 76: 3416) of the genes for human Ig heavy chains to chromosome 14.     

Cloning non-transformed sheep B cells

The capacity to clone B cells and establish permanent B cell lines has greatly facilitated a wide variety of studies characterising the growth, differentiation, and gene expression of murine and human B cells. Similar investigations of B cell biology for other species have been severely restricted by an inability to culture or clone B cells. This is the first report of a method to clone non-transformed sheep B cells using a culture system based on murine CD154 and a combination of human gamma chain-common cytokines. Sheep Peyer's patch B cells were cultured for 120 days and then cloned by limiting dilution culture. The parental B cell culture contained both surface immunoglobulin (sIg)M(+) and sIgG1(+) B cells and both types of B cell were cloned. Clonality was confirmed by PCR analysis of Ig heavy chain (HC) and light chain (LC) expression and DNA sequencing of HC V genes. There was agreement between the PCR and flow cytometric analyses of HC isotype expression on the B cell clones but the available monoclonal antibodies specific for sheep lambda and kappa LC did not react with all clones. Soluble Ig was detected in the culture supernatant of sIgG1(+) clones but not sIgM(+) clones. The B cell clones remained dependent upon CD154 and gamma chain-common cytokine co-stimulation for sustained growth and maintained stable Ig expression. The cloning of non-transformed sheep B cells should provide a valuable tool for studying sheep B cell biology, establishing Ig HC- and LC-specific monoclonal antibodies, analysing the B cell Ig repertoire, and may be used to produce sheep monoclonal antibodies.     

Stimulation of human B lymphocytes by antibodies to IgM and IgG: Functional evidence for the expression of IgG on B-lymphocyte surface membranes

Isolated human tonsillar and peripheral blood B lymphocytes were induced to proliferate in vitro after exposure to F(ab′)₂ fragments of affinity purified antibodies to IgM, IgG, Fab, κ, and λ chain determinants. Low levels of DNA synthesis were observed in cultures containing anti-IgA antibodies, whereas DNA synthesis was not detected in cultures stimulated with anti-IgD. Divalent antibodies were essential for the generation of blastogenesis. These proliferative responses were T-cell independent and susceptible to suppression by excess numbers of monocytes. In addition, they were elicitable in cultures not containing FCS or 2-mercaptoethanol. Highly specific antibodies to IgG induced marked proliferation and this was similar in degree to that induced by anti-IgM. Subfractionation studies demonstrated that the anti-IgG responsive cells were contained to a major extent within the surface IgM⁺ B-cell population. None of the antibody preparations elicited B-cell differentiation to antibody producing cells. Moreover, anti-μ antibodies completely abrogated Ig synthesis by pokeweed mitogen-stimulated cultures of unseparated tonsillar mononuclear cells. Anti-IgG antibodies similarly suppressed PWM-induced antibody production, although this was most apparent on the IgG response. In contrast anti-IgD antibodies both failed to suppress Ig production and in certain instances resulted in an increased level of Ig synthesis. These functional studies suggest that IgG molecules are intimately associated with the surface membrane of some B cells and that coexpression of IgG with IgM occurs. In addition, the observations emphasize further the divergent functional roles which surface IgM and IgG vs surface IgD have in B-cell proliferation and differentiation.     

Monoclonal antibodies specific for kappa chain, lambda chain, and IgG1 of human gammaglobulin

Hybrid cell lines secreting antibodies specific for human gammaglobulin (HGG) were prepared by cell fusion and cloning techniques. The monoclonal antibodies were tested for their antibody reacts with a different antigenic determinant of HGG. One reacts with isolated kappa (kappa) light chains, one with isolated lambda (lambda) light chains, and one with the Fc fragment of IgG1 molecules. The reactivity patterns of two additional monoclonal antibodies are more complex. One reacts with a determinant present on the Fc of all IgG subclasses and the other binds to a determinant on the Fab of IgG molecules. The two monoclonal antibodies reactive with light chains also bind to surface components of human B cells. The murine immunoglobulin (Ig) class of each clone product was identified.     

L and H cells of nodular lymphocyte predominant Hodgkin''s disease show immunoglobulin light-chain restriction.

The nodular form of lymphocyte predominant Hodgkin's disease (NLPHD) is widely accepted to be a B-cell-derived neoplasm. Despite this consensus, previous studies have not shown monotypic immunoglobulin (Ig) light-chain expression by the putatively malignant L & H cells. We have studied paraffin-embedded tissue from 19 cases of NLPHD for the presence of Ig light and heavy chains and J chain. In addition, frozen tissue, available from one case, was examined with antibodies to light chains. In paraffin material, in each case L & H cells in individual nodules were examined for the presence of Ig light chain restriction. Kappa-light-chain restriction was detected in all L & H cells in all nodules in 5/19 cases (26.5%) and this was confirmed in frozen sections available from one case. Of the remaining 14 cases, 13 (68.5%) showed kappa-light-chain restriction in a proportion of nodules and in 1 case light-chain restriction was not observed. Of 14 cases stained with antibodies to Ig heavy chains 13 contained IgG, and in 1 case no heavy chains were demonstrable. Most L & H cells in 15 cases examined contained J chain. Our finding of monotypic L & H cells in NLPHD in 95% of our investigated cases provides strong evidence for the neoplastic nature of L & H cells and supports the hypothesis that NLPHD is a malignant nonHodgkin's B-cell lymphoma.     

A role for human heavy chain binding protein in the developmental regulation of immunoglobin transport

Human EBV transformed lymphoblastoid cell lines and lymphomas representing various stages of B cell development were examined for heavy chain binding protein (BiP) expression and its association with immunoglobin (Ig) heavy chains. Human BiP was shown to migrate with an apparent mol. wt of 79,000 and to have a pI of approximately 5.5 in all the human cell lines examined. Both the mum and the mus heavy chains synthesized in a pre-B cell line (mu+, LC-) remained associated with BiP and were all found to be endo H sensitive, suggesting that this association occurred in the endoplasmic reticulum (ER). Surface Ig+ B cell lines produce membrane type heavy chains which are expressed on the cell surface and secretory type heavy chains which remain intracellular. The membrane type mu heavy chains produced by a surface Ig+ B cell line were not associated with BiP after assembling with light chains and processing in the Golgi. However, the secretory type mu heavy chains synthesized by these same cells did not combine efficiently with LC and a significant quantity remained associated with BiP and were not secreted suggesting that BiP is involved in the divergent transport of membrane and secretory mu heavy chains in surface Ig+ B cell lines. In Ig secreting plasmacytoid lines the heavy chains were only associated with BiP prior to assembling with LC. When LC assembly was inhibited, the association of heavy chains with BiP was prolonged and Ig secretion was blocked. Therefore, BiP was found to participate in the post-translational processing of mu heavy chains synthesized by human lymphoid cell lines representing all stages of B cell development. Further, heavy chains that remained associated with BiP were not transported to the cell surface or secreted while heavy chains that were only transiently associated with BiP chains were expressed on the cell surface or secreted.     

Monoclonal antibodies specific for murine IgM. II. Activation of B lymphocytes by monoclonal antibodies specific for the four constant domains of IgM

Seventeen monoclonal antibodies specific for IgM and one kappa light chain-specific antibody were used to test the effect of immunoglobulin (Ig)-specific antibodies on B cell activation. In soluble form, either alone or together with T cell-derived growth and maturation factors, none of the antibodies stimulated resting B cells to divide or secrete Ig. The soluble antibodies inhibited lipopolysaccharide-induced B cell activation. The inhibitory effect of the antibodies was independent of their Fc part. When immobilized, the same antibodies could activate B cells to proliferate and together with T cell-derived maturation factors to mature to plasma cells. Occupation by immobilized antibody of determinants on any of the four constant region domains and on the light chain of surface Ig can lead to the stimulation of B cells.     

Triggering of monoclonal human lymphoma B cells with antibodies to IgM heavy chains: differences of response obtained with monoclonal as compared to polyclonal antibodies

A comparative study of human B lymphoma cells activation by monoclonal (murine hybridoma) antibodies to mu heavy chains (Ma-mu) as compared to polyclonal (rabbit) antibodies to mu heavy chains (Ra-mu) has been carried out. Early events related to calmodulin activation such as 86Rb influx and changes in cell volume at 4 h could be induced by Ma-mu. One antibody (AF6) approached Ra-mu with regard to the strength of response obtained. However, Ma-mus including AF6 were deficient in inducing DNA synthesis under conditions where this was achieved with Ra-mu. Studies in one lymphoma, where stimulation of re-expressed surface IgM could be studied, revealed that Ma-mu was deficient in stimulating re-expressed sIgM. These findings raise questions with regard to polyclonal antibody to surface Ig as a model for B cell triggering by antigen and suggest that antigen-induced B cell triggering may be more complex than indicated by previous studies with polyclonal antibody.     

Modulation and High Frequency Expression of Autoantibody-Associated Cross-Reactive Idiotypes linked to the VhI Subgroup in CD5-Expressing B Lymphocytes from Patients with Chronic Lymphocytic Leukaemia (B-CLL)

Leukaemic B cells from patients with chronic lymphocytic leukaemia (B-CLL) are known to express the pan T-cell marker CD5 and a restricted set of immunoglobulin (Ig) variable region heavy (V_h) and light (V_L) chains encoded by germline or minimally mutated germline genes. We have studied surface expression of certain V_H and V_K gene products on peripheral blood B lymphocytes from 23 patients with B-CLL, using a panel of monoclonal antibodies (MoAbs) recognizing germline encoded cross-reactive idiotypes (CRI) associated with V_HI (G6, G8), V_HIII (B6, D12), V_KIIIb (17–109) and an epitope linked to the V_KIII light chain subgroup (C7). While only 1.7–3.2% of peripheral blood B lymphocytes from normal individuals expressed the V_HI-associated CRI (V_hI-CRI), these CRI were expressed on virtually all the leukaemic B cells from 17–22% of the CLL patients. The V_HIII-associated CRI (V_HIII-CRI), however, were found in 8.5–13% of the CLL B cells. Fifty per cent of the IgMK-expressing CLL cells (7/14) expressed the V_RIII light chain subgroup of which only one expressed the V_KIIIb-associated CRI (V_KIIIb-CRI), 17–109. The anti-V_HI-associated CRI antibodies were used to study their regulatory effect on in vitro Ig synthesis by the leukaemic cells. A significant suppression of spontaneous and mitogen-driven Ig production was observed in all cases studied. These results demonstrate an over-expression of V_HI and V_KIII gene products in B-CLL and suggest that B cells expressing these CRI are particularly susceptible to lymphoproliferative stimuli. The anti-CRI antibodies can be used to modulate Ig production by the leukaemic cells and may be of potential value for selective immunotherapy.     

Demonstration of immunoglobulin-related molecules on human peripheral blood T lymphocytes using chicken anti-F(ab'')2 and anti-IgM antibodies.

A mean of 98% of lymphocytes in T-enriched preparations of human peripheral blood bound purified chicken anti-human F(ab')2 antibodies to their surface membranes as demonstrated by the mixed antiglobulin rosetting reaction (MARR). By reverse passive hemagglutination these antibodies reacted strongly with kappa light chains and with Fd gamma but gel diffusion analyses, absorption on affinity columns and inhibition experiments established that the anti-F(ab')2 antibodies were not isotype specific and that there was extensive serological cross-reactivity between Fd gamma and kappa and probably also between Fd gamma and mu chains. The antigenic determinants recognized on the T-cells surfaces were not shared by Ig fractions from several other mammalian species. Trypsin treatment of lymphocytes removed all determinants recognized by anti-F(ab')2 antibodies from approximately two-thirds of the cells but these cells almost completely re-expressed these determinants during in vitro culture; this indicates that the T-cell determinants seen by anti-F(ab')2 antibodies are T cell products and do not represent adsorbed Ig. Complete inhibition of the MARR by human F(ab')2 excludes false positive rosette formation due to contaminating specificities directed against non-Ig molecules on T cells. Together the various findings are consistent with the conclusion that most human peripheral blood T cells express Ig or Ig-related molecules in their surface membranes. A mean of 13% of lymphocytes in T-enriched preparations were reactive with mu-chain specific chicken antibodies; it may be that a minority of T cells express mu chain determinants in addition to those recognized by anti-F(ab')2 antibodies.     

Re-expression of RAG-1 and RAG-2 genes and evidence for secondary rearrangements in human germinal center B lymphocytes

V(D)J recombination occurs in immature B cells within primary lymphoid organs. However, recent evidence demonstrated that the recombination activating genes RAG-1 and RAG-2 can also be expressed in murine germinal centers (GC) where they can mediate secondary rearrangements. This finding raises a number of interesting questions, the most important of which is what is the physiological role, if any, of secondary immunoglobulin (Ig) gene rearrangements. In the present report, we provide evidence that human GC B cells that have lost surface immunoglobulin re-express RAG-1 and RAG-2, suggesting that they may be able to undergo Ig rearrangement. Furthermore, we describe two mature B cell clones in which secondary rearrangements have possibly occurred, resulting in light chain replacement. The two clones carry both κ and λ light chains productively rearranged, but fail to express the κ chain on the cell surface due to a stop codon acquired by somatic mutation. Interestingly, the analysis of the extent of somatic mutations accumulated by the two light chains might suggest that the λ chain could have been acquired through a secondary rearrangement. Taken together, these data suggest that secondary Ig gene rearrangements leading to replacement may occur in human GC and may contribute to the peripheral B cell repertoire.