Epstein-Barr virus immortalization of normal cells of B cell lineage with nonproductive, rearranged immunoglobulin genes

Most continuous cell lines derived by EBV immortalization of peripheral blood cells are composed of phenotypically mature B lymphocytes, and secrete Ig. Occasionally, EBV-immortalized cell lines have failed to secrete Ig. Expansion and characterization of one of these EBV-induced cell lines, VDS-O, showed that in addition to a lack of Ig secretion, surface and intracytoplasmic Ig were absent. Cell surface phenotyping revealed that VDS-O belongs to the B cell lineage, because it expresses the B cell restricted antigens B1 and B4, while it lacks T cell and monocyte-associated determinants. Analysis of the Ig gene organization in VDS-O revealed that the Ig genes are rearranged for both heavy (gamma) and light (kappa) chains. However, the expected gamma-heavy chain and/or kappa-light chain RNA species were not detected. These findings demonstrate the existence in normal peripheral blood of cells of B cell lineage susceptible to EBV immortalization that have Ig genes that are rearranged but are nonproductive.     

IgM expressed by leukemic CD5(+) B cells binds mouse immunoglobulin light chain

Mouse immunoglobulin (Ig) molecules have previously been shown to bind to the surface of CD5(+) B cells from patients with B-cell chronic lymphocytic leukemia (B-CLL). The results indicated that surface IgM was involved in the interaction and suggested the phenomenon was an example of the polyreactive binding capacity of the surface Ig (sIg) expressed by these malignant cells. This article describes the further characterization of the interaction between human IgM and mouse Ig molecules and subunits. Mouse Ig molecules of both kappa and lambda light chain classes bound to the B-CLL cell surface. The dissociation constant for the interaction of mouse IgG1 (K121) with the B-CLL cell surface was 3.6 x 10(-7) M. To confirm the involvement of the human IgM expressed by the B-CLL cells in the interaction, the malignant cells were stimulated in vitro to induce secretion of human IgM. Enzyme immunoassay was used to show that secreted human IgM bound to intact mouse Ig, as occurred with the cell surface analysis. The mouse Ig epitope recognized by the purified secreted human IgM was shown by Western blot analysis to be located on the light chain of the mouse Ig molecule and to be conformationally dependent. K121 light chain was cloned and expressed in E. coli and the recombinant light chain bound to the surface of CLL B cells. The results confirm that human IgM is the reactive ligand in the interaction with mouse Ig and indicate that the interaction of polyreactive IgM with mouse IgG occurs via the light chain component of IgG.     

Mycoplasma contamination of membrane associated measles antigens. Inability to demonstrate in vitro lymphocyte responsiveness to measles.

A marked suppression of protein synthesis including IgA was demonstrated in BALB/c mouse thymocytes incubated in short-term tissue culture in the presence of anti-mouse α chain antibodies. In C57BL mouse thymocytes a suppression of protein synthesis including IgM was obtained by incubating the cells in tissue culture containing anti-mouse μ chain antibodies. The association of anti-α chain antibodies or anti-κ chain antibodies, at 4 °, to the Ig subunits on the surface of BALB/c mouse thymocytes was demonstrated following radioiodination of the antibody treated cells. The anti-mouse Ig antibodies attached to the surface α chains or κ chains at 4 ° were radioiodinated on the cell surface, rendering the corresponding surface mouse Ig subunits inaccessible to radioiodination by lactoperoxidase. The α chains or κ chains complexed with their specific antibodies were independently removed from the cell surface upon heating to 37 °. However, it was found that each of the antibodies attached first to its Ig subunit on the cell surface interferes with the subsequent binding of antibodies to the second subunit. The antibody-surface Ig complexes, although cleared from the cell membrane at 37 °, were not released into the culture medium, leading to the conclusion that these complexes were endocytosed.     

Quantitative monitoring of lymphoid malignancies. Application and findings in chronic lymphocytic leukemia

The present study investigated the surface immunoglobulin (Ig) phenotypic pattern in 64 cases of chronic lymphocytic leukemia (CLL). The fluorescence activated cell sorter techniques were modified to provide sensitive and highly reproducible detection and quantification of the otherwise faint surface immunoglobulins on the cells in CLL. In over 98% of the cases of CLL, light chain of the surface Ig could be identified and measured. Serial measurements were shown to be highly reproducible. The phenotypic pattern and density identified on the surface of the circulating lymphocytes precisely reflected the findings in any given patient when other lymphoid tissue (bone marrow, lymph node or spleen) was sampled. Intraclonal heterogeneity detected by surface Ig was seen in some cases of CLL in spite of a relatively uniform morphology by other classical techniques. Three patterns of cell-to-cell variation were seen: 1) that of a non-Gaussian distribution of surface light Ig staining intensity 2) that of the presence of increased surface light Ig chain on a portion of the cells with a subpopulation of CLL cells showing an additional class of heavy chain, and 3) that of anisotropy where the surface Ig quantitatively did not correlate with cell size. Immunoglobulin phenotypic characterization of the cases of CLL was correlated with their clinical stage of disease activity. The distribution of surface light chain phenotype did not relate to any pattern of clinical stage of activity of the disease. By contrast, cases where the cells had a predominance of surface IgM were associated with a more advanced stage of CLL and a poorer clinical prognosis. When surface IgG was predominant, a lesser degree of clinical activity of disease was identified. The phenotypic pattern of the surface Ig on the lymphocytes in CLL mirrors the pattern of differentiation in the murine model of B-cell differentiation, and clinical aggressiveness appears to correlate with the character and degree of B-cell differentiation.     

MRC OX-2 antigen: a lymphoid/neuronal membrane glycoprotein with a structure like a single immunoglobulin light chain.

The MRC OX-2 antigen is a rat cell surface glycoprotein of mol. wt. 41 000-47 000 found on neurones, thymocytes, B cells, follicular dendritic cells and endothelium. We now report the amino sequence for this antigen as deduced from the nucleotide sequence of cDNA clones detected by use of an oligonucleotide probe. The sequence contains 248 amino acid residues of which 202 residues are likely to be outside the cell with two domains that show homology with immunoglobulins. The N-terminal domain fits best with Ig V domains and Thy-1 antigen while the C-terminal part is like an Ig C domain. Thus the structure overall is similar to an Ig light chain or the T cell receptor beta chain. Three glycosylation sites are identified on each of the MRC OX-2 antigen domains.     

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.     

Enhanced transcription in a pre-B-like cell line transformed with cloned antibody genes after antigen-specific stimulation

The transducing vector, pSV2-neo, carrying the rearranged immunoglobulin (Ig) heavy (mu) and light (kappa) chain genes specific for the hapten 2,4,6-trinitrophenyl (TNP) was introduced into a pre-B cell line. The transformants expressed the TNP-specific IgM receptor on the surface. Furthermore, the addition of TNP-bovine serum albumin (hapten-carrier conjugate) to the culture media activated the expression of the transferred Ig genes and several endogenous genes such as v-abl and beta-tubulin. However, expression of the beta2-microglobulin gene was not affected. The results presented in this paper show that transfection of cloned Ig genes into B cells is a useful system for establishing monoclonal B cell lines expressing functional Ig receptor molecules with defined hapten specificity.     

Production, characterisation and applicability of monoclonal antibodies to immunoglobulin of Japanese flounder (Paralichthys olivaceus)

Immunoglobulin (Ig) of Japanese flounder (Paralichthys olivaceus) was purified by a combination of salting-out and DEAE Sepharose Column chromatography. The purified immunoglobulin had an apparent molecular weight of 74 kDa (heavy chain) and 24 kDa (light chain) in SDS-PAGE. Eighteen hybridomas secreting monoclonal antibodies (MAbs) against Japanese flounder Ig were obtained by immunisation of Balb/C mice with purified Ig preparations, which were selected on the basis of the double indirect enzyme-linked immunosorbent assay (D-ELISA). Two of them designated as 2D8 and 2H1 were cloned by limiting dilution and characterised with western blotting, indirect immunofluorescence assay test (IIFAT) and fluorescence-activated cell sorter (FACS) analysis. Under reducing conditions in western blotting, both MAb 2D8 and MAb 2H1 were specific for the heavy chain of Japanese flounder Ig. MAb 2D8 was used to identify surface Ig-positive lymphocytes in the peripheral blood, spleen and pronephros of healthy Japanese flounder by flow cytometry. FACS analysis revealed that 40.48% of lymphocytes in the peripheral blood, 17.32% in the spleen and 9.67% in the pronephros were reactive to 2D8.     

Crosslinking by ligands to surface immunoglobulin triggers mobilization of intracellular 45Ca2+ in B lymphocytes

Detailed studies of steady-state ion fluxes in murine lymphocytes were used to examine for possible ionic changes generated by surface Ig, the antigen receptor of B lymphocytes. When bound by ligands, surface Ig triggered the mobilization and release of 45Ca2+ from the cell interior by a transmembrane process requiring crosslinking of the bound receptors. This ionic event was unique for two reasons: (a) it did not occur when other common lymphocyte surface macromolecules were bound with rabbit anti-lymphocyte antibodies; and (b) it was not accompanied by a general perturbation of lymphocyte ionic properties such as a change in 42K+ fluxes nor did it depend on the presence of extracellular ions. Capping of surface Ig shares the same time sequence, dose response, requirement for crosslinking, and lack of dependence on extracellular ions. These correlations suggest that mobilization of intracellular Ca2+ may represent an early ionic signal for the contractile activation of lymphocytes that generates capping of surface Ig.     

Deletion of the immunoglobulin kappa chain intron enhancer abolishes kappa chain gene rearrangement in cis but not lambda chain gene rearrangement in trans.

Immunoglobulins (Ig) secreted from a plasma cell contain either kappa or lambda light chains, but not both. This phenomenon is termed isotypic kappa-lambda exclusion. While kappa-producing cells have their lambda chain genes in germline configuration, in most lambda-producing cells the kappa chain genes are either non-productively rearranged or deleted. To investigate the molecular mechanism for isotypic kappa-lambda exclusion, in particular the role of the Ig kappa intron enhancer, we replaced this enhancer by a neomycin resistance (neoR) gene in embryonic stem (ES) cells. B cells heterozygous for the mutation undergo V kappa-J kappa recombination exclusively in the intact Ig kappa locus but not in the mutated Ig kappa locus. Homozygous mutant mice exhibited no rearrangements in their Ig kappa loci. However, splenic B cell numbers were only slightly reduced as compared with the wild-type, and all B cells expressed lambda chain bearing surface Ig. These findings demonstrate that rearrangement in the Ig kappa locus is not essential for lambda gene rearrangement. We also generated homozygous mutant mice in which the neoR gene was inserted at the 3' end of the Ig kappa intron enhancer. Unexpectedly, mere insertion of the neoR gene showed some suppressive effect on V kappa-J kappa recombination. However, the much more pronounced inhibition of V kappa-J kappa recombination by the replacement of the Ig kappa intron enhancer suggests that this enhancer is essential for V kappa-J kappa recombination.     

Immunoglobulin mu heavy chains do not mediate tyrosine phosphorylation of Ig alpha from the ER-cis-Golgi

Signals delivered by Ig receptors guide the development of functional B lymphocytes. For example, clonal expansion of early mu heavy chain ( mu HC)-positive pre-B cells requires the assembly of a signal-competent pre-B cell receptor complex (pre-BCR) consisting of a mu HC, a surrogate L chain, and the signal dimer Ig alpha beta. However, only a small fraction of the pre-BCR is transported to the cell surface, suggesting that pre-BCR signaling initiates already from an intracellular compartment, e.g., the endoplasmic reticulum (ER). The finding that differentiation of pre-B cells and allelic exclusion at the IgH locus take place in surrogate L chain-deficient mice further supports the presence of a mu HC-mediated intracellular signal pathway. To determine whether a signal-competent Ig complex can already be assembled in the ER, we analyzed the consequence of pervanadate on tyrosine phosphorylation of Ig alpha in J558L plasmacytoma and 38B9 pre-B cells transfected with either a transport-competent IgL chain-pairing or an ER-retained nonpairing micro HC. Flow cytometry, combined Western blot-immunoprecipitation-kinase assays, and confocal microscopy revealed that both the nonpairing and pairing mu HC assembled with the Ig alpha beta dimer; however, in contrast to a pairing mu HC, the nonpairing mu HC was retained in the ER-cis-Golgi compartment, and neither colocalized with the src kinase lyn nor induced tyrosine phosphorylation of Ig alpha after pervanadate treatment of cells. On the basis of these findings, we propose that a signal-competent Ig complex consisting of mu HC, Ig alpha beta, and associated kinases is assembled in a post-ER compartment, thereby supporting the idea that a pre-BCR must be transported to the cell surface to initiate pre-BCR signaling.     

Basal immunoglobulin signaling actively maintains developmental stage in immature B cells

In developing B lymphocytes, a successful V(D)J heavy chain (HC) immunoglobulin (Ig) rearrangement establishes HC allelic exclusion and signals pro-B cells to advance in development to the pre-B stage. A subsequent functional light chain (LC) rearrangement then results in the surface expression of IgM at the immature B cell stage. Here we show that interruption of basal IgM signaling in immature B cells, either by the inducible deletion of surface Ig via Cre-mediated excision or by incubating cells with the tyrosine kinase inhibitor herbimycin A or the phosphatidylinositol 3-kinase inhibitor wortmannin, led to a striking “back-differentiation” of cells to an earlier stage in B cell development, characterized by the expression of pro-B cell genes. Cells undergoing this reversal in development also showed evidence of new LC gene rearrangements, suggesting an important role for basal Ig signaling in the maintenance of LC allelic exclusion. These studies identify a previously unappreciated level of plasticity in the B cell developmental program, and have important implications for our understanding of central tolerance mechanisms.     

B cell abnormalities induced by a mu Ig transgene extend to L chain isotype usage

We have analyzed the phenotype of B cell populations from mice transgenic for a rearranged Ig mu H chain gene. We find a decrease in the number of B cells in the spleens of these mice. Transgenic B cells have decreased surface levels of both IgM and IgD. The circulating IgM in these mice is 3- to 10-fold enriched in lambda L chains, compared with that in non-transgenic mice. Analysis of IgM-producing hybridomas, from transgenic mice that express the transgene at high levels, demonstrates that this higher lambda frequency is observed in transgene-nonexpressing as well as transgene-expressing hybridomas. A partial loss of L chain isotype exclusion is also noted in these hybridomas, and a significant proportion of primary B cells expressing both kappa and lambda L chains on their surface can be demonstrated. These findings suggest an ability of the transgenic Ig H chain to affect events in B cell ontogeny beyond the H chain locus. Our results support a quantitative model of exclusion for both the H chain alleles and the L chain isotypes.     

In vivo survival of autoreactive B cells: characterization of long-lived B cells

To determine the effects of chronic Ag stimulation on B cell survival and phenotype, we compared survival and surface markers of hen egg lysozyme (HEL)-specific B cells in Ig transgenic (Tgn) mice, which lack HEL, and in HEL-Ig transgenic mice, which express soluble HEL. Serum HEL levels were maximized in HEL-Ig Tgn mice by feeding them zinc, which activates the metallothionein promoter that regulates HEL expression. B cell age was characterized by expression of heat-stable Ag, and B220 and B cell survival was studied by evaluating changes in B cell number when lymphopoiesis was suppressed with anti-IL-7 mAb and by identifying newly generated B cells through 5-bromo-2'-deoxyuridine incorporation. Our observations show that the mean B cell life span is considerably reduced in HEL-Ig Tgn compared with Ig Tgn mice, but also demonstrate that some HEL-Ig Tgn B cells survive to maturity. Some of these surviving B cells have undergone receptor editing (substitution of an endogenous Ig light chain for the transgenic Ig light chain), so that their ability to bind HEL is decreased or absent. Surviving HEL-Ig Tgn B cells that retain HEL specificity express decreased mIgD and little or no mIgM. mIgD expression progressively decreases with increasing HEL-Ig Tgn B cell age. These observations suggest that self Ag-specific B cells can survive in the presence of soluble self Ag by down-regulating mIg expression, which should limit B cell signaling by Ag that might otherwise cause deletion of these cells.     

Expression of VHIII-associated cross-reactive idiotype on human B lymphocytes. Association with staphylococcal protein A binding and Staphylococcus aureus Cowan I stimulation.

It has been demonstrated that staphylococcal protein A (SPA) has an "alternative" binding site with specificity for human Ig H chain V region of the VHIII subgroup. Because the major mitogenic component of Staphylococcus aureus Cowan I (SAC) is SPA, it is possible that SAC stimulates a subpopulation of B cells expressing Ig of the VHIII H chain subgroup. In the present study, we have investigated further the relationship between SPA binding and the expression of VHI- or VHIII-associated cross-reactive idiotype (CRI) on the surface of tonsillar B lymphocytes enriched for the expression or nonexpression of the CRI, and we examined the Ig secreted by cell lines established from these populations of B cells by EBV transformation. The VHIII CRI (D12)-enriched population yielded 21 cell lines, with 67% of them secreting SPA-reactive Ig; in contrast, only 6% (1 of 16) of VHI CRI-expressing lines secreted SPA-reactive Ig. The CRI-negative B cell population yielded 54 cell lines, of which 20% secreted SPA-reactive Ig, as might be anticipated because a majority of VHIII Ig+ B cells will be CRI-. SAC stimulation of CRI+ and CRI- populations showed preferential stimulation of the D12 population. These data support the proposal that SAC stimulation of human B cells is mediated through binding of SPA by its alternative binding site to IgV regions of the VHIII subgroup.     

Identification of IL-7-dependent bone marrow-derived Thy-1-B220- lymphoid cell clones that rearrange and express both Ig and T cell receptor genes.

Bone marrow stromal cell lines and lymphoid cell lines were co-established from the Whitlock-Witte type of long term liquid cultures of MRL/1 and C57BL/10 (B10) (Thy-1.1) bone marrow cells. The present study investigates the immunologic nature of parental and cloned lymphoid cell lines. Both strains of parental lines and their clones did not grow alone but proliferated on the monolayers of co-established parental stromal cell lines from a syngeneic or alternative strain. When various lymphokines or cytokines were tested for their capacity to support the growth of these lymphoid cell clones, only IL-7 could substitute for the growth-promoting function of stromal cells. These IL-7-dependent clones expressed neither Thy-1 nor B220 Ag. However, all of them from two strains were found to rearrange synchronously H chain of Ig as well as gamma chain of TCR genes. Some of the clones transcribed a mature size of IgH mRNA. Co-expression of mRNA for lambda 5 but not for IgL chain (kappa, lambda) genes resulted in the generation of cell surface mu chain in these clones. Other clones expressed a smaller size of IgH mRNA without exhibiting surface mu chain. Irrespective of the differences in IgH rearrangements and its mRNA expression, a mature size TCR gamma mRNA was detected in all of the clones. Thus, these results demonstrate the existence of untransformed (IL-7-dependent) immature lymphoid cells rearranging both Ig and TCR genes. Their unique features concerning cell surface markers (B220- mu+), specific growth factor requirement, and various modes of Ig/TCR gene rearrangements are discussed in the context of early lymphoid development.     

Association of Ig L chain-like protein lambda 5 with a 16-kilodalton protein in mouse pre-B cell lines is not dependent on the presence of Ig H chain protein

Using a polyclonal rabbit antiserum against recombinant mouse lambda 5 protein, we determined that the pre-B cell specific mouse lambda 5 gene encodes a 22-kDa protein. The lambda 5 protein, which is related to conventional Ig lambda L chain proteins forms a complex with Ig mu H chain protein and an as yet unidentified 16-kDa protein (p16) in mu+ pre-B cell lines carrying a functionally rearranged VH-DH-JH allele. In pre-B cell lines which carry DH-JH rearrangements and do not express mu H chain protein, lambda 5 protein is associated with p16. Thus the expression of lambda 5 protein precedes the expression of intact mu H chain protein. This suggests the existence of developmentally regulated protein complexes involving the Ig L chain-like protein lambda 5 and p16 in mu- pre-B cells; lambda 5, p16, and Ig H chain protein in mu+ pre-B cells and Ig H chain and conventional Ig L chain proteins in B cells and plasma cells.