A human myeloma cell line that does not express immunoglobulin but yields a high frequency of antibody-secreting hybridomas

We selected an 8-azaguanine-resistant variant of a human myeloma cell line (RPMI 8226) by cloning the parental cells on a feeder layer of mouse spleen cells in the presence of increasing concentrations of 8-azaguanine. Culture media and cellfree extracts of both the parental and variant (8226 AR/NIP4-1) cell lines were assayed for production of immunoglobulin heavy and light chains by double immunodiffusion and for lambda-chain by radioimmunoassay. Secretion of free lambda-chain by the parental cell line was confirmed. In contrast, no immunoglobulin heavy or light chains were detected in culture medium of the variant cell line by either immunodiffusion or radioimmunoassay. No intracellular lambda-chain could be detected in the variant cells by radioimmunoassay of cellfree extracts or by immunofluorescence of fixed cells. Hybridomas were produced by fusion of 8226AR/NIP4-1 cells with lymphocytes from a mesenteric lymph node recovered at surgery from a hypertransfused renal transplant recipient. Twenty hybrid culture supernatants were assayed for immunoglobulin by double immunodiffusion, and 15 contained either IgG (lambda) or IgG (kappa). None produced IgM or IgA. An IgG (kappa)-producing hybridoma was shown by immunofluorescence not to express lambda-chain. A second fusion between the variant cell line and spleen cells from a renal transplant patient produced a stable hybridoma secreting IgM (lambda) antibody specific for the I antigen.     

Establishment and characterization of a clonal human T-cell line, MKB-1 derived from a patient with acute myeloblastic leukemia

A human T-cell line, designated as MKB-1, was established by cloning procedures in a suspension culture from a peripheral blood of a 17-year-old female patient with acute myeloblastic leukemia. The immunological marker profile of MKB-1 indicated that unlike a myeloid phenotype of the original leukemic cells, the cells were positive for CD3 (both cell surface and cytoplasm), T cell receptor (TcR) alpha/beta heterodimer, CD4, CD5, CD7, CD10, CD57 (Leu7), SN-1 and the cytoplasmic TcR beta chain. These findings indicate the T cell nature of the established cells. Terminal deoxynucleotidyl transferase (TdT) was also detected in 60%. We did not detect markers of human myeloid and B cell associated antigens, HLA-class II or immunoglobulin chains. Cytogenetic study revealed that the MKB-1 cells had a female hypo-tetraploid karyotype with chromosomal abnormalities including a translocation between chromosomes 10 and 14. The breakpoint of chromosome 14 of this translocation, 14q11.2, is known to be the location of TcR alpha and delta genes; t(10; 14) (q26; q11.2) is a variant type of a T cell neoplasm-associated translocation, t (10; 14) (q24; q11.2). The MKB-1 cell line is unusual in that its T cell characteristics are phenotypically and cytogenetically distinct from the original myeloid leukemia cells.     

Co-expression of an epitope on human free kappa-light chains and on a cytoplasmic component in activated T cells

K-1-21 is a murine monoclonal antibody that reacts with human kappa-light chains in free form but not when they are associated with immunoglobulin heavy chains. K-1-21 was unexpectedly shown to bind to a determinant, STA (Sezary T cell antigen), detected by immunofluorescence in the cytoplasm but not on the surface of Sezary T cells isolated from peripheral blood (4/4 cases) and in Sezary T cells from lymph node and bone marrow (one patient). STA was detected in F2/F7, CCRF-CEM, Molt-4, and CCRF-HSB (four human T ALL cell lines), in JURKAT (a human T cell leukemia line), and in MLA144 (a Gibbon T cell lymphoma line). It also occurred in Leu-3a+ antigen-specific T cell clones (6/6 tested). Moreover, although STA was absent from freshly isolated normal T cells, its expression could be evoked in E+ cells from peripheral blood by in vitro culture with phytohemagglutinin. Thus, STA appears to be a cytoplasmic marker for activated T cells. Cytoplasmic inhibition immunofluorescence studies indicated that K-1-21 binding to STA in Sezary cells or T cell lines was inhibited by preincubation of the K-1-21 antibody with purified kappa-Bence Jones protein. STA from radiolabeled MLA144 cell lysates was immunoprecipitated by K-1-21 and was identified on polyacrylamide gel electrophoresis under reducing conditions as a protein of m.w. 57,000. Additional experiments are underway to define the molecular basis of the interesting cross-reactivity between a determinant in T cells and the K-1-21 reactive epitope on free kappa-light chains.     

Equine cutaneous amyloidosis derived from an immunoglobulin lambda-light chain. Immunohistochemical, immunochemical and chemical results.

Amyloid deposits from equine cutaneous nodular amyloidosis associated with extramedullary plasmacytoma were classified immunohistochemically as equine immunoglobulin lambda-light chain-derived and designated eA lambda (HIP). For chemical identification, the amyloid fibril proteins were separated on Sephadex G-100 in 6M guanidine.HCl. Polypeptides of predominantly 24 kDa and 50 kDa were found by polyacrylamide gel electrophoresis. They have preponderance of immunoglobulin lambda-antigenic determinants as detected by immunodiffusion and immunoblotting. Since the N-terminus of the major proteins was blocked, peptides were generated with trypsin and endoproteinase Asp-N and then isolated using reversed-phase high-performance liquid chromatography. Automatic amino-acid sequence determination of seven peptides showed novel sequences. Data bank comparison indicated that these peptides were derived from a monoclonal immunoglobulin lambda-light and a gamma-heavy chain. The light chain was considered to be the leading amyloidogenic polypeptide, since it was the predominant component in a virtually pure amyloid fibril preparation. Thus, immunoglobulin lambda-light chain-derived amyloidosis, so far established only in man and cat, has now also been identified in the horse.     

Detection of lecithin: cholesterol acyltransferase (LCAT) in a human hepatoma cell line

A human hepatoma cell line (HepG-2) was probed for the presence of lecithin: cholesterol acyltransferase (LCAT) using an antiserum to human plasma LCAT. Double immunodiffusion analysis using antiserum to human plasma LCAT revealed a single precipitin line in the sonicated cell homogenate. This precipitin line showed a reaction of identity with highly purified plasma LCAT. The presence of LCAT within the hepatoma cells was also confirmed by an immunofluorescence test. In contrast, the cell culture supernate showed a weak and inconsistent precipitin line. These data suggest that HepG-2 cells synthesize LCAT but secretion of the enzyme by these cells into the culture medium may be partially or totally impaired.     

Cloning of mouse myeloma cells and detection of rare variants

Cultured mouse myeloma cells have been cloned in soft agar using a modification of the method established by Pluznik and Sachs ('65, '66) and by Bradley and Metcalf ('66). A linear relationship existed between the number of cells plated and the number of colonies produced. Conditions for obtaining optimum cloning efficiency and colony size were determined for the MPC-11 cell line. Feeder cells of mouse, human and rabbit origin and conditioned growth medium obtained from mouse cultures and had an enhancing effect on colony formation. Immunoglobulin production by cloned cells was detected by overlaying the clones with anti-immunoglobulin antiserum. The antiserum had no adverse effect on cloning efficiency or colony size. A reconstruction experiment was performed to show that the plate assay could reliably detect rare variants of immunoglobulin producing cells. The plate assay was validated by studying immunoglobulin production following recovery of clones from dishes and their growth to mass suspension culture. Immunoglobulin formation in these cultures was assessed by a Ouchterlony immunodiffusion of the supernatant medium, and by incubating the cells with radioactive amino acids and analyzing the intracellular and secreted immunoglobulin on polyacrylamide gels.     

The pathology and homing of a transplantable murine B cell leukemia (BCL1).

The pathology and homing characteristics of a murine B cell leukemia are described. Experiments utilizing autoradiography to determine the early homing pattern of the leukemic cells revealed a pronounced localization of the labeled cells to the spleen. The cells that were seen in the white pulp showed preferential localization to the follicles or B cell domains. Tissue section immunofluorescence with antibodies to kappa- and lambda-light chains was used to study the initial mouse with this disease as well as to study the mice that were injected with in vivo passaged cells. These mice also showed predominant involvement of the spleen. Although the initial mouse with this disease had 200,000 lambda-bearing B lymphocytes per mm3 in the peripheral blood and closely resembled a human chronic lymphocytic leukemia patient, the studies described suggest that this murine B cell neoplasm is a lymphoma with a striking predilection for splenic involvement. The other organs including the bone marrow as well as the peripheral blood appeared to be involved secondarily. This unusual spontaneously occurring murine B cell disease provides a useful model for the investigation of certain commonly occurring human lymphomas and leukemias.     

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.     

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.     

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.     

Human granulocyte-specific nuclear antigen(s). II. Detection of antigens in human proliferative syndromes

Antisera raised to dehistonized chromatin from isolated normal human granulocytes revealed the presence of chromatin-associated antigens specific for the human neutrophils that appear during late stages of myeloid cellular differentiation. Immunological specificity was demonstrated by C fixation, immunodiffusion, and immunocytochemical reactions. Chromatin prepared from both normal granulocytes and specimens of myeloid leukemia showed immunologic reactivity. Although the normal antigens were detected in a specimen of CML, the position of immunodiffusion precipitin lines was different from that obtained with normal granulocyte chromatin. In addition, chromatin prepared from the myeloid leukemic cell line HL-60 expressed only one of the three precipitin bands normally found in immunodiffusion. The immunocytochemical staining reaction was confined to the nucleus of mature neutrophils in normal peripheral blood smears. Greater than 90% of cells in peripheral blood specimens of CML showed positive immunocytochemical nuclear staining. In other types of leukaemia, the normal mature granulocyte reacted with antiserum, but the nonmyeloid leukemic cells in these specimens did not. The specificity of immunologic reactions described here suggests the usefulness of nuclear antigens as cell markers.     

Alteration of immunoglobulin-bearing lymphoma cells by fixation.

Four large cell lymphomas known to be monoclonal B-cell proliferations were studied with immunofluorescent and immunohistochemical methods for the detection of kappa- and lambda-light chains. Frozen sections of lymphoma tissues as well as formalin and B-5-fixed tissues embedded in paraffin were studied. Both immunofluorescent and immunohistochemical methods gave similar results on frozen sections; however, a number of discrepancies were noted between the results obtained on fixed tissues and those obtained on frozen tissues. In an effort to identify a fixative which did not alter immunoglobulin (Ig), mouse lymph nodes were fixed in different fixatives before Ig detection; but all of the fixatives tested destroyed the Ig present on normal cortical B lymphocytes. Immunoglobulin-bearing normal and neoplastic lymphocytes are better detected on frozen sections than on paraffin sections after routine fixation.     

The V lambda J lambda repertoire in human fetal spleen: evidence for positive selection and extensive receptor editing

VlambdaJlambda rearrangements obtained from genomic DNA of individual IgM(+) B cells from human fetal spleen were analyzed. A nonrandom pattern of lambda gene rearrangements that differed from the adult Vlambda repertoire was found. The Vlambda distal genes 8A and 4B were absent from the nonproductive fetal repertoire, whereas 2E and 3L were overrepresented and 1B was underrepresented in the productive fetal repertoire. Positive selection of the Vlambda gene, 2E, along with Vlambda rearrangements employing homologous VlambdaJlambda joins were observed in the fetal, but not in the adult Vlambda repertoire. Overrepresentation of Jlambda distal cluster C genes rearranging to the Vlambda distal J segment, Jlambda7, in both productive and nonproductive fetal repertoires suggested that receptor editing/replacement was more active in the fetus than in adults. Numerous identical VlambdaJlambda junctions were observed in both the productive and nonproductive repertoire of the fetus and adult, but were significantly more frequent in the productive repertoire of the fetus, suggesting expansion of B cells expressing particular lambda-light chains in both stages of development, with more profound expansion in the fetal repertoire. Notably, B cells expressing identical lambda-light chains expressed diverse heavy chains. These data demonstrate that three mechanisms strongly influence the shaping of the human fetal lambda-chain repertoire that are less evident in the adult: positive selection, receptor editing, and expansion of B cells expressing specific lambda-light chains. These events imply that the expressed fetal repertoire is shaped by exposure to self Ags.     

A new parental cell line for human x human hybridoma production

The potential of a new HAT-sensitive human lymphoblastoid cell line TK6 TG^r.P1. as a fusion partner was assessed, by comparison with the established human parental cell line UC729.6. Both of these cell lines were fused with the peripheral blood mononuclear cells of a patient with B-chronic lymphocytic leukaemia. The hybridomas generated in these fusion experiments were analysed by the fluorescence activated cell sorter and karyotyping. An anti-idiotype ELISA assay detected the presence of the patient's characteristic idiotype bearing immunoglobulin in the supernatant of a number of the hybridoma cell lines generated in both fusions.     

Anti-lambda-light chain-peptide antibodies are suitable for the immunohistochemical classification of AL amyloid

We aimed to test whether antibodies raised against recombinant peptides corresponding to the variable region of immunoglobulin light chains are suitable for the immunohistochemical classification of amyloid. The Entrez database of the National Center for Biotechnology Information (NCBI) was searched for all protein sequence entries which met the search criteria "amyloid" and "lambda light chain". Sixty-four different lambda-light chain-derived amyloid protein sequences were retrieved, aligned and categorized into the V region subgroups of lambda-light chain detailed by the NCBI, i.e. subgroup I (21 protein sequences), II (14), III (6), IV (1), V (1) and VI (21). V region subgroup I was chosen for epitope sequence selection and two rabbits were immunized with the following peptides: NH2-ISCSGSSSNIGSNTV-CONH2 and NH2-QRPSG VPDRFSGSKSGTS-CONH2. Sensitivity and specificity of the IgG-purified antibodies was tested by Western blotting using amyloid A- (AA), ALlambda- and ALkappa-amyloid proteins, and by immunohistochemistry on tissue microarrays with 110 different amyloid containing tissue samples obtained at autopsy from 22 patients, and on 27 biopsy specimens from a series of 24 patients. Our peptide antibodies specifically stained AL amyloid lambda-light chain-origin, in both Western blots and formalin-fixed and paraffin-embedded tissue sections, confirming that peptide-antibodies directed against immunoglobulin-derived lambda-light chain proteins can be applied for the immunohistochemical classification of amyloid. This offers the opportunity to generate a large set of anti-lambda-light chain protein-antibodies for the immunohistochemical classification of amyloid independently from native human tissue sources.     

Naturally soluble tumor antigens from guinea pig hepatomas: isolation and partial characterization.

Naturally soluble tumor antigens were detected in the ascites fluid of guinea pigs bearing an ascites tumor and from exhausted tissue culture media of cultured tumor cells. Two antigenically distinct cell lines of diethylnitrosamine-induced strain-2 guinea pig hepatomas (line-10 and line-1) served as the source of tumor antigens. Tumor antigen activity was detected by four different techniques: immunodiffusion, inhibition of complement-mediated cytotoxicity, inhibition of membrane immunofluorescence, and delayed cutaneous hypersensitivity. With syngeneic tumor-specific antiserum, line-10 guinea pig tumor antigens were detected by immunofluorescence in the concentrated ascites and tissue culture fluids. With a xenogenic antiserum, demonstrated to be tumor specific, line-10 tumor antigens were detected not only in the concentrated ascites and tissue culture fluids but also in two of the partially purified fractions of these fluids. When the line-10 concentrated ascites and its fraction I were subjected to ultracentrifugation at 300,000 x G for 1 hr, the antigen activity was retained in the supernatant and thus by this criterion the tumor antigens detected in these samples are soluble. Immunodiffusion data indicate that more than one antigen is present in the line-10 system since three lines of precipitation were detected when line-10 concentrated ascites was reacted with the line-10 tumor-specific antiserum. In contrast to this, the line-10-concentrated tissue culture fluid displayed only one line of precipitation. Although tumor antigens could not be demonstrated in the other antigenically distinct tumor cell line, line-1, by immunodiffusion or inhibition of membrane immunofluorescence, inhibition of complement-mediated cytotoxicity was able to detect tumor antigens in the line-1 concentrated ascites and tissue culture fluids.     

Regulation of lymphocyte blastogenesis and antibody production by soluble factor released by a human B-lymphoblastoid cell line

Yam 1B, a human B lymphoblastoid cell line, spontaneously produced an immunoregulatory factor, which suppresses blastogenesis and antibody formation by human lymphocytes. The Yam 1B cells, which were derived from the peripheral blood of an adult T-cell leukemia patient, have been established and maintained in our laboratory since 1985. This cell line expressed mature B-cell surface antigens including surface immunoglobulin M (IgM), CD23, and HLA-DR; had cytoplasmic IgM; and secreted small amounts of IgM in the culture supernatants. Yam 1B was positive for Epstein-Barr virus-associated antigen (EBNA) but negative for adult T-cell-associated antigen (ATLA). The serum-free Yam 1B culture supernatants (SN) inhibited the expression of transferrin R, but neither the expression of interleukin 2 (IL-2) R(CD25) nor the production of IL-2 in the lymphocytes stimulated with phytohemagglutin. Yam 1B SN also inhibited DNA synthesis by human T and B lymphocytes and immunoglobulin generation by normal B cells as well as by Epstein-Barr virus-transformed human B lymphoblastoid cell lines. The inhibitory activity of Yam 1B SN was inactivated at 56 degrees C and at pH 10 but was relatively stable at pH 2. It was abrogated by digestion with pronase and was partially stable by digestion with trypsin. Fractions collected from a Sephacryl S-300 gel filtration column (Pharmacia Fine Chemicals, Uppsala, Sweden) were found to have a peak of inhibitory activity of cell proliferation associated with molecules of apparent MWr of 43,000 to 67,000. The inhibitory activity of Yam 1B SN was not blocked by the anti-transforming growth factor beta antibody.(ABSTRACT TRUNCATED AT 250 WORDS)     

Expression of an unidentified immunoglobulin isotype on rabbit Ig-bearing lymphocytes.

A non-IgM immunoglobulin molecule was found on most rabbit Ig-bearing lymphocytes isolated from mesenteric lymph nodes. Membrane bound immunoglobulin light chains and heavy chains were detected by immunofluorescence and by rosetting with antibody-coated erythrocytes on mesenteric lymph node cells stripped of IgM by anti-IgM allotype antibodies. The percentage of cells bearing these residual immunoglobulin molecules was similar to the percentage of cells bearing immunoglobulin before "stripping" with anti-IgM antibody. These residual immunoglobulin molecules were not IgA nor IgG and are believed to be the rabbit analogue of human IgD.     

Expression of immunoglobulin lambda light chain by the promyelocytic cell line HL-60

The HL-60 cell line, established from a patient with acute promyelocytic leukemia, can be induced to undergo differentiation along the granulocyte or monocyte/macrophage line, depending on the particular inducer that is used. In this communication we provide evidence that HL-60 cells also have B lymphoid characteristics because by flow cytometry and clonal excess calculations, these cells are found to express immunoglobulin (Ig) lambda light chains on their surface. Furthermore, HL-60 cells contain poly(A)+ RNA that hybridizes with a DNA fragment encoding the constant region of Ig lambda chains and comigrates with lambda mRNA on RNA blots. Treatment of HL-60 cells with a phorbol ester that induces monocyte/macrophage differentiation resulted in the loss of surface Ig lambda chains and lambda RNA.     

A newly established human acute lymphoblastic leukemia cell line with characteristics of the earliest B-cell maturation

A new human acute lymphoblastic leukemia (ALL) cell line, designated HBL-3, was established from the bone marrow of a patient with non-T-ALL. The HBL-3 cell line expressed B4 (CD 19), BA-1 (CD 24) and HLA-DR antigens, but not surface immunoglobulin (SIg) or cytoplasmic immunoglobulin (CIg). The cell line lacked the common acute lymphoblastic leukemia antigen (CALLA) and antigenic markers characteristic of T-cell and myeloid cell lineages. The HBL-3 cells had structural rearrangements of both the homologous chromosome 9s, including a translocation with chromosome 1 which has been reported in a patient with common ALL. The cell line had rearranged immunoglobulin heavy chain genes but retained germ-line kappa light chain genes and germ-line T-cell receptor beta- and gamma-chain genes. The HBL-3 cell line was strongly positive for terminal deoxynucleotidyl transferase (TdT). These findings indicate that the HBL-3 cell line is derived from the earliest B-cell committed to B-cell lineage.