Somatic Cell Genetics of Antibody-secreting Cells: Studies of Clonal Diversification and Analysis by Cell Fusion

Milstein, C.; Adetugbo, Kayode; Cowan, Nicholas J.; Köhler, Georges; Secher, David S.; Wilde, C. Deborah

doi:10.1101/sqb.1977.041.01.090

Cited by 67 publications

(15 citation statements)

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“…Based on the results of the myeloma-myeloma fusion experiments (15,25) it was assumed that the antibody-secreting hybrids were due to fusion of myeloma cells with mature antibody-secreting cells ("like-like" fusion) (34). The results of the present study suggest, however, that myeloma cells may also fuse and induce antibody formation in a less-mature normal B lymphocyte.…”

Section: Frequency Of Recoverable Hybridmentioning

confidence: 53%

Induction of amplified synthesis and secretion of IgM by fusion of murine 'b lymphoma with myeloma cells.

Laskov¹,

Kim²,

Asofsky³

1979

Proc. Natl. Acad. Sci. U.S.A.

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Murine B lymphoma cultured cell lines bearing membrane IgM and lacking the ability to secrete measurable amounts of IgM were fused with drug-resistant cell lines derived from the IgG2b-producing MPC-1I myeloma. Many of the hybrid clones synthesized and secreted large amounts of IgM Stimulation of B lymphocytes by immunogens or by mitogens results in a series of maturational events leading to the formation of antibody-secreting cells (1). At the molecular level, these changes include the transition from the expression of minute quantities of IgM on the surface of B lymphocytes to the production and secretion of large quantities of IgM by the mature antibody-secreting cells. Evidence also exists that individual antigen-stimulated B cells or their progeny may express more than one class of Ig molecules of similar antigen-binding specificity (2)(3)(4)(5)(6). Therefore, in the same cell, one type of amino-terminal variable region is able to associate with more than one class of the heavy chains. The mechanisms responsible for these steps in antigen-induced B-cell maturation are not well understood. Attempts to elucidate the biochemical events occurring during the maturation of normal B lymphocytes are hindered by the heterogeneous nature of the population of B cells involved.Recently, murine lymphomas that bear membrane IgM and having other properties of B lymphocytes have been characterized in several laboratories (7-10). These tumors are presumably arrested at various stages of differentiation and are considered to be of monoclonal origin. Successful induction of maturation in these tumors may provide a suitable experimental model system for study of the molecular events involved in B-cell maturation.Attempts to induce the differentiation of B lymphomas by using various mitogens (11) and dimethyl sulfoxide (12) have previously resulted at most in partial maturation of these cells. Recently, some human B-lymphoid cell lines were induced by lipopolysaccharide and normal T cells to secrete low quantities of Ig (13).In the present work, we have used an alternative approach to induce the maturation of membrane IgM-bearing murine B lymphomas. It Two clones of the MPC-11 myeloma, kindly provided by Matthew Scharff (Albert Einstein College of Medicine) were used for fusion: clone 4TOO. 1 producing IgG2b and clone 4T00.1L1 which produces only light chains. Both clones were resistant to 6-thioguanine and ouabain (14).Fusion Procedure. This was done by using polyethylene glycol (PEG-1000, Sigma) essentially according to Margulies' et al. (14,15). The cells were seeded into either 96-microwell or 24-well Linbro plates, and the hybrids were selected in growth medium containing 100MM hypoxanthine, 1 MuM aminopterin, 30 AtM thymidine, and 1.5 mM ouabain (15).Preparation of Monospecific Anti-Ig Sera. Antisera were raised in rabbits and goats against purified myeloma proteins of various Ig classes (16). The antisera were made Ig class-specific by absorption with other myeloma proteins as described (16).Analysis of Secre...

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Section: Frequency Of Recoverable Hybridmentioning

confidence: 53%

Induction of amplified synthesis and secretion of IgM by fusion of murine 'b lymphoma with myeloma cells.

Laskov¹,

Kim²,

Asofsky³

1979

Proc. Natl. Acad. Sci. U.S.A.

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“…Several workers have failed to find V region variants at high frequency in myeloma and hybridoma proteins (23)(24)(25)(26), although there exists one case where the mutator system may still be active (27). To determine whether the mutator system is active at the plasma cell stage, we fused subclone A3.2, a y2b chain producer (Fig.…”

Section: Resultsmentioning

confidence: 99%

Hypermutation at the immunoglobulin heavy chain locus in a pre-B-cell line.

Wabl¹,

Burrows²,

Gabain³

et al. 1985

Proc. Natl. Acad. Sci. U.S.A.

141

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Most cells in the well-known pre-B-lymphocyte line 18-81 have correctly assembled genes for both alleles at the immunoglobulin heavy chain locus. Only one allele is "active"; the other, "silent" allele contains an amber termination codon. The rate of reversion of this amber codon was determined to be 0.3_1 x 10-4 per cell generation. This high rate is termed hypermutation.Higher vertebrates can synthesize a vast number of antibody molecules that are formed by combining immunoglobulin heavy (H) and light (L) chains, each of which bears one out of a vast number of variable (V) regions. In the germ line, there are no complete genes for the Ig chains, only gene segments. At the H chain locus in the mouse, there are ca. 100 VH, ca. 20 diversity (DH), and 4 joining (JH) gene segments.

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“…Labeled intracellular immunoglobulin was prepared as described previously (30), except that the labeling medium contained no serum. [35], for anti-K precipitation). PC700 culture supernatant was used as a source of carrier IgM in the precipitation.…”

mentioning

confidence: 99%

Mutations affecting the structure and function of immunoglobulin M.

Shulman¹,

Heusser²,

Filkin³

et al. 1982

Mol. Cell. Biol.

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Using a hybridoma cell line which secretes hapten-specific immunoglobulin M (IgM), we have isolated a variety of mutants which produce abnormal immunoglobulin. Immunoglobulin was tested for the size and composition of the component heavy and light chains and for variable and constant region related functional and serological activities. Some mutants secrete IgM which seems to be defective in hapten binding; others make IgM which appears not to activate complement. Many of the mutants secrete monomeric as opposed to pentameric IgM. In some cases, the defect apparently correlates with structural alterations in the mu heavy chain: partial deletion, polypeptide addition, and abnormal glycosylation have been observed. These mutant cell lines provide a means of identifying the structural basis of IgM function and of studying the biochemistry of IgM synthesis and processing.

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Somatic Cell Genetics of Antibody-secreting Cells: Studies of Clonal Diversification and Analysis by Cell Fusion

Cited by 67 publications

References 0 publications

Induction of amplified synthesis and secretion of IgM by fusion of murine 'b lymphoma with myeloma cells.

Induction of amplified synthesis and secretion of IgM by fusion of murine 'b lymphoma with myeloma cells.

Hypermutation at the immunoglobulin heavy chain locus in a pre-B-cell line.

Mutations affecting the structure and function of immunoglobulin M.

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