Induction of Ig somatic hypermutation and class switching in a human monoclonal IgM+ IgD+ B cell line in vitro: definition of the requirements and modalities of hypermutation.
Partly because of the lack of a suitable in vitro model, the trigger(s) and the mechanism(s) of somatic hypermutation in Ig genes are largely unknown. We have analyzed the hypermutation potential of human CL-01 lymphocytes, our monoclonal model of germinal center B cell differentiation. These cells are surface IgM+ IgD+ and, in the absence of T cells, switch to IgG, IgA, and IgE in response to CD40:CD40 ligand engagement and exposure to appropriate cytokines. We show here that CL-01 cells can be induced to effectively mutate the expressed VHDJH-C mu, VHDJH-C delta, VHDJH-C gamma, VHDJH-C alpha, VHDJH-C epsilon, and V lambda J lambda-C lambda transcripts before and after Ig class switching in a stepwise fashion. In these cells, induction of somatic mutations required cross-linking of the surface receptor for Ag and T cell contact through CD40:CD40 ligand and CD80: CD28 coengagement. The induced mutations showed intrinsic features of Ig V(D)J hypermutation in that they comprised 110 base substitutions (97 in the heavy chain and 13 in the lambda-chain) and only 2 deletions and targeted V(D)J, virtually sparing CH and C lambda. These mutations were more abundant in secondary VHDJH-C gamma than primary VHDJH-C mu transcripts and in V(D)J-C than V lambda J lambda-C lambda transcripts. These mutations were also associated with coding DNA strand polarity and showed an overall rate of 2.42 x 10(-4) base changes/cell division in VHDJH-CH transcripts. Transitions were favored over transversions, and G nucleotides were preferentially targeted, mainly in the context of AG dinucleotides. Thus, in CL-01 cells, Ig somatic hypermutation is readily inducible by stimuli different from those required for class switching and displays discrete base substitution modalities.
Zan
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Department of Pathology, Weill Medical College, Cornell University, New York 10021, USA.
Cerutti
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Dramitinos
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Schaffer
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Li
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Casali
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D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000235:genetics*; D001402:B-Lymphocytes / Q000276:immunology / Q000378:metabolism*; D018122:B7-1 Antigen / Q000378:metabolism; D001482:Base Composition / Q000235:genetics / Q000276:immunology; D001483:Base Sequence; D018106:CD28 Antigens / Q000378:metabolism; D019013:CD40 Antigens / Q000378:metabolism; D023201:CD40 Ligand; D002460:Cell Line; D002999:Clone Cells / Q000276:immunology; D003062:Codon / Q000235:genetics / Q000276:immunology; D004252:DNA Mutational Analysis; D005803:Genes, Immunoglobulin / Q000235:genetics*; D006801:Humans; D007070:Immunoglobulin A / Q000235:genetics; D017578:Immunoglobulin Class Switching / Q000235:genetics*; D007072:Immunoglobulin D / Q000096:biosynthesis / Q000235:genetics*; D007073:Immunoglobulin E / Q000235:genetics; D007074:Immunoglobulin G / Q000235:genetics; D007143:Immunoglobulin Heavy Chains / Q000235:genetics; D007144:Immunoglobulin J-Chains / Q000235:genetics; D007075:Immunoglobulin M / Q000096:biosynthesis / Q000235:genetics*; D007135:Immunoglobulin Variable Region / Q000235:genetics; D008024:Ligands; D008562:Membrane Glycoproteins / Q000378:metabolism; D008969:Molecular Sequence Data; D017354:Point Mutation / Q000276:immunology*
D000911:Antibodies, Monoclonal; D018122:B7-1 Antigen; D018106:CD28 Antigens; D019013:CD40 Antigens; D003062:Codon; D007070:Immunoglobulin A; D007072:Immunoglobulin D; D007074:Immunoglobulin G; D007143:Immunoglobulin Heavy Chains; D007144:Immunoglobulin J-Chains; D007075:Immunoglobulin M; D007135:Immunoglobulin Variable Region; D008024:Ligands; D008562:Membrane Glycoproteins; D023201:CD40 Ligand; D007073:Immunoglobulin E