In vitro induction of somatic mutations in human peripheral B lymphocytes.
The major cellular and molecular factors that lead to the induction of somatic mutations in human B cells remain unclear. This study describes an approach which allowed us to single-cell culture antigen-specific B cells after in vitro immunization. Using single strand conformational polymorphism analysis on the descendant cultures we were able to demonstrate that the Ig gene of B cells was induced to mutate. Three clones were isolated from a single cell culture of naive B cells immunized against the semi-conserved region of V3 of HIV-1, which contained two point mutations at the CDR2 region leading to amino acid codon change. Two clones were also obtained from a single cell culture of memory B cells immunized against the recall antigen tetanus toxoid. These clones showed one point mutation at the CDR1-FR2 border region leading to amino acid codon change. Fab constructs from the mutated culture of naive B cells immunized against V3 were cloned to a procaryotic expression vector. The expression products of two clones showed anti-V3 specificity in ELISA using three different conjugates. Our results suggest that somatic mutations can be induced in human peripheral B cells through specific interaction with antigen and antigen-activated T cells.
Zafiropoulos
A
A
Department of Applied Immunology and Biochemistry, Institute of Molecular Biology and Biotechnology, FORTH, Heraklion, Crete, Greece.
Kandilogiannaki
M
M
Dahlenborg
C
C
Borrebaeck
C A
CA
Krambovitis
E
E
D000595:Amino Acid Sequence; D000818:Animals; D001402:B-Lymphocytes / Q000166:cytology / Q000276:immunology / Q000378:metabolism*; D001483:Base Sequence; D002999:Clone Cells / Q000166:cytology / Q000276:immunology / Q000378:metabolism; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000737:chemistry / Q000235:genetics; D015870:Gene Expression; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000235:genetics / Q000276:immunology; D007143:Immunoglobulin Heavy Chains / Q000235:genetics / Q000276:immunology; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics*; D018807:Polymorphism, Single-Stranded Conformational; D017422:Sequence Analysis, DNA; D014407:Tumor Cells, Cultured
D018076:DNA, Complementary; D007140:Immunoglobulin Fab Fragments; D007143:Immunoglobulin Heavy Chains