In vitro induction of somatic mutations in human peripheral B lymphocytes

The major cellular and molecular factors that lead to the induction of soma tic mutations in human B cells remain unclear. This study describes an appr oach which allowed us to single-cell culture antigen-specific B cells after in vitro immunization. Using single strand conformational polymorphism ana lysis on the descendant cultures we were able to demonstrate that the Ig ge ne of B cells was induced to mutate. Three clones were isolated from a sing le cell culture of naive B cells immunized against the semi-conserved regio n of V3 of HIV-1, which contained two point mutations at the CDR2 region le ading to amino acid codon change. Two clones were also obtained from a sing le cell culture of memory B cells immunized against the recall antigen teta nus toroid. These clones showed one point mutation at the CDR1-FR2 border r egion leading to amino acid codon change. Fab constructs from the mutated c ulture of naive B cells immunized against V3 were cloned to a procaryotic e xpression vector. The expression products of two clones showed anti-V3 spec ificity in ELISA using three different conjugates. Our results suggest that somatic mutations can be induced in human peripheral B cells through speci fic interaction with antigen and antigen-activated T cells.