Signals sustaining human immunoglobulin V gene hypermutation in isolated germinal centre B cells

Affinity maturation of antibody responses depends on somatic hypermutation of the immunoglobulin V genes. Hypermutation is initiated specifically in p roliferating B cells in lymphoid germinal centres but the signals driving t his process remain unknown. This study identifies signals that promote V ge ne mutation in human germinal centre (GC) B cells in vitro. Single GC B cel ls were cultured by limiting dilution to allow detection of mutations arisi ng during proliferation in vitro. Cells were first cultured in the presence of CD32L cell transfectants and CD40 antibody (the 'CD40 system') suppleme nted with combinations of cytokines capable of supporting similar levels of CD40-dependent GC B-cell growth [interleukin (IL)-10 + IL-1 beta + IL-2 an d IL-10 + IL-7 + IL-4]. Components of the 'EL4 system' were then added to d rive differentiation, providing sufficient immunoglobulin mRNA for analysis . Analysis of VH3 genes from cultured cells by reverse transcription-polyme rase chain reaction (RT-PCR)-based single-strand conformation polymorphism indicated that the combination IL-10 + IL-1 beta + IL-2 promoted active V g ene mutation whereas IL-10 + IL-7 + IL-4 was ineffective. This was confirme d by sequencing which also revealed that the de novo generated mutations we re located in framework and complementarity-determining regions and shared characteristics with those arising in vivo. Somatic mutation in the target GC B-cell population may therefore be actively cytokine driven and not simp ly a consequence of continued proliferation. The experimental approach we d escribe should facilitate further studies of the mechanisms underlying V ge ne hypermutation.