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 V(H)DJ(H)-Cμ, V(H)DJ(H)-Cδ, V(H)DJ(H)- Cγ, V(H)DJ(H)-Cα, V(H)DJ(H)-Cε, and VλJλ-Cλ 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 λ- chain) and only 2 deletions and targeted V(D)J, virtually sparing C(H) and Cλ. These mutations were more abundant in secondary V(H)DJ(H)-Cγ than primary V(H)DJ(H)-Cμ transcripts and in V(D)J-C than VλJλ-Cλ 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 V(H)DJ(H)-C(H) 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.