An activation-induced cytidine deaminase-independent mechanism of secondary V_H gene rearrangement in preimmune human B cells

V_H replacement is a form of IgH chain receptor editing that is believed to be mediated by recombinase cleavage at cryptic recombination signal sequences (cRSS) embedded in V_H genes. Whereas there are several reports of V_H replacement in primary and transformed human B cells and murine models, it remains unclear whether V_H replacement contributes to the normal human B cell repertoire. We identified V _H→V_H(D)J_H compound rearrangements from fetal liver, fetal bone marrow, and naive peripheral blood, all of which involved invading and recipient V_H4 genes that contain a cryptic heptamer, a 13-bp spacer, and nonamer in the 5′ portion of framework region 3. Surprisingly, all pseudohybrid joins lacked the molecular processing associated with typical V_H(D)J_H recombination or nonhomologous end joining. Although inefficient compared with a canonical recombination signal sequences, the V_H4 cRSS was a significantly better substrate for in vitro RAG-mediated cleavage than the V_H3 cRSS. It has been suggested that activation-induced cytidine deamination (AICDA) may contribute to V_H replacement. However, we found similar secondary rearrangements using V_H4 genes in AICDA-deficient human B cells. The data suggest that V_H4 replacement in preimmune human B cells is mediated by an AICDA-independent mechanism resulting from inefficient but selective RAG activity.