VH replacement is unlikely to contribute significantly to receptor editing due to an ineffectual embedded recombination signal sequence

Abstract

Receptor editing is a process consisting of replacement of pre-existing H or L chain rearrangements by secondary rearrangements. This process could serve to remove autoreactive specificities, or to rescue loci with non-functional rearrangements. At the H chain locus, functional replacement of a V HDJ H rearrangement by an upstream V H requires the presence of an embedded RSS located in reverse orientation near the 3′ end of the V H segment. Although most V H genes contain a fairly consensus embedded heptamer, the nonamer sequence bears little resemblance to the consensus RSS nonamer. Therefore, the physiologic rate of H chain editing by V H replacement is yet unknown. In this study, we used both conventional and sensitive competition recombination substrate assays to determine the recombination frequency of the V H81X embedded RSS relative to consensus and non-consensus RSS’s. Results show no detectable recombination of the 81X embedded RSS in a recombination substrate, and the competition substrate allows us to estimate that the 81X embedded RSS recombines at least 1300 fold less often than a consensus RSS. This suggests that V H gene replacement is not responsible for the decrease in representation of the 81X gene during differentiation. Furthermore, since the sequence of the embedded RSS is very similar for many V H genes, our results suggest that receptor editing of the H chain will be an infrequent event, leaving L chain editing as the main mode of avoiding autoreactive specificities in vivo.