Restriction of endogenous T cell antigen receptor rearrangements to V 14 through selective recombination signal sequence modifications

T cell antigen receptor (TCR) V(D)J variable region exon assembly is ordered, with D to J rearrangements occurring before joining of V s to a DJ complex. Germ-line V(D)J segments are flanked by recombination signal (RS) sequences, which consist of heptamers and nonamers separated by a spacer of 12 (12-RS) or 23 (23-RS) bp. V(D)J recombination is restricted by the 12/23 rule; joining occurs only between gene segments flanked by 12-RSs and 23-RSs. V segments have 23-RSs and J segments 12-RSs, which based on the 12/23 rule should allow direct joining. However, V segments rearrange only to DJ complexes and not J segments, because of restrictions beyond 12/23 (B12/23) that make the V 23-RS incompatible with the J 12-RS. To determine whether direct V to J joining occurs if flanking RSs are B12/23 compatible, we generated mice whose lymphocytes contained replacement of the V 1412-RS with the 3 D 112-RS on a TCR allele lacking D segments (the J 1M6 allele). Mice heterozygous for the J 1M6 allele had dramatically increased V 14 thymocyte and T cell numbers and decreased numbers of cells expressing other V s. This altered V repertoire resulted from direct V 14 to J 1 rearrangements on the J 1M6 allele. Mice harboring lymphocytes homozygous for J 1M6 allele developed normal thymocyte and T cell numbers with all expressing V 14. Our findings show that selective RS modifications enforce rearrangement of a specific V gene segment and demonstrate the importance of B12/23 mechanisms for ensuring generation of diverse TCR repertoires.