Restriction of endogenous T cell antigen receptor beta rearrangements to Vbeta14 through selective recombination signal sequence modifications.

T cell antigen receptor (TCR)beta V(D)J variable region exon assembly is ordered, with Dbeta to Jbeta rearrangements occurring before joining of Vbetas to a DJbeta 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. Vbeta segments have 23-RSs and Jbeta segments 12-RSs, which based on the 12/23 rule should allow direct joining. However, Vbeta segments rearrange only to DJbeta complexes and not Jbeta segments, because of restrictions beyond 12/23 (B12/23) that make the Vbeta23-RS incompatible with the Jbeta12-RS. To determine whether direct Vbeta to Jbeta joining occurs if flanking RSs are B12/23 compatible, we generated mice whose lymphocytes contained replacement of the Vbeta1412-RS with the 3'Dbeta112-RS on a TCRbeta allele lacking Dbeta segments (the Jbeta1(M6) allele). Mice heterozygous for the Jbeta1(M6) allele had dramatically increased Vbeta14(+) thymocyte and T cell numbers and decreased numbers of cells expressing other Vbetas. This altered Vbeta repertoire resulted from direct Vbeta14 to Jbeta1 rearrangements on the Jbeta1(M6) allele. Mice harboring lymphocytes homozygous for Jbeta1(M6) allele developed normal thymocyte and T cell numbers with all expressing Vbeta14. Our findings show that selective RS modifications enforce rearrangement of a specific Vbeta gene segment and demonstrate the importance of B12/23 mechanisms for ensuring generation of diverse TCRbeta repertoires.

T cell receptor (TCR) alpha/delta locus enhancer identity and position are critical for the assembly of TCR delta and alpha variable region genes.

T cell receptor (TCR) delta and alpha variable region genes are assembled from germ-line gene segments located in a single chromosomal locus in which TCR delta segments are situated between TCR alpha segments. The TCR alpha enhancer (E alpha) located at the 3' end of the TCR alpha/delta locus functions over a long chromosomal distance to promote TCR alpha rearrangement and maximal TCR delta expression; whereas the TCR delta enhancer (E delta) is located among the TCR delta segments and functions with additional element(s) to mediate TCR delta rearrangement. We used gene-targeted mutation to evaluate whether the identity of E alpha and the position of E delta are critical for the developmental stage-specific assembly of TCR delta and alpha variable region genes. Specific replacement of E alpha with E delta, the core E alpha element (E alpha C), or the Ig heavy chain intronic enhancer (iE mu), all of which promote accessibility in the context of transgenic V(D)J recombination substrates, did not promote a significant level of TCR alpha rearrangement beyond that observed in the absence of E alpha. Therefore, the identity and full complement of E alpha-binding sites are critical for promoting accessibility within the TCR alpha locus. In the absence of the endogenous E delta element, specific replacement of E alpha with E delta also did not promote TCR delta rearrangement. However, deletion of intervening TCR alpha/delta locus sequences to restore the inserted E delta to its normal chromosomal position relative to 5' sequences rescued TCR delta rearrangement. Therefore, unlike E alpha, E delta lacks ability to function over the large intervening TCR alpha locus and or E delta function requires proximity to additional upstream element(s) to promote TCR delta accessibility.

Dramatically increased rearrangement and peripheral representation of Vbeta14 driven by the 3'Dbeta1 recombination signal sequence.

V(D)J recombination is targeted by short recombination signal (RS) sequences that are relatively conserved but exhibit natural sequence variations. To evaluate the potential of RS sequence variations to determine the primary and peripheral TCRbeta repertoire, we generated mice containing specific replacement of the endogenous Vbeta14 RS with the 3'Dbeta1 RS (Vbeta14/3'DbetaRS). These mice exhibited a dramatic increase in Vbeta14(+) thymocyte numbers at the expense of thymocytes expressing other Vbetas. In addition, the percentage of peripheral Vbeta14(+) alphabeta T lymphocytes was similarly increased. Strikingly, this altered Vbeta repertoire resulted predominantly from a higher relative level of primary Vbeta14/3'DbetaRS rearrangement to DbetaJbeta complexes, despite the ability of the 3'Dbeta1 RS to break B12/23 restriction and allow direct rearrangement of Vbeta14/3'DbetaRS to Jbeta segments.