Vbeta cluster sequences reduce the frequency of primary Vbeta2 and Vbeta14 rearrangements

Abstract

T-cell receptor (TCR) beta variable region exons are assembled from numerous gene segments in a highly ordered and regulated manner. To elucidate mechanisms and identify cis-acting elements that control Vbeta rearrangement, we generated an endogenous TCR-beta allele with only the Vbeta2, Vbeta4, and Vbeta14 segments. We found that alphabeta T lineage cells containing this Vbeta(2-4-14) allele and a wild-type TCR-beta allele developed normally, but exhibited a significant increase in Vbeta2(+) and Vbeta14(+) cells. To quantify Vbeta rearrangements on the Vbeta(2-4-14) allele, we generated alphabeta T-cell hybridomas and analyzed TCR-beta rearrangements. Despite the deletion of almost all Vbeta segments and 234 kb of Vbeta cluster sequences, the Vbeta(2-4-14) allele exhibited only a slight decrease in Vbeta rearrangement as compared with the wild-type TCR-beta allele. Thus, cis-acting control elements essential for directing Vbeta rearrangement across large chromosomal distances are not located within the Vbeta cluster. We also found a significant increase in the frequency of Vbeta rearrangements involving Vbeta2 and Vbeta14, but not Vbeta4, on the Vbeta(2-4-14) allele. Collectively, our data suggest that Vbeta cluster sequences reduce the frequency of Vbeta2 and Vbeta14 rearrangements by competing with the productive coupling of accessible Vbeta2 and Vbeta14 segments with DJbeta1 complexes.

Figure 1. Schematic representation of the Vβ^ω and Vβ^2-4-14 alleles

(A) Schematic diagrams of the entire TCRβ loci on the Vβ^ω and Vβ^2-4-14 alleles. The loxP sites inserted in place of the DJβ2 clusters and during gene-targeting simplification of the Vβ cluster are indicated by triangles. The relative size of the Vβ cluster and the Dβ-Jβ-Cβ regions are not drawn to scale. (B) Schematic diagrams of the EcoRI restriction fragment length polymorphisms created on the Vβ^ω and Vβ^2-4-14 alleles. Black bars indicate the locations of the Vβ4 and 3'Vβ18 probes.

Figure 2. Normal αβ T cell development in Vβ^2-4-14/ω chimeric mice

Shown are representative CD4-PE and CD8-FITC FACS analyses of cells isolated from the thymuses or lymph nodes of Vβ^ω/ω and Vβ^2-4-14/ω mice.

Figure 3. Altered Vβ repertoire in Vβ^2-4-14/ω thymocytes and peripheral αβ T cells

(A) Shown are representative TCRβ-PE and Vβ5-FITC, Cβ-PE and Vβ14-FITC, Cβ-PE and Vβ2-FITC, Cβ-PE and Vβ4-FITC FACS analyses of cells isolated from the lymph nodes of Vβ^ω/ω and Vβ^2-4-14/ω mice. The percentage of TCRβ positive αβ T cells that express each particular Vβ is indicated. (B) Bar graphs showing the average percentage of TCRβ positive thymocytes and lymph node cells that express Vβ2, Vβ4, Vβ5, or Vβ14.