The Role of Recombination Activating Gene (RAG) Reinduction in Thymocyte Development in Vivo

Assembly of T cell receptor (TCR)α/β genes by variable/diversity/joining (V[D]J) rearrangement is an ordered process beginning with recombination activating gene (RAG) expression and TCRβ recombination in CD4⁻CD8⁻CD25⁺ thymocytes. In these cells, TCRβ expression leads to clonal expansion, RAG downregulation, and TCRβ allelic exclusion. At the subsequent CD4⁺CD8⁺ stage, RAG expression is reinduced and V(D)J recombination is initiated at the TCRα locus. This second wave of RAG expression is terminated upon expression of a positively selected α/β TCR. To examine the physiologic role of the second wave of RAG expression, we analyzed mice that cannot reinduce RAG expression in CD4⁺CD8⁺ T cells because the transgenic locus that directs RAG1 and RAG2 expression in these mice is missing a distal regulatory element essential for reinduction. In the absence of RAG reinduction we find normal numbers of CD4⁺CD8⁺ cells but a 50–70% reduction in the number of mature CD4⁺CD8⁻ and CD4⁻CD8⁺ thymocytes. TCRα rearrangement is restricted to the 5′ end of the Jα cluster and there is little apparent secondary TCRα recombination. Comparison of the TCRα genes expressed in wild-type or mutant mice shows that 65% of all α/β T cells carry receptors that are normally assembled by secondary TCRα rearrangement. We conclude that RAG reinduction in CD4⁺CD8⁺ thymocytes is not required for initial TCRα recombination but is essential for secondary TCRα recombination and that the majority of TCRα chains expressed in mature T cells are products of secondary recombination.