The Journal of Experimental Medicine
ImmunoPrecise
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Published online 20 August 2001. doi:10.1084/jem.194.4.471
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© The Rockefeller University Press, 0022-1007/2001/8/471/ $5.00
The Journal of Experimental Medicine, Volume 194, Number 4, August 20, 2001 471-480

Original Article

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

Nikos Yannoutsosa, Patrick Wilsona, Wong Yua, Hua Tang Chend, Andre Nussenzweigd, Howard Petriec, and Michel C. Nussenzweiga,b

a Laboratory of Molecular Immunology, The Rockefeller University, New York, NY 10021
b Howard Hughes Medical Institute, The Rockefeller University, New York, NY 10021
c Immunology Program, Memorial Sloan-Kettering Cancer Center, New York, NY 10021
d Experimental Immunology Branch, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892
The Rockefeller University, Box 220, 1230 York Ave., New York, NY 10021.212-327-8370212-327-8068

yannoun{at}mail.rockefeller.edu

Assembly of T cell receptor (TCR){alpha}/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 TCR{alpha} locus. This second wave of RAG expression is terminated upon expression of a positively selected {alpha}/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. TCR{alpha} rearrangement is restricted to the 5' end of the J{alpha} cluster and there is little apparent secondary TCR{alpha} recombination. Comparison of the TCR{alpha} genes expressed in wild-type or mutant mice shows that 65% of all {alpha}/β T cells carry receptors that are normally assembled by secondary TCR{alpha} rearrangement. We conclude that RAG reinduction in CD4+CD8+ thymocytes is not required for initial TCR{alpha} recombination but is essential for secondary TCR{alpha} recombination and that the majority of TCR{alpha} chains expressed in mature T cells are products of secondary recombination.

Key Words: T cell receptor {alpha} chain • gene rearrangement • regulation of gene expression • T cell receptor editing • recombination activating gene

The online version of this article contains supplemental material.

Abbreviations used in this paper: APC, allophycocyanin; BAC, bacterial artificial chromosome; DN, double negative; DP, double positive; NBS, Nijmegen breakage syndrome; SP, single positive; RAG, recombination activating gene; RT, reverse transcription; TEA, T early {alpha} promoter; YAC, yeast artificial chromosome.

© 2001 The Rockefeller University Press


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