The requirement for pre-TCR during thymic differentiation enforces a developmental pause that is essential for V-DJß rearrangement.

ABSTRACT

T cell development occurs in the thymus and is critically dependent on productive TCRß rearrangement and pre-TCR expression in DN3 cells. The requirement for pre-TCR expression results in the arrest of thymocytes at the DN3 stage (ß checkpoint), which is uniquely permissive for V-DJß recombination; only cells expressing pre-TCR survive and develop beyond the DN3 stage. In addition, the requirement for TCRß rearrangement and pre-TCR expression enforces suppression of TCRß rearrangement on a second allele, allelic exclusion, thus ensuring that each T cell expresses only a single TCRß product. However, it is not known whether pre-TCR expression is essential for allelic exclusion or alternatively if allelic exclusion is enforced by developmental changes that can occur in the absence of pre-TCR. We asked if thymocytes that were differentiated without pre-TCR expression, and therefore without pause at the ß checkpoint, would suppress all V-DJß rearrangement. We previously reported that premature CD28 signaling in murine CD4(-)CD8(-) (DN) thymocytes supports differentiation of CD4(+)CD8(+) (DP) cells in the absence of pre-TCR expression. The present study uses this model to define requirements for TCRß rearrangement and allelic exclusion. We demonstrate that if cells exit the DN3 developmental stage before TCRß rearrangement occurs, V-DJß rearrangement never occurs, even in DP cells that are permissive for D-Jß and TCRα rearrangement. These results demonstrate that pre-TCR expression is not essential for thymic differentiation to DP cells or for V-DJß suppression. However, the requirement for pre-TCR signals and the exclusion of alternative stimuli such as CD28 enforce a developmental "pause" in early DN3 cells that is essential for productive TCRß rearrangement to occur.