Unexpectedly late expression of intracellular CD3ε and TCR γδ proteins during adult thymus development

During adult thymus development immature CD4(-)CD8(-) [double-negative (DN)] precursor cells pass through four phenotypically distinct stages defined by expression of CD44 and CD25: CD44(hi)CD25(-) (DN1), CD44(hi)CD25(+) (DN2), CD44(lo)CD25(+) (DN3) and CD44(lo)CD25(-) (DN4). Although it is well established that the TCR beta, gamma and delta genes are rearranged and expressed in association with the CD3 components in DN thymocytes, the precise timing of expression of the TCR and CD3 proteins has not been determined. In this report we have utilized a sensitive intracellular (ic) staining technique to analyze the expression of ic CD3 epsilon, TCR beta and TCR gamma delta proteins in immature DN subsets. As expected from previous studies of TCR beta rearrangement and mRNA expression, icTCR beta(+) cells were first detected in the DN3 subset and their proportion increased thereafter. Surprisingly, however, both icCD3 epsilon(+) and icTCR gamma delta(+) cells were detected at later stages of development than was predicted by molecular studies. In particular icCD3 epsilon protein expression coincided with the transition from the DN2 to DN3 stage of development, whereas icTCR gamma delta protein expression was only detected in a minor subset of DN4 cells. The implications of these findings for alpha beta lineage divergence will be discussed.