The thymus generates two divergent types of lymphocytes, innate and adaptive T cells. Innate T cells such as invariant NKT cells provide immediate immune defense, whereas adaptive T cells require a phase of expansion and functional differentiation outside the thymus. Naive adaptive T lymphocytes should not proliferate much after positive selection in the thymus to ensure a highly diverse TCR repertoire. In contrast, oligoclonal innate lymphocyte populations are efficiently expanded through intrathymic proliferation. For CD4(+)Foxp3(+) regulatory T cells (Tregs), which are thought to be generated by agonist recognition, it is not clear whether they proliferate upon thymic selection. In this study, we investigated thymic and peripheral T cell proliferation by genetic pulse labeling. To this end, we used a mouse model in which all developing αβ thymocytes were marked by expression of a histone 2B-enhanced GFP (H2BeGFP) fusion-protein located within the Tcrd locus (TcrdH2BeGFP). This reporter gene was excised during TCR α-chain VJ-recombination, and the retained H2BeGFP signal was thus diluted upon cell proliferation. We found that innate T cells such as CD1d-restricted invariant NKT cells all underwent a phase of intense intrathymic proliferation, whereas adaptive CD4(+) and CD8(+) single-positive thymocytes including thymic Tregs cycled, on average, only once after final selection. After thymic exit, retention or loss of very stable H2BeGFP signal indicated the proliferative history of peripheral αβ T cells. There, peripheral Tregs showed lower levels of H2BeGFP compared with CD4(+)Foxp3(-) T cells. This further supports the hypothesis that the Treg repertoire is shaped by self-Ag recognition in the steady-state.
