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Early in T-cell development, cells proceed through stages that are critically dependent on signaling through the Notch receptor. As cells mature, thymocytes transition from being Notch dependent to being Notch independent, but the stage of development during which this transition occurs is unknown. We used an in vitro differentiation system in which thymocytes can be cultured in the presence or absence of a Notch ligand to identify the stage of development in which thymocytes transition from being Notch responsive to Notch non-responsive. We identified the immature single-positive (ISP) CD8(+) stage of T-cell development as being this transition point. ISP thymocytes were responsive to Notch, but ISP cells responded to Notch ligation in a manner that was distinct from the response by double-negative (DN) thymocytes. Fewer ISP thymocytes proliferated and more ISP cells died in culture than DN thymocytes. Further, fewer double-positive (DP) thymocytes generated by culturing ISP thymocytes were in the S, G2 or M phase of the cell cycle as compared with DP thymocytes derived from DN thymocytes. These data indicate that the DP population created varied depending on the input population. In summary, the data presented here indicate that ISP thymocytes responded to Notch differently than DN thymocytes and ISP thymocytes represent the transition stage from Notch-dependent survival and proliferation to Notch-independent survival and proliferation.

Immature single-positive CD8+ thymocytes represent the transition from Notch-dependent to Notch-independent T-cell development