Proliferation Kinetics Associated with T Cell Receptor-β Chain Selection of Fetal Murine Thymocytes

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Abstract
After productive rearrangement of a TCRβ chain gene, CD4−8− double negative (DN) thymocytes express TCRβ polypeptide chains on the cell surface together with pre-Tα and the CD3 complex forming the pre-TCR. Signals transmitted through the pre-TCR select TCRβ+ DN thymocytes for further maturation to the CD4+8+ double positive stage, whereas DN cells that fail to generate a productive TCRβ gene rearrangement do not continue in development. This process is termed TCRβ chain selection. Although it is likely that differences between proliferation dynamics of TCRβ+ and TCRβ− cells may play a role, the exact mechanisms of TCRβ chain selection have not been elucidated. We therefore studied the proliferation dynamics of TCRβ+ and TCRβ− thymocytes during fetal development, i.e., when TCRβ chain selection takes place for the first time. We analyzed in situ accumulation of TCRβ+ thymocytes by confocal microscopy, and determined cell cycle and division parameters of TCRβ+ and TCRβ− populations by flow cytometry. About 600 TCRβ+ cells/thymic lobe are generated by independent induction events between days of gestation (dg) 13.5. and 15.5. As of dg 14.5, most TCRβ+ cells have entered S/G2 phase of cell cycle, followed by seven to eight rapid cell divisions in fetal thymic organ culture, suggesting a corresponding burst of nine cell divisions within 4 d in vivo. By dg 18.5, the division rate of TCRβ+ cells has slowed down to less than 1/d. About three quarters of TCRβ− cells divide at a slow rate of 1/d on dg 14.5, the proportion of nondividing cells increasing to 50% within the following four d. From dg 16.5 onwards, TCRβ− cells, but not TCRβ+ cells, contain a significant proportion of apoptotic cells. The results suggest that failure to become selected results in shutdown of proliferation and eventual programmed cell death of fetal TCRβ− cells. Positive selection of fetal TCRβ+ cells is achieved by an increased rate of cell divisions lasting for approximately 4 d.