Scheduled kinetics of cell proliferation and phenotypic changes during immature thymocyte generation

Precursor CD4(-)CD8(-) (DN) thymocytes rearrange their TCR-beta genes, and only those which succeed in beta -selection subsequently expand and differe ntiate into immature CD4(+)CD8(+) (DP) thymocytes. The cell subsets corresp onding to the successive steps of this transition can be defined in terms o f CD44 and CD25 expression. We partially synchronized the differentiation p rocess by eliminating cycling cells with the anti-mitotic agent demecolcine . Using in vivo pulse labeling with bromodeoxyuridine, we determined the or der of entry into DNA synthesis of the different DN and transitory (CD4(-/l o)CD8(+)) cell subsets. Two independent proliferation phases were identifie d. The first cells to enter the cell cycle were CD44(-)CD25(lo), and CD4/CD 8/TCR-/BrdU four-color staining showed that they all expressed a low densit y of the TCR-beta chain, an element of the pre-TCR (the TCR-alpha locus is still in germ-line configuration at this stage). Cycling of CD44(+)CD25(+) cells was detected later, and no starting point was observed at the CD44(-) CD25(hi) stage. CD8 expression was immediately detectable in cycling cells, but they took 24 In to reach the DID stage. The study of TCR-C alpha -defi cient mice showed that P gene rearrangement occurred once proliferation had ceased at the DP stage, and that it had no influence on the DN-DP transiti on. These data show that precursor thymocytes undergo two independent waves of expansion, and that the second wave is restricted to cells capable of p re-TCR expression.