TREC levels in FACS-purified thymocyte subsets from the human thymus.

TREC levels were measured in FACS-sorted cells from a fresh thymus of a newborn infant. For CD3⁻ cells, the thymocytes were first depleted of CD3⁺ cells by MACS and then sorted by FACS for the subsets based on CD4 and CD8 expression (dot plot, upper right) to more precisely determine the timing of peak TREC production. Cells were also sorted for CD1a^+/++ cells so as to exclude the CD1a⁻CD3⁻ subset, which may contain precursors for NK cells and thymic dendritic cells. The sorted cells were subjected to TREC assay. The CD3⁺-depleted fraction (shown as R4 in the histogram, upper left) contained cells expressing CD3 at the intermediate level (referred to as CD3^low) as well as CD3⁻ cells. On the other hand, for measuring the frequency of each subpopulation in CD3⁻ and CD3^−/low populations, we analyzed whole thymocytes from the same thymus sample using gate R1. For CD3⁺ subsets, the whole thymocytes were analyzed for frequency measurement using gates R2 or R3 and then sorted for the TREC assay. Therefore, it should be noted that the actual frequency of CD3^−/lowCD4⁺CD8⁺ and CD3⁺CD4⁺CD8⁺ cells should be slightly higher and lower, respectively, than the values shown here (36.4% and 35.7%, respectively). The TREC values are expressed as copies per 10 000 cells. These data represent 3 different experiments.