Identification of nonviable cells in immunofluorescently stained cell populations is essential for obtaining accurate data. Fluorescent non‐vital DNA dyes, particularly propidium iodide (PI), have been used routinely in flow cytometry for discrimination of dead cells from viable cells on the basis of fluorescence. We describe here the use of an alternative DNA dye, 7‐amino‐actinomycin D (7‐AAD), which can replace PI for the exclusion of nonviable cells. As an example, we present in this paper the utilization of 7‐AAD on various leukemic cell lines for dead cell exclusion whenever the viable cell population could not be discriminated reliably from nonviable cells on the light scatter histogram; 7‐AAD is suitable for dead cell discrimination in lengthy experiments because it is efficiently excluded by intact cells and has a high DNA binding constant. In addition, the dye is valuable in combination with phycoerythrin (PE)‐fluorescence dual‐color flow cytometry on a single argon laser instrument, since its emission in the far red can easily be separated from the emission of PE; 7‐AAD was used on fluoresceinisothiocyanate (FITC) and PE surface‐labeled human thymocytes for characterization of the dying subpopulation of cells which is undergoing programmed cell death. In this heterogeneous cell preparation, the spectral properties of the dye permitted the classification of viable and nonviable cell subpopulations by multiparameter analysis.

Galectin-1, a β-galactoside binding protein, is produced by thymic epithelial cells and binds to human thymocytes. We have previously reported that galectin-1 induces the apoptosis of activated T lymphocytes. Because the majority of thymocytes die via apoptosis while still within the thymus, we tested whether galectin-1 could induce the apoptosis of these cells. We now report that in vitro exposure to galectin-1 induced apoptosis of two subsets of CD4lo CD8lo thymocytes. The phenotypes of susceptible thymocytes were consistent with that of both negatively selected and nonselected cells. Galectin-1–induced apoptosis was enhanced by preexposure of thymocytes to antibody to CD3, suggesting that galectin-1 may be a participant in T-cell– receptor mediated apoptosis. In contrast, pretreatment of thymocytes with dexamethasone had no effect on galectin-1 susceptibility. We noted that 71% of the cells undergoing apoptosis after galectin-1 treatment had a DNA content greater than 2N, indicating that proliferating thymocytes were most sensitive to galectin-1. We propose that galectin-1 plays a role in the apoptosis of both negatively selected and nonselected thymocytes, and that the susceptibility of thymocytes to galectin-1 is regulated, in part, by entry or exit from the cell cycle.

We found previously that Id3, which inhibits transcriptional activities of many basic helix-loop-helix transcription factors, blocked T and B cell development but stimulated natural killer (NK) cell development. Here we report that ectopic expression of Id3 and another Id protein, Id2, strongly inhibited the development of primitive CD34+CD38− progenitor cells into CD123high dendritic cell (DC)2 precursors. In contrast, development of CD34+CD38− cells into CD4+CD14+ DC1 precursors and mature DC1 was not affected by ectopic Id2 or Id3 expression. These observations support the notion of a common origin of DC2 precursors, T and B cells. As Id proteins did not block development of NK cells, a model presents itself in which these proteins drive common lymphoid precursors to develop into NK cells by inhibiting their options to develop into T cells, B cells, and pre-DC2.

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Tumor suppressor p53 plays an important role in regulating cell cycle progression and apoptosis. Here we applied RNA interference to study the role of p53 in human hematopoietic development in vivo. An siRNA construct specifically targeting the human tumor-suppressor gene p53 was introduced into human CD34 ؉ progenitor cells by lentivirus-mediated gene transfer, which resulted in more than 95% knockdown of p53. We adapted the human-SCID mouse model to opti- IntroductionThe tumor suppressor p53 plays an important role in regulating the cell cycle and apoptosis in response to DNA damage caused by irradiation or exposure to genotoxic mediators. In addition, p53 can mediate several cellular responses, including cell cycle arrest, senescence, differentiation, and apoptosis, depending on the cell type and the microenvironment. 1 Although mutations occur in the gene encoding p53 in human cancers, including tumors of hematopoietic origin, its function in normal human hematopoietic development remains largely unexplored.Recently, we obtained evidence that p53 plays a role in regulating the replicative lifespan of mature human T cells in vitro through the suppression of human telomerase reverse transcriptase (hTERT) (R.G., E.W., R. Beijersbergen, and H.S., manuscript in preparation). Telomeres are DNA repeats at the distal ends of the chromosomes that protect against chromosome end-to-end fusion. 2 Telomeres are shortened at each cell division, and cells with critically short telomeres undergo cell cycle arrest and become senescent. [3][4][5] hTERT, which prevents telomere shortening, is transiently up-regulated in T cells on stimulation through the T-cell receptor (TCR), 4,5 and expression of a dominant-negative mutant of hTERT significantly decreased the lifespan of CD4 ϩ and CD8 ϩ T cells, 5 indicating that hTERT plays a regulatory role in the lifespan of human T cells. Recently, we observed that the down-regulation of p53 by RNA interference (RNAi) extends the lifespan of mature human T cells and neutralizes the inhibition by dominant-negative hTERT, indicating that p53 regulates hTERT expression in primary human T cells. Given the function of p53 in mature human T cells, we asked whether the p53 loss would affect the homeostatic proliferation of T cells. Mice deficient in p53 did not show obvious defects in T-cell homeostasis. However, T cells from inbred mouse strains have longer telomeres than T cells from humans, resulting in a delay in the onset of replicative senescence and in an extended lifespan of these murine T cells. It is, therefore, of interest to test the effect of p53 inactivation on human T-cell homeostasis.In addition, we examined the role of p53 in thymic T-cell development because studies in the mouse have revealed that p53 is induced after the initiation of TCR rearrangement 6 and that it plays an important role in early T-cell development, specifically in pre-T-cell receptor signaling. 7 Results obtained in mice cannot always be extrapolated to humans, and the role of p53 in human T-cell developm...