Human T lymphocytes were stimulated using phorbol myristate acetate and ionomycin. Twenty‐four hours post‐activation the cells were harvested for DNA content and for measurements using a newly ...developed cell profiling system employing dielectrophoresis. This system provides individual cell size and dielectrophoresis data for statistically relevant numbers of control and activated cells. From this it was determined that the mean membrane specific capacitance decreased from 13.49 (± 4.72) mF/m2 to 10.62 (± 5.13) mF/m2. This can be related to a 21.3% reduction in the effective membrane surface area associated with membrane topography (e.g. reduction of membrane associated microvilli, blebs and folding), or to other changes of membrane architecture, following cell activation. From cytometric determinations of DNA content, it was concluded that these effects were related to a 3.0‐fold decrease of cells in S‐phase, and a 1.5‐fold increase in G1 cells. This work demonstrates the powerful potential of using dielectrophoresis as a noninvasive tool to follow physiological changes that accompany transmembrane signaling events.
nu/nu mice fail to develop a thymus and mature T cells due to a defect in the whn gene encoding a transcription factor necessary for terminal epithelial cell differentiation. We investigated whether ...early T cell progenitor development in the nu/nu bone marrow is also defective. We demonstrated a maturation arrest of nu/nu marrow T cell progenitors associated with a lack of pTalpha gene expression and a failure to give rise to mature T cells in adoptive euthymic hosts. Wild-type hemopoietic stem cells rapidly matured into functional T cell progenitors in the marrow of euthymic or thymectomized but not nu/nu hosts. We show that defects in bone marrow prethymic T cell development can also contribute to T cell deficiency in nu/nu mice.
Introduction Hematopoietic stem cells (HSCs) follow a genetically programmed pattern of migration during development. Extracellular matrix and adhesion molecules, as well as chemokines and their ...receptors, are important in adult HSC migration. However, little is known about the role these molecules play at earlier developmental stages. Methods We have analyzed by quantitative polymerase chain reaction (qPCR) array the expression pattern of extracellular matrix and adhesion molecules as well as chemokines and chemokine receptors in Lineage.sup.-.sup.Sca-1.sup.+.sup.c-Kit.sup.+ .sup.(LSK) cells at different stages of development, in order to characterize the role played by these molecules in LSK. Data were represented by volcano plots to show the differences in expression pattern at the time points studied. Results Our results show marked changes in the expression pattern of extracellular matrix, adhesion molecules, chemokines and their receptors with developmental age, particularly in later stages of development. Ten molecules were significantly increased among the LSK populations studied. Our screen identified the upregulation of Col4a1, as well as molecules involved in its degradation (Mmp2, Timp2), with development. Other genes identified were Sell, Tgfbi, and Entpd1. Furthermore, we show that the expression of the chemokines Ccl4, Ccl9, Il18 and the chemokine receptor Cxcr4 increases in LSK cells during development. Conclusions Several genes are upregulated in the LSK population in their transition to the bone marrow microenvironment, increasing at later stages of development. This gene pattern should be emulated by embryonic stem cell-derived hematopoietic progenitors in order to improve their properties for clinical applications such as engraftment.
We searched for clonable committed T cell progenitors in the adult mouse bone marrow and isolated rare (≈0.05%) cells with the Thy-1hiCD2-CD16+CD44hiCD25-Lin-phenotype. In vivo experiments showed ...that these cells were progenitors committed only to reconstituting the T cell lineage of irradiated Ly5 congenic hosts. Reconstitution of the thymus was minimal compared with that of the bone marrow, spleen, and lymph nodes. At limiting dilutions, donor T cell reconstitution of the spleen frequently occurred without detectable donor cells in the thymus. Progenitors were capable of rapidly reconstituting athymic hosts. In conclusion, the clonable bone marrow progenitors were capable of T cell reconstitution predominantly by means of an extrathymic pathway.
T cell apoptosis has been proposed as an Important contributor to the functional defects and depletion of T cells in HIV-lnfected Individuals. However, the mechanisms Involved in this apoptosis have ...not been elucidated. We recently showed that peripheral blood T cells from HIV-infected individuals are especially susceptible to Fas antigen-induced apoptosis. In this study we examine the role of Fas, CTLA-4, tumor necrosis factor (TNF) receptors (TNFR) and CD30, receptors known to be involved In T cell activation-induced cell death (AICD), in the spontaneous and activation (anti-CD3)-lnduced apoptosis of peripheral blood T cells from asymptomatic HIV-infected individuals. We report here that spontaneous and activation-Induced T cell apoptosis cannot be inhibited by reagents that block interactions of Fas, CTLA-4, p55 and p75 TNFR and CD30 with their respective ligands. We also show that IL-12, IFN-γ, IL-4 and IL-10 cannot modify spontaneous, activation- and anti-Fas-induced apoptosis. Anti-Fas preferentially Induced CD4+ T cell apoptosis whereas AICD induced apoptosis equally In CD4+ and CD8+ T cells. We conclude that T cell AICD In HIV infection Is not mediated by Fas, thus Indicating that Fas-Induced and activation-Induced T cell apoptosis are independent mechanisms of apoptosis which may play different roles in the pathogenesls of HIV infection.
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