Scientific Resources

To search for new indicators of self-renewing hematopoietic stem cells (HSCs),highly purified populations were isolated from adult mouse marrow,micromanipulated into a specially designed microscopic array,and cultured for 4 days in 300 ng/ml Steel factor,20 ng/ml IL-11,and 1 ng/ml flt3-ligand. During this period,each cell and its progeny were imaged at 3-min intervals by using digital time-lapse photography. Individual clones were then harvested and assayed for HSCs in mice by using a 4-month multilineage repopulation endpoint (textgreater1% contribution to lymphoid and myeloid lineages). In a first experiment,6 of 14 initial cells (43%) and 17 of 61 clones (28%) had HSC activity,demonstrating that HSC self-renewal divisions had occurred in vitro. Characteristics associated with HSC activity included longer cell-cycle times and the absence of uropodia on a majority of cells within the clone during the final 12 h of culture. Combining these criteria maximized the distinction of clones with HSC activity from those without and identified a subset of 27 of the 61 clones. These 27 clones included all 17 clones that had HSC activity; a detection efficiency of 63% (2.26 times more frequently than in the original group). The utility of these characteristics for discriminating HSC-containing clones was confirmed in two independent experiments where all HSC-containing clones were identified at a similar 2- to 3-fold-greater efficiency. These studies illustrate the potential of this monitoring system to detect new features of proliferating HSCs that are predictive of self-renewal divisions. View Publication

MICA is a stress-regulated molecule recognized by the NK cell-activating receptor NKG2D. Previously,we demonstrated that MICA is induced on activated T cells but regulation by mitogenic cytokines and its biological consequences remain unexplored. Here,we show that IL-2,IL-4,and IL-15 but not TNF-alpha or IFN-alpha induced MICA expression in T lymphocytes present in peripheral blood mononuclear cells (PBMCs),as assessed by Western blot. IL-2 effect involved Jak3/STAT5,p38 MAPK,p70(56) kinase,Lck/fyn kinases,and NF-kappaB. MICA expression was also observed in Th1 and Th2 cells. However,surface expression was not detected. T lymphocytes present in PBMCs and isolated CD4+ T lymphocytes stimulated with phorbol-12-myristate-13-acetate and ionomycin also induced MICA expression as assessed by Western blot,but only low levels were expressed at the cell surface. Activated but not resting CD4+ T lymphocytes were lysed by IL-15- or IL-2-stimulated NK cells,and susceptibility was increased when HLA class I molecules were blocked. Also,cytokine-stimulated NK cells produced more IFN-gamma after culture with activated CD4+ T lymphocytes. However,the participation of MICA in these responses,if any,was marginal. Confocal microscopy revealed that MICA is retained mostly inside activated CD4+ T cells. Our results suggest that low surface expression of MICA on activated CD4+ T lymphocytes might be a safeguard mechanism to protect them from NK cells in an inflammatory,virus-infected,or tumor microenvironment,where NK and activated CD4+ T cells are recruited. View Publication

CD4+CD25+ regulatory T cells play an important role in peripheral tolerance. Upon T cell receptor (TCR)-mediated activation,the cells fail to proliferate but are induced to have a suppressor function. The intracellular signaling events that lead to their responses have not been elucidated. In this study,we have examined the proximal TCR signaling events in freshly isolated human CD4+CD25+ regulatory T cells after TCR ligation. In contrast to CD4+CD25- T cells,TCR ligation of CD4+CD25+ regulatory T cells by anti-CD3 cross-linking resulted in a lower calcium influx and extracellular signal-regulated kinase 1/2 phosphorylation. Examination of the CD3zeta chain phosphorylation status indicated that CD4+CD25+ regulatory T cells have poor phosphorylation of the protein and consequently,reduced recruitment of zeta-associated protein-70 to the TCR immunoreceptor tyrosine motif. The adaptor protein,Src homology 2 domain-containing leukocyte phosphoprotein of 76 kDa,which relays signals to downstream signaling components,also showed reduced phosphorylation,which correlated with reduced VAV guanine nucleotide exchange factors association. Consistent with other findings,the defect is accompanied with impaired actin cap formation,implicating a failure of actin remodeling of the cells. Together,our results demonstrate that CD4+CD25+ regulatory T cells have altered TCR proximal signaling pathways,which could be critical for inducing the distinct behavior of these cells. View Publication

The t(16:16) and inv(16) are associated with FAB M4Eo myeloid leukemias and result in fusion of the CBFB gene to the MYH11 gene (encoding smooth muscle myosin heavy chain [SMMHC]). Knockout of CBFbeta causes embryonic lethality due to lack of definitive hematopoiesis. Although knock-in of CBFB-MYH11 is not sufficient to cause disease,expression increases the incidence of leukemia when combined with cooperating events. Although mouse models are valuable tools in the study of leukemogenesis,little is known about the contribution of CBFbeta-SMMHC to human hematopoietic stem and progenitor cell self-renewal. We introduced the CBFbeta-MYH11 cDNA into human CD34+ cells via retroviral transduction. Transduced cells displayed an initial repression of progenitor activity but eventually dominated the culture,resulting in the proliferation of clonal populations for up to 7 months. Long-term cultures displayed a myelomonocytic morphology while retaining multilineage progenitor activity and engraftment in NOD/SCID-B2M-/- mice. Progenitor cells from long-term cultures showed altered expression of genes defining inv(16) identified in microarray studies of human patient samples. This system will be useful in examining the effects of CBFbeta-SMMHC on gene expression in the human preleukemic cell,in characterizing the effect of this oncogene on human stem cell biology,and in defining its contribution to the development of leukemia. View Publication