Medical Biology - Research Publications

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    Pre-T cell receptor (TCR) and TCR-controlled checkpoints in T cell differentiation are set by Ikaros.
    Winandy, S ; Wu, L ; Wang, JH ; Georgopoulos, K (Rockefeller University Press, 1999-10-18)
    T cell differentiation relies on pre-T cell receptor (TCR) and TCR signaling events that take place at successive steps of the pathway. Here, we show that two of these T cell differentiation checkpoints are regulated by Ikaros. In the absence of Ikaros, double negative thymocytes can differentiate to the double positive stage without expression of a pre-TCR complex. Subsequent events in T cell development mediated by TCR involving transition from the double positive to the single positive stage are also regulated by Ikaros. Nonetheless, in Ikaros-deficient thymocytes, the requirement of pre-TCR expression for expansion of immature thymocytes as they progress to the double positive stage is still maintained, and the T cell malignancies that invariably arise in the thymus of Ikaros-deficient mice are dependent on either pre-TCR or TCR signaling. We conclude that Ikaros regulates T cell differentiation, selection, and homeostasis by providing signaling thresholds for pre-TCR and TCR.
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    Developmental potential of the earliest precursor cells from the adult mouse thymus.
    Wu, L ; Antica, M ; Johnson, GR ; Scollay, R ; Shortman, K (Rockefeller University Press, 1991-12-01)
    A new, numerically minute population of cells representing the earliest T precursor cells in the adult mouse thymus has recently been isolated. This population has been shown to be similar to bone marrow hemopoietic stem cells in surface antigenic phenotype and to express moderate levels of CD4. We now show, by fluorescence-activated cell sorting and intrathymic transfer to irradiated mice, that this apparently homogeneous population differs from multipotent stem cells in expressing the surface stem cell antigen 2 (Sca-2), that it differs from most early B lineage cells in lacking B220 and class II major histocompatibility complex expression, and that it binds rhodamine 123 like an activated rather than a quiescent cell. Irradiated recipient mice differing at the Ly 5 locus were used to compare the developmental potential of these early intrathymic precursors with bone marrow stem cells. Only T lineage product cells were detected when the intrathymic precursor population was transferred back into an irradiated thymus. However, when the intrathymic precursor population was transferred intravenously, it displayed the capacity to develop into both B and T lymphoid cells in recipient bone marrow, spleen, and lymph nodes, but no donor-derived myeloid cells were detected. The absence of myeloid and erythroid precursor activity was confirmed by showing that the intrathymic precursor population was unable to develop into myeloid or erythroid spleen colonies on intravenous transfer or to form colonies in an agar culture. These findings indicate that this earliest intrathymic precursor population has become restricted (or strongly biased) to lymphoid lineage development, but not exclusively to T lymphocytes.
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    Thymic dendritic cell precursors: relationship to the T lymphocyte lineage and phenotype of the dendritic cell progeny.
    Wu, L ; Li, CL ; Shortman, K (Rockefeller University Press, 1996-09-01)
    Successive T-precursors isolated from adult mouse thymus were examined for their developmental potential, by transfer to irradiated Ly 5-disparate recipients. The earliest, "low CD4" precursors formed T, B, and dendritic cells (DC), but not myeloid cells, in accordance with earlier studies. Surprisingly, the next downstream CD4-8-3 44+25+ precursor population still formed DC as well as T cells although it no longer formed B or myeloid cells. Further down-stream, the CD4-8 3-44-25+ population formed only T cells. The thymic and splenic DC progeny of the early thymic precursors all expressed high levels of CD8 alpha, in contrast with normal splenic DC and the splenic DC progeny of bone marrow stem cells, which consisted of both CD8 and CD8+ DC. A common precursor of T cells and of a subclass of DC is proposed, with CD8 alpha as a marker of the lymphoid-related DC lineage.
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    The surface phenotype of dendritic cells purified from mouse thymus and spleen: investigation of the CD8 expression by a subpopulation of dendritic cells.
    Vremec, D ; Zorbas, M ; Scollay, R ; Saunders, DJ ; Ardavin, CF ; Wu, L ; Shortman, K (Rockefeller University Press, 1992-07-01)
    A new procedure for rapid isolation of dendritic cells (DC) was devised, involving collagenase digestion of tissues, dissociation of lymphoid-DC complexes, selection of light-density cells, then depletion of lymphocytes and other non-DC by treatment with a mixture of lineage-specific monoclonal antibodies (mAbs) and removal with anti-immunoglobulin-coupled magnetic beads. This enriched population (approximately 80% DC) was further purified when required by fluorescence-activated cell sorting for cells expressing high levels of class II major histocompatibility complex (MHC). The isolated DC were characterized by immunofluorescent staining using a panel of 30 mAbs. Thymic DC were surface positive for a number of markers characteristic of T cells, but they were distinct from T-lineage cells in expressing high levels of class II MHC, in lacking expression of the T cell receptor (TCR)-CD3 complex, and having TCR beta and gamma genes in germline state. Splenic DC shared many markers with thymic DC, but were negative for most T cell markers, with the exception of CD8. A substantial proportion of DC from both thymus and spleen expressed CD8 at high levels, comparable with that on T cells. This appeared to be authentic CD8, and was produced by the DC themselves, since they contained CD8 alpha mRNA. Thymic DC presented both the CD8 alpha and beta chains on the cell surface (Ly-2+3+), although the alpha chain was in excess; the splenic DC expressed only the CD8 alpha chain (Ly-2+3-). It is suggested that the expression of CD8 could endow certain antigen-presenting DC with a veto function.
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    Dendritic cell development in culture from thymic precursor cells in the absence of granulocyte/macrophage colony-stimulating factor.
    Saunders, D ; Lucas, K ; Ismaili, J ; Wu, L ; Maraskovsky, E ; Dunn, A ; Shortman, K (Rockefeller University Press, 1996-12-01)
    The earliest lymphoid precursor population in the adult mouse thymus had previously been shown to produce not only T cells, but also dendritic cell (DC) progeny on transfer to irradiated recipients. In this study, culture of these isolated thymic precursors with a mixture of cytokines induced them to proliferate and to differentiate to DC, but not to T lineage cells. At least 70% of the individual precursors had the capacity to form DC. The resultant DC were as effective as normal thymic DC in the functional test of T cell stimulation in mixed leukocyte cultures. The cultured DC also expressed high levels of class I and class II major histocompatibility complex, together with CD11c, DEC-205, CD80, and CD86, markers characteristic of mature DC in general. However, they did not express CD8 alpha or BP-1, markers characteristic of normal thymic DC. The optimized mixture of five to seven cytokines required for DC development from these thymic precursors did not include granulocyte/macrophage colony stimulating factor (GM-CSF), usually required for DC development in culture. The addition of anti-GM-CSF antibody or the use of precursors from GM-CSF-deficient mice did not prevent DC development. Addition of GM-CSF was without effect on DC yield when interleukin (IL) 3 and IL-7 were present, although some stimulation by GM-CSF was noted in their absence. In contrast, DC development was enhanced by addition of the Flt3/Flk2 ligand, in line with the effects of the administration of this cytokine in vivo. The results indicate that the development of a particular lineage of DC, probably those of lymphoid precursor origin, may be independent of the myeloid hormone GM-CSF.
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    Mouse hepatitis virus A59-induced demyelination can occur in the absence of CD8+ T cells.
    Gombold, JL ; Sutherland, RM ; Lavi, E ; Paterson, Y ; Weiss, SR (Elsevier BV, 1995-03)
    Mouse hepatitis virus causes a chronic demyelinating disease in C57BL/6 mice. While early studies suggested demyelination is due to direct cytolytic effects of virus on oligodendrocytes, there is increasing evidence for the involvement of the immune system in the mechanism of demyelination. In this study we have asked whether demyelination can occur in the absence of functional MHC class I expression and CD8+ T cells. We infected transgenic mice lacking expression of beta 2 microglobulin (beta 2 M -/- mice) with MHV-A59. In beta 2M-/- mice, virus was much more lethal than in either of the parental strains used to produce the mice; furthermore, while clearance from the CNS did occur in beta 2M-/- mice, it was slower than in C57BL/6 mice. This is consistent with the importance of CD8+ cells in viral clearance. Because of the increased sensitivity of the beta 2M-/- mice to infection, only low levels of virus could be used to evaluate chronic disease. Even at these low levels, demyelination did occur in some animals. To compare infection in beta 2M-/- and C57BL/6 mice we used a higher dose of an attenuated variant of MHV-A59, C12. The attenuated variant induced less demyelination in C57BL/6 mice compared to wild type A59, but the levels observed were not significantly different from those seen in beta 2M-/- mice. Thus, MHV-induced demyelination can occur in some animals in the absence of MHC class I and CD8+ cells.
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    RAPID UP-REGULATION OF MDR1 EXPRESSION BY ANTHRACYCLINES IN A CLASSICAL MULTIDRUG-RESISTANT CELL-LINE
    HU, XF ; SLATER, A ; WALL, DM ; KANTHARIDIS, P ; PARKIN, JD ; COWMAN, A ; ZALCBERG, JR (STOCKTON PRESS, 1995-05-01)
    Studies were carried out in a variant human multidrug-resistant (MDR) cell line CEM/A7R, which expresses very low levels of mdr1 mRNA and P-glycoprotein (P-gp). The induction of mdr1 RNA expression by three anthracyclines, (doxorubicin, daunorubicin, epirubicin), VP-16 and two vinca alkaloids (vincristine, vinblastine) was semiquantitatively assessed by scanning Northern blots on a phosphorimager. The relative level of mdr1 expression was expressed as ratio of mdr1 to the internal RNA (actin). A significant increase (P < 0.02) in expression of mdr1 was noted within 4 hrs of exposure to 1.5 micrograms ml-1 daunorubicin or epirubicin. Neither vinblastine nor vincristine had any effect on mdr1 levels after an 8 h exposure. With increasing concentrations of daunorubicin or epirubicin in a fixed 24 h time period, mdr1 expression increased, although a biphasic response was seen. Based on MRK 16 binding, an increase in P-gp levels was seen in the CEM/A7R line after a 24 h exposure to 1 microgram ml-1 daunorubicin or epirubicin. The rapid increase in mdr1 expression after a short period of exposure to doxorubicin, daunorubicin or epirubicin suggests that induction of mdr1 expression may have an important role in the development of drug-resistant tumours.
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    The role of gp130-mediated signals in osteoclast development: Regulation of interleukin 11 production by osteoblasts and distribution of its receptor in bone marrow cultures
    Romas, E ; Udagawa, N ; Zhou, H ; Tamura, T ; Saito, M ; Taga, T ; Hilton, DJ ; Suda, T ; Ng, KW ; Martin, TJ (ROCKEFELLER UNIV PRESS, 1996-06-01)
    Interleukin (IL)-11 is a multifunctional cytokine whose role in osteoclast development has not been fully elucidated. We examined IL-11 production by primary osteoblasts and the effects of rat monoclonal anti-mouse glycoprotein 130 (gp130) antibody on osteoclast formation, using a coculture of mouse osteoblasts and bone marrow cells. IL-1, TNF alpha, PGE2, parathyroid hormone (PTH) and 1 alpha,25-dihydroxyvitamin D3 (1 alpha,25(OH)2D3) similarly induced production of IL-11 by osteoblasts, but IL-6, IL-4, and TGF beta did not. Primary osteoblasts constitutively expressed mRNAs for both IL-11 receptor (IL-11R alpha) and gp130. Osteotropic factors did not modulate IL-11R alpha mRNA at 24 h, but steady-state gp130 mRNA expression in osteoblasts was upregulated by 1 alpha,25(OH)2D3, PTH, or IL-1. In cocultures, the formation of multinucleated osteoclast-like cells (OCLs) in response to IL-11, or IL-6 together with its soluble IL-6 receptor was dose-dependently inhibited by rat monoclonal anti-mouse gp130 antibody. Furthermore, adding anti-gp130 antibody abolished OCL formation induced by IL-1, and partially inhibited OCL formation induced by PGE2, PTH, or 1 alpha,25(OH)2D3. During osteoclast formation in marrow cultures, a sequential relationship existed between the expression of calcitonin receptor mRNA and IL-11R alpha mRNA. Osteoblasts as well as OCLs expressed transcripts for IL-11R alpha, as indicated by RT-PCR analysis and in situ hybridization. These results suggest a central role of gp130-coupled cytokines, especially IL-11, in osteoclast development. Since osteoblasts and mature osteoclasts expressed IL-11R alpha mRNA, both bone-forming and bone-resorbing cells are potential targets of IL-11.
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    B lymphocytes differentially use the Rel and nuclear factor kappa B1 (NF-kappa B1) transcription factors to regulate cell cycle progression and apoptosis in quiescent and mitogen-activated cells
    Grumont, RJ ; Rourke, IJ ; O'Reilly, LA ; Strasser, A ; Miyake, K ; Sha, W ; Gerondakis, S (ROCKEFELLER UNIV PRESS, 1998-03-02)
    Rel and nuclear factor (NF)-kappaB1, two members of the Rel/NF-kappaB transcription factor family, are essential for mitogen-induced B cell proliferation. Using mice with inactivated Rel or NF-kappaB1 genes, we show that these transcription factors differentially regulate cell cycle progression and apoptosis in B lymphocytes. Consistent with an increased rate of mature B cell turnover in naive nfkb1-/- mice, the level of apoptosis in cultures of quiescent nfkb1-/-, but not c-rel-/-, B cells is higher. The failure of c-rel-/- or nfkb1-/- B cells to proliferate in response to particular mitogens coincides with a cell cycle block early in G1 and elevated cell death. Expression of a bcl-2 transgene prevents apoptosis in resting and activated c-rel-/- and nfkb1-/- B cells, but does not overcome the block in cell cycle progression, suggesting that the impaired proliferation is not simply a consequence of apoptosis and that Rel/NF-kappaB proteins regulate cell survival and cell cycle control through independent mechanisms. In contrast to certain B lymphoma cell lines in which mitogen-induced cell death can result from Rel/NF-kappaB-dependent downregulation of c-myc, expression of c-myc is normal in resting and stimulated c-rel-/- B cells, indicating that target gene(s) regulated by Rel that are important for preventing apoptosis may differ in normal and immortalized B cells. Collectively, these results are the first to demonstrate that in normal B cells, NF-kappaB1 regulates survival of cells in G0, whereas mitogenic activation induced by distinct stimuli requires different Rel/NF-kappaB factors to control cell cycle progression and prevent apoptosis.
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    Early function of Pax5 (BSAP) before the pre-B cell receptor stage of B lymphopoiesis
    Thevenin, C ; Nutt, SL ; Busslinger, M (ROCKEFELLER UNIV PRESS, 1998-08-17)
    The formation of the pre-B cell receptor (BCR) corresponds to an important checkpoint in B cell development that selects pro-B (pre-BI) cells expressing a functionally rearranged immunoglobulin mu (Igmu) heavy chain protein to undergo the transition to the pre-B (pre-BII) cell stage. The pre-BCR contains, in addition to Igmu, the surrogate light chains lambda5 and VpreB and the signal transducing proteins Igalpha and Igbeta. The absence of one of these pre-BCR components is known to arrest B cell development at the pre-BI cell stage. Disruption of the Pax5 gene, which codes for the B cell-specific activator protein (BSAP), also blocks adult B lymphopoiesis at the pre-BI cell stage. Moreover, expression of the mb-1 (Igalpha) gene and VH-to-DHJH recombination at the IgH locus are reduced in Pax5-deficient B lymphocytes approximately 10- and approximately 50-fold, respectively. Here we demonstrate that complementation of these deficiencies in pre-BCR components by expression of functionally rearranged Ig mu and chimeric Igmu-Igbeta transgenes fails to advance B cell development to the pre-BII cell stage in Pax5 (-/-) mice in contrast to RAG2 (-/-) mice. Furthermore, the pre-BCR is stably expressed on cultured pre-BI cells from Igmu transgenic, Pax5-deficient bone marrow, but is unable to elicit its normal signaling responses. In addition, the early developmental block is unlikely to be caused by the absence of a survival signal, as it could not be rescued by expression of a bcl2 transgene in Pax5-deficient pre-BI cells. Together, these data demonstrate that the absence of Pax5 arrests adult B lymphopoiesis at an early developmental stage that is unresponsive to pre-BCR signaling.