技术资料

Human T-cell leukemia virus type 1 (HTLV-1) and HTLV-2 are highly related viruses that differ in disease manifestation. HTLV-1 is the etiologic agent of adult T-cell leukemia and lymphoma,an aggressive clonal malignancy of human CD4-bearing T lymphocytes. Infection with HTLV-2 has not been conclusively linked to lymphoproliferative disorders. We previously showed that human hematopoietic progenitor (CD34(+)) cells can be infected by HTLV-1 and that proviral sequences were maintained after differentiation of infected CD34(+) cells in vitro and in vivo. To investigate the role of the Tax oncoprotein of HTLV on hematopoiesis,bicistronic lentiviral vectors were constructed encoding the HTLV-1 or HTLV-2 tax genes (Tax1 and Tax2,respectively) and the green fluorescent protein marker gene. Human hematopoietic progenitor (CD34(+)) cells were infected with lentivirus vectors,and transduced cells were cultured in a semisolid medium permissive for the development of erythroid,myeloid,and primitive progenitor colonies. Tax1-transduced CD34(+) cells displayed a two- to fivefold reduction in the total number of hematopoietic clonogenic colonies that arose in vitro,in contrast to Tax2-transduced cells,which showed no perturbation of hematopoiesis. The ratio of colony types that developed from Tax1-transduced CD34(+) cells remained unaffected,suggesting that Tax1 inhibited the maturation of relatively early,uncommitted hematopoietic stem cells. Since previous reports have linked Tax1 expression with initiation of apoptosis,lentiviral vector-mediated transduction of Tax1 or Tax2 was investigated in CEM and Jurkat T-cell lines. Ectopic expression of either Tax1 or Tax2 failed to induce apoptosis in T-cell lines. These data demonstrate that Tax1 expression perturbs development and maturation of pluripotent hematopoietic progenitor cells,an activity that is not displayed by Tax2,and that the suppression of hematopoiesis is not attributable to induction of apoptosis. Since hematopoietic progenitor cells may serve as a latently infected reservoir for HTLV infection in vivo,the different abilities of HTLV-1 and -2 Tax to suppress hematopoiesis may play a role in the respective clinical outcomes after infection with HTLV-1 or -2. View Publication

Germ line mutations in the Adenomatous polyposis coli tumor suppressor gene cause a hereditary form of intestinal tumorigenesis in both mice and man. Here we show that in Apc(Min/+) mice,which carry a heterozygous germ line mutation at codon 850 of Apc,there is progressive loss of immature and mature thymocytes from approximately 80 days of age with complete regression of the thymus by 120 days. In addition,Apc(Min/+) mice show parallel depletion of splenic natural killer (NK) cells,immature B cells,and B progenitor cells in bone marrow due to complete loss of interleukin 7 (IL-7)-dependent B-cell progenitors. Using bone marrow transplantation experiments into wild-type recipients,we have shown that the capacity of transplanted Apc(Min/+) bone marrow cells for T- and B-cell development appears normal. In contrast,although the Apc(Min/+) bone marrow microenvironment supported short-term reconstitution with wild-type bone marrow,Apc(Min/+) animals that received transplants subsequently underwent lymphodepletion. Fibroblast colony-forming unit (CFU-F) colony assays revealed a significant reduction in colony-forming mesenchymal progenitor cells in the bone marrow of Apc(Min/+) mice compared with wild-type animals prior to the onset of lymphodepletion. This suggests that an altered bone marrow microenvironment may account for the selective lymphocyte depletion observed in this model of familial adenomatous polyposis. View Publication

Expression of the nuclear hormone receptor peroxisome proliferator-activated receptor alpha (PPARalpha) in resting lymphocytes was recently established,although the physiologic role(s) played by this nuclear hormone receptor in these cell types remains unresolved. In this study,we used CD4(+) T cells isolated from PPARalpha(-/-) and wild-type mice,as well as cell lines that constitutively express PPARalpha,in experiments designed to evaluate the role of this hormone receptor in the regulation of T cell function. We report that activated CD4(+) T cells lacking PPARalpha produce increased levels of IFN-gamma,but significantly lower levels of IL-2 when compared with activated wild-type CD4(+) T cells. Furthermore,we demonstrate that PPARalpha regulates the expression of these cytokines by CD4(+) T cells in part,through its ability to negatively regulate the transcription of T-bet. The induction of T-bet expression in CD4(+) T cells was determined to be positively influenced by p38 mitogen-activated protein (MAP) kinase activation,and the presence of unliganded PPARalpha effectively suppressed the phosphorylation of p38 MAP kinase. The activation of PPARalpha with highly specific ligands relaxed its capacity to suppress p38 MAP kinase phosphorylation and promoted T-bet expression. These results demonstrate a novel DNA-binding independent and agonist-controlled regulatory influence by the nuclear hormone receptor PPARalpha. View Publication

We evaluated the effects of ectopic granulocyte/macrophage colony-stimulating factor (GM-CSF) signals on hematopoietic commitment and differentiation. Lineage-restricted progenitors purified from mice with the ubiquitous transgenic human GM-CSF receptor (hGM-CSFR) were used for the analysis. In cultures with hGM-CSF alone,hGM-CSFR-expressing (hGM-CSFR+) granulocyte/monocyte progenitors (GMPs) and megakaryocyte/erythrocyte progenitors (MEPs) exclusively gave rise to granulocyte/monocyte (GM) and megakaryocyte/erythroid (MegE) colonies,respectively,providing formal proof that GM-CSF signals support the GM and MegE lineage differentiation without affecting the physiological myeloid fate. hGM-CSFR transgenic mice were crossed with mice deficient in interleukin (IL)-7,an essential cytokine for T and B cell development. Administration of hGM-CSF in these mice could not restore T or B lymphopoiesis,indicating that enforced GM-CSF signals cannot substitute for IL-7 to promote lymphopoiesis. Strikingly,textgreater50% hGM-CSFR+ common lymphoid progenitors (CLPs) and textgreater20% hGM-CSFR+ pro-T cells gave rise to granulocyte,monocyte,and/or myeloid dendritic cells,but not MegE lineage cells in the presence of hGM-CSF. Injection of hGM-CSF into mice transplanted with hGM-CSFR+ CLPs blocked their lymphoid differentiation,but induced development of GM cells in vivo. Thus,hGM-CSF transduces permissive signals for myeloerythroid differentiation,whereas it transmits potent instructive signals for the GM differentiation to CLPs and early T cell progenitors. These data suggest that a majority of CLPs and a fraction of pro-T cells possess plasticity for myelomonocytic differentiation that can be activated by ectopic GM-CSF signals,supporting the hypothesis that the down-regulation of GM-CSFR is a critical event in producing cells with a lymphoid-restricted lineage potential. View Publication

We investigated the in vivo role of CD69 by analyzing the susceptibility of CD69-/- mice to tumors. CD69-/- mice challenged with MHC class I- tumors (RMA-S and RM-1) showed greatly reduced tumor growth and prolonged survival compared with wild-type (WT) mice. The enhanced anti-tumor response was NK cell and T lymphocyte-mediated,and was due,at least in part,to an increase in local lymphocytes. Resistance of CD69-/- mice to MHC class I- tumor growth was also associated with increased production of the chemokine MCP-1,diminished TGF-beta production,and decreased lymphocyte apoptosis. Moreover,the in vivo blockade of TGF-beta in WT mice resulted in enhanced anti-tumor response. In addition,CD69 engagement induced NK and T cell production of TGF-beta,directly linking CD69 signaling to TGF-beta regulation. Furthermore,anti-CD69 antibody treatment in WT mice induced a specific down-regulation in CD69 expression that resulted in augmented anti-tumor response. These data unmask a novel role for CD69 as a negative regulator of anti-tumor responses and show the possibility of a novel approach for the therapy of tumors. View Publication

PURPOSE: Immunotherapy holds promise as a new strategy for the eradication of residual cells in acute myeloid leukaemia (AML). Leukaemic antigen presenting cells (APCs) combining optimal antigen presentation and tumour antigenicity could be used as potent T cell activators. For clinical purposes it is desirable to culture APCs under serum-free conditions. Therefore,we compared morphological,immunophenotypical and functional outcome of the serum-free culture of AML-APCs to their serum-enriched culture. METHODS: AML blasts (n=19) were cultured in the presence of either a cytokine mix or calcium ionophore (CI) for 14 and 2 days,respectively,in FCS-containing medium (FCS),StemSpan serum-free medium (SP) and CellGro serum-free medium (CG). After culture relative yields were calculated and immunophenotypic analysis of APC markers was performed. The mixed leukocyte reaction (MLR) was used to determine T cell stimulating capacity. RESULTS: Serum-free culture of AML-APCs resulted in comparable morphology,relative yields and immunophenotype to serum-enriched culture. By comparing both serum-free media we observed a trend towards a more mature phenotype of CI-cultured AML-APCs in SP. MLR showed that serum-free cultured cells have equal T cell stimulatory capacity in comparison with serum-enriched culture. CONCLUSION: These data show that the serum-free culture of AML-APCs is feasible and that these APCs are comparable to serum-enriched cultured AML-APCs with regard to morphological,immunophenotypical and functional characteristics. These AML-APCs are suitable for the development of active specific immunisation protocols which meet the criteria for good clinical practise (GCP). View Publication

Essential thrombocythemia (ET) and polycythemia vera (PV) are clonal myeloproliferative disorders that are often difficult to distinguish from other causes of elevated blood cell counts. Assays that could reliably detect clonal hematopoiesis would therefore be extremely valuable for diagnosis. We previously reported 3 X-chromosome transcription-based clonality assays (TCAs) involving the G6PD,IDS,and MPP1 genes,which together were informative in about 65% of female subjects. To increase our ability to detect clonality,we developed simple TCA for detecting the transcripts of 2 additional X-chromosome genes: Bruton tyrosine kinase (BTK) and 4-and-a-half LIM domain 1 (FHL1). The combination of TCA established the presence or absence of clonal hematopoiesis in about 90% of female subjects. We show that both genes are subject to X-chromosome inactivation and are polymorphic in all major US ethnic groups. The 5 TCAs were used to examine clonality in 46 female patients along with assays for erythropoietin-independent erythroid colonies (EECs) and granulocyte PRV-1 mRNA levels to discriminate polycythemias and thrombocytoses. Of these,all 19 patients with familial polycythemia or thrombocytosis had polyclonal hematopoiesis,whereas 22 of 26 patients with clinical evidence of myeloproliferative disorder and 1 patient with clinically obscure polycythemia were clonal. Interestingly,interferon alpha therapy in 2 patients with PV was associated with reversion of clonal to polyclonal hematopoiesis. EECs were observed in 14 of 14 patients with PV and 4 of 12 with ET,and increased granulocyte PRV-1 mRNA levels were found in 9 of 13 patients with PV and 2 of 12 with ET. Thus,these novel clonality assays are useful in the diagnosis and follow-up of polycythemic conditions and disorders with increased platelet levels. View Publication

Patients with mutations of either RAG-1 or RAG-2 genes suffer from severe combined immunodeficiency (SCID) characterized by the lack of T and B lymphocytes. The only curative treatment today consists of hematopoietic stem cell (HSC) transplantation,which is only partially successful in the absence of an HLA genoidentical donor,thus justifying research to find an alternative therapeutic approach. To this end,RAG-2-deficient mice were used to test whether retrovirally mediated ex vivo gene transfer into HSCs could provide long-term correction of the immunologic deficiency. Murine RAG-2-/-Sca-1(+) selected bone marrow cells were transduced with a modified Moloney leukemia virus (MLV)-based MND (myeloproliferative sarcoma virus enhancer,negative control region deleted,dl587rev primer-binding site substituted) retroviral vector containing the RAG-2 cDNA and transplanted into RAG-2-/- sublethally irradiated mice (3Gy). Two months later,T- and B-cell development was achieved in all mice. Diverse repertoire of T cells as well as proliferative capacity in the presence of mitogens,allogeneic cells,and keyhole limpet hemocyanin (KLH) were shown. B-cell function as shown by serum Ig levels and antibody response to a challenge by KLH also developed. Lymphoid subsets and function were shown to be stable over a one-year period without evidence of any detectable toxicity. Noteworthy,a selective advantage for transduced lymphoid cells was evidenced by comparative provirus quantification in lymphoid and myeloid lineages. Altogether,this study demonstrates the efficiency of ex vivo RAG-2 gene transfer in HSCs to correct the immune deficiency of RAG-2-/- mice,constituting a significant step toward clinical application. View Publication

In mice there are two families of MHC class I-specific receptors,namely the Ly49 and CD94/NKG2 receptors. The latter receptors recognize the nonclassical MHC class I Qa-1(b) and are thought to be responsible for the recognition of missing-self and the maintenance of self-tolerance of fetal and neonatal NK cells that do not express Ly49. Currently,how NK cells acquire individual CD94/NKG2 receptors during their development is not known. In this study,we have established a multistep culture method to induce differentiation of embryonic stem (ES) cells into the NK cell lineage and examined the acquisition of CD94/NKG2 by NK cells as they differentiate from ES cells in vitro. ES-derived NK (ES-NK) cells express NK cell-associated proteins and they kill certain tumor cell lines as well as MHC class I-deficient lymphoblasts. They express CD94/NKG2 heterodimers,but not Ly49 molecules,and their cytotoxicity is inhibited by Qa-1(b) on target cells. Using RT-PCR analysis,we also report that the acquisition of these individual receptor gene expressions during different stages of differentiation from ES cells to NK cells follows a predetermined order,with their order of acquisition being first CD94; subsequently NKG2D,NKG2A,and NKG2E; and finally,NKG2C. Single-cell RT-PCR showed coexpression of CD94 and NKG2 genes in most ES-NK cells,and flow cytometric analysis also detected CD94/NKG2 on most ES-NK cells,suggesting that the acquisition of these receptors by ES-NK cells in vitro is nonstochastic,orderly,and cumulative. View Publication

In early life,a high susceptibility to infectious diseases as well as a poor capacity to respond to vaccines are generally observed as compared with observations in adults. The mechanisms underlying immune immaturity have not been fully elucidated and could be due to the immaturity of the T/B cell responses and/or to a defect in the nature and quality of Ag presentation by the APC. This prompted us to phenotypically and functionally characterize early life murine dendritic cells (DC) purified from spleens of 7-day-old mice. We showed that neonatal CD11c(+) DC express levels of costimulatory molecules and MHC molecules similar to those of adult DC and are able to fully maturate after LPS activation. Furthermore,we demonstrated that neonatal DC can efficiently take up,process,and present Ag to T cells in vitro and induce specific CTL responses in vivo. Although a reduced number of these cells was observed in the spleen of neonatal mice as compared with adults,this study clearly shows that neonatal DC have full functional capacity and may well prime Ag-specific naive T cells in vivo. View Publication

The purpose of the present study was to characterize primitive epithelial progenitor populations present in adult normal human mammary tissue using a combination of flow cytometry and in vitro colony assay procedures. Three types of human breast epithelial cell (HBEC) progenitors were identified: luminal-restricted,myoepithelial-restricted and bipotent progenitors. The first type expressed epithelial cell adhesion molecule (EpCAM),alpha6 integrin and MUC1 and generated colonies composed exclusively of cells positive for the luminal-associated markers keratin 8/18,keratin 19,EpCAM and MUC1. Bipotent progenitors produced colonies containing a central core of cells expressing luminal markers surrounded by keratin 14+ myoepithelial-like cells. Single cell cultures confirmed the bipotentiality of these progenitors. Their high expression of alpha6 integrin and low expression of MUC1 suggests a basal position of these cells in the mammary epithelium in vivo. Serial passage in vitro of an enriched population of bipotent progenitors demonstrated that only myoepithelial-restricted progenitors could be readily generated under the culture conditions used. These results support a hierarchical branching model of HBEC progenitor differentiation from a primitive uncommitted cell to luminal- and myoepithelial-restricted progenitors. View Publication

Genetic modification of hematopoietic stem cells often results in the expression of foreign proteins in pluripotent progenitor cells and their progeny. However,the potential for products of foreign genes introduced into hematopoietic stem cells to induce host immune responses is not well understood. Gene marking and induction of immune responses to enhanced green fluorescent protein (eGFP) were examined in rhesus macaques that underwent nonmyeloablative irradiation followed by infusions of CD34(+) bone marrow cells transduced with a retroviral vector expressing eGFP. CD34(+) cells were obtained from untreated animals or from animals treated with recombinant human granulocyte colony-stimulating factor (G-CSF) alone or G-CSF and recombinant human stem cell factor. Levels of eGFP-expressing cells detected by flow cytometry peaked at 0.1% to 0.5% of all leukocytes 1 to 4 weeks after transplantation. Proviral DNA was detected in 0% to 17% of bone marrow--derived colony-forming units at periods of 5 to 18 weeks after transplantation. However,5 of 6 animals studied demonstrated a vigorous eGFP-specific cytotoxic T lymphocyte (CTL) response that was associated with a loss of genetically modified cells in peripheral blood,as demonstrated by both flow cytometry and polymerase chain reaction. The eGFP-specific CTL responses were MHC-restricted,mediated by CD8(+) lymphocytes,and directed against multiple epitopes. eGFP-specific CTLs were able to efficiently lyse autologous CD34(+) cells expressing eGFP. Antibody responses to eGFP were detected in 3 of 6 animals. These data document the potential for foreign proteins expressed in CD34(+) hematopoietic cells and their progeny to induce antibody and CTL responses in the setting of a clinically applicable transplantation protocol. (Blood. 2001;97:1951-1959) View Publication