Gene transfer into hemopoietic stem cells using retroviral vectors

Gene transfer to hemopoietic cells offers a variety of new approaches to the experimental hematologist as well as potentially providing a means for correcting a number of genetic disorders of humans. From the experimental viewpoint, gene transfer utilizing retroviral vectors introduces new methods for analyzing hemopoietic cell lineages, and the effects of over-expression of genes affecting hemopoietic cell proliferation and differentiation. The unique properties of retroviral vectors and the optimized methods currently in use to infect hemopoietic cells are represented as a brief review of a rapidly expanding new field of experimental hematology.     

Cellular FLIP (long isoform) overexpression in T cells drives Th2 effector responses and promotes immunoregulation in experimental autoimmune encephalomyelitis

Cellular FLIP (c-FLIP) is an endogenous inhibitor of death receptor-induced apoptosis through the caspase 8 pathway. It is an NF-kappaB-inducible protein thought to promote the survival of T cells upon activation, and its down-regulation has been implicated in activation-induced cell death. We have generated transgenic mice overexpressing human c-FLIP long form (c-FLIP(L)) specifically in T cells using the CD2 promoter (TgFLIP(L)). TgFLIP(L) mice exhibit increased IgG1 production upon stimulation by a T cell-dependent Ag and a markedly enhanced contact hypersensitivity response to allergen. In addition to showing augmented Th2-type responses, TgFLIP(L) mice are resistant to the development of myelin oligodendrocyte glycoprotein 35-55 peptide-induced experimental autoimmune encephalomyelitis, a Th1-driven autoimmune disease. In vitro analyses revealed that T cells of TgFLIP(L) mice proliferate normally, but produce higher levels of IL-2 and show preferential maturation of Th2 cytokine-producing cells in response to antigenic stimulation. After adoptive transfer, these (Th2) cells protected wild-type recipient mice from experimental autoimmune encephalomyelitis induction. Our results show that the constitutive overexpression of c-FLIP(L) in T cells is sufficient to drive Th2 polarization of effector T cell responses and indicate that it might function as a key regulator of Th cell differentiation.     

Expression of the human gamma-globin gene after retroviral transfer to transformed erythroid cells.

Regulation of the human fetal (gamma) globin gene and a series of mutant gamma-globin genes was studied after retroviral transfer into erythroid cells with fetal or adult patterns of endogenous globin gene expression. Steady-state RNA from a virally transferred A gamma-globin gene with a normal promoter increased after induction of erythroid maturation of murine erythroleukemia cells and comprised from 2% to 23% of the mouse beta maj-globin RNA level. RNA expression from the virally transferred A gamma-globin gene comprised 23% of the endogenous G gamma- + A gamma-globin expression in K 562 cells after treatment with hemin. Expression from a virally transferred gamma- or beta-globin gene exceeded endogenous gamma- or beta-globin expression by a factor of 6 or more in the human erythroleukemia line KMOE, in which the endogenous globin genes are weakly inducible. In these experiments, no difference in expression was observed between the gene with the normal promoter and an A gamma-globin gene with a point mutation in its promoter (-196 C-to-T) that has been associated with hereditary persistence of fetal hemoglobin (HPFH). To test for cis-acting determinants located within the introns of the gamma-globin gene, expression was measured from a set of gamma-globin genes configured with either intron alone or with neither intron. In contrast to an intronless beta-globin gene, which is not expressed in MEL cells, the intronless gamma-globin gene was expressed in MEL cells at 24% of the level of an intron-containing gene.(ABSTRACT TRUNCATED AT 250 WORDS)     

Autophagy regulates the therapeutic potential of mesenchymal stem cells in experimental autoimmune encephalomyelitis

Mesenchymal stem cell (MSC)-based therapy is a promising approach to treat various inflammatory disorders including multiple sclerosis. However, the fate of MSCs in the inflammatory microenvironment is largely unknown. Experimental autoimmune encephalomyelitis (EAE) is a well-studied animal model of multiple sclerosis. We demonstrated that autophagy occurred in MSCs during their application for EAE treatment. Inflammatory cytokines, e.g., interferon gamma and tumor necrosis factor, induced autophagy in MSCs synergistically by inducing expression of BECN1/Beclin 1. Inhibition of autophagy by knockdown of Becn1 significantly improved the therapeutic effects of MSCs on EAE, which was mainly attributable to enhanced suppression upon activation and expansion of CD4⁺ T cells. Mechanistically, inhibition of autophagy increased reactive oxygen species generation and mitogen-activated protein kinase 1/3 activation in MSCs, which were essential for PTGS2 (prostaglandin-endoperoxide synthase 2 [prostaglandin G/H synthase and cyclooxygenase]) and downstream prostaglandin E2 expression to exert immunoregulatory function. Furthermore, pharmacological treatment of MSCs to inhibit autophagy increased their immunosuppressive effects on T cell-mediated EAE. Our findings indicate that inflammatory microenvironment-induced autophagy downregulates the immunosuppressive function of MSCs. Therefore, modulation of autophagy in MSCs would provide a novel strategy to improve MSC-based immunotherapy.     

Expression of the human beta-globin gene after retroviral transfer into murine erythroleukemia cells and human BFU-E cells.

Replication-defective amphotropic retrovirus vectors containing either the human beta-globin gene with introns or an intronless beta-globin minigene were constructed and used to study beta-globin expression following gene transfer into hematopoietic cells. The beta-globin genes were marked by introducing a 6-base-pair insertion into the region corresponding to the 5' untranslated region of the beta-globin mRNA to allow detection of RNA encoded by the new gene in human cells expressing normal human beta-globin RNA. Introduction of a virus containing the beta-globin gene with introns into murine erythroleukemia cells resulted in inducible expression of human beta-globin RNA and protein, while the viruses containing the minigene were inactive. The introduced human beta-globin gene was 6 to 110% as active as the endogenous mouse beta maj-globin genes in six randomly chosen cell clones. Introduction of the viruses into human BFU-E cells, followed by analysis of marked and unmarked globin RNAs in differentiated erythroid colonies, revealed that the introduced beta-globin gene was about 5% as active as the endogenous genes in these normal human erythroid cells and that again the minigene was inactive. These data are discussed in terms of the potential treatment of genetic disease by gene therapy.     

Quantitative analysis of retroviral and lentiviral gene transfer to murine embryonic stem cells

The effectiveness of retrovirus or lentivirus transduction of embryonic stem (ES) cells is often limited because transgene expression is silenced or variegated. We wondered if other steps of transduction, in addition to gene expression, were restricted in ES cells. We quantitatively compared (1) the amount of virus binding, (2) the number of integrated transgenes, and (3) the resulting level of gene expression. We found that three- to fourfold fewer retroviruses and lentiviruses bound to R1 mES cells than to NIH 3T3 cells, suggesting that both types of viruses bind less efficiently to mES cells. Retroviruses and lentiviruses differed in the efficiency with which they completed post-binding steps of transduction. In R1 mES cells, we detected 3-fold fewer integrated retrovirus transgenes and 11-fold lower expression levels than in NIH 3T3 cells, which suggests that the primary limitation to retrovirus transduction may be low levels of transgene expression. In contrast, we detected 10-fold fewer integrated lentivirus transgenes and 8-fold lower expression levels in R1 mES cells than in NIH 3T3 cells, which suggests that lentivirus transduction may be limited by inefficient intracellular post-binding steps of transduction. The implications of our findings for developing improved viral vectors for transducing mES cells are discussed.     

Transduction of human embryonic stem cells by ecotropic retroviral vectors

The steadily increasing availability of human embryonic stem (hES) cell lines has created strong interest in applying available tools for gene transfer in murine cells to human systems. Here we present a method for the transduction of hES cells with ecotropic retroviral vectors. hES cells were transiently transfected with a construct carrying the murine retrovirus receptor mCAT1. Subsequently, the cells were exposed to replication-deficient Moloney murine leukemia virus (MoMuLV) derivatives or pseudotyped lentiviral vectors. With oncoretroviral vectors, this procedure yields overall transduction efficiencies of up to 20% and permits selection of permanently transduced clones with high frequency. Selected clones maintained expression of pluripotency-associated markers and exhibited multi-germ layer differentiation both in vitro and in vivo. HES cell-derived somatic cells including neural progeny maintained high levels of transgene expression. Lentiviral vectors pseudotyped with the MoMuLV envelope could be introduced in the same manner with efficiencies of up to 33%. Transgene expression of lentivirally transduced hES cells remained permanent after differentiation even without selection pressure. Bypassing the regulatory issues associated with the use of amphotropic retroviral systems and exploiting the large pool of existing murine vectors, this method provides a safe and versatile tool for gene transfer and lineage analysis in hES cells and their progeny.     

Functional expression of human CD8 in fully reconstituted mice after retroviral-mediated gene transfer of hemopoietic stem cells.

Retroviral-mediated gene transfer has been used in an attempt to efficiently and stably express functional cell-surface molecules in lymphoid and myeloid cells. The human CD8 molecule is a T cell-specific surface receptor that is intimately involved in class I MHC-restricted Ag recognition and subsequent T cell activation. After infection with a recombinant, replication-defective retrovirus containing the human CD8 alpha cDNA, bone marrow cells were transplanted into lethally irradiated recipients. The majority of lymphoid and myeloid cells of reconstituted animals expressed high levels of human CD8 for at least 8 months after transplantation. Transfer of bone marrow and spleen cells from these recipients 100 days after transplantation into secondary recipients also resulted in long term expression of CD8 in lymphoid and myeloid cells. CD8 expressed in splenic T cells associated with the lymphoid-specific tyrosine protein kinase p56lck, participated in T cell activation and conferred an increased xenogeneic response to human MHC class I Ag. Thus, retroviral-mediated gene transfer allows the long term, functional expression of cell-surface molecules in normal murine lymphoid and myeloid cells.     

Suppression of experimental autoimmune encephalomyelitis by dermatan sulfate

The effect of dermatan sulfate (DS) on the treatment of Lewis rats with experimental autoimmune encephalomyelitis (EAE) was examined. DS, a sulfated glycosaminoglycan, has been reported to exhibit anticoagulant and fibrinolytic activities. DS treatment (50 mg/kg/day) facilitates recovery from the clinical manifestations of EAE. In this study, the fibrinolytic activity was higher in DS-treated rats than in saline-treated rats. Although the degree of perivascular mononuclear cell infiltration in the spinal cord was not suppressed in DS-treated rats compared to that in saline-treated rats, perivascular fibrin deposition was markedly suppressed in DS-treated rats. These findings suggest that DS would act as an effective therapeutic agent for EAE by preventing fibrin deposition.     

The effects of transplanted mesenchymal stem cells treated with 17-b estradiol on experimental autoimmune encephalomyelitis

Molecular Biology Reports - The present study was conducted aimed at exploring the modulatory effects of 17-b estradiol (17-bED) on mesenchymal stem cells (MSCs) in the EAE (experimental autoimmune...     

Expression of the GM-CSF gene after retroviral transfer in hematopoietic stem cell lines induces synchronous granulocyte-macrophage differentiation

Multipotent murine stem cell lines (FDC-Pmix) depend on IL-3 for self-renewal and proliferation and can be induced to differentiate into multiple hematopoietic lineages. Single FDC-Pmix cells infected with retroviral vectors expressing GM-CSF are induced to differentiate into granulocytes and macrophages. This results in a complete loss of clonogenic cells if IL-3 is not exogenously supplied; however, multipotent variants can be selected that do not terminally differentiate if cells are kept in the presence of IL-3. Unidirectional and synchronous granulocyte and macrophage differentiation accompanied with loss of self-renewal capacity is induced when IL-3 is removed. Our data indicate that activation of the GM-CSF receptor induces differentiation of stem cells by an instructive mechanism that can be blocked by the activated IL-3 receptor. A model of how receptors can induce proliferation and cell-specific differentiation by two separate pathways is discussed.     

Prevention of experimental autoimmune encephalomyelitis by transfer of embryonic stem cell-derived dendritic cells expressing myelin oligodendrocyte glycoprotein peptide along with TRAIL or programmed death-1 ligand

Experimental autoimmune encephalomyelitis (EAE) is caused by activation of myelin Ag-reactive CD4+ T cells. In the current study, we tested a strategy to prevent EAE by pretreatment of mice with genetically modified dendritic cells (DC) presenting myelin oligodendrocyte glycoprotein (MOG) peptide in the context of MHC class II molecules and simultaneously expressing TRAIL or Programmed Death-1 ligand (PD-L1). For genetic modification of DC, we used a recently established method to generate DC from mouse embryonic stem cells (ES cells) in vitro (ES-DC). ES cells were sequentially transfected with an expression vector for TRAIL or PD-L1 and an MHC class II-associated invariant chain-based MOG epitope-presenting vector. Subsequently, double-transfectant ES cell clones were induced to differentiate to ES-DC, which expressed the products of introduced genes. Treatment of mice with either of the double-transfectant ES-DC significantly reduced T cell response to MOG, cell infiltration into spinal cord, and the severity of MOG peptide-induced EAE. In contrast, treatment with ES-DC expressing MOG alone, irrelevant Ag (OVA) plus TRAIL, or OVA plus PD-L1, or coinjection with ES-DC expressing MOG plus ES-DC-expressing TRAIL or PD-L1 had no effect in reducing the disease severity. In contrast, immune response to irrelevant exogenous Ag (keyhole limpet hemocyanin) was not impaired by treatment with any of the genetically modified ES-DC. The double-transfectant ES-DC presenting Ag and simultaneously expressing immune-suppressive molecules may well prove to be an effective therapy for autoimmune diseases without inhibition of the immune response to irrelevant Ag.     

A reversible form of axon damage in experimental autoimmune encephalomyelitis and multiple sclerosis

In multiple sclerosis, a common inflammatory disease of the central nervous system, immune-mediated axon damage is responsible for permanent neurological deficits. How axon damage is initiated is not known. Here we use in vivo imaging to identify a previously undescribed variant of axon damage in a mouse model of multiple sclerosis. This process, termed 'focal axonal degeneration' (FAD), is characterized by sequential stages, beginning with focal swellings and progressing to axon fragmentation. Notably, most swollen axons persist unchanged for several days, and some recover spontaneously. Early stages of FAD can be observed in axons with intact myelin sheaths. Thus, contrary to the classical view, demyelination-a hallmark of multiple sclerosis-is not a prerequisite for axon damage. Instead, focal intra-axonal mitochondrial pathology is the earliest ultrastructural sign of damage, and it precedes changes in axon morphology. Molecular imaging and pharmacological experiments show that macrophage-derived reactive oxygen and nitrogen species (ROS and RNS) can trigger mitochondrial pathology and initiate FAD. Indeed, neutralization of ROS and RNS rescues axons that have already entered the degenerative process. Finally, axonal changes consistent with FAD can be detected in acute human multiple sclerosis lesions. In summary, our data suggest that inflammatory axon damage might be spontaneously reversible and thus a potential target for therapy.     

CD4+ suppressor cells differentially affect the production of IFN-gamma by effector cells of experimental autoimmune encephalomyelitis

Spleen cells from rats that have recovered from experimental autoimmune encephalomyelitis (EAE) suppress the production of IFN-gamma by effector T cells of EAE in an Ag-specific manner. These postrecovery suppressor cells also inhibit EAE in vivo. Fractionation of the postrecovery suppressor spleen cells on nylon wool and OX-8 coated plates yields a nylon wool-adherent CD4+ suppressor cell population that, when cocultured with effector T cells, suppresses IFN-gamma production by these effector cells. In contrast, the nylon wool-adherent, CD4+ postrecovery suppressor cell population fails to inhibit the production of IL-2 by the effector T cells. In further experiments, the effector T cell population was depleted of CD8+ cells and cocultured with the nylon wool-adherent, CD4+ postrecovery suppressor cells, and the supernatants were assayed for IFN-gamma and IL-2. IFN-gamma production was inhibited in these cultures but IL-2 production was not inhibited. Irradiated effector T cells were cocultured with CD4+ postrecovery suppressor cells, without myelin basic protein, in an effort to determine whether the mechanism of differential lymphokine suppression involved an anti-idiotypic response against effector T cells. No IL-2 was produced, indicating that there was no CD4+ suppressor cell mediated anti-idiotypic response against effector T cells. These studies suggest that the suppressor cell is a nylon wool adherent, CD4+ T cell that functions to down-regulate EAE effector T cells by differential inhibition of lymphokine production.     

Expression of human urokinase in inoculated rodent cells. Loss of introns during gene transfer using a retroviral vector]

Expression of urokinase in murine and rat cells was performed by two recombinant constructs, one containing cDNA and the other--hybrid (cDNA/genome) variant of human urokinase gene conserving 7 introns of 10, in the eukaryotic retrovirus vector pPS-3-neo. DNA of both constructs was introduced into packaging cell line psi 2 by a standard Ca-phosphate transfection technique. Infection of mouse and rat fibroblasts BALB/c 3T3 and Rat I with virus particles, produced by transfected psi 2 cells, led to an integration into the host genome of one or two recombinant proviral copies. Stable expression and secretion into the culture medium of glycosylated high molecular weight human urokinase was observed for both cell types. For the hybrid gene construct, precise excision of intervening sequences was shown during transferring of genetic material from packaging to recipient cells.     

Peripheral T cells are the therapeutic targets of glucocorticoids in experimental autoimmune encephalomyelitis

High-dose glucocorticoid (GC) therapy is widely used to treat multiple sclerosis (MS), but the underlying mechanisms remain debatable. In this study, we investigated the impact of GC administration on experimental autoimmune encephalomyelitis using different GC receptor (GR)-deficient mutants. Heterozygous GR knockout mice were less sensitive to dexamethasone therapy, indicating that the expression level of the receptor determines therapeutic efficacy. Mice reconstituted with homozygous GR knockout fetal liver cells showed an earlier onset of the disease and were largely refractory to GC treatment, indicating that the GR in hematopoietic cells is essential for the beneficial effects of endogenous GCs and dexamethasone. Using cell-type specific GR-deficient mice, we could demonstrate that GCs mainly act on T cells, while modulation of macrophage function was largely dispensable in this context. The therapeutic effects were achieved through induction of apoptosis and down-regulation of cell adhesion molecules in peripheral T(H)17 and bystander T cells, while similar effects were not observed within the spinal cord. In addition, dexamethasone inhibited T cell migration into the CNS, confirming that peripheral but not CNS-residing T lymphocytes are the essential targets of GCs. Collectively, our findings reveal a highly selective mechanism of GC action in experimental autoimmune encephalomyelitis and presumably multiple sclerosis.