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1.
Blood ; 96(3): 885-93, 2000 Aug 01.
Article in English | MEDLINE | ID: mdl-10910901

ABSTRACT

Retroviral vectors based on the Moloney murine leukemia virus (MuLV) have become the primary tool for gene delivery into hematopoietic cells, but clinical trials have been hampered by low transduction efficiencies. Recently, we and others have shown that gene transfer of MuLV-based vectors into T cells can be significantly augmented using a fibronectin-facilitated protocol. Nevertheless, the relative abilities of naive (CD45RA(+)) and memory (CD45RO(+)) lymphocyte subsets to be transduced has not been assessed. Although naive T cells demonstrate a restricted cytokine profile following antigen stimulation and a decreased susceptibility to infection with human immunodeficiency virus, it was not clear whether they could be efficiently infected with a MuLV vector. This study describes conditions that permitted gene transfer of an enhanced green fluorescent protein-expressing retroviral vector in more than 50% of naive umbilical cord (UC) blood and peripheral blood (PB) T cells following CD3/CD28 ligation. Moreover, treatment of naive T cells with interleukin-7 resulted in the maintenance of a CD45RA phenotype and gene transfer levels approached 20%. Finally, it was determined that parameters for optimal transduction of CD45RA(+) T cells isolated from PB and UC blood differed: transduction of the UC cells was significantly increased by the presence of autologous mononuclear cells (24.5% versus 56.5%). Because naive T cells harbor a receptor repertoire that allows them to respond to novel antigens, the development of protocols targeting their transduction is crucial for gene therapy applications. This approach will also allow the functions of exogenous genes to be evaluated in primary nontransformed naive T cells.


Subject(s)
Gene Transfer Techniques , Genetic Vectors , Leukemia Virus, Murine , T-Lymphocyte Subsets/physiology , Genes, Reporter , Green Fluorescent Proteins , Humans , Immunophenotyping , Luminescent Proteins , Lymphocyte Activation
2.
J Biol Chem ; 275(21): 15832-8, 2000 May 26.
Article in English | MEDLINE | ID: mdl-10748099

ABSTRACT

ZAP-70-deficient patients present with nonfunctional CD4+ T cells in the periphery. We find that a subset of primary ZAP-70-deficient T cells, expressing high levels of the related protein-tyrosine kinase Syk, can proliferate in vitro. These cells (denoted herein as Syk(hi)/ZAP-70(-) T cells) provide a unique model in which the contribution of Syk to TCR-mediated responses can be explored in a nontransformed background. Importantly, CD3-induced responses, such as tyrosine phosphorylation of cellular substrates (LAT, SLP76, and PLC-gamma1), as well as calcium mobilization, which are defective in T cells expressing neither ZAP-70 nor Syk, are observed in Syk(hi)/ZAP-70(-) T cells. However, Syk(hi)/ZAP-70(-) T cells differ from control T cells with respect to the T cell antigen receptor (TCR)-mediated activation of the MAPK cascades: extracellular signal-regulated kinase activity and recruitment of the JNK and p38 stress-related MAPK pathways are diminished. This distinct phenotype of Syk(hi)/ZAP-70(-) T cells is associated with a profound decrease in CD3-mediated interleukin 2 secretion and proliferation relative to control T cells. Thus, ZAP-70 and Syk appear to play distinct roles in transducing a TCR-mediated signal.


Subject(s)
Adaptor Proteins, Signal Transducing , Enzyme Precursors/metabolism , Membrane Proteins , Protein-Tyrosine Kinases/deficiency , Protein-Tyrosine Kinases/metabolism , Receptors, Antigen, T-Cell/metabolism , Base Sequence , CD3 Complex/metabolism , Calcium/metabolism , Carrier Proteins/metabolism , Cell Division , Humans , Interleukin-2/metabolism , Intracellular Signaling Peptides and Proteins , Isoenzymes/metabolism , JNK Mitogen-Activated Protein Kinases , Jurkat Cells , Mitogen-Activated Protein Kinases/metabolism , Molecular Sequence Data , Mutation , Phospholipase C gamma , Phosphoproteins/metabolism , Phosphorylation , Phosphotyrosine/metabolism , Protein-Tyrosine Kinases/genetics , Signal Transduction/genetics , Syk Kinase , Type C Phospholipases/metabolism , ZAP-70 Protein-Tyrosine Kinase
3.
Leukemia ; 14(1): 188-97, 2000 Jan.
Article in English | MEDLINE | ID: mdl-10637495

ABSTRACT

We have previously reported obtaining two monoclonal antibodies (mAb) against the human gp130 interleukin-6 (IL-6) transducer which made possible the dimerization of gp130 and the activation of several IL-6-driven functions when used together. We report here that these mAb induce gp130-mediated signaling in human myeloma cells and support the survival and the long-term growth of five IL-6-dependent human myeloma cell lines. Their agonist activity is not affected by neutralizing antibodies to IL-6 or IL-6R. These mAb induce a transient proliferation of primary myeloma cells from most patients with multiple myeloma. Again, IL-6 inhibitors do not affect this agonist activity. By using highly purified primary myeloma cells, we found that these anti-gp130 mAb supported the long-term survival of primary myeloma cells from five patients with primary plasma cell leukemia but failed to induce their long-term growth. For patients with fulminant disease and secondary extramedullary proliferation, the antibodies supported a long-term survival and growth, and anti-gp130 mAb-dependent cell lines were obtained. For patients with medullary involvement only, a co-stimulatory signal is necessary, together with gp130 activation, to trigger cell survival and cycling. Leukemia (2000) 14, 188-197.


Subject(s)
Antibodies, Monoclonal/immunology , Antigens, CD/immunology , Growth Substances/physiology , Membrane Glycoproteins/immunology , Multiple Myeloma/pathology , Signal Transduction , Blotting, Western , Cell Division , Cell Survival/immunology , Cytokine Receptor gp130 , Flow Cytometry , Humans , Middle Aged , Multiple Myeloma/immunology
4.
Hum Gene Ther ; 10(1): 5-14, 1999 Jan 01.
Article in English | MEDLINE | ID: mdl-10022526

ABSTRACT

The success of gene therapy strategies for congenital and acquired blood disorders requires high levels of gene transfer into hematopoietic cells. Retroviral vectors have been extensively used to deliver foreign genes to mammalian cells and improvement of transduction protocols remains dependent on markers that can be rapidly monitored and used for efficient selection of transduced cells. The enhanced green fluorescent protein (EGFP) is a suitable reporter molecule for gene expression because of its lack of cytotoxicity and stable fluorescence signal that can be readily detected by flow cytometry. However, attempts to adapt the GFP system to stable transduction of human lymphocytes have not been satisfactory. In this article, transductions of primary human T lymphocytes were performed using cell-free supernatants from a PG13 packaging cell line in which a retroviral vector expressing EGFP was pseudotyped with the gibbon ape leukemia virus (GALV) envelope. Using this system combined with a fibronectin-facilitated protocol, primary lymphocytes were transduced with a mean gene transfer efficiency of 27.5% following a 2-day stimulation with either PHA or anti-CD3/CD28 antibodies. Conditions that increased the entry of lymphocytes into cell cycle did not consistently correlate with enhanced gene transfer, indicating that factors other than proliferation are important for optimal retroviral gene transfer. These results demonstrate the utility of EGFP as a marker for human T cell transduction and will enable further optimization of T cell gene therapy protocols.


Subject(s)
Fibronectins/pharmacology , Gene Transfer Techniques , Luminescent Proteins/genetics , T-Lymphocytes/physiology , Cell Cycle , Flow Cytometry , Genetic Vectors , Green Fluorescent Proteins , Humans , Indicators and Reagents/analysis , Jurkat Cells , Leukemia Virus, Gibbon Ape/metabolism , Luminescent Proteins/analysis , Retroviridae/genetics , Transduction, Genetic
5.
Blood ; 91(12): 4727-37, 1998 Jun 15.
Article in English | MEDLINE | ID: mdl-9616171

ABSTRACT

Agonist antihuman gp130 transducer monoclonal antibodies (MoAbs) were used in SCID mice to grow myeloma cells whose survival and proliferation is dependent on gp130 transducer activation. The agonist anti-gp130 MoAbs neither bound to murine gp130 nor activated murine cells and, as a consequence, did not induce interleukin-6 (IL-6)-related toxicities in mice. They have a 2-week half-life in vivo when injected in the peritoneum. The agonist antibodies made possible the in vivo growth of exogenous IL-6-dependent human myeloma cells as well as that of freshly explanted myeloma cells from 1 patient with secondary plasma cell leukemia. Tumors occurred 4 to 10 weeks after myeloma cell graft and weighed 3 to 5 g. They grew as solid tumors in the peritoneal cavity and metastasized to the different peritoneal organs: liver, pancreas, spleen, and intestine. Tumoral cells were detected in blood and bone marrow of mice grafted with the XG-2 myeloma cells. Tumoral cells grown in SCID mice had kept the phenotypic characteristics of the original tumoral cells and their in vitro growth required the presence of IL-6 or agonist anti-gp130 MoAbs. Myeloma cells from 4 patients with medullary involvement persisted for more than 1 year as judged by detectable circulating human Ig. However, no tumors were detected, suggesting a long-term survival of human myeloma cells without major proliferation. These observations paralleled those made in in vitro cultures as well as the tumor growth pattern in these patients. This gp130 transducer-dependent SCID model of multiple myeloma should be useful to study various therapeutical approaches in multiple myeloma in vivo.


Subject(s)
Antibodies, Monoclonal/immunology , Interleukin-6/immunology , Multiple Myeloma , Neoplasms, Experimental , Animals , Antibodies, Monoclonal/administration & dosage , Cell Division/drug effects , Cell Division/immunology , Disease Models, Animal , Humans , Interleukin-6/administration & dosage , Mice , Mice, SCID , Multiple Myeloma/immunology , Multiple Myeloma/pathology , Neoplasms, Experimental/immunology , Neoplasms, Experimental/pathology
6.
Leukemia ; 12(4): 610-8, 1998 Apr.
Article in English | MEDLINE | ID: mdl-9557621

ABSTRACT

The function of CD28 molecules that are present on malignant plasma cells of human myeloma cell lines (HMCL) was studied. First, myeloma cells expressed a similar density of CD28 antigen to that of normal T cells. The myeloma CD28 molecules were able to bind B7-Ig molecules as well as L cells transfected with a B7-1 cDNA, and anti-CD28 mAb inhibited the binding. Myeloma cells did not express B7-1 antigens but a low density of B7-2 antigens. The myeloma B7-2 molecules of two HMCL were able to bind CTLA-4 protein. No autocrine CD28:B7-2 activation could be evidenced as we found no spontaneous binding of the p85 subunit of PI-3 kinase to CD28 molecules. In addition, a blocking anti-CD28 mAb did not affect the IL-6-dependent or autonomous proliferation of the HMCL. The activation of myeloma CD28 molecules with or without TPA stimulation did not affect the proliferation, survival, differentiation, expression of activation antigens and cytokine receptors or cytokine production of myeloma cells. However, the triggering of myeloma CD28 molecules by B7-1 transfectant cells resulted in binding of the p85 subunit of PI-3 kinase to CD28 molecules as previously shown for T cell CD28 molecules. This expression of a large density of CD28 molecules able to bind B7 molecules might contribute to a downregulation of the immune control of myeloma cells.


Subject(s)
CD28 Antigens/physiology , Multiple Myeloma/metabolism , Plasma Cells/metabolism , B7-1 Antigen/metabolism , CD28 Antigens/biosynthesis , CD28 Antigens/metabolism , Enzyme Activation , Humans , Lymphocyte Activation/physiology , Multiple Myeloma/pathology , Phosphatidylinositol 3-Kinases/metabolism , T-Lymphocytes/metabolism , T-Lymphocytes/physiology , Tumor Cells, Cultured
7.
J Interferon Cytokine Res ; 17(1): 17-26, 1997 Jan.
Article in English | MEDLINE | ID: mdl-9041467

ABSTRACT

We have established a cDNA library from interferon (IFN)-treated human lymphoblastoid Daudi cells and made use of differential screening to search for yet unidentified IFN-regulated genes. In the course of these studies, we have isolated a human cDNA coding for the glycosyl-phosphatidylinositol-linked (GPI) membrane glycoprotein CD48 (TCT-1, Blast-1). Various studies demonstrated that the murine CD48 is the predominant counterreceptor for the mouse CD2 and is involved in the regulation of T cell activation. Since the murine CD48 is functionally homologous to the human CD2 ligand LFA-3 (CD48), the function of the human CD48 remains unknown. In this report, we show that both Hu-IFN-alpha/beta and Hu-IFN-gamma increase the level of CD48 mRNA and upregulate the expression of CD48 proteins at the surface of various cultured human cell lines. However, the IFN have no effect on the expression of LFA-3. In addition, we show that IFN increase CD48 expression on peripheral blood mononuclear CD3+, CD14+, and CD19+ subpopulations. These data suggest that in addition to modulation of the conventional MHC class I and class II-restricted interactions, the IFN might promote MHC-unrestricted interactions of target cells with the immune cells by inducing the expression of the cell surface CD48 molecule.


Subject(s)
Antigens, CD/biosynthesis , Antigens, Surface/biosynthesis , Antineoplastic Agents/pharmacology , Interferon Type I/pharmacology , Interferon-gamma/pharmacology , Lymphoma/drug therapy , Antigens, CD/blood , Antigens, Surface/blood , CD48 Antigen , DNA, Complementary/isolation & purification , Genetic Code , Histocompatibility Antigens Class I/immunology , Histocompatibility Antigens Class II/immunology , Humans , Leukocytes, Mononuclear/immunology , Lymphoma/immunology , RNA, Messenger/biosynthesis , Tumor Cells, Cultured , Up-Regulation
8.
J Immunol ; 156(2): 859-65, 1996 Jan 15.
Article in English | MEDLINE | ID: mdl-8543843

ABSTRACT

mAbs that bind to the Ig CDR3-like region in D1 domain of the CD4 molecule can inhibit the HIV-1 life cycle in CD4-positive T cells and lymphoblastoid cell lines at the stage of transcription. This antiviral effect requires the integrity of the cytoplasmic tail of CD4, which acts as a signal transduction region through its association with protein tyrosine kinases such as p56Ick. Here we investigated the role of p56Ick in the cascade of molecular events that control HIV-1 transcription in cells treated with anti-CD4 mAb directed against the Ig CDR3-like region. The Ig CDR3-like region-specific mAb, 13B8-2, blocked HIV-1 production in CD4-positive/p56Ick-negative HTLV-I-producing MT2 cells superinfected by HIV-1Lai, but had no effect on HTLV-I production, although it did inhibit Tax-induced NF-kappa B translocation. These results raise the possibility that an as yet unidentified tyrosine kinase may be capable of associating with CD4 and mediating intracellular signaling.


Subject(s)
Antibodies, Monoclonal/pharmacology , Antiviral Agents/pharmacology , CD4 Antigens/immunology , HIV-1/immunology , Human T-lymphotropic virus 1/physiology , Protein-Tyrosine Kinases/physiology , Signal Transduction , Virus Replication/drug effects , src-Family Kinases/physiology , Antibodies, Monoclonal/immunology , Base Sequence , CD4 Antigens/biosynthesis , CD4 Antigens/chemistry , Cell Line, Transformed , Epitopes/chemistry , Epitopes/immunology , Gene Products, tax/metabolism , HIV-1/drug effects , HIV-1/physiology , Humans , Lymphocyte Specific Protein Tyrosine Kinase p56(lck) , Molecular Sequence Data , NF-kappa B/metabolism , Polymerase Chain Reaction , Protein Structure, Tertiary , Recombinant Fusion Proteins/metabolism , beta 2-Microglobulin/immunology , src-Family Kinases/analysis , src-Family Kinases/deficiency
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