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1.
Curr Protoc Immunol ; Chapter 7: Unit7.10, 2011 Aug.
Article in English | MEDLINE | ID: mdl-21809319

ABSTRACT

Measurement of proliferative responses of human lymphocytes is a fundamental technique for the assessment of their biological responses to various stimuli. Most simply, this involves measurement of the number of cells present in a culture before and after the addition of a stimulating agent. This unit contains several different prototype protocols to induce proliferation in lymphocytes following exposure to mitogens, antigens, allogeneic or autologous cells, or soluble factors. Each of these protocols can be used in conjunction with an accompanying protocol, which contains methods to determine cell proliferation by incorporation of [(3)H]thymidine into DNA by nonradioactive methods, e.g., reduction of tetrazolium salts (MTT or WST-1). These protocols provide an estimate of cell proliferation indirectly by measuring DNA synthesis, and cell metabolic activity in an entire cell population, but no data on individual cells is obtained. A protocol for CFSE labeling allows direct detection of single proliferating cells and facilitates the quantification of cell divisions by flow cytometry according to the respective CFSE-dilution, and following costaining with fluorescent labeled antibodies, the characterization of subpopulations in the cell culture.


Subject(s)
Lymphocytes/cytology , Antigens/pharmacology , Cell Proliferation/drug effects , Cells, Cultured , Humans , Lymphocyte Count , Lymphocytes/drug effects , Lymphokines/pharmacology , Mitogens/pharmacology
2.
Curr Protoc Immunol ; Chapter 7: Unit 7.10.1-7.10.24, 2008 Aug.
Article in English | MEDLINE | ID: mdl-18729064

ABSTRACT

Measurement of proliferative responses of human lymphocytes is a fundamental technique for the assessment of their biological responses to various stimuli. Most simply, this involves measurement of the number of cells present in a culture before and after the addition of a stimulating agent. This unit contains several different prototype protocols to measure the proliferative response of lymphocytes following exposure to mitogens, antigens, allogeneic or autologous cells, or soluble factors. Each of these protocols can be used in conjunction with an accompanying support protocol which contains methods for pulsing cultures with [3H]thymidine and determining incorporation of [3H]thymidine into DNA or assessing cell proliferation by nonradioactive methods, e.g., reduction of tetrazolium salts (MTT). The protocols described here provide an estimate of DNA synthesis and cell proliferation in an entire cell population, but do not provide information on the proliferation of individual cells. A protocol for CFSE labeling allows specific subpopulations of cells to be separated viably for further analysis.


Subject(s)
Cytological Techniques/methods , Lymphocytes/cytology , Fluoresceins/metabolism , Humans , Lymphocyte Activation , Lymphocytes/immunology , Lymphocytes/metabolism , Succinimides/metabolism , Tetrazolium Salts/metabolism , Thymidine/metabolism
3.
Curr Gene Ther ; 7(5): 361-8, 2007 Oct.
Article in English | MEDLINE | ID: mdl-17979682

ABSTRACT

Gene-modified T cells were the first gene therapy tool used in clinical gene transfer trials. After the first applications in immunodeficiency diseases, T cell gene therapy has been extended to HIV infection and cancer. The primary obstacle to successful T cell gene therapy has proven to be the robust immune responses elicited by the gene-modified T cells even in severely immunosuppressed patients. The potent antibody and cytotoxic immune responses have interfered with the expression and persistence of the therapeutic transgene. In this review we will address each of the components of T cell gene therapy -- culture conditions, vector, and transgene -- that have elicited these immune responses and the strategies used to minimize them.


Subject(s)
Antibody Formation/immunology , Genetic Therapy/methods , Genetic Vectors/immunology , Host vs Graft Reaction/immunology , Immunotherapy, Adoptive , T-Lymphocytes/immunology , T-Lymphocytes/transplantation , Animals , Cell Culture Techniques , Cell- and Tissue-Based Therapy , Child , Cytotoxicity, Immunologic/immunology , Dendritic Cells , Gene Transfer Techniques , Genetic Vectors/administration & dosage , Host vs Graft Reaction/genetics , Humans , Immunity, Cellular , Metabolic Diseases/therapy , Mice , Neoplasms/therapy , T-Lymphocytes/metabolism , Transduction, Genetic , Transgenes/immunology
4.
Blood ; 101(7): 2563-9, 2003 Apr 01.
Article in English | MEDLINE | ID: mdl-12456496

ABSTRACT

The first human gene therapy experiment begun in September 1990 used a retroviral vector containing the human adenosine deaminase (ADA) cDNA to transduce mature peripheral blood lymphocytes from patients with ADA deficiency, an inherited disorder of immunity. Two patients who had been treated with intramuscular injections of pegylated bovine ADA (PEG-ADA) for 2 to 4 years were enrolled in this trial and each received a total of approximately 10(11) cells in 11 or 12 infusions over a period of about 2 years. No adverse events were observed. During and after treatment, the patients continued to receive PEG-ADA, although at a reduced dose. Ten years after the last cell infusion, approximately 20% of the first patient's lymphocytes still carry and express the retroviral gene, indicating that the effects of gene transfer can be remarkably long lasting. On the contrary, the persistence of gene-marked cells is very low (< 0.1%), and no expression of the transgene is detectable in lymphocytes from the second patient who developed persisting antibodies to components of the gene transfer system. Data collected from these original patients have provided novel information about the longevity of T lymphocytes in humans and persistence of gene expression in vivo from vectors driven by the Moloney murine leukemia virus long-terminal repeat (LTR) promoter. This long-term follow-up has also provided unique evidence supporting the safety of retroviral-mediated gene transfer and illustrates clear examples of both the potential and the pitfalls of gene therapy in humans.


Subject(s)
Adenosine Deaminase/deficiency , Adenosine Deaminase/genetics , Antibody Formation , Genetic Therapy/methods , Purine-Pyrimidine Metabolism, Inborn Errors/therapy , Adenosine Deaminase/administration & dosage , Adenosine Deaminase/biosynthesis , Animals , Antibodies, Heterophile/blood , Antibodies, Viral/blood , Cattle , Gene Expression , Gene Transfer Techniques , Genetic Vectors/immunology , Humans , Longitudinal Studies , Moloney murine leukemia virus/genetics , Moloney murine leukemia virus/immunology , Receptors, Antigen, T-Cell/analysis , T-Lymphocytes/cytology , T-Lymphocytes/immunology , T-Lymphocytes/metabolism
5.
Hum Gene Ther ; 13(13): 1605-10, 2002 Sep 01.
Article in English | MEDLINE | ID: mdl-12228015

ABSTRACT

The first approved clinical gene therapy trial for adenosine deaminase (ADA) deficiency employed autologous T cells grown in fetal calf serum (FCS)-supplemented medium and transduced with a retroviral vector (LASN) also produced in the presence of FCS. Ten years after their enrollment, both patients have circulating T cells containing vector DNA. However, whereas approximately 20% of the circulating T cells from patient 1 are still vector positive, less than 1% of patient 2's T cells have detectable vector. This difference appears to be not only a function of the original transduction efficiency and cell expansion capability in vitro, but also of the immune response that patient 2 developed to FCS components during the course of her treatment. In this study, serum samples from each patient were tested for antibodies to FCS by enzyme-linked immunosorbent assay and anti-FCS responses were demonstrated in both patients. Analysis of immunoglobulin classes revealed comparable levels of IgA and IgM anti-FCS titers. Patient 2, however, had significantly higher IgG responses to FCS than did patient 1. Investigation of the development of anti-FCS responses by IgG subclasses indicated that there was a different pattern in the development of IgG immunity to FCS between the two patients. In addition, significant antibody response to bovine lipoprotein was detected in patient 2, but not in patient 1 or in control samples. These findings suggest that the unique immune response mounted by patient 2 may have influenced the outcome of the gene transfer treatments in this patient.


Subject(s)
Genetic Therapy , Severe Combined Immunodeficiency/immunology , Severe Combined Immunodeficiency/therapy , T-Lymphocytes/immunology , Adenosine Deaminase/deficiency , Animals , Blood Proteins/immunology , Blood Transfusion, Autologous , Cattle , Enzyme-Linked Immunosorbent Assay , Genetic Vectors , Humans , Retroviridae , Severe Combined Immunodeficiency/genetics , T-Lymphocytes/transplantation , T-Lymphocytes/virology , Transduction, Genetic
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