Measurement of proliferative responses of cultured lymphocytes.

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.

Somatic mosaicism in the Wiskott-Aldrich syndrome: molecular and functional characterization of genotypic revertants.

The reasons underlying the occurrence of multiple revertant genotypes in Wiskott-Aldrich syndrome (WAS) patients remain unclear. We have identified more than 30 revertant genotypes in a C995T WAS patient having 10-15% revertant, WAS protein (WASp)-expressing circulating lymphocytes. Of 497 allospecific T-cell clones generated from the peripheral blood, 47.

Tpl2 kinase regulates T cell interferon-gamma production and host resistance to Toxoplasma gondii.

Tpl2 (Tumor progression locus 2), also known as Cot/MAP3K8, is a hematopoietically expressed serine-threonine kinase. Tpl2 is known to have critical functions in innate immunity in regulating tumor necrosis factor-alpha, Toll-like receptor, and G protein-coupled receptor signaling; however, our understanding of its physiological role in T cells is limited. We investigated the potential roles of Tpl2 in T cells and found that it was induced by interleukin-12 in human and mouse T cells in a Stat4-dependent manner.

Measurement of proliferative responses of cultured lymphocytes.

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.

Unprecedented diversity of genotypic revertants in lymphocytes of a patient with Wiskott-Aldrich syndrome.

Spontaneous somatic reversions of inherited mutations are poorly understood phenomena that are thought to occur uncommonly in a variety of genetic disorders. When molecularly characterized, revertant cells have rarely exhibited more than one revertant genotype per patient. We analyzed individual allospecific T-cell clones derived from a Wiskott-Aldrich syndrome (WAS) patient identified by flow cytometry to have 10% to 15% revertant, WAS protein-expressing lymphocytes in his blood.

Immune responses to gene-modified T cells.

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.

Bovine apolipoprotein B-100 is a dominant immunogen in therapeutic cell populations cultured in fetal calf serum in mice and humans.

Recent studies have demonstrated that cell populations intended for therapeutic purposes that are cultured in heterologous animal products can acquire xenoantigens, potentially limiting their utility. In investigations of the immune response to murine embryonic stem cells, we found that a strong antibody response was generated after the second infusion. Both polyclonal and monoclonal antibody responses, derived from immunized mice, were found to be specific for bovine apolipoprotein B-100, which binds to abundant low-density lipoprotein receptors on the cell surface and is internalized.

Persistence and expression of the adenosine deaminase gene for 12 years and immune reaction to gene transfer components: long-term results of the first clinical gene therapy trial.

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.

Immune response to fetal calf serum by two adenosine deaminase-deficient patients after T cell gene therapy.

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.

Flow cytometry analysis of adenosine deaminase (ADA) expression: a simple and reliable tool for the assessment of ADA-deficient patients before and after gene therapy.

Clinical gene therapy trials for adenosine deaminase (ADA) deficiency have shown limited success of corrective gene transfer into autologous T lymphocytes and CD34(+) cells. In these trials, the levels of gene transduction and expression in hematopoietic cells have been assessed by DNA- or RNA-based assays and measurement of ADA enzyme activity. Although informative, these methods are rarely applied to clonal analysis.