The analysis of costimulatory receptor signaling cascades in normal T lymphocytes using in vitro gene transfer and reporter gene analysis.
Ligation of the antigen receptor and costimulatory receptors on the surface of T lymphocytes initiates intracellular signals that regulate cell-cycle progression and cell differentiation. To effectively manipulate the activation of T cells for immunotherapeutic applications, it will be important to understand how these signaling pathways are integrated to control specific gene transcription events. Here we describe a novel transient transfection procedure that efficiently introduces DNA into non-dividing normal human and murine T lymphocytes while maintaining high cell viability. Using this technique, reporter genes can be introduced to characterize intracellular signaling pathways that regulate specific gene transcription events in normal T-lymphocyte populations. We show that the CD28 receptor can be differentially coupled to downstream signaling pathways in different T-lymphocyte populations. In addition, we demonstrate that a gene encoding a tagged constitutively active mitogen-activated kinase kinase-1 protein can be transfected and rapidly expressed to regulate the expression of Bcl-2 in normal thymocytes.
Bell
M P
MP
Department of Immunology, Mayo Clinic, Rochester, Minnesota, USA.
Huntoon
C J
CJ
Graham
D
D
McKean
D J
DJ
D000818:Animals; D018106:CD28 Antigens / Q000235:genetics / Q000276:immunology*; D004789:Enzyme Activation; D005260:Female; D015870:Gene Expression; D005786:Gene Expression Regulation; D018014:Gene Transfer Techniques; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins; D006801:Humans; D019169:Jurkat Cells; D008164:Luminescent Proteins / Q000235:genetics*; D048369:MAP Kinase Kinase 1; D051379:Mice; D008810:Mice, Inbred C57BL; D020929:Mitogen-Activated Protein Kinase Kinases / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000235:genetics / Q000276:immunology; D015398:Signal Transduction / Q000276:immunology*; D013601:T-Lymphocytes / Q000276:immunology*; D014162:Transfection
D018106:CD28 Antigens; D008164:Luminescent Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D049452:Green Fluorescent Proteins; D017346:Protein Serine-Threonine Kinases; D048369:MAP Kinase Kinase 1; C482125:MAP2K1 protein, human; C482127:Map2k1 protein, mouse; D020929:Mitogen-Activated Protein Kinase Kinases