This research project is directed toward establishing experimental systems to enable the discovery of cytokine response (CR) genes, and to determine which CR genes function to facilitate or inhibit T cell cycle progression, cellular survival, cytokine production, and surface protein expression.
The aims of the project are focused on interleukin-2 (IL-2) and T cells, but the methods to be developed should be applicable to other cytokines and cells, and to other ligands, including transcription factors. The proposed research follows from the hypothesis that IL-2 promotes the expression of specific genes, the products of which regulate lymphocyte proliferation and differentiation.
Two Specific Aims test this hypothesis: 1) To create a more rapid and efficient system to identify additional CR genes, by coupling our methods to enrich for CR genes via sulfhydryl labeling and affinity purification (SLAP) to the technique of serial analysis of gene expression (SAGE); and 2) To employ retroviral-mediated gene transfer in T lymphocytes, and flow cytometry-based assay systems to screen for CR genes which affect cell cycle progression, apoptosis, cytokine production, and immunomodulatory surface protein expression. The SLAP method yields a 40-fold enrichment of CR genes, so that approximately 98 percent of constitutively-expressed, """"""""housekeeping"""""""" genes are removed. The SAGE method to identify genes by sequencing 9 base pair """"""""tags"""""""" also improves efficiency by 40-fold. Therefore, the combination of these 2 synergistic methods will reduce the time necessary to identify new CR genes from several months to a few weeks. To enable the functional analysis of each CR gene product identified, a gibbon ape leukemia virus- pseudotyped, bicistronic retroviral vector will be used. This system allows co-expression of the CR gene with a selectable marker, green fluorescent protein (GFP), in peripheral blood T cells. Functional analyses of infected cells may then be performed, using flow cytometry with gating on the GFP-positive population. The combination of retrovirus-mediated gene transfer into normal T cells, and sensitive flow cytometry assays for cellular proliferation, apoptosis, cytokine production, and surface protein expression provide a powerful means to assess CR gene functions. Consequently, this research project will contribute to our understanding of the molecular mechanisms responsible for fundamental lymphocyte responses to cytokines, and to the diagnosis and therapeutic manipulation of immune system disorders including immunodeficiency as well as hypersensitivity disease states.
| Beadling, C; Cereseto, A; Fan, W et al. (2001) Cytokine response gene 8 (CR8) regulates the cell cycle G1-S phase transition and promotes cellular survival. Oncogene 20:1771-83 |
