FLOURESCENT MARKERS OF T CELL ACTIVATION

Emory University, Atlanta GA

Investigators

Linked publications & trials

Abstract

DESCRIPTION (adapted from applicant's abstract): Understanding the kinetics of activation and differentiation of T cells during immune responses is vital to the development of vaccines, regiments for transplantation, and in the ability to provide immune-based therapies for cancer and autoimmunity. Proliferation and the cytokine profile that the T cells secrete define activation and differentiation of T cells. The cytokine profile of an activated T cell determines the nature of the immune response. Important cytokines include, but are not limited to, interleukin-2 (IL-2), interleukin-4 (IL-4), and interferon-gamma (IFN-g). The current technology to measure cytokines from individual cells has several disadvantages, including limited sensitivity, expense, difficulty, and the death of cells being assayed. Because these measurements are critical in most immunological models that employ Tg and gene disrupted strains of mice, other assays are required. The specific goal of this project is to design, construct, and test Tg mice and cell lines that could be used to rapidly determine T cell cytokine expression profiles. The investigators propose to develop a novel system using the IL-2, IL-4, and IFN-g promoters to drive a series of FP reporter genes for detection of multiple cytokines that is rapid and does not alter the physiology of the cell. A result of this system will be a series of markers within living mice that can be crossed and manipulated for immunological testing and evaluation. By using different alleles of the green FP (GFP), multiple colors will be visualized in the same cell and, therefore, provide a profile of the cytokine expression pattern. The proposed system has several advantages over the current technologies that either evaluate populations of cells or individual cells through intracellular cytokine staining. These advantages include ease of use, sensitivity, cost, and the ability to sort and use the sorted cells that are positive for cytokine expression in adoptive transfer and cell differentiation experiments. The applicants will develop this system by creating stably transfected cell lines and Tg animals that carry the cytokine-FP reporters in their genomes. The cytokine-FP reporters will be evaluated in model hybridoma systems, TCR-Tg animal model systems, and in two infectious disease models that skew the immune responses toward one phenotype of T cell helper, Thl or Th2.