Apoptosis, or programmed cell death, is a process of selective cell death characterized by nuclear condensation and endogenous endonuclease activation. It is the most common form of physiological cell death. In the immune system, apoptosis appears to occur physiologically during T and B lymphocyte repertoire selection and during the death of target cells induced by cytolytic T cells. A defect in the process can lead to autoimmunity. The long term goal of this project is to determine how cell death is regulated during normal T cell development and activation. Fas (APO-1) is a cell surface molecule of the tumor necrosis factor/nerve growth factor/nerve growth factor receptor family, that can trigger programmed cell death. This molecule is expressed in the thymus and on activated mature T cells. A defect in the Fas gene (the lpr mutation) causes abnormal lymphocyte development and autoimmunity in mice, suggesting that this molecule plays an important role in T cell development and tolerance. We would like to know whether the putative Fas-ligand interaction is involved in physiological T cell apoptosis and, if so 1) how it regulates cell death and 2) how it defects leads to autoimmunity. We will determine which T cell subpopulations express Fas in the thymus and periphery and biochemically characterize Fas expressed by each of those subpopulations, using anti-mouse Fas monoclonal antibodies (mAb) generated in our laboratory. We will also determine whether Fas mediated apoptosis occurs by the same pathway as anti-T cell receptor mAb or steroid induced death. Next, We will identify the ligand for Fas. By analogy with other proteins of the TNF/NGF receptor family, we expect that Fas has a membrane associated ligand. We have developed a Fas-immunoglobulin (Ig) fusion protein as a probe for this putative ligand. We show that this Fas-Ig protein specifically binds to cell surface molecules expressed on B cells. We will characterize this Fas binding molecule and molecularly clone its cDNA. We will also determine whether mice carrying the gld mutation, which induce a disease similar to Gas defective lpr mice, have a defect in the Fas ligand. Finally, we will characterize the role of Fas and its ligand in normal T cell death, differentiation, and function. We will modulate development of thymocytes, activation of mature T cells, and the lysis of target cells by cytolytic T cells with anit-Fas MAb and/or Fas-Ig fusion protein. We will also dysregulate the expression of Fas on T cell clones and hybridomas to study its influence on the biological function of T cells. Lastly, we will study the interaction between Fas pathways and other cell death related molecule such as bcl-2, by backcrossing bcl-2 transgenic mice to lpr/lpr and gld/gld mice and characterize T cell development, function and autoimmunity in these progeny.
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