As part of a search for genes expressed """"""""late"""""""" (3-5 days) after T cell activation, our group identified, cloned, and characterized two genes, granulysin and RANTES, that function as immunoregulatory molecules. Granulysin is a cytolytic molecule produced by cytotoxic T lymphocytes (CTL) and natural killer (NK) cells with activity against a variety of microbes and tumors. It also functions as a chemoattractant and activating molecule for a variety of lymphoid cells, including memory T cells, NK cells, and immature dendritic cells. Granulysin kills human tumor cells by initiating a cascade of intracellular events that involve the influx of calcium, efflux of potassium, damage to mitochondria, and apoptosis. The mechanisms by which granulysin kills microbes have not yet been elucidated. We prepared a panel of synthetic peptides based on the primary sequence of granulysin. Some of these are potent antimicrobial agents while others selectively kill tumor cells. Since granulysin is not found in mice, we generated mice expressing the human granulysin gene to evaluate its effects in vivo.In August 2007, my group relocated from Stanford University to the NCI. We equipped the laboratory and hired key personnel by early 2008. We began experiments in October 2007 and are actively pursuing studies in the following areas:1. Determine the role of granulysin in vivo using granulysin transgenic mice (mice do not express granulysin or a homolog). We recently found that perforin is required for granulysin killing in vivo. This was not the case in vitro.2. Investigate the roles of the 15 and 18kD forms of granulysin. Alternative splicing and post-translational modifications give rise to alternate forms of granulysin. We just found that 15kD granulysin does not kill but induces dendritic cell/macrophage differentiation and antigen processing. An 18kD form is phosphorylated and only briefly expressed in T cells and NK cells but is the major form in neutrophils. We are in the process of expressing 18kD granulysin to define its function..3. Examine granulysin as a therapeutic in preclinical models. The CT 26 tumor model is being tested in granulysin transgenic mice. We recently showed that granulysin transgenic mice have smaller tumors, increased dendritic cells and cytokines compared to wild type controls.4. Examine the expression and function of granulysin in neutrophils. This has been a controversial area but we recently showed that granulysin mRNA and 18kD protein are expressed in human neutrophils. The granulysin transgenic mice are being used to characterize the role of neutrophil granulysin in vivo. We find dramatically increased cell numbers and markedly improved survival in granulysin transgenic mice compared to controls. Granulysin transgenic neutrophils kill bacteria better than wildtype controls.These studies are aimed at designing new therapeutics for human diseases as diverse as cancer, tuberculosis, malaria, AIDS, and autoimmune diseases including diabetes, rheumatoid arthritis, and mutliple sclerosis.
