The long-term goal of this proposal is to understand the role of intracellular calcium signals in the function of cytotoxic T lymphocytes (CTLs). These critical effectors of the immune system kill virus-infected cells and cancer cells and play a major role in the immune response to transplanted tissues; inappropriate killing can cause autoimmune diseases such as Lupus, certain forms of diabetes, and arthritis. Understanding CTL function is therefore important for preventing and treating naturally occurring viral diseases such as AIDS and influenza, and viral diseases such as smallpox used as biological weapons. It is also important for understanding and treating cancers and autoimmune diseases. Finally, the ability to suppress CTL function is vital for successful organ transplantation. One of the main mechanisms CTLs use to kill is the perforin pathway, which involves the exocytotic release of pore-forming peptides and hydrolytic enzymes contained in specialized lytic granules into an area of close apposition formed with the target. Granule exocytosis is known absolutely to require increased intracellular calcium caused by influx across the plasma membrane. However, the specific role(s) of calcium in granule exocytosis are unknown, the number of calcium-dependent steps is unclear, and molecules that confer calcium-dependence have not been identified.
The specific aims of this proposal will use a battery of techniques, including novel fluorescence imaging methodologies we have developed, to: 1) determine whether bulk cytosolic calcium increases are sufficient to support granule exocytosis, or whether higher-than-cytosolic calcium increases in microdomains are required. 2) Investigate the calcium dependence of granule reorientation and of reorientation-independent exocytosis. 3) Determine whether immunological synapse formation is calcium dependent, and acts as a slow step in granule reorientation. 4) Investigate the role of the calcium-dependent phosphatase calcineurin in granule exocytosis. These studies will significantly further our understanding of the role of calcium influx in lytic granule exocytosis.

National Institute of Health (NIH)
National Institute of Allergy and Infectious Diseases (NIAID)
Research Project (R01)
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Cell Development and Function Integrated Review Group (CDF)
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Nasseri, M Faraz
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University of Colorado Denver
Schools of Medicine
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Pores-Fernando, Arun T; Zweifach, Adam (2009) Calcium influx and signaling in cytotoxic T-lymphocyte lytic granule exocytosis. Immunol Rev 231:160-73
Pores-Fernando, Arun T; Ranaghan, Michelle Y D; Zweifach, Adam (2009) No specific subcellular localization of protein kinase C is required for cytotoxic T cell granule exocytosis. J Biol Chem 284:25107-15
Pores-Fernando, Arun T; Gaur, Surabhi; Doyon, Michelle Y et al. (2009) Calcineurin-dependent lytic granule exocytosis in NK-92 natural killer cells. Cell Immunol 254:105-9
Pores-Fernando, Arun T; Gaur, Surabhi; Grybko, Michael J et al. (2008) ERK activation is only one role of PKC in TCR-independent cytotoxic T cell granule exocytosis. Biochem Biophys Res Commun 371:630-4
Grybko, Michael J; Pores-Fernando, Arun T; Wurth, Georjeana A et al. (2007) Protein kinase C activity is required for cytotoxic T cell lytic granule exocytosis, but the theta isoform does not play a preferential role. J Leukoc Biol 81:509-19
Grybko, Michael J; Bartnik, Jakub P; Wurth, Georjeana A et al. (2007) Calcineurin activation is only one calcium-dependent step in cytotoxic T lymphocyte granule exocytosis. J Biol Chem 282:18009-17
Pores-Fernando, Arun T; Bauer, Roslyn A; Wurth, Georjeana A et al. (2005) Exocytic responses of single leukaemic human cytotoxic T lymphocytes stimulated by agents that bypass the T cell receptor. J Physiol 567:891-903