My laboratory is employing molecular genetic and biochemical techniques to investigate how T-lymphocytes perform their specific functions. The major focus at present is aimed at understanding how cytotoxic T-cells kill their targets. We reasoned that "cytotoxicity-related proteins" would be expressed specifically in these cells. Using recombinant DNA technology, we identified a number of genes which are expressed uniquely in cytotoxic cells. We identified a novel family of serine proteinases (now called granzymes), whose expression correlated with cytotoxicity, and are contained in cytoplasmic granules. Taken together our results suggest that these genes play a key role in cytotoxicity. Current efforts are directed at understanding what part they do play in the killing mechanism, determining how the genes are organized and investigating how they are regulated (i.e., determination of important DNA regulatory sequences and detection of transacting regulatory proteins). Other mechanisms of CTL-mediated lysis are also under investigation.
T-lymphocytes play a key role in the body's defenses against disease, and are important effectors in organ transplant rejection. A knowledge of how they function may well lead to the development of new forms of rational immunotherapy. Upon interaction with a virus infected, tumor or transplanted cell, the granzymes are exocytosed and enter the cytoplasm of the target. The enzymes then cleave critical substrates that initiate both apoptotic and necrotic cell death. We have recently discovered that uptake of granzyme is in via a specific receptor. Modulation of the levels of this protein may be very useful to increase or decrease sensitivity to lymphocyte-mediated killing.
Brenda Duggan, Technologist
Nancy Ehrman, Technologist
Diane Marganski, Technologist
1. Design and characterization of a novel human Granzyme B inhibitor. Marcet-Palacios M, Ewen C, Pittman E, Duggan B, Carmine-Simmen K, Fahlman RP, Bleackley RC. Protein Eng Des Sel. 2015 Jan;28(1):9-17.
2. Cloning CTL-Specific Genes (And Now for Something Completely Differential). Bleackley RC. Front Immunol. 2014 Oct 14;5:509.
3. Granzyme B degradation by autophagy decreases tumor cell susceptibility to natural killer-mediated lysis under hypoxia. Baginska J, Viry E, Berchem G, Poli A, Noman MZ, van Moer K, Medves S, Zimmer J, Oudin A, Niclou SP, Bleackley RC, Goping IS, Chouaib S, Janji B. Proc Natl Acad Sci U S A. 2013 Oct 22;110(43):17450-5.
4. Granzyme B inhibits vaccinia virus production through proteolytic cleavage of eukaryotic initiation factor 4 gamma 3. Marcet-Palacios M, Duggan BL, Shostak I, Barry M, Geskes T, Wilkins JA, Yanagiya A, Sonenberg N, Bleackley RC. PLoS Pathog. 2011 Dec;7(12):e1002447.
5. Perforin pores in the endosomal membrane trigger the release of endocytosed granzyme B into the cytosol of target cells. Thiery J, Keefe D, Boulant S, Boucrot E, Walch M, Martinvalet D, Goping IS, Bleackley RC, Kirchhausen T, Lieberman J. Nat Immunol. 2011 Jun 19;12(8):770-7.
6. Granzyme H induces cell death primarily via a Bcl-2-sensitive mitochondrial cell death pathway that does not require direct Bid activation. Ewen CL, Kane KP, Bleackley RC. Mol Immunol. 2013 Jul;54(3-4):309-18.