Characterization of bovine granzymes and studies of the role of granzyme B in killing of Theileria-infected cells by CD8+ T cells
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Yang2012.doc (5.175Mb)
Date
30/06/2012Author
Yang, Jie
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Abstract
Previous studies have shown that cytotoxic CD8+ T cells are important mediators of
immunity against the bovine intracellular protozoan parasite T. parva. The present
study set out to determine the role of granule enzymes in mediating killing of
parasitized cells, first by characterising the granzymes expressed by bovine
lymphocytes and, second, by investigating their involvement in killing of target
cells.
Experiments using the perforin inhibitor concanamycin A confirmed that CD8+ T
cell killing of T. parva-infected cells is dependent on granule exocytosis, a process
that involves release of granzymes into the target cell, resulting in activation of
apoptotic pathways. Analysis of the bovine genome sequence identified orthologues
of granzymes A, B, H, K and M, as well as another gene O, most closely related to
granzyme A. The genes were found within 3 loci in the genome. Using specific PCR
assays, all of these granzymes were shown to be expressed in Theileria-specific
CD8+ T cells. Further studies were undertaken to study the role of granzyme B in
killing. DNA constructs encoding functional and non-functional forms of bovine
granzyme B were produced and the proteins expressed in COS cells were used to
establish an enzymatic assay to detect and quantify expression of functional
granzyme B protein. Using this assay, the levels of killing of different T. parvaspecific
CD8+ T cell clones were found to be significantly correlating with levels of
granzyme B protein expression. Moreover, the granzyme B inhibitor III, Z-IETDFMK
was shown to inhibit killing by CD8+ T cell clones.