Characterization of bovine granzymes and studies of the role of granzyme B in killing of Theileria-infected cells by CD8+ T cells

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.