Abstract
There is established evidence that cytotoxic CD8+ T cells are important mediators of immunity against the bovine intracellular protozoan parasite Theileria parva However, the mechanism by which the specific CD8+ T cells kill parasitized cells is not understood. Although the predominant pathway used by human and murine CD8+ T cells to kill pathogen-infected cells is granule exocytosis, involving the release of perforin and granzyme B, there is to date a lack of published information on the biological activities of bovine granzyme B. The present study set out to define the functional activities of bovine granzyme B and determine its role in mediating the killing of T. parva-parasitized cells. DNA constructs encoding functional and nonfunctional forms of bovine granzyme B were produced, and the proteins expressed in Cos-7 cells were used to establish an enzymatic assay to detect and quantify the expression of functional granzyme B protein. Using this assay, the levels of killing of different T. parva-specific CD8+ T cell clones were found to be significantly correlated with the levels of granzyme B protein but not the levels of mRNA transcript expression. Experiments using inhibitors specific for perforin and granzyme B confirmed that CD8+ T cell killing of parasitized cells is dependent on granule exocytosis and, specifically, granzyme B. Further studies showed that the granzyme B-mediated death of parasitized cells is independent of caspases and that granzyme B activates the proapoptotic molecule Bid.
Highlights
There is established evidence that cytotoxic CD8ϩ T cells are important mediators of immunity against the bovine intracellular protozoan parasite Theileria parva
In this study, we aimed to examine the role of bovine granzyme B in the cytotoxic function of T. parva-specific CD8ϩ T cell responses
We showed that the levels of killing of different T. parva-specific CD8ϩ T cell clones are significantly correlated with the levels of granzyme B protein and that the killing of infected cells by bovine CD8ϩ T cells is mediated by the granule exocytosis pathway and critically requires granzyme B for induction of cell death
Summary
There is established evidence that cytotoxic CD8ϩ T cells are important mediators of immunity against the bovine intracellular protozoan parasite Theileria parva. The present study set out to define the functional activities of bovine granzyme B and determine its role in mediating the killing of T. parva-parasitized cells. Further studies showed that the granzyme B-mediated death of parasitized cells is independent of caspases and that granzyme B activates the proapoptotic molecule Bid. Antigen-specific CD8ϩ T cell responses have been shown to play a key role in immunity to a number of viral, bacterial, and parasitic infections. Antigen-specific CD8ϩ T cell responses have been shown to play a key role in immunity to a number of viral, bacterial, and parasitic infections One such parasite is the tick-borne protozoan Theileria parva. Understanding the mechanisms of killing of T. parva-infected cells by bovine CD8 T cells is required to identify relevant molecular markers that can be used to monitor vaccine-induced immune responses and accelerate vaccine development. Extrapolation of findings in mice to other mammalian species is complicated by the finding of differences in protein substrate specificity between murine and human granzyme B; in contrast to human granzyme B, mouse granzyme B is inefficient at cleaving Bid and is believed to rely largely on the direct activation of caspases [16]
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