Eur J Immunogenet 25:365C370

Eur J Immunogenet 25:365C370. parasite-specific CD8+ T cell response directed against the parasitized lymphoblasts (3, 4), and transfer of purified CD8+ T cells from immune to naive twin calves has been shown to confer immunity to parasite challenge (5). The mechanism by which CD8+ T cells mediate protection against is usually poorly comprehended. They exhibit strong major histocompatibility complex (MHC)-restricted cytotoxic activity and secrete gamma interferon and tumor necrosis factor alpha; however, unlike other intracellular protozoa (6, 7), these cytokines do not appear to have a direct effector role against the parasite (8). Hence, cytotoxicity is considered likely to have an important role in immunity, although direct evidence for this is usually lacking, and at present, there is no information around the molecular mediators of cell killing. As an initial step toward investigating the development of subunit vaccines, studies have exhibited that granzyme B induces target cell death by two main pathways, one including direct proteolytic activation of caspases (leading to DNA damage) and the other by triggering outer mitochondrial membrane permeabilization via cleavage of the proapoptotic protein BH3-interaction domain death agonist (Bid) (14). The relative physiological roles of these activities remain unclear, particularly in view of the potential functional redundancy among the granzymes. Nevertheless, gene knockout mice deficient in granzyme B have been shown to have reduced levels of CD8+ T cell-mediated cytotoxicity and have increased susceptibility to some viral infections. Despite the residual ability of CD8+ T cells from granzyme B?/? mice to kill target cells, they were unable to induce DNA fragmentation (15). Extrapolation of findings in mice to other mammalian species is also complicated by the obtaining of differences in protein substrate specificity between murine and human granzyme B; in contrast to human granzyme B, mouse granzyme B is usually inefficient at cleaving Bid and is therefore believed to rely largely around the direct activation of caspases (16). In view of the potential importance of CD8+ T cell-mediated cytotoxicity as an effector mechanism against assay of granzyme B activity. In order to assess the role of granzyme B in the killing of values of 0.05 were considered significant. Relationship of cytotoxic activity and level of granzyme B protein expression. A series of CD8+ T cell clones specific for the same epitope in the Tp1 antigen from residues 214 to 224 (Tp1214C224) was used to examine the relationship between killing activity and granzyme B protein expression. These CD8+ clones exhibited maximal levels of killing of infected target cells ranging from 1% to 47% at effector-to-target cell ratios of 1 1:1 or greater (observe Fig. S1 in the supplemental material). Assays of granzyme B were conducted at a standard effector-to-target cell ratio of 2:1 to ensure maximal killing activity (Fig. 3A). Granzyme B activity in the culture supernatants and in the cell lysates of these clones following incubation with infected cells was measured using the substrate-specific assay established in the present study and explained above. As shown in Fig. 3A, the T cell clones showed variable levels of granzyme B release following exposure to antigen-expressing cells (which prior assays had confirmed do not express granzyme B protein; data not shown). The levels of granzyme activity in cell supernatants showed a highly significant correlation with the levels of granzyme protein in lysates of the respective clones (values of 0.05 were considered significant. The cytotoxic activity of T cells is dependent on perforin and granzyme B. The involvement of lytic granule exocytosis and, specifically, the role of granzyme B Talaporfin sodium in cell killing by bovine CD8+ T cells were investigated by screening the effects of specific inhibitors of perforin and granzyme B. Cytotoxicity assays were first conducted in the presence of a range of concentrations of concanamycin A (CMA), an inhibitor of vacuolar-type H+-ATPase (17), which raises the pH of the lytic granule and, thus, induces the degradation of perforin (18). The effect of CMA on cytotoxic activity was examined using an uncloned CD8+ T cell line assayed on either antigen Tp2 from residues 49 to 59 (Tp249C59) (1,000?ng/ml). (C) Three cloned CD8+ T cell lines (1??104) were preincubated for 1 h with 40 M Z-IETD-FMK and a negative control,.The mechanism by which CD8+ T cells mediate protection against is poorly understood. cell, ensuring that infection is retained in both daughter cells. Hence, the parasite remains in an intracellular location during this stage of development. Cattle that recover from infection with are solidly immune to subsequent challenge with the same parasite strain but show variable susceptibility to other parasite strains (2). Development of immunity is associated with a potent parasite-specific CD8+ T cell response directed against the parasitized lymphoblasts (3, 4), and transfer of purified CD8+ T cells from immune to naive twin calves has been shown to confer immunity to parasite challenge (5). The mechanism by which Talaporfin sodium CD8+ T cells mediate protection against is poorly understood. They exhibit strong major histocompatibility complex (MHC)-restricted cytotoxic activity and secrete gamma interferon and tumor necrosis factor alpha; however, unlike other intracellular protozoa (6, 7), these cytokines do not appear to have a direct effector role against the parasite (8). Hence, cytotoxicity is considered likely to have an important role in Talaporfin sodium immunity, although direct evidence for this is lacking, and at present, there is no information on the molecular mediators of cell killing. As an initial step toward investigating the development of subunit vaccines, studies have demonstrated that granzyme B induces target cell death by two main pathways, one involving direct proteolytic activation of caspases (leading to DNA damage) and the other by triggering outer mitochondrial membrane permeabilization via cleavage of the proapoptotic protein BH3-interaction domain death agonist (Bid) (14). The relative physiological roles of these activities remain unclear, particularly in view of the potential functional redundancy among the granzymes. Nevertheless, gene knockout mice deficient in granzyme B Talaporfin sodium have been shown to have reduced levels of CD8+ T cell-mediated cytotoxicity and have increased susceptibility to some viral infections. Despite the residual ability of CD8+ T cells from granzyme B?/? mice to kill target cells, they were unable to induce DNA fragmentation (15). Extrapolation of findings in mice to other mammalian species is also 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 therefore believed to rely largely on the direct activation of caspases (16). In view of the potential importance of CD8+ T cell-mediated cytotoxicity as an effector mechanism against assay of granzyme B activity. In order to assess the role of granzyme B in the killing of values of 0.05 were considered significant. Relationship of cytotoxic activity and level of granzyme B protein expression. A series of CD8+ T cell clones specific for the same epitope in the Tp1 antigen from residues 214 to 224 (Tp1214C224) was used to examine the relationship between killing activity and granzyme B protein expression. These CD8+ clones exhibited maximal levels of killing of infected target cells ranging from 1% to 47% at effector-to-target cell ratios of 1 1:1 or greater (see Fig. S1 in the supplemental material). Assays of granzyme B were conducted at a standard effector-to-target cell ratio of 2:1 to ensure maximal killing activity (Fig. 3A). Granzyme B activity in the culture supernatants and in the cell lysates of these clones following incubation with infected cells was measured using the substrate-specific assay established in the present study and described above. As shown in Fig. 3A, the T cell clones showed variable levels of granzyme B release following exposure to antigen-expressing cells (which prior assays had confirmed do not express granzyme B protein; data not shown). The levels of granzyme activity in cell supernatants showed a highly significant correlation with the levels of granzyme protein in lysates of the respective clones (values of 0.05 were considered significant. The cytotoxic CEACAM8 activity of T cells is dependent on perforin and granzyme B. The involvement of lytic granule exocytosis and, specifically, the role of granzyme B in cell killing by bovine CD8+ T cells were investigated by testing the effects of specific inhibitors of perforin and granzyme B. Cytotoxicity assays were first conducted in the presence of a range of concentrations of concanamycin A (CMA), an inhibitor of.