Granzyme B Is an Essential Mediator in CD8⁺T Cell Killing ofTheileria parva-Infected Cells

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.

Acquired cancer cell resistance to T cell bispecific antibodies and CAR T targeting HER2 through JAK2 down-modulation

“…Each well contained 25 μl of lysis supernatant, granzyme B substrate Ac-IEPD-pNA (#368057, Sigma-Aldrich) at a final concentration of 300 μM and reaction buffer (0.1 M HEPES pH 7.0, 0.3 M NaCl, 1 mM EDTA) in a total volume of 250 μl/well. Mixtures were incubated at 37 °C overnight and color reaction generated by the cleavage of the pNA substrate was measured at a wavelength of 405 nm with the Infinite M200 PRO (Tecan) plate reader 36 .…”

Immune Response to Tick-Borne Hemoparasites: Host Adaptive Immune Response Mechanisms as Potential Targets for Therapies and Vaccines

“…Nevertheless, how they kill parasitized cells is still poorly known. As already reported for human and murine CD8 + T cells, and also in bovine, probably, CD8 + T cells act through granule exocytosis and, specifically, granzyme B [88]. Cytotoxic T lymphocytes (CTLs) usually elicit responses restricted to the parasite strain and this is related to the issue of cross-protection towards different strains.…”

Interaction Networks Converging on Immunosuppressive Roles of Granzyme B: Special Niches Within the Tumor Microenvironment

“…In addition to AICD-induced “suicide,” granzyme B could be either intentionally secreted to extracellular space or randomly escape from the immunological synapse between cytotoxic lymphocytes and their target cells during immune surveillance. This diffusive granzyme B would adsorb onto the cell membrane of other bystanders and being endocytosed inside the cells by different mechanisms to induce cell death ( 76 , 77 ). The randomly escaped granzyme B would flow with body fluid and initiate an indiscriminate attack to any cells it makes contact with, leading to the increased inflammatory status, which might facilitate tumor progression ( 6 ).…”

“…Besides, they can kill DCs within TDLNs and TME in a contact-dependent way where Tregs recognize tumor-specific antigens presented by class 2 MHC ligand on DCs and release granzyme B/perforin granules to eliminate them. Hence GrB + Tregs could both impair autoimmunity and prevent the onset of DC-mediated adaptive immunity ( 77 ).…”