Entry and Trafficking of Granzyme B in Target Cells During Granzyme B-Perforin–Mediated Apoptosis

Blood ◽  
1998 ◽  
Vol 92 (3) ◽  
pp. 1044-1054 ◽  
Author(s):  
Michael J. Pinkoski ◽  
Marita Hobman ◽  
Jeffrey A. Heibein ◽  
Kevin Tomaselli ◽  
Feng Li ◽  
...  
Keyword(s):  
Cell Death ◽  
Indirect Immunofluorescence ◽  
Target Cell ◽  
Granzyme B ◽  
Fluorescein Isothiocyanate ◽  
Target Cells ◽  
Cytotoxic Lymphocytes ◽  
Direct Visualization ◽  
New Model ◽  
American Society

Abstract In the widely accepted model of granule-mediated killing by cytotoxic lymphocytes, granzyme B entry into the target cell is facilitated by the pore forming molecule, perforin. Using indirect immunofluorescence and also direct visualization of fluorescein isothiocyanate (FITC)-conjugated granzyme B, we demonstrate internalization in the absence of perforin. Induction of the lytic pathway, however, required a second signal that was provided by perforin or adenovirus (Ad2). The combination of agents also resulted in a dramatic relocalization of the granzyme. Microinjection of granzyme B directly into the cytoplasm of target cells resulted in apoptosis without the necessity of a second stimulus. This suggested that the key event is the presence of granzyme B in the cytoplasm, and that when the enzyme is internalized by a target cell, it trafficks to an intracellular compartment and accumulates until release is stimulated by the addition of perforin. We found that the proteinase passed through rab5-positive vesicles and then accumulated within a novel compartment. On the basis of these results, we propose a new model for granzyme-perforin–induced target cell lysis in which granzyme B is subjected to trafficking events in the target cell that control and contribute to cell death. © 1998 by The American Society of Hematology.

Entry and Trafficking of Granzyme B in Target Cells During Granzyme B-Perforin–Mediated Apoptosis

Blood ◽  
1998 ◽  
Vol 92 (3) ◽  
pp. 1044-1054 ◽  
Author(s):  
Michael J. Pinkoski ◽  
Marita Hobman ◽  
Jeffrey A. Heibein ◽  
Kevin Tomaselli ◽  
Feng Li ◽  
...  
Keyword(s):  
Cell Death ◽  
Indirect Immunofluorescence ◽  
Target Cell ◽  
Granzyme B ◽  
Fluorescein Isothiocyanate ◽  
Target Cells ◽  
Cytotoxic Lymphocytes ◽  
Direct Visualization ◽  
New Model ◽  
American Society

In the widely accepted model of granule-mediated killing by cytotoxic lymphocytes, granzyme B entry into the target cell is facilitated by the pore forming molecule, perforin. Using indirect immunofluorescence and also direct visualization of fluorescein isothiocyanate (FITC)-conjugated granzyme B, we demonstrate internalization in the absence of perforin. Induction of the lytic pathway, however, required a second signal that was provided by perforin or adenovirus (Ad2). The combination of agents also resulted in a dramatic relocalization of the granzyme. Microinjection of granzyme B directly into the cytoplasm of target cells resulted in apoptosis without the necessity of a second stimulus. This suggested that the key event is the presence of granzyme B in the cytoplasm, and that when the enzyme is internalized by a target cell, it trafficks to an intracellular compartment and accumulates until release is stimulated by the addition of perforin. We found that the proteinase passed through rab5-positive vesicles and then accumulated within a novel compartment. On the basis of these results, we propose a new model for granzyme-perforin–induced target cell lysis in which granzyme B is subjected to trafficking events in the target cell that control and contribute to cell death. © 1998 by The American Society of Hematology.

scholarly journals NOVEL IMMUNOTHERAPEUTIC TARGETED GRANZYME DELIVERY SYSTEMS IN TREATMENT OF MALIGNANT TUMORS

2021 ◽  
Vol 20 (2) ◽  
pp. 31-41
Author(s):  
I. V. Yarosh ◽  
V. A. Misyurin ◽  
I. I. Krasnyuk
Keyword(s):  
Cell Death ◽  
Target Cell ◽  
Serine Proteases ◽  
Malignant Tumors ◽  
Granzyme B ◽  
Killer Cell ◽  
Cascade Reactions ◽  
Target Cells ◽  
Cell Property ◽  
Infected Cells

Cytotoxicity is the main human killer cell property. The cytotoxicity reaction of human killer cells is achieved through a complex of molecules, including perforins, granzyme, cathepsin and others. However, only one molecule is enough for target cell death: granzyme. Other molecules are intended for granzyme activation and its delivery to the target cell cytoplasm. Granzymes are a whole family of serine proteases that perform their function in the human body as integral cytolytic effectors during programmed cell death of cancer and pathogen-infected cells. Secreted mainly by cytotoxic T-lymphocytes and NK-cells, granzymes initiate apoptosis via caspase-dependent and caspase-independent pathways. These natural properties make granzymes one of the most promising human enzymes for use in the development of targeted therapeutic strategies in the treatment of various types of cancer.The most promising is granzyme B, because it has the most powerful effector properties. Due to the initiation of cascade reactions that activate apoptosis, granzyme is attractive as a basis for the development of medicines applicable in clinical oncology. At this time, several approaches have been developed for delivering granzyme molecules to tumor cells and facilitating its penetration through the cell membrane. Moreover, some solutions are proposed to overcome the resistance of target cells to granzyme-mediated apoptosis. These approaches are discussed in this review.The purpose of this review was to systematize information on the use of granzyme B as a nanostructured drug delivery system in the treatment of solid and hematological malignancies. In addition, this review discusses ways to overcome the resistance of granzyme penetration into target cells.

scholarly journals The target cell nucleus is not required for cell-mediated granzyme- or Fas-based cytotoxicity.

1995 ◽  
Vol 181 (5) ◽  
pp. 1905-1909 ◽  
Author(s):  
H Nakajima ◽  
P Golstein ◽  
P A Henkart
Keyword(s):  
Cell Death ◽  
Target Cell ◽  
Cytotoxic T Lymphocyte ◽  
Target Cells ◽  
Cytotoxic Lymphocytes ◽  
Cell Nuclei ◽  
Effector Cells ◽  
Cell Death Pathways ◽  
Mast Cell Tumors ◽  
Nuclear Damage

The requirement for target cell nuclei in the two apoptotic death pathways used by cytotoxic lymphocytes was tested using model effector systems in which the granzyme and Fas pathways of target damage are isolated. Mast cell tumors expressing granzymes A and B in addition to cytolysin/perforin lysed tumor target cells about 10-fold more efficiently than comparable effector cells without granzymes. Enucleated cytoplast targets derived from these cells were also lysed with a similar 10-fold effect of granzymes. In contrast to cytoplasts, effector granzyme expression did not influence lysis of red cell targets. The Fas pathway was assessed using the selected cytotoxic T lymphocyte hybridoma subline d11S, which lysed target cells expressing Fas but not those lacking Fas. Similarly, cytoplasts derived from Fas+ but not Fas- cells were also readily lysed by these effector cells. Thus, neither the nucleus itself nor the characteristic apoptotic nuclear damage associated with the two major cell death pathways used by cytotoxic lymphocytes are required for cell death per se.

scholarly journals A clathrin/dynamin- and mannose-6-phosphate receptor–independent pathway for granzyme B–induced cell death

2003 ◽  
Vol 160 (2) ◽  
pp. 223-233 ◽  
Author(s):  
Joseph A. Trapani ◽  
Vivien R. Sutton ◽  
Kevin Y.T. Thia ◽  
Yu Qin Li ◽  
Christopher J. Froelich ◽  
...  
Keyword(s):  
Cell Death ◽  
Hela Cells ◽  
Granzyme B ◽  
Dominant Negative ◽  
Target Cells ◽  
Mouse Tumor ◽  
Cytotoxic Lymphocytes ◽  
L Cells ◽  
Mannose 6 Phosphate ◽  
B Uptake

The 280-kD cation-independent mannose-6-phosphate receptor (MPR) has been shown to play a role in endocytic uptake of granzyme B, since target cells overexpressing MPR have an increased sensitivity to granzyme B–mediated apoptosis. On this basis, it has been proposed that cells lacking MPR are poor targets for cytotoxic lymphocytes that mediate allograft rejection or tumor immune surveillance. In the present study, we report that the uptake of granzyme B into target cells is independent of MPR. We used HeLa cells overexpressing a dominant-negative mutated (K44A) form of dynamin and mouse fibroblasts overexpressing or lacking MPR to show that the MPR/clathrin/dynamin pathway is not required for granzyme B uptake. Consistent with this observation, cells lacking the MPR/clathrin pathway remained sensitive to granzyme B. Exposure of K44A-dynamin–overexpressing and wild-type HeLa cells to granzyme B with sublytic perforin resulted in similar apoptosis in the two cell populations, both in short and long term assays. Granzyme B uptake into MPR-overexpressing L cells was more rapid than into MPR-null L cells, but the receptor-deficient cells took up granzyme B through fluid phase micropinocytosis and remained sensitive to it. Contrary to previous findings, we also demonstrated that mouse tumor allografts that lack MPR expression were rejected as rapidly as tumors that overexpress MPR. Entry of granzyme B into target cells and its intracellular trafficking to induce target cell death in the presence of perforin are therefore not critically dependent on MPR or clathrin/dynamin-dependent endocytosis.

scholarly journals Single-fluorescent protein reporters allow parallel quantification of NK cell-mediated granzyme and caspase activities in single target cells

2018 ◽  
Author(s):  
Clarissa Liesche ◽  
Patricia Sauer ◽  
Maren Claus ◽  
Roland Eils ◽  
Joël Beaudouin ◽  
...  
Keyword(s):  
Cell Death ◽  
Nk Cells ◽  
Caspase 3 ◽  
Fluorescent Protein ◽  
Nk Cell ◽  
Granzyme B ◽  
Target Cells ◽  
Caspase 8 ◽  
Cytotoxic Lymphocytes ◽  
Cell Death Mechanisms

1.AbstractNatural killer (NK) cells eliminate infected and tumorigenic cells through delivery of granzymes via perforin pores or by activation of caspases via death receptors. In order to understand how NK cells combine different cell death mechanisms it is important to quantify target cell responses on a single cell level. However, currently existing reporters do not allow the measurement of several protease activities inside the same cell. Here we present a strategy for the comparison of two different proteases at a time inside individual target cells upon engagement by NK cells. We developed single-fluorescent protein reporters containing the RIEAD or the VGPD cleavage site for the measurement of granzyme B activity. We show that these two granzyme B reporters can be applied in combination with caspase-8 or caspase-3 reporters. While we did not find that caspase-8 was activated by granzyme B, our method revealed that caspase-3 activity follows granzyme B activity with a delay of about 6 minutes. Finally, we illustrate the comparison of several different reporters for granzyme A, M, K and H. The here presented approach is a valuable means for the investigation of the temporal evolution of cell death mediated by cytotoxic lymphocytes.

scholarly journals Selective Regulation of Apoptosis: the Cytotoxic Lymphocyte Serpin Proteinase Inhibitor 9 Protects against Granzyme B-Mediated Apoptosis without Perturbing the Fas Cell Death Pathway

1998 ◽  
Vol 18 (11) ◽  
pp. 6387-6398 ◽  
Author(s):  
Catherina H. Bird ◽  
Vivien R. Sutton ◽  
Jiuru Sun ◽  
Claire E. Hirst ◽  
Andrea Novak ◽  
...  
Keyword(s):  
Proteinase Inhibitor ◽  
Granzyme B ◽  
Target Cells ◽  
Cytotoxic Lymphocytes ◽  
Caspase Inhibitor ◽  
Caspase Activation ◽  
Cytotoxic Lymphocyte ◽  
Transfected Cells ◽  
Cell Death Pathway ◽  
Proteinase Inhibitor 9

ABSTRACT Cytotoxic lymphocytes (CLs) induce caspase activation and apoptosis of target cells either through Fas activation or through release of granule cytotoxins, particularly granzyme B. CLs themselves resist granule-mediated apoptosis but are eventually cleared via Fas-mediated apoptosis. Here we show that the CL cytoplasmic serpin proteinase inhibitor 9 (PI-9) can protect transfected cells against apoptosis induced by either purified granzyme B and perforin or intact CLs. A PI-9 P1 mutant (Glu to Asp) is a 100-fold-less-efficient granzyme B inhibitor that no longer protects against granzyme B-mediated apoptosis. PI-9 is highly specific for granzyme B because it does not inhibit eight of the nine caspases tested or protect transfected cells against Fas-mediated apoptosis. In contrast, the P1(Asp) mutant is an effective caspase inhibitor that protects against Fas-mediated apoptosis. We propose that PI-9 shields CLs specifically against misdirected granzyme B to prevent autolysis or fratricide, but it does not interfere with homeostatic deletion via Fas-mediated apoptosis.

scholarly journals Granzyme B Is a Biomarker for Suspicion of Malignant Seromas Around Breast Implants

2020 ◽  
Author(s):  
Marshall E Kadin ◽  
John Morgan ◽  
Haiying Xu ◽  
Caroline Glicksman ◽  
David Sieber ◽  
...  
Keyword(s):  
Early Detection ◽  
Tumor Cells ◽  
Breast Implant ◽  
Granzyme B ◽  
Breast Implants ◽  
Large Cell ◽  
Target Cells ◽  
Cytotoxic Lymphocytes ◽  
Tumor Cell Death ◽  
Level Of Evidence

Abstract Background Granzyme B (GrB) is a serine protease secreted, along with pore-forming perforin, by cytotoxic lymphocytes to mediate apoptosis in target cells. GrB has been detected in tumor cells associated with systemic and breast implant–associated anaplastic large cell lymphoma (BIA-ALCL) but its potential use for detection of early BIA-ALCL has not been fully investigated. Objectives Prompted by the increased incidence of BIA-ALCL, the aim of this study was to assess GrB as a new biomarker to detect early disease in malignant seromas and to better understand the nature of the neoplastic cell. Methods A Human XL Cytokine Discovery Magnetic Luminex 45-plex Fixed Panel Performance Assay was used to compare cytokine levels in cell culture supernatants of BIA-ALCL and other T-cell lymphomas, as well as malignant and benign seromas surrounding breast implants. Immunohistochemistry was employed to localize GrB to cells in seromas and capsular infiltrates. Results Differences in GrB concentrations between malignant and benign seromas were significant (P < 0.001). GrB was found in and around apoptotic tumor cells, suggesting that the protease may be involved in tumor cell death. Conclusions GrB is a useful marker for early detection of malignant seromas and to identify tumor cells in seromas and capsular infiltrates. Because there is an overlap between the lowest concentrations of soluble GrB in malignant seromas and the highest concentrations of GrB in benign seromas, it is recommended that GrB be used only as part of a panel of biomarkers for the screening and early detection of BIA-ALCL. Level of Evidence: 5

scholarly journals Participation of the H-2 antigens of tumor cells in their lysis by syngeneic T cells.

1976 ◽  
Vol 143 (3) ◽  
pp. 601-614 ◽  
Author(s):  
J W Schrader ◽  
G M Edelman
Keyword(s):  
T Cells ◽  
Tumor Cell ◽  
Tumor Cells ◽  
Target Cell ◽  
Cytotoxic T Cells ◽  
Hybrid Origin ◽  
Target Cells ◽  
Cytotoxic Lymphocytes ◽  
Effector Cells

Cytotoxic T lymphocytes were generated in vitro against H-2 compatible or syngeneic tumor cells. In vitro cytotoxic activity was inhibited by specific anti-H2 sera, suggesting that H-2 antigens are involved in cell lysis. Two observations directly demonstrated the participation of the H-2 antigens on the tumor cells in their lysis by H-2-compatible T cells. First, coating of the H-2 antigens on the target tumor cell reduced the number of cells lysed on subsequent exposure to cytotoxic T cells. Second, when cytotoxic T cells were activated against an H-2 compatible tumor and assayed against an H-2-incompatible tumor, anti-H-2 serum that could bind to the target cell, but not to the cytotoxic lymphocyte, inhibited lysis. H-2 antigens were also shown to be present on the cytotoxic lymphocytes. Specific antisera reacting with these H-2 antigens, but not those of the target cell, failed to inhibit lysis when small numbers of effector cells were assayed against H-2-incompatible target cells or when effector cells of F1-hybrid origin and bearing two H-2 haplotypes were assayed against a tumor cell of one of the parental strains. These findings suggest that it is the H-2 antigens on the tumor cell and not those on the cytotoxic lymphocytes that are important in cell-mediated lysis of H-2-compatible tumor cells.

scholarly journals Granzyme B-induced Cell Death Involves Induction of p53 Tumor Suppressor Gene and Its Activation in Tumor Target Cells

2007 ◽  
Vol 282 (45) ◽  
pp. 32991-32999 ◽  
Author(s):  
Franck Meslin ◽  
Jerome Thiery ◽  
Catherine Richon ◽  
Abdelali Jalil ◽  
Salem Chouaib
Keyword(s):  
Cell Death ◽  
Tumor Suppressor ◽  
Tumor Suppressor Gene ◽  
Granzyme B ◽  
Suppressor Gene ◽  
Target Cells ◽  
Tumor Target ◽  
P53 Tumor Suppressor ◽  
P53 Tumor Suppressor Gene

Fas-based d1OS-mediated cytotoxicity requires macromolecular synthesis for effector cell activation but not for target cell death

1994 ◽  
Vol 345 (1313) ◽  
pp. 303-309 ◽  
Keyword(s):  
Cell Death ◽  
T Cell ◽  
Cell Surface ◽  
Target Cell ◽  
Cell Activation ◽  
Effector Cell ◽  
Apoptotic Cell ◽  
Target Cells ◽  
Macromolecular Synthesis ◽  
Effector Cells

Two main mechanisms seem at play in T cell-mediated cytotoxicity, a process in which target cell death often follows an apoptotic cell death pattern. One of these involves Fas at the target cell surface and a Fas ligand at the effector cell surface. This allowed us to reinvestigate the long-standing question of macromolecular synthesis requirement in T cell-mediated cytotoxicity, using the dlOS model cell line which is cytotoxic apparently only via the Fas molecularly defined mechanism. We showed, first, that induction of cytotoxic activity of effector cells, obtained by preincubating these effector cells with a phorbol ester and a calcium ionophore, could be inhibited by macromolecular synthesis inhibitors (cycloheximide, actinomycin D, DRB). We then investigated whether macromolecular synthesis was required, when effector and target cells were mixed, to obtain target cell death. Preincubating already activated effector cells for 30 min with macromolecular synthesis inhibitors, then adding target cells and performing the 51 Cr release cytotoxicity test in the presence of these inhibitors, did not significantly decrease subsequent target cell death, indicating that already activated effector cells could kill without further requirement for macromolecular synthesis. In addition, target cell preincubation for up to 3 h in the presence of one of these inhibitors did not decrease cell death. The high sensitivity of mouse thymocytes to this type of cytotoxicity enabled us to devise the following experiment. As previously shown by others, thymocyte death induced by dexamethasone (DEX) could be blocked by coincubation with cycloheximide (CHX). Such DEX-treated CHX-rescued thymocytes, the survival of which was an internal control of efficiency of protein synthesis inhibition, were then subjected to effector cells in the presence of CHX, and were shown to die. Thus, there is no requirement for macromolecular synthesis at the target cell level in this variety of apoptotic cell death. Altogether, in this defined mechanism of T cell-mediated cytotoxicity, macromolecular synthesis is required for dlOS effector cell activation, but not for lysis by already activated effector cells nor for target cell death.

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