Granzyme B Activity in Target Cells Detects Attack by Cytotoxic Lymphocytes

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.

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

Aesthetic Surgery Journal ◽

10.1093/asj/sjaa302 ◽

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

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

Blood ◽

10.1182/blood.v92.3.1044.415k12_1044_1054 ◽

1998 ◽

Vol 92 (3) ◽

pp. 1044-1054 ◽

Cited By ~ 3

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.

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

Blood ◽

10.1182/blood.v92.3.1044 ◽

1998 ◽

Vol 92 (3) ◽

pp. 1044-1054 ◽

Cited By ~ 136

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.

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

The Journal of Cell Biology ◽

10.1083/jcb.200210150 ◽

2003 ◽

Vol 160 (2) ◽

pp. 223-233 ◽

Cited By ~ 70

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.

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Faculty Opinions recommendation of Granzyme B activity in target cells detects attack by cytotoxic lymphocytes.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1090831.544165 ◽

2007 ◽

Author(s):

Eckhard Podack

Keyword(s):

Granzyme B ◽

Target Cells ◽

Cytotoxic Lymphocytes

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

10.1101/328260 ◽

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.

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Orphan Granzymes Downstream from Granzyme B Are Important for Tumor Clearance In Vivo and in Vitro.

Blood ◽

10.1182/blood.v104.11.2653.2653 ◽

2004 ◽

Vol 104 (11) ◽

pp. 2653-2653

Author(s):

Xuefang Cao ◽

Paula A. Revell ◽

William J. Grossman ◽

Dori A. Thomas ◽

Zhi Hong Lu ◽

...

Keyword(s):

Tumor Cells ◽

Serine Proteases ◽

Granzyme B ◽

Lak Cells ◽

Target Cells ◽

Cytotoxic Lymphocytes ◽

Cytotoxic Lymphocyte ◽

Deficient Mice

Abstract Cytotoxic lymphocytes (Natural Killer cells and Cytotoxic T lymphocytes) can utilize the perforin/granzyme pathway as a major mechanism to kill pathogen-infected cells and tumor cells. Perforin is responsible for delivering and/or trafficking the granzymes (a family of neutral serine proteases) to the target cells. In the target cell cytoplasm and nucleus, the granzymes deliver the lethal hits. Granzymes A and B are the best characterized granzymes, and they can cleave a variety of important protein substrates to execute the target cells. However, some tumors and viruses have developed potent granzyme inhibitors that may allow them to evade cytotoxic lymphocyte-induced death. Interestingly, additional granzyme genes downstream from granzyme B (C, F, G, and D) on murine chromosome 14 are also expressed in cytotoxic lymphocytes, and are referred to as “orphans” since their functions have not been defined. We have developed two kinds of granzyme B knockout mice in the 129/SvJ background (H-2b) and examined their expression of granzyme B and orphan granzymes using quantitative RT-PCR and Western Blotting. In the first mouse (Gzm B−/−/+PGK-neo) a PGK-neo cassette was retained in the granzyme B gene, which caused a neighborhood effect, with significantly reduced expression of orphan granzymes C and F in cytotoxic lymphocytes (this mouse is referred to as “B cluster” deficient); In the second mouse (Gzm B−/−/ΔPGK-neo) the PGK-neo cassette was removed by Cre/loxP technology, which restored expression of granzymes C and F in cytotoxic lymphocytes (referred to as “B only” deficient). Both mutations completely abolish granzyme B expression. Using a Flow-based Killing Assay (FloKA), we have examined the cytotoxic functions of lymphocytes derived from mixed lymphocyte reactions (MLR) and 10-day lymphokine activated killer (LAK) cultures. We have found that granzyme B cluster-deficient cytotoxic lymphocytes (H-2b) generated by MLR kill allogeneic P815 or TA-3 tumor cells (H-2d) less efficiently than those deficient for granzyme B only (e.g. P815 killing at 3 hours, WT: 35%±1%, B only-deficient: 24%±5%, B cluster-deficient: 14%±3%, p<0.001). The reduction in granzyme B cluster-deficient killing is also seen with LAK cells against YAC-1 and RMA-S target cells (e.g. RMA-S killing at 4 hours, WT: 26%±1%, B only-deficient: 24%±1%, B cluster-deficient: 18%±1%, p<0.001). These results suggest that both allogeneic CTL and LAK cells require orphan granzymes (C and/or F) for optimal tumor cell killing. The defects in cytotoxicity detected by the FloKA assay have been confirmed to be biologically relevant (Revell et al, Blood2003, 102 (11): 1022) since granzyme B cluster-deficient mice cleared P815 cells less efficiently than either WT or granzyme B only-deficient mice (p<0.02). These studies suggest that the orphan granzymes are important for cytotoxic lymphocyte functions, and that they may provide a source of functional redundancy that would help protect from pathogens or tumor cells that express inhibitors of granzyme A or B.

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Cytotoxic lymphocytes require granzyme B for the rapid induction of DNA fragmentation and apoptosis in allogeneic target cells

Cell ◽

10.1016/0092-8674(94)90376-x ◽

1994 ◽

Vol 76 (6) ◽

pp. 977-987 ◽

Cited By ~ 594

Author(s):

Jonathan W. Heusel ◽

Robin L. Wesselschmidt ◽

Sujan Shresta ◽

John H. Russell ◽

Timothy J. Ley

Keyword(s):

Dna Fragmentation ◽

Granzyme B ◽

Target Cells ◽

Cytotoxic Lymphocytes ◽

Rapid Induction

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Granzyme B and perforin: constitutive expression in human polymorphonuclear neutrophils

Blood ◽

10.1182/blood-2003-04-1069 ◽

2004 ◽

Vol 103 (3) ◽

pp. 1099-1104 ◽

Cited By ~ 71

Author(s):

Christof Wagner ◽

Christof Iking-Konert ◽

Birgit Denefleh ◽

Sabine Stegmaier ◽

Friederike Hug ◽

...

Keyword(s):

Granzyme B ◽

Constitutive Expression ◽

Jurkat Cells ◽

Polymorphonuclear Neutrophils ◽

Target Cells ◽

Dependent Manner ◽

Cellular Cytotoxicity ◽

Antibody Dependent Cellular Cytotoxicity ◽

Cytotoxic Molecules ◽

Chicken Erythrocytes

AbstractPolymorphonuclear neutrophils (PMNs) produce an abundance of bactericidal and cytotoxic molecules consistent with their role as first-line defense against bacterial infection. PMNs, however, also cause efficient cellular cytotoxicity when targeted through Fc receptors to appropriate antibody-coated target cells. Although this so-called antibody-dependent cellular cytotoxicity (ADCC) was described many years ago, the mechanism of killing is still elusive. We now have found that PMNs contain perforin and granzyme B, the 2 molecules known as the cytotoxic entity of natural killer cells and of cytotoxic T lymphocytes as well. Lysates of PMNs were lytic for chicken erythrocytes in a time-, temperature-, and Ca2+-dependent manner. Moreover, apoptosis of Jurkat cells was induced, consistent with the observation that the PMN lysates contain enzymatically active granzyme B. Taken together, our data provide evidence for the presence of perforin and granzyme B within the cytotoxic arsenal of PMNs. (Blood. 2004;103:1099-1104)

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