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

Blood ◽  
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.

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 In Vitro and In Vivo Comparison of Lymphocytes Transduced with a Human CD16 or with a Chimeric Antigen Receptor Reveals Potential Off-Target Interactions due to the IgG2 CH2-CH3 CAR-Spacer

2015 ◽  
Vol 2015 ◽  
pp. 1-13 ◽  
Author(s):  
Béatrice Clémenceau ◽  
Sandrine Valsesia-Wittmann ◽  
Anne-Catherine Jallas ◽  
Régine Vivien ◽  
Raphaël Rousseau ◽  
...  
Keyword(s):  
Chimeric Antigen Receptor ◽  
Tumor Regression ◽  
Critical Role ◽  
Antigen Receptor ◽  
Target Cells ◽  
Cytotoxic Lymphocytes ◽  
Her2 Positive ◽  
Positive Target

The present work was designed to compare two mechanisms of cellular recognition based on Ab specificity: firstly, when the anti-HER2 mAb trastuzumab bridges target cells and cytotoxic lymphocytes armed with a Fc receptor (ADCC) and, secondly, when HER2 positive target cells are directly recognized by cytotoxic lymphocytes armed with a chimeric antigen receptor (CAR). To compare these two mechanisms, we used the same cellular effector (NK-92) and the same signaling domain (FcεRIγ). The NK-92 cytotoxic cell line was transfected with either a FcγRIIIa-FcεRIγ(NK-92CD16) or a trastuzumab-based scFv-FcεRIγchimeric receptor (NK-92CAR). In vitro, the cytotoxic activity against HER2 positive target cells after indirect recognition byNK-92CD16was always inferior to that observed after direct recognition byNK-92CAR. In contrast, and somehow unexpectedly, in vivo, adoptive transfer ofNK-92CD16+ trastuzumab but not ofNK-92CARinduced tumor regression. Analysis of the in vivo xenogeneic system suggested that the human CH2-CH3 IgG2 used as a spacer in our construct was able to interact with the FcR present at the cell surface of the few NSG-FcR+ remaining immune cells. This interaction, leading to blockage of theNK-92CARin the periphery of the engrafted tumor cells, stresses the critical role of the composition of the spacer domain.

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.

Macrophage Antitumor Activity Induced by the Antigenic Lymphoma L5178Y/DTIC Subline

1982 ◽  
Vol 68 (5) ◽  
pp. 365-371 ◽  
Author(s):  
Ornella Marelli ◽  
Alberto Mantovani ◽  
Paola Franco ◽  
Angelo Nicotin
Keyword(s):  
Antitumor Activity ◽  
Tumor Cell ◽  
Tumor Cells ◽  
Peritoneal Macrophages ◽  
Leukemic Cells ◽  
Target Cells ◽  
Tumor Target ◽  
Growth Inhibitory

Murine leukemic cells, after in vivo treatment with antineoplastic drugs, have been shown to express new antigenic specificities that were not detectable on parental cells and that were heritable after the withdrawal of drug treatment. A study was conducted of macrophage antitumor activity triggered by LY/DTIC cells, a subline of LY murine lymphoma, antigenically altered by the drug DTIC. In vitro non-specific inhibition of tumor cell growth was exhibited by spleen and peritoneal macrophages from mice previously challenged with viable LY/DTIC. Peritoneal macrophages from LY/DTIC immune animals showed moderate, although significant lytic activity against unrelated tumor target cells. Supernatants from mixed lymphocyte-tumor cell cultures, in which LY/DTIC immune lymphocytes and LY/DTIC tumor cells had been cultured, rendered normal macrophages non-specifically growth inhibitory for tumor cells.

scholarly journals The role of granzyme B in murine models of acute graft-versus-host disease and graft rejection

Blood ◽  
1996 ◽  
Vol 87 (4) ◽  
pp. 1232-1237 ◽  
Author(s):  
TA Graubert ◽  
JH Russell ◽  
TJ Ley
Keyword(s):  
Nk Cells ◽  
Graft Versus Host Disease ◽  
Graft Rejection ◽  
Granzyme B ◽  
Cytotoxic Lymphocytes ◽  
Host Disease ◽  
Graft Versus Host ◽  
Hybrid Resistance

A complete molecular description of the syndromes of graft-versus-host disease (GVHD) and graft rejection could have a significant impact on clinical bone marrow transplantation. Recent in vitro experiments (Heusel et al, Cell 76:977, 1994 and Shresta et al, Proc Natl Acad Sci USA 92:5679, 1995) have shown that the putative mediators of these two syndromes, cytotoxic lymphocytes (CTL) and natural killer (NK) cells, respectively, initiate a program of cell death (apoptosis) in susceptible target tissues in a manner critically dependent on the serine protease Granzyme B (gzm B). In the present study, we have analyzed the phenotype of gzm B-deficient mice using experimental transplant models designed to isolate their CD8+ CTL, CD4+ CTL, and NK compartments. We found a significant impairment in class I-dependent GVHD mediated by gzm B -/- CD8+ CTL, whereas class II-dependent GVHD was not altered using gzm B -/- CD4+ effectors. In a hybrid resistance model, gzm B -/- hosts rejected haplo-identical marrow grafts as efficiently as did their wild-type littermates. This result is surprising in light of a severe defect in the ability of gzm B -/- NK cells to induce apoptosis in susceptible targets in vitro. These in vivo data define significant role for gzm B in cytotoxicity mediated by CD8+ CTL, but not by CD4+ CTL. Furthermore, these results do not support a model of hybrid resistance in which NK cells play a pivotal role.

scholarly journals NF-kB c-Rel is dispensable for the development but is required for the cytotoxic function of NK cells

2021 ◽  
Author(s):  
Y Vicioso ◽  
K Zhang ◽  
Parameswaran Ramakrishnan ◽  
Reshmi Parameswaran
Keyword(s):  
Nk Cells ◽  
Nk Cell ◽  
Granzyme B ◽  
Conditional Knockout ◽  
Cytotoxic Lymphocytes ◽  
And Control ◽  
Cytotoxic Function

AbstractNatural Killer (NK) cells are cytotoxic lymphocytes critical to the innate immune system. We found that germline deficiency of NF-kB c-Rel results in a marked decrease in cytotoxic function of NK cells, both in vitro and in vivo, with no significant differences in the stages of NK cell development. We found that c-Rel binds to the promoters of perforin and granzyme B, two key proteins required for NK cytotoxicity, and controls their transactivation. We generated a NK cell specific c-Rel conditional knockout to study NK cell intrinsic role of c-Rel and found that both global and conditional c-Rel deficiency leads to decreased perforin and granzyme B expression and thereby cytotoxic function. We also confirmed the role of c-Rel in perforin and granzyme B expression in human NK cells. c-Rel reconstitution rescued perforin and granzyme B expressions in c-Rel deficient NK cells and restored their cytotoxic function. Our results show a previously unknown role of c-Rel in transcriptional regulation of perforin and granzyme B expressions and control of NK cell cytotoxic function.

Antitumor activity against established intracerebral gliomas exhibited by cytotoxic T lymphocytes, but not by lymphokine-activated killer cells

1992 ◽  
Vol 77 (5) ◽  
pp. 757-762 ◽  
Author(s):  
Frank P. Holladay ◽  
Teresa Heitz ◽  
Gary W. Wood
Keyword(s):  
Brain Tumor ◽  
T Lymphocytes ◽  
Tumor Cells ◽  
Cytotoxic T Lymphocytes ◽  
Lak Cells ◽  
Lymphokine Activated Killer Cells ◽  
Killer Cells ◽  
Brain Tumor Cells

✓ Specific immune responses against malignant brain tumors have been difficult to demonstrate. Moreover, immunotherapy has met with little success, despite using lymphocytes with high levels of cytotoxicity against brain tumor cells. Lymphokine-activated killer (LAK) cells that nonspecifically kill brain tumor cells are produced by stimulating resting precursors with high concentrations of interleukin-2 (IL-2). Cytotoxic T lymphocytes that specifically kill brain tumor cells are produced by stimulating antigen receptor-positive immune-cell precursors with tumor cells. In an attempt to gain insight into immune cell function against brain tumors, the present study compared the in vitro and in vivo activities of LAK cells and cytotoxic T lymphocytes produced against RT2, a fast-growing rat glioma cell line. Lymphokine-activated killer cells were produced by stimulating normal rat spleen cells with 1000 units of IL-2, and RT2-specifie cytotoxic T lymphocytes were produced by priming them in vivo with RT2 and Corynebacterium parvum and restimulating primed spleen cells with RT2 in vitro. Lymphokine-activated killer cells were highly cytotoxic for a panel of syngeneic and allogeneic brain tumor and non-brain tumor target cells, including RT2, as measured in a 4-hour 51Cr release assay. Cytotoxic T lymphocytes were highly cytotoxic only for syngeneic brain tumor target cells. Lymphokine-activated killer cells and cytotoxic T lymphocytes were tested for in vivo antitumor activity against intracerebral RT2 by intravenous adoptive transfer of activated lymphocytes. Untreated rats died in approximately 2 weeks. Lymphokine-activated killer cells plus IL-2 failed to affect survival when treatment was initiated as early as 1 day following tumor inoculation. Cytotoxic T lymphocytes and IL-2 administered as late as Day 5 rejected progressing intracerebral tumor. Thus, although both cytotoxic T lymphocytes and LAK cells exhibited high levels of in vitro killing of glioma cells, only cytotoxic T lymphocytes rejected progressing intracerebral tumors.

Expression of the granzyme B inhibitor, protease inhibitor 9, by tumor cells in patients with non-Hodgkin and Hodgkin lymphoma: a novel protective mechanism for tumor cells to circumvent the immune system?

Blood ◽  
2002 ◽  
Vol 99 (1) ◽  
pp. 232-237 ◽  
Author(s):  
Bellinda A. Bladergroen ◽  
Chris J. L. M. Meijer ◽  
Rosita L. ten Berge ◽  
C. Erik Hack ◽  
Jettie J. F. Muris ◽  
...  
Keyword(s):  
T Cell ◽  
T Lymphocytes ◽  
B Cell ◽  
Hodgkin Lymphoma ◽  
Tumor Cells ◽  
Nk Cell ◽  
Granzyme B ◽  
Protective Mechanism ◽  
Cytotoxic Lymphocytes

In tumor cells, the serine protease granzyme B is the primary mediator of apoptosis induced by cytotoxic T lymphocytes (CTLs)/natural killer (NK) cells. The human intracellular serpin proteinase inhibitor 9 (PI9) is the only known human protein able to inhibit the proteolytic activity of granzyme B. When present in the cytoplasm of T lymphocytes, PI9 is thought to protect CTLs against apoptosis induced by their own misdirected granzyme B. Based on the speculation that tumors may also express PI9 to escape CTL/NK cell surveillance, immunohistochemical studies on the expression of PI9 in various lymphomas were performed. Ninety-two cases of T-cell non-Hodgkin lymphoma (NHL), 75 cases of B-cell NHL, and 57 cases of Hodgkin lymphomas were stained with a PI9-specific monoclonal antibody. In T-cell NHL, highest PI9 expression was found in the extranodal T-cell NHL. In nearly 90% of enteropathy-type T-cell NHLs and 80% of NK/T-cell, nasal-type lymphomas, the majority of the tumor cells expressed PI9. In nodal T-anaplastic large cell lymphomas and peripheral T-cell lymphomas (not otherwise specified), PI9 expression occurred less frequently. In B-cell NHL, PI9 expression was associated with high-grade malignancy; 43% of diffuse large B-cell lymphomas showed PI9+ tumor cells. Finally, PI9 expression was also found in 10% of Hodgkin lymphomas. This is the first report describing the expression of the granzyme B inhibitor PI9 in human neoplastic cells in vivo. Expression of this inhibitor is yet another mechanism used by tumor cells to escape their elimination by cytotoxic lymphocytes.

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