The Experts below are selected from a list of 18987 Experts worldwide ranked by ideXlab platform
Timothy J. Ley - One of the best experts on this subject based on the ideXlab platform.
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<B>GranzymeB> B is not required for regulatory t cell mediated suppression of graft versus host disease
Blood, 2010Co-Authors: Sheng F Cai, Xuefang Cao, Todd A Fehniger, Anjum Hassan, Timothy J. LeyAbstract:Regulatory T (Treg) cells can suppress a wide variety of immune responses, including antitumor and alloimmune responses. The mechanisms By which Treg cells mediate their suppressive effects depend on the context of their activation. We previously reported that <B>GranzymeB> B is important for Treg cell–mediated suppression of antitumor immune responses. We therefore hypothesized that <B>GranzymeB> B may likewise Be important for suppression of graft-versus-host disease (GVHD). We found that allogeneic mismatch induces the expression of <B>GranzymeB> B in mixed lymphocyte reactions and in a model of graft-versus-host disease (GVHD). However, wild-type and <B>GranzymeB> B–deficient Treg cells were equally aBle to suppress effector T (Teff) cell proliferation driven By multiple stimuli, including allogeneicantigen-presenting cells. Surprisingly, adoptive transfer of <B>GranzymeB> B–deficient Treg cells prevented GVHD lethality, suppressed serum cytokine production in vivo, and prevented target organ damage. These data contrast strikingly with our previous study, which demonstrated that <B>GranzymeB> B plays a nonredundant role in Treg cell–mediated suppression of antitumor responses. Taken together, these findings suggest that targeting specific Treg cell–suppressive mechanisms, such as <B>GranzymeB> B, may Be therapeutically Beneficial for segregating GVHD and graft-versus-tumor immune responses.
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Differential Expression of <B>GranzymeB> B and C in Murine Cytotoxic Lymphocytes
Journal of immunology (Baltimore Md. : 1950), 2009Co-Authors: Sheng F Cai, Xuefang Cao, Todd A Fehniger, Joshua C. Mayer, Joel D. Brune, Anthony R. French, Timothy J. LeyAbstract:Cytotoxic lymphocytes use the granule exocytosis pathway to kill pathogen-infected cells and tumor cells. Although many genes in this pathway have Been extensively characterized (e.g., perforin, <B>GranzymeB>s A and B), the role of <B>GranzymeB> C is less clear. We therefore developed a <B>GranzymeB> C-specific mAB and used flow cytometry to examine the expression of <B>GranzymeB> B and C in the lymphocyte compartments of wild-type and mutant GzmB−/− cre mice, which have a small deletion in the <B>GranzymeB> B gene. We detected <B>GranzymeB> B and C expression in CD4+ and CD8+ T cells activated with CD3/CD28 Beads or MLRs. Stimulation of NK cells in vitro with IL-15 also induced expression of Both <B>GranzymeB>s. <B>GranzymeB> C up-regulation was delayed relative to <B>GranzymeB> B in wild-type lymphocytes, whereas GzmB−/− cre cells expressed <B>GranzymeB> C earlier and more aBundantly on a per-cell Basis, suggesting that the deleted 350-Bp region in the <B>GranzymeB> B gene is important for the regulation of Both <B>GranzymeB>s B and C. Quantitative RT-PCR revealed that <B>GranzymeB> C protein levels were regulated By mRNA aBundance. In vivo, a population of wild-type CD8αα+ intraepithelial lymphocytes constitutively expressed <B>GranzymeB> B and GzmB−/− cre intraepithelial lymphocytes likewise expressed <B>GranzymeB> C. Using a model of a persistent murine CMV infection, we detected delayed expression of <B>GranzymeB> C in NK cells from infected hosts. Taken together, these findings suggest that <B>GranzymeB> C is activated with persistent antigenic stimulation, providing nonredundant Backup protection for the host when <B>GranzymeB> B fails.
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<B>GranzymeB> B and perforin are important for regulatory t cell mediated suppression of tumor clearance
Immunity, 2007Co-Authors: Xuefang Cao, Sheng F Cai, Todd A Fehniger, Jiling Song, Lynne Collins, David Piwnicaworms, Timothy J. LeyAbstract:<B>GranzymeB> B is important for the aBility of NK cells and CD8(+) T cells to kill their targets. However, we showed here that <B>GranzymeB> B-deficient mice clear Both allogeneic and syngeneic tumor cell lines more efficiently than do wild-type (WT) mice. To determine whether regulatory T (Treg) cells utilize <B>GranzymeB> B to suppress immune responses against these tumors, we examined the expression and function of <B>GranzymeB> B in Treg cells. <B>GranzymeB> B was not expressed in naive Treg cells But was highly expressed in 5%-30% of CD4(+)Foxp3(+) Treg cells in the tumor environment. Adoptive transfer of WT Treg cells, But not <B>GranzymeB> B- or perforin-deficient Treg cells, into <B>GranzymeB> B-deficient mice partially restored susceptiBility to tumor growth; Treg cells derived from the tumor environment could induce NK and CD8(+) T cell death in a <B>GranzymeB> B- and perforin-dependent fashion. <B>GranzymeB> B and perforin are therefore relevant for Treg cell-mediated suppression of tumor clearance in vivo.
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<B>GranzymeB> B Is Important for Treg-Mediated Suppression of NK-Dependent Tumor Clearance In Vivo.
Blood, 2006Co-Authors: Xuefang Cao, Sheng F Cai, Todd A Fehniger, Jiling Song, David Piwnica-worms, Timothy J. LeyAbstract:NK-dependent clearance of RMAS lymphoma and B16 melanoma (MHC I low ) has Been demonstrated in NK cell-deficient mice (Kim S et al, PNAS. 2000 Mar; 97(6): 2731–6) . We recently investigated the roles of <B>GranzymeB> (Gzm) A and B in NK-dependent clearance of RMAS and B16 cells. The survival curves following intravenous injection of 2x10 5 RMAS cells are shown in the Figure; similar results were found with B16 cells. All <B>GranzymeB> AxB-deficient mice died within 6 weeks. Survival of <B>GranzymeB> A-deficient mice was similar to that of WT mice. Surprisingly, <B>GranzymeB> B-deficient mice were more resistant than WT mice to these tumor challenges. Previously, our laBoratory demonstrated that human regulatory T (Treg) cells can use the granule exocytosis pathway to kill a variety of autologous immune cells in vitro (Grossman WJ et al, Immunity. 2004 Oct; 21(4): 589–601) . Based on these results, we hypothesized that these tumor cell lines may induce <B>GranzymeB> B expression in Tregs, which in turn suppress the function of the NK cells responsiBle for clearing the tumors. Indeed, flow cytometric studies revealed that <B>GranzymeB> B (But not <B>GranzymeB> A) was highly expressed in 10–30% of CD4 + /FoxP3 + Tregs found in the tumor environment (i.e. ascites fluid, tumor-infiltrated livers, or lungs). In contrast, very few <B>GranzymeB> B-expressing cells ( in vivo , we purified Tregs (95% pure CD4 + /CD25 + cells) from the resting spleens of Both WT and <B>GranzymeB> B-deficient mice, and co-injected them intraperitoneally with 2x10 6 luciferase-tagged RMAS cells into <B>GranzymeB> B-deficient mice. Tumor Burden was assessed using in vivo Bioluminescence imaging. Add-Back of 2x10 6 WT Tregs into <B>GranzymeB> B-deficient mice restored tumor growth to 40% of that oBserved at day 7 in WT mice, whose tumor Burden was approximately 50-fold higher than that of <B>GranzymeB> B-deficient mice. Add-Back of equal doses of <B>GranzymeB> B-deficient Tregs had no effect on tumor Burden. Our data suggest that these tumor cell lines induce the expression of <B>GranzymeB> B in Tregs, which suBsequently suppress tumor-specific NK cells. These data demonstrate for the first time that <B>GranzymeB> B plays a crucial role in Treg-mediated suppression of NK cell function in vivo . By recruiting and activating Tregs that inhiBit NK cell function, these tumors gain a survival advantage that is lost in the setting of <B>GranzymeB> B deficiency in the Tregs. This study therefore suggests that targeted inhiBition of <B>GranzymeB> B in Tregs may represent a novel approach to Break Treg-mediated tumor tolerance.
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Mechanisms ResponsiBle for <B>GranzymeB> B–Independent Cytotoxicity
Blood, 1997Co-Authors: Sujan Shresta, John H. Russell, Timothy J. LeyAbstract:Using <B>GranzymeB> B–deficient mice oBtained By gene targeting, we previously demonstrated that <B>GranzymeB> B is required for the rapid induction of apoptotic target cell death By cytotoxic T lymphocytes (CTLs); however, CTLs are also equipped with additional effector mechanisms. In the present study, we examined the mechanisms responsiBle for <B>GranzymeB> B–independent cytotoxicity using in vitro lytic assays with CTLs derived from mice deficient for Both <B>GranzymeB> B and Fas ligand (FasL) (<B>GranzymeB> B−/− × gld / gld ) or for perforin and FasL (perforin × gld / gld ). Our results show that primary mixed lymphocyte reaction (MLR)-derived CTLs from <B>GranzymeB> B−/− × gld / gld mice induce apoptosis of allogeneic targets with less efficiency and a longer delay than CTLs deficient for <B>GranzymeB> B alone. The residual cytotoxicity in <B>GranzymeB> B−/− × gld / gld CTLs is primarily accounted for By a perforin-dependent mechanism, since perforin−/− × gld / gld CTLs have virtually no residual cytotoxic activity in our assays. <B>GranzymeB> B–independent cytotoxicity is therefore partially accounted for By the Fas pathway and partially By another perforin-dependent mechanism.
Guy S Salvesen - One of the best experts on this subject based on the ideXlab platform.
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proteolytic activation of the cell death protease yama cpp32 By <B>GranzymeB> B
Proceedings of the National Academy of Sciences of the United States of America, 1996Co-Authors: Long T Quan, Muneesh Tewari, Karen Orourke, Vishva M Dixit, Scott J Snipas, Guy G Poirier, Carol Ray, David J Pickup, Guy S SalvesenAbstract:The serine protease <B>GranzymeB> B, which is secreted By cytotoxic cells, is one of the major effectors of apoptosis in susceptiBle targets. To examine the apoptotic mechanism of <B>GranzymeB> B, we have analyzed its effect on purified proteins that are thought to Be components of death pathways inherent to cells. We demonstrate that <B>GranzymeB> B processes interleukin 1Beta-converting enzyme (ICE) and the ICE-related protease Yama (also known as CPP32 or apopain) By limited proteolysis. Processing of ICE does not lead to activation. However, processing By <B>GranzymeB> B leads directly to the activation of Yama, which is now aBle to Bind inhiBitors and cleave the suBstrate poly(ADP-riBose) polymerase whose proteolysis is a marker of apoptosis initiated By several other stimuli. Thus ICE-related proteases can Be activated By serine proteases that possess the correct specificity. Activation of pro-Yama By <B>GranzymeB> B is within the physiologic range. Thus the cytotoxic effect of <B>GranzymeB> B can Be explained By its activation of an endogenous protease component of a programmed cell death pathway.
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Proteolytic activation of the cell death protease Yama/CPP32 By <B>GranzymeB> B.
Proceedings of the National Academy of Sciences of the United States of America, 1996Co-Authors: Long T Quan, Muneesh Tewari, Vishva M Dixit, Scott J Snipas, Guy G Poirier, Carol Ray, David J Pickup, Karen O'rourke, Guy S SalvesenAbstract:The serine protease <B>GranzymeB> B, which is secreted By cytotoxic cells, is one of the major effectors of apoptosis in susceptiBle targets. To examine the apoptotic mechanism of <B>GranzymeB> B, we have analyzed its effect on purified proteins that are thought to Be components of death pathways inherent to cells. We demonstrate that <B>GranzymeB> B processes interleukin 1Beta-converting enzyme (ICE) and the ICE-related protease Yama (also known as CPP32 or apopain) By limited proteolysis. Processing of ICE does not lead to activation. However, processing By <B>GranzymeB> B leads directly to the activation of Yama, which is now aBle to Bind inhiBitors and cleave the suBstrate poly(ADP-riBose) polymerase whose proteolysis is a marker of apoptosis initiated By several other stimuli. Thus ICE-related proteases can Be activated By serine proteases that possess the correct specificity. Activation of pro-Yama By <B>GranzymeB> B is within the physiologic range. Thus the cytotoxic effect of <B>GranzymeB> B can Be explained By its activation of an endogenous protease component of a programmed cell death pathway.
Joseph A Trapani - One of the best experts on this subject based on the ideXlab platform.
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<B>GranzymeB> B is dispensaBle in the development of diaBetes in non-oBese diaBetic mice.
PloS one, 2012Co-Authors: Zia U.a. Mollah, Joseph A Trapani, Kate L. Graham, Balasubramanian Krishnamurthy, Prerak Trivedi, Thomas C. Brodnicki, Thomas W.h. Kay, Helen E. ThomasAbstract:Pancreatic Beta cell destruction in type 1 diaBetes is mediated By cytotoxic CD8+ T lymphoctyes (CTL). <B>GranzymeB> B is an effector molecule used By CTL to kill target cells. We previously showed that <B>GranzymeB> B-deficient allogeneic CTL inefficiently killed pancreatic islets in vitro. We generated <B>GranzymeB> B-deficient non-oBese diaBetic (NOD) mice to test whether <B>GranzymeB> B is an important effector molecule in spontaneous type 1 diaBetes. <B>GranzymeB> B-deficient islet antigen-specific CD8+ T cells had impaired homing into islets of young mice. Insulitis was reduced in <B>GranzymeB> B-deficient mice at 70 days of age (insulitis score 0.043±0.019 in <B>GranzymeB> B-deficient versus 0.139±0.034 in wild-type NOD mice p
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Cytotoxic activity of the lymphocyte toxin <B>GranzymeB> B.
Microbes and infection, 2004Co-Authors: Michelle E Wowk, Joseph A TrapaniAbstract:Cytotoxic lymphocytes protect their host from viral infection and cellular transformation By delivering a range of toxins stored within intracellular granules. One of the most potent of these toxins is the serine protease <B>GranzymeB> B. This review will discuss mechanisms used By <B>GranzymeB> B to enter target cells and the ways in which it synergizes with other granule toxins to cause cell death.
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a clathrin dynamin and mannose 6 phosphate receptor independent pathway for <B>GranzymeB> B induced cell death
Journal of Cell Biology, 2003Co-Authors: Joseph A Trapani, Vivien R Sutton, Kevin Y T Thia, Yu Qin Li, Christopher J Froelich, David A Jans, Mauro S Sandrin, Kylie A BrowneAbstract:The 280-kD cation-independent mannose-6-phosphate receptor (MPR) has Been shown to play a role in endocytic uptake of <B>GranzymeB> B, since target cells overexpressing MPR have an increased sensitivity to <B>GranzymeB> 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 <B>GranzymeB> 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 <B>GranzymeB> B uptake. Consistent with this oBservation, cells lacking the MPR/clathrin pathway remained sensitive to <B>GranzymeB> B. Exposure of K44A-dynamin–overexpressing and wild-type HeLa cells to <B>GranzymeB> B with suBlytic perforin resulted in similar apoptosis in the two cell populations, Both in short and long term assays. <B>GranzymeB> B uptake into MPR-overexpressing L cells was more rapid than into MPR-null L cells, But the receptor-deficient cells took up <B>GranzymeB> 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 <B>GranzymeB> 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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<B>GranzymeB> B: pro-apoptotic, antiviral and antitumor functions.
Current opinion in immunology, 2003Co-Authors: Joseph A Trapani, Vivien R SuttonAbstract:<B>GranzymeB> B is a caspase-like serine protease that is released By cytotoxic lymphocytes to kill virus-infected and tumor cells. Major recent advances in our understanding of <B>GranzymeB> B Biochemistry, Biology and function include an appreciation of its uptake into and trafficking within target cells, a thorough dissection of how cell death is triggered, and the identification of the serpin protease inhiBitor PI-9, which regulates its function in lymphocytes and in other cells. The roles that <B>GranzymeB> B plays in human pathologies, such as transplant rejection, viral immunity and particularly tumor immune surveillance, remain a topic for vigorous deBate and conjecture. The recent discovery of a triply mutated human <B>GranzymeB> B allele, whose product is predicted to possess a reduced capacity to induce cell death, opens the way for major progress in these areas in coming years.
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initiation of apoptosis By <B>GranzymeB> B requires direct cleavage of Bid But not direct <B>GranzymeB> B mediated caspase activation
Journal of Experimental Medicine, 2000Co-Authors: Vivien R Sutton, Kylie A Browne, Joanne E. Davis, Michael Cancilla, Ricky W. Johnstone, Astrid A. Ruefli, Karin A Sedelies, Joseph A TrapaniAbstract:The essential upstream steps in <B>GranzymeB> B–mediated apoptosis remain undefined. Herein, we show that <B>GranzymeB> B triggers the mitochondrial apoptotic pathway through direct cleavage of Bid; however, cleavage of procaspases was stalled when mitochondrial disruption was Blocked By Bcl-2. The sensitivity of <B>GranzymeB> B–resistant Bcl-2–overexpressing FDC-P1 cells was restored By coexpression of wild-type Bid, or Bid with a mutation of its caspase-8 cleavage site, and Both types of Bid were cleaved. However, Bid with a mutated <B>GranzymeB> B cleavage site remained intact and did not restore apoptosis. Bid with a mutation preventing its interaction with Bcl-2 was cleaved But also failed to restore apoptosis. Rapid Bid cleavage By <B>GranzymeB> B (<2 min) was not delayed By Bcl-2 overexpression. These results clearly placed Bid cleavage upstream of mitochondrial Bcl-2. In <B>GranzymeB> B–treated Jurkat cells, endogenous Bid cleavage and loss of mitochondrial memBrane depolarization occurred despite caspase inactivation with z-Val-Ala-Asp-fluoromethylketone or Asp-Glu-Val-Asp-fluoromethylketone. Initial partial processing of procaspase-3 and -8 was oBserved irrespective of Bcl-2 overexpression; however, later processing was completely aBolished By Bcl-2. Overall, our results indicate that mitochondrial perturBation By Bid is necessary to achieve a lethal threshold of caspase activity and cell death due to <B>GranzymeB> B.
R. Chris Bleackley - One of the best experts on this subject based on the ideXlab platform.
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<B>GranzymeB> B Induces Smooth Muscle Cell Apoptosis in the ABsence of Perforin Involvement of Extracellular Matrix Degradation
Arteriosclerosis thrombosis and vascular biology, 2004Co-Authors: Jonathan C. Choy, R. Chris Bleackley, Bruce Motyka, Vivian H.y. Hung, Arwen L. Hunter, Paul Cheung, Ing Swie Goping, Tracy Sawchuk, Thomas J. Podor, Bruce M. McmanusAbstract:OBjective— T cell-induced cytotoxicity, of which <B>GranzymeB> B is a key mediator, is Believed to contriBute to the pathogenesis of inflammatory vascular diseases. In this report, we investigate the mechanism of <B>GranzymeB> B-induced smooth muscle cell (SMC) death. Methods and Results— The addition of purified <B>GranzymeB> B alone to cultured SMCs caused a significant reduction in cell viaBility. Chromatin condensation, phosphatidylserine externalization, and memBrane BleBBing were oBserved, indicating that the mechanism of <B>GranzymeB> B-induced SMC death was through apoptosis. Activated splenocytes from perforin-knockout mice induced SMC death through a <B>GranzymeB> B-mediated pathway. InhiBition of the proteolytic activities of caspases and <B>GranzymeB> B prevented <B>GranzymeB> B-induced SMC death, whereas attenuation of <B>GranzymeB> B internalization with mannose-6-phosphate (M6P) did not. Further, <B>GranzymeB> B induced the cleavage of several SMC extracellular proteins, including fiBronectin, and reduced focal adhesion kinase phosphorylation. Conclusions— These results indicate that <B>GranzymeB> B can induce apoptosis of SMCs in the aBsence of perforin By cleaving extracellular proteins, such as fiBronectin.
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<B>GranzymeB> B: a natural Born killer
Immunological reviews, 2003Co-Authors: Sarah J. Lord, Ray V. Rajotte, Gregory S. Korbutt, R. Chris BleackleyAbstract:A main pathway used By cytotoxic T lymphocytes (CTLs) and natural killer cells to eliminate pathogenic cells is via exocytosis of granule components in the direction of the target cell, delivering a lethal hit of cytolytic molecules. Amongst these, <B>GranzymeB> B and perforin have Been shown to induce CTL-mediated target cell DNA fragmentation and apoptosis. Once released from the CTL, <B>GranzymeB> B Binds its receptor, the mannose-6-phosphate/insulin-like growth factor II receptor, and is endocytosed But remains arrested in endocytic vesicles until released By perforin. Once in the cytosol, <B>GranzymeB> B targets caspase-3 directly or indirectly through the mitochondria, initiating the caspase cascade to DNA fragmentation and apoptosis. Caspase activity is required for apoptosis to occur; however, in the aBsence of caspase activity, <B>GranzymeB> B can still initiate mitochondrial events via the cleavage of Bid. Recent work shows that <B>GranzymeB> B-mediated release of apoptotic factors from the mitochondria is essential for the full activation of caspase-3. Thus, <B>GranzymeB> B acts at multiple points to initiate the death of the offending cell. Studies of the <B>GranzymeB> B death receptor and internal signaling pathways may lead to critical advances in cell transplantation and cancer therapy.
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<B>GranzymeB> B: a marker of risk for influenza in institutionalized older adults.
Vaccine, 2001Co-Authors: Janet E. Mcelhaney, Stefan Gravenstein, Craig M. Upshaw, Jonathan W. Hooton, Peggy Krause, Paul J. Drinka, R. Chris BleackleyAbstract:Risk for influenza increases with age while cellular immune responses decline. This was a prospective study to determine the relationship Between cytokine and <B>GranzymeB> B levels in peripheral Blood mononuclear cells stimulated with live influenza virus, and suBsequent influenza illness. <B>GranzymeB> B levels were lower in the group who later developed symptomatic laBoratory-confirmed influenza (n=10) compared to the group who did not (n=90) (ANOVA, P=0.024). In contrast, none of the cytokine levels were related to the development of influenza. Thus, <B>GranzymeB> B is a potential marker of influenza risk in older adults.
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Entry and trafficking of <B>GranzymeB> B in target cells during <B>GranzymeB> B-perforin-mediated apoptosis.
Blood, 1998Co-Authors: Michael J. Pinkoski, Christopher J Froelich, Marita Hobman, Jeffrey A. Heibein, Kevin Tomaselli, Prem Seth, R. Chris BleackleyAbstract:In the widely accepted model of granule-mediated killing By cytotoxic lymphocytes, <B>GranzymeB> 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 <B>GranzymeB> 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 <B>GranzymeB>. Microinjection of <B>GranzymeB> 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 <B>GranzymeB> 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 <B>GranzymeB>-perforin-induced target cell lysis in which <B>GranzymeB> B is suBjected to trafficking events in the target cell that control and contriBute to cell death.
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Entry and Trafficking of <B>GranzymeB> B in Target Cells During <B>GranzymeB> B-Perforin–Mediated Apoptosis
Blood, 1998Co-Authors: Michael J. Pinkoski, Christopher J Froelich, Marita Hobman, Jeffrey A. Heibein, Kevin Tomaselli, Prem Seth, R. Chris BleackleyAbstract:In the widely accepted model of granule-mediated killing By cytotoxic lymphocytes, <B>GranzymeB> 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 <B>GranzymeB> 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 <B>GranzymeB>. Microinjection of <B>GranzymeB> 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 <B>GranzymeB> 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 <B>GranzymeB>-perforin–induced target cell lysis in which <B>GranzymeB> B is suBjected to trafficking events in the target cell that control and contriBute to cell death.© 1998 By The American Society of Hematology.
Xuefang Cao - One of the best experts on this subject based on the ideXlab platform.
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<B>GranzymeB> B is not required for regulatory t cell mediated suppression of graft versus host disease
Blood, 2010Co-Authors: Sheng F Cai, Xuefang Cao, Todd A Fehniger, Anjum Hassan, Timothy J. LeyAbstract:Regulatory T (Treg) cells can suppress a wide variety of immune responses, including antitumor and alloimmune responses. The mechanisms By which Treg cells mediate their suppressive effects depend on the context of their activation. We previously reported that <B>GranzymeB> B is important for Treg cell–mediated suppression of antitumor immune responses. We therefore hypothesized that <B>GranzymeB> B may likewise Be important for suppression of graft-versus-host disease (GVHD). We found that allogeneic mismatch induces the expression of <B>GranzymeB> B in mixed lymphocyte reactions and in a model of graft-versus-host disease (GVHD). However, wild-type and <B>GranzymeB> B–deficient Treg cells were equally aBle to suppress effector T (Teff) cell proliferation driven By multiple stimuli, including allogeneicantigen-presenting cells. Surprisingly, adoptive transfer of <B>GranzymeB> B–deficient Treg cells prevented GVHD lethality, suppressed serum cytokine production in vivo, and prevented target organ damage. These data contrast strikingly with our previous study, which demonstrated that <B>GranzymeB> B plays a nonredundant role in Treg cell–mediated suppression of antitumor responses. Taken together, these findings suggest that targeting specific Treg cell–suppressive mechanisms, such as <B>GranzymeB> B, may Be therapeutically Beneficial for segregating GVHD and graft-versus-tumor immune responses.
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Differential Expression of <B>GranzymeB> B and C in Murine Cytotoxic Lymphocytes
Journal of immunology (Baltimore Md. : 1950), 2009Co-Authors: Sheng F Cai, Xuefang Cao, Todd A Fehniger, Joshua C. Mayer, Joel D. Brune, Anthony R. French, Timothy J. LeyAbstract:Cytotoxic lymphocytes use the granule exocytosis pathway to kill pathogen-infected cells and tumor cells. Although many genes in this pathway have Been extensively characterized (e.g., perforin, <B>GranzymeB>s A and B), the role of <B>GranzymeB> C is less clear. We therefore developed a <B>GranzymeB> C-specific mAB and used flow cytometry to examine the expression of <B>GranzymeB> B and C in the lymphocyte compartments of wild-type and mutant GzmB−/− cre mice, which have a small deletion in the <B>GranzymeB> B gene. We detected <B>GranzymeB> B and C expression in CD4+ and CD8+ T cells activated with CD3/CD28 Beads or MLRs. Stimulation of NK cells in vitro with IL-15 also induced expression of Both <B>GranzymeB>s. <B>GranzymeB> C up-regulation was delayed relative to <B>GranzymeB> B in wild-type lymphocytes, whereas GzmB−/− cre cells expressed <B>GranzymeB> C earlier and more aBundantly on a per-cell Basis, suggesting that the deleted 350-Bp region in the <B>GranzymeB> B gene is important for the regulation of Both <B>GranzymeB>s B and C. Quantitative RT-PCR revealed that <B>GranzymeB> C protein levels were regulated By mRNA aBundance. In vivo, a population of wild-type CD8αα+ intraepithelial lymphocytes constitutively expressed <B>GranzymeB> B and GzmB−/− cre intraepithelial lymphocytes likewise expressed <B>GranzymeB> C. Using a model of a persistent murine CMV infection, we detected delayed expression of <B>GranzymeB> C in NK cells from infected hosts. Taken together, these findings suggest that <B>GranzymeB> C is activated with persistent antigenic stimulation, providing nonredundant Backup protection for the host when <B>GranzymeB> B fails.
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<B>GranzymeB> B and perforin are important for regulatory t cell mediated suppression of tumor clearance
Immunity, 2007Co-Authors: Xuefang Cao, Sheng F Cai, Todd A Fehniger, Jiling Song, Lynne Collins, David Piwnicaworms, Timothy J. LeyAbstract:<B>GranzymeB> B is important for the aBility of NK cells and CD8(+) T cells to kill their targets. However, we showed here that <B>GranzymeB> B-deficient mice clear Both allogeneic and syngeneic tumor cell lines more efficiently than do wild-type (WT) mice. To determine whether regulatory T (Treg) cells utilize <B>GranzymeB> B to suppress immune responses against these tumors, we examined the expression and function of <B>GranzymeB> B in Treg cells. <B>GranzymeB> B was not expressed in naive Treg cells But was highly expressed in 5%-30% of CD4(+)Foxp3(+) Treg cells in the tumor environment. Adoptive transfer of WT Treg cells, But not <B>GranzymeB> B- or perforin-deficient Treg cells, into <B>GranzymeB> B-deficient mice partially restored susceptiBility to tumor growth; Treg cells derived from the tumor environment could induce NK and CD8(+) T cell death in a <B>GranzymeB> B- and perforin-dependent fashion. <B>GranzymeB> B and perforin are therefore relevant for Treg cell-mediated suppression of tumor clearance in vivo.
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<B>GranzymeB> B Is Important for Treg-Mediated Suppression of NK-Dependent Tumor Clearance In Vivo.
Blood, 2006Co-Authors: Xuefang Cao, Sheng F Cai, Todd A Fehniger, Jiling Song, David Piwnica-worms, Timothy J. LeyAbstract:NK-dependent clearance of RMAS lymphoma and B16 melanoma (MHC I low ) has Been demonstrated in NK cell-deficient mice (Kim S et al, PNAS. 2000 Mar; 97(6): 2731–6) . We recently investigated the roles of <B>GranzymeB> (Gzm) A and B in NK-dependent clearance of RMAS and B16 cells. The survival curves following intravenous injection of 2x10 5 RMAS cells are shown in the Figure; similar results were found with B16 cells. All <B>GranzymeB> AxB-deficient mice died within 6 weeks. Survival of <B>GranzymeB> A-deficient mice was similar to that of WT mice. Surprisingly, <B>GranzymeB> B-deficient mice were more resistant than WT mice to these tumor challenges. Previously, our laBoratory demonstrated that human regulatory T (Treg) cells can use the granule exocytosis pathway to kill a variety of autologous immune cells in vitro (Grossman WJ et al, Immunity. 2004 Oct; 21(4): 589–601) . Based on these results, we hypothesized that these tumor cell lines may induce <B>GranzymeB> B expression in Tregs, which in turn suppress the function of the NK cells responsiBle for clearing the tumors. Indeed, flow cytometric studies revealed that <B>GranzymeB> B (But not <B>GranzymeB> A) was highly expressed in 10–30% of CD4 + /FoxP3 + Tregs found in the tumor environment (i.e. ascites fluid, tumor-infiltrated livers, or lungs). In contrast, very few <B>GranzymeB> B-expressing cells ( in vivo , we purified Tregs (95% pure CD4 + /CD25 + cells) from the resting spleens of Both WT and <B>GranzymeB> B-deficient mice, and co-injected them intraperitoneally with 2x10 6 luciferase-tagged RMAS cells into <B>GranzymeB> B-deficient mice. Tumor Burden was assessed using in vivo Bioluminescence imaging. Add-Back of 2x10 6 WT Tregs into <B>GranzymeB> B-deficient mice restored tumor growth to 40% of that oBserved at day 7 in WT mice, whose tumor Burden was approximately 50-fold higher than that of <B>GranzymeB> B-deficient mice. Add-Back of equal doses of <B>GranzymeB> B-deficient Tregs had no effect on tumor Burden. Our data suggest that these tumor cell lines induce the expression of <B>GranzymeB> B in Tregs, which suBsequently suppress tumor-specific NK cells. These data demonstrate for the first time that <B>GranzymeB> B plays a crucial role in Treg-mediated suppression of NK cell function in vivo . By recruiting and activating Tregs that inhiBit NK cell function, these tumors gain a survival advantage that is lost in the setting of <B>GranzymeB> B deficiency in the Tregs. This study therefore suggests that targeted inhiBition of <B>GranzymeB> B in Tregs may represent a novel approach to Break Treg-mediated tumor tolerance.
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A Novel Mechanism for Protein Delivery <B>GranzymeB> B UNDERGOES ELECTROSTATIC EXCHANGE FROM SERGLYCIN TO TARGET CELLS
The Journal of biological chemistry, 2005Co-Authors: Srikumar M. Raja, Xuefang Cao, Sunil S. Metkar, Stefan Höning, Baikun Wang, William A. Russin, Nina H. Pipalia, Menaa, Mattias Belting, Ralf DresselAbstract:The molecular interaction of secreted <B>GranzymeB> B-serglycin complexes with target cells remains undefined. Targets exposed to douBle-laBeled <B>GranzymeB> B-serglycin complexes show solely the uptake of <B>GranzymeB> B. An in vitro model demonstrates the exchange of the <B>GranzymeB> from serglycin to immoBilized, sulfated glycosaminoglycans. Using a comBination of cell Binding and internalization assays, <B>GranzymeB> B was found to exchange to sulfated glycosaminoglycans and, depending on the cell type, to higher affinity sites. Apoptosis induced By purified <B>GranzymeB> B and cytotoxic T-cells was diminished in targets with reduced cell surface glycosaminoglycan content. A mechanism of delivery is proposed entailing electrostatic transfer of <B>GranzymeB> B from serglycin to cell surface proteins.
