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Transcriptomic profiling of adjuvant colorectal cancer identifies three key prognostic biological processes and a disease specific role for granzyme B. PLoS One 2022; 16:e0262198. [PMID: 34972191 PMCID: PMC8719661 DOI: 10.1371/journal.pone.0262198] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/23/2020] [Accepted: 11/25/2021] [Indexed: 12/20/2022] Open
Abstract
Background Colorectal cancer (CRC) is a leading cause of cancer-related deaths, with a 5% 5-year survival rate for metastatic disease, yet with limited therapeutic advancements due to insufficient understanding of and inability to accurately capture high-risk CRC patients who are most likely to recur. We aimed to improve high-risk classification by identifying biological pathways associated with outcome in adjuvant stage II/III CRC. Methods and findings We included 1062 patients with stage III or high-risk stage II colon carcinoma from the prospective three-arm randomized phase 3 AVANT trial, and performed expression profiling to identify a prognostic signature. Data from validation cohort GSE39582, The Cancer Genome Atlas, and cell lines were used to further validate the prognostic biology. Our retrospective analysis of the adjuvant AVANT trial uncovered a prognostic signature capturing three biological functions—stromal, proliferative and immune—that outperformed the Consensus Molecular Subtypes (CMS) and recurrence prediction signatures like Oncotype Dx in an independent cohort. Importantly, within the immune component, high granzyme B (GZMB) expression had a significant prognostic impact while other individual T-effector genes were less or not prognostic. In addition, we found GZMB to be endogenously expressed in CMS2 tumor cells and to be prognostic in a T cell independent fashion. A limitation of our study is that these results, although robust and derived from a large dataset, still need to be clinically validated in a prospective study. Conclusions This work furthers our understanding of the underlying biology that propagates stage II/III CRC disease progression and provides scientific rationale for future high-risk stratification and targeted treatment evaluation in biomarker defined subpopulations of resectable high-risk CRC. Our results also shed light on an alternative GZMB source with context-specific implications on the disease’s unique biology.
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Lu CC, Wu TS, Hsu YJ, Chang CJ, Lin CS, Chia JH, Wu TL, Huang TT, Martel J, Ojcius DM, Young JD, Lai HC. NK cells kill mycobacteria directly by releasing perforin and granulysin. J Leukoc Biol 2014; 96:1119-29. [DOI: 10.1189/jlb.4a0713-363rr] [Citation(s) in RCA: 72] [Impact Index Per Article: 6.5] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/27/2022] Open
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CD94 surface density identifies a functional intermediary between the CD56bright and CD56dim human NK-cell subsets. Blood 2009; 115:274-81. [PMID: 19897577 DOI: 10.1182/blood-2009-04-215491] [Citation(s) in RCA: 183] [Impact Index Per Article: 11.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/21/2022] Open
Abstract
Human CD56(bright) natural killer (NK) cells possess little or no killer immunoglobulin-like receptors (KIRs), high interferon-gamma (IFN-gamma) production, but little cytotoxicity. CD56(dim) NK cells have high KIR expression, produce little IFN-gamma, yet display high cytotoxicity. We hypothesized that, if human NK maturation progresses from a CD56(bright) to a CD56(dim) phenotype, an intermediary NK cell must exist, which demonstrates more functional overlap than these 2 subsets, and we used CD94 expression to test our hypothesis. CD94(high)CD56(dim) NK cells express CD62L, CD2, and KIR at levels between CD56(bright) and CD94(low)CD56(dim) NK cells. CD94(high)CD56(dim) NK cells produce less monokine-induced IFN-gamma than CD56(bright) NK cells but much more than CD94(low)CD56(dim) NK cells because of differential interleukin-12-mediated STAT4 phosphorylation. CD94(high)CD56(dim) NK cells possess a higher level of granzyme B and perforin expression and CD94-mediated redirected killing than CD56(bright) NK cells but lower than CD94(low)CD56(dim) NK cells. Collectively, our data suggest that the density of CD94 surface expression on CD56(dim) NK cells identifies a functional and likely developmental intermediary between CD56(bright) and CD94(low)CD56(dim) NK cells. This supports the notion that, in vivo, human CD56(bright) NK cells progress through a continuum of differentiation that ends with a CD94(low)CD56(dim) phenotype.
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4
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Voskoboinik I, Smyth MJ, Trapani JA. Perforin-mediated target-cell death and immune homeostasis. Nat Rev Immunol 2007; 6:940-52. [PMID: 17124515 DOI: 10.1038/nri1983] [Citation(s) in RCA: 432] [Impact Index Per Article: 24.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/30/2022]
Abstract
The granule exocytosis pathway of cytotoxic lymphocytes is crucial for immune surveillance and homeostasis. The trafficking of granule components, including the membrane-disruptive protein perforin, to the immunological synapse leads to the delivery of granule proteases (granzymes) into the target cell and its destruction through apoptosis. Several independent molecular abnormalities associated with defects of either granule trafficking or perforin function can cause cytotoxic lymphocyte dysfunction. In humans, inherited perforin mutations result in severe immune dysregulation that manifests as familial haemophagocytic lymphohistiocytosis. This Review describes recent progress in defining the structure, function, biochemistry and cell biology of perforin.
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Affiliation(s)
- Ilia Voskoboinik
- Cancer Immunology Program, Peter MacCallum Cancer Centre, St. Andrew's Place, East Melbourne, Victoria 3002, Australia.
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5
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Lettau M, Schmidt H, Kabelitz D, Janssen O. Secretory lysosomes and their cargo in T and NK cells. Immunol Lett 2006; 108:10-9. [PMID: 17097742 DOI: 10.1016/j.imlet.2006.10.001] [Citation(s) in RCA: 62] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/06/2006] [Accepted: 10/10/2006] [Indexed: 11/22/2022]
Abstract
Secretory lysosomes are specialized organelles that combine catabolic functions of conventional lysosomes with an inducible secretory potential. They are present in various hematopoietic cell types commonly characterized by the need for rapid mobilization and secretion of effector proteins. As an example, the cytotoxic effector function of T cells and natural killer cells strictly depends on the activation-dependent mobilization of such vesicles to the cytotoxic immunological synapse. This review focuses on some molecules that have been identified as cargo of secretory lysosomes and which play a major role in effector function of CTL and NK cells. We also briefly point to the fact that the dysregulation of formation and transport of secretory vesicles is causative for severe immunodeficiencies and autoimmunity observed in patients and also in mice that have been used as representative model systems to analyze the pathophysiological relevance of secretory vesicles in vivo.
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Affiliation(s)
- Marcus Lettau
- Institute of Immunology, Medical Center Schleswig-Holstein Campus Kiel, Michaelisstr. 5, D-24105 Kiel, Germany
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Savas B, Kerr PE, Pross HF. Lymphokine-activated killer cell susceptibility and adhesion molecule expression of multidrug resistant breast carcinoma. Cancer Cell Int 2006; 6:24. [PMID: 17081316 PMCID: PMC1635735 DOI: 10.1186/1475-2867-6-24] [Citation(s) in RCA: 12] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/18/2005] [Accepted: 11/03/2006] [Indexed: 11/25/2022] Open
Abstract
Reports showing susceptibility of multidrug resistant (MDR) cancer cells to immune effectors, together with P-glycoprotein (P-gp) expression in immune effector subsets, including immature natural killer (NK) cells, and some activated T cells, suggest P-gp or some changes associated with it, have implications in immune-mediated mechanisms. A series of experiments were done to determine the nature of alterations associated with susceptibility to immune effector cells of MDR tumor cells. A cell line isolated from the malignant pleural effusion of a breast cancer patient was transfected with human and murine MDR1 genes, and four variants with different levels of MDR were obtained. Lymphokine-activated killer (LAK) activity was measured by a 51Chromium release, and conjugate formation assays. MDR1 transfectant P-gp+ breast carcinoma lines had increased LAK susceptibility compared to their parent line. Some part of the increased LAK susceptibility of drug-resistant cell lines was at the binding/recognition level as shown by conjugate formation assays. This suggests that differences may exist between paired cell lines with respect to the expression of cell adhesion molecules (CAMs). Monoclonal antibodies (mAbs) to CAMs and flow cytometry were used to quantitate these antigens. The CAMs studied were those previously found to be upregulated by stimulating NK cells with (interleukin-2) IL-2; ICAM-1 (CD54), LFA-3 (CD58), N-CAM (CD56), and the β chain of LFA-1 (CD18). Although no differences in these CAMs were found between the breast carcinoma line and its MDR1-transfected variants, the target susceptibility results given above suggest that IL-2 treatment could be effective in combination with current protocols using chemotherapeutics, monoclonal antibodies (mAbs) and stem cell transplantation.
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Affiliation(s)
- Burhan Savas
- Dept. of Oncology, Akdeniz University, Antalya, Turkiye
- Dept. of Microbiology and Immunology, Queen's University, Kingston, Canada
| | - Pauline E Kerr
- Dept. of Microbiology and Immunology, Queen's University, Kingston, Canada
| | - Hugh F Pross
- Dept. of Microbiology and Immunology, Queen's University, Kingston, Canada
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7
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Abstract
Perforin is a cytolytic protein stored in secretory granules of CTL and NK cells. It synergizes with proapoptotic serine proteases, granzymes, to deliver the lethal hit to virus-infected or transformed target cells. The mechanism of perforin action has not been described beyond its original characterization in the 1980s, and its role in human disease has remained elusive. This article addresses recent key advances in genetic, clinical and biochemical studies that have reignited the current interest in perforin biology.
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Affiliation(s)
- Ilia Voskoboinik
- Cancer Immunology Program, Peter MacCallum Cancer Centre, Melbourne, Victoria, Australia.
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8
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Catlin R, Shah H, Bankhurst AD, Whalen MM. Dibutyltin exposure decreases granzyme B and perforin in human natural killer cells. ENVIRONMENTAL TOXICOLOGY AND PHARMACOLOGY 2005; 20:395-403. [PMID: 21783618 DOI: 10.1016/j.etap.2005.03.012] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Received: 11/10/2004] [Accepted: 03/15/2005] [Indexed: 05/31/2023]
Abstract
Natural killer (NK) cells are a subset of lymphocytes that are capable of killing tumor and virally-infected cells. Dibutyltin (DBT) is a catalyst in the production of PVC plastics and a breakdown product of tributyltin (TBT). DBT is a significant environmental contaminant. This study investigates the mechanism by which DBT exposure decreases the immune function of human NK cells. NK cells destroy their target cells by releasing cytotoxic proteins, perforin, and granzyme B. We examined the effect of DBT exposures on the levels of cytotoxic proteins and their mRNAs. Exposure of NK cells to DBT for 1h caused significant decreases in the mRNAs for granzyme B and perforin but not in protein levels. A 24h exposure to DBT decreased mRNAs as well as protein levels for both granzyme B and perforin. Exposure to DBT for 1h followed by either a 24 or 48h period in DBT-free media, decreased levels of granzyme B and perforin. The results indicate that decreases in granzyme B and perforin levels in NK cells are consequences of DBT exposure. Additionally, DBT causes rapid decreases in mRNAs for perforin and granzyme B, suggesting decreases in transcription and/or increases in mRNA degradation.
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Affiliation(s)
- Reetta Catlin
- Department of Biological Sciences, Tennessee State University, Nashville, TN 37209, USA
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9
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Thomas LD, Shah H, Bankhurst AD, Whalen MM. Effects of interleukins 2 and 12 on the levels of granzyme B and perforin and their mRNAs in tributyltin-exposed human natural killer cells. Arch Toxicol 2005; 79:711-20. [PMID: 16032371 DOI: 10.1007/s00204-005-0002-z] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/21/2005] [Accepted: 06/01/2005] [Indexed: 10/25/2022]
Abstract
Natural killer (NK) cells are a subset of lymphocytes that are capable of killing tumor cells, virally infected cells and antibody coated cells. Tributyltin (TBT) is a toxic chemical used for various industrial purposes such as: slime control in paper mills, disinfection of circulating industrial cooling waters, anti-fouling agents, and the preservation of wood. TBT can be found in edible items such as fish. A previous study showed that a 1 h exposure of NK cells to TBT caused persistent inhibition of NK-cell ability to destroy tumor cells in the 24 and 48 h periods following exposure and that this loss of function could be significantly prevented and/or reversed if the NK-stimulatory interleukins (IL) 2 or 12 were present during the 24 and 48 h periods. We had also shown that TBT exposure was able to significantly decrease the protein and mRNA levels of the cytotoxic proteins, granzyme B and perforin, and the phosphorylation of cAMP-response-element-binding protein (CREB) under these conditions. In this study we address the effects of IL-2 and IL-12 on the TBT-induced decreases in NK-cell levels of the cytotoxic proteins, their mRNAs, and CREB phosphorylation. IL-2 appeared to prevent/reverse TBT-induced declines in perforin protein levels and the mRNA for perforin seen in the 24 h period following a 1 h exposure to 300 nM TBT. However, the TBT-induced decreases in the levels of perforin and perforin mRNA seen in the 48 h period following a 1 h exposure to TBT were not statistically significantly prevented/reversed by IL-2. Additionally, the TBT-induced decreases in granzyme B, granzyme B mRNA, and CREB phosphorylation were not statistically significantly reversed by either IL-2 or IL-12 after 24 or 48 h.
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Affiliation(s)
- LeeShawn D Thomas
- Department of Biological Sciences, Tennessee State University, Nashville, TN 37209-1561, USA
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10
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Thomas LD, Shah H, Green SA, Bankhurst AD, Whalen MM. Tributyltin exposure causes decreased granzyme B and perforin levels in human natural killer cells. Toxicology 2004; 200:221-33. [PMID: 15212818 DOI: 10.1016/j.tox.2004.04.002] [Citation(s) in RCA: 29] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/09/2004] [Revised: 03/13/2004] [Accepted: 04/10/2004] [Indexed: 10/26/2022]
Abstract
Natural Killer (NK) cells are a subset of lymphocytes that are capable of killing tumor cells, virally infected cells and antibody coated cells. Tributyltin (TBT) is a toxic chemical used for various industrial purposes such as: slime control in paper mills, disinfection of circulating industrial cooling waters, anti-fouling agents in shower curtains and the preservation of wood. TBT can be found in edible items such as dairy products and fish. This study investigates the mechanism by which TBT exposure decreases the immune function of human NK cells, in vitro. Cytotoxic function, the expression of the cytotoxic proteins (granzyme B and perforin), and cAMP response element binding protein (CREB) phosphorylation were examined. NK cells exposed to 300 nM TBT for 1 h showed no significant decrease in cytotoxic function, levels of granzyme B and perforin, or phosphorylation of CREB. However, mRNA levels for the cytotoxic proteins were significantly decreased. A 24 h exposure to 200 nM TBT caused significant decreases in cytotoxic function, levels of granzyme B and perforin, and levels of granzyme B and perforin mRNA. When NK cells were exposed to 300 nM TBT for 1h followed by a 24 h period in TBT-free media, again there were significant decreases in NK cell cytotoxic function, levels of granzyme B and perforin and their mRNA. A 1h exposure to 300 nM TBT followed by a 48 h period in TBT-free media showed similar changes in cytotoxic function and levels of granzyme B and perforin as seen after 24 h in TBT-free media. Additionally, both of these exposures showed significant decreases in phosphorylation of CREB. These results indicate that TBT exposures can disrupt the transcription of granzyme B and perforin and that this disruption cannot be entirely accounted for by a decrease in phosphorylated CREB (phosphoCREB) levels.
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Affiliation(s)
- LeeShawn D Thomas
- Department of Medicine, University of New Mexico School of Medicine, Albuquerque, NM 87131, USA
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11
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Acute myeloblastic leukemic cells acquire cellular cytotoxicity under genotoxic stress: implication of granzyme B and perforin. Blood 2000. [DOI: 10.1182/blood.v96.5.1914.h8001914_1914_1920] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022] Open
Abstract
Granzyme B (GrB) and perforin (PFN) are the major components of cytoplasmic granules contained in immune cellular effectors. The granule secretory pathway is one of the mechanisms by which these cells exert their cellular cytotoxicity. Recently, it has been reported that GrB and PFN are also present in circulating hemopoietic CD34+ progenitor cells mobilized by chemotherapy and granulocyte-colony stimulating factor, whereas these proteins are undetected in steady-state peripheral CD34+ cells. In this study, we hypothesized that anticancer agents may increase GrB and PFN expression in immature myeloid leukemic cells and that these treated leukemic cells become cellular effectors through a granule-dependent mechanism. Our results show that KG1a, HEL, and TF-1 CD34+acute myeloblastic leukemia cells expressed both GrB and PFN. Moreover, ionizing radiation, aracytine, and etoposide not only increase GrB and PFN expression but also conferred potent cellular cytotoxicity to these cells toward various cellular targets. Cellular cytotoxicity required cell-cell contact, was not influenced by anti-tumor necrosis factor α or anti-Fas blocking antibodies, and was abrogated by GrB inhibitors or antisense. These results suggest that, when exposed to genotoxic agents, immature leukemic cells acquire potent GrB- and PFN-dependent cellular cytotoxicity that can be potentially directed against normal residual myeloid progenitors or immune effectors.
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12
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Acute myeloblastic leukemic cells acquire cellular cytotoxicity under genotoxic stress: implication of granzyme B and perforin. Blood 2000. [DOI: 10.1182/blood.v96.5.1914] [Citation(s) in RCA: 20] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022] Open
Abstract
AbstractGranzyme B (GrB) and perforin (PFN) are the major components of cytoplasmic granules contained in immune cellular effectors. The granule secretory pathway is one of the mechanisms by which these cells exert their cellular cytotoxicity. Recently, it has been reported that GrB and PFN are also present in circulating hemopoietic CD34+ progenitor cells mobilized by chemotherapy and granulocyte-colony stimulating factor, whereas these proteins are undetected in steady-state peripheral CD34+ cells. In this study, we hypothesized that anticancer agents may increase GrB and PFN expression in immature myeloid leukemic cells and that these treated leukemic cells become cellular effectors through a granule-dependent mechanism. Our results show that KG1a, HEL, and TF-1 CD34+acute myeloblastic leukemia cells expressed both GrB and PFN. Moreover, ionizing radiation, aracytine, and etoposide not only increase GrB and PFN expression but also conferred potent cellular cytotoxicity to these cells toward various cellular targets. Cellular cytotoxicity required cell-cell contact, was not influenced by anti-tumor necrosis factor α or anti-Fas blocking antibodies, and was abrogated by GrB inhibitors or antisense. These results suggest that, when exposed to genotoxic agents, immature leukemic cells acquire potent GrB- and PFN-dependent cellular cytotoxicity that can be potentially directed against normal residual myeloid progenitors or immune effectors.
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Luo DZ, Vermijlen D, Ahishali B, Triantis V, Plakoutsi G, Braet F, Vanderkerken K, Wisse E. On the cell biology of pit cells, the liver-specific NK cells. World J Gastroenterol 2000; 6:1-11. [PMID: 11819514 PMCID: PMC4723571 DOI: 10.3748/wjg.v6.i1.1] [Citation(s) in RCA: 21] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 09/22/1999] [Revised: 11/02/1999] [Accepted: 11/15/1999] [Indexed: 02/06/2023] Open
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Affiliation(s)
- A J Darmon
- Medical Research Council Laboratory for Molecular Cell Biology, University College London, UK
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15
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Sayers TJ, Brooks AD, Lee JK, Fenton RG, Komschlies KL, Wigginton JM, Winkler-Pickett R, Wiltrout RH. Molecular Mechanisms of Immune-Mediated Lysis of Murine Renal Cancer: Differential Contributions of Perforin-Dependent Versus Fas-Mediated Pathways in Lysis by NK and T Cells. THE JOURNAL OF IMMUNOLOGY 1998. [DOI: 10.4049/jimmunol.161.8.3957] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/01/2023]
Abstract
Abstract
Mice bearing the experimental murine renal cancer Renca can be successfully treated with some forms of immunotherapy. In the present study, we have investigated the molecular pathways used by NK and T cells to lyse Renca cells. Renca cells normally express low levels of Fas that can be substantially enhanced by either IFN-γ or TNF-α, and the combination of IFN-γ + TNF-α synergistically enhances cell-surface Fas expression. In addition, cells pretreated with IFN-γ and TNF-α are sensitive to lysis mediated by Fas ligand (FasL)-expressing hybridomas (dllS), cross-linking of anti-Fas Abs or soluble Fas (FasL). Lysis via Fas occurs by apoptosis, since Renca shows all the typical characteristics of apoptosis. No changes in levels of bcl-2 were observed after cytokine treatments. We also examined cell-mediated cytotoxic effects using activated NK cells and T cells from gld FasL-deficient mice, and perforin-deficient mice, as well as wild-type C57BL/6 and BALB/c mice. Interestingly, the granule-mediated pathway predominated in killing of Renca by activated NK cells, while the Fas/FasL pathway contributed significantly to cell-mediated killing of Renca by activated T cells. These results suggest that killing of Renca tumor cells by immune effector cells can occur by both granule and Fas-mediated cytotoxicity. However, for the Fas-mediated pathway to function, cell surface levels of Fas need to be increased beyond a critical threshold level by proinflammatory cytokines such as IFN-γ and TNF-α.
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Affiliation(s)
- Thomas J. Sayers
- *Intramural Research Support Program, Science Applications International Corporation-Frederick,
| | - Alan D. Brooks
- *Intramural Research Support Program, Science Applications International Corporation-Frederick,
| | - Jong-Keuk Lee
- †Laboratory of Experimental Immunology, Division of Basic Sciences, National Cancer Institute-Frederick Cancer Research and Development Center, and
| | - Robert G. Fenton
- ‡Department of Experimental Transplantation and Immunology, Division of Clinical Sciences, National Cancer Institute-Frederick Cancer Research and Development Center, Frederick, MD 21702; and
| | - Kristin L. Komschlies
- *Intramural Research Support Program, Science Applications International Corporation-Frederick,
| | - Jon M. Wigginton
- §Pediatric Oncology Branch, DCS, National Cancer Institute, Bethesda, MD 20892
| | - Robin Winkler-Pickett
- †Laboratory of Experimental Immunology, Division of Basic Sciences, National Cancer Institute-Frederick Cancer Research and Development Center, and
| | - Robert H. Wiltrout
- †Laboratory of Experimental Immunology, Division of Basic Sciences, National Cancer Institute-Frederick Cancer Research and Development Center, and
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Trapani JA. Dual mechanisms of apoptosis induction by cytotoxic lymphocytes. INTERNATIONAL REVIEW OF CYTOLOGY 1998; 182:111-92. [PMID: 9522460 DOI: 10.1016/s0074-7696(08)62169-5] [Citation(s) in RCA: 57] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 02/06/2023]
Abstract
Cytotoxic T lymphocytes and natural killer cells together comprise the means by which the immune system detects and rids higher organisms of virus-infected or transformed cells. Although differing considerably in the way they detect foreign or mutated antigens, these cells utilize highly analogous mechanisms for inducing target cell death. Both types of effector lymphocytes utilize two principal contact-dependent cytolytic mechanisms. The first of these, the granule exocytosis mechanism, depends on the synergy of a calcium-dependent pore-forming protein, perforin, and a battery of proteases (granzymes), and it results in penetration by effector molecules into the target cell cytoplasm and nucleus. The second, which requires binding of FasL (CD95L) on the effector cell with trimeric Fas (CD95) molecules on receptive target cells, is calcium independent and functions by generating a death signal at the inner leaflet of the target cell membrane. Exciting recent developments have indicated that both cytolytic mechanisms impinge on an endogenous signaling pathway that is strongly conserved in species as diverse as helminths and humans and dictates the death or survival of all cells.
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Affiliation(s)
- J A Trapani
- John Connell Cellular Cytotoxicity Laboratory, Austin Research Institute, Heidelberg, Australia
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17
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Jans DA, Jans P, Briggs LJ, Sutton V, Trapani JA. Nuclear transport of granzyme B (fragmentin-2). Dependence of perforin in vivo and cytosolic factors in vitro. J Biol Chem 1996; 271:30781-9. [PMID: 8940058 DOI: 10.1074/jbc.271.48.30781] [Citation(s) in RCA: 92] [Impact Index Per Article: 3.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/03/2023] Open
Abstract
Cytotoxic T and natural killer cells are able to kill their target cells through synergistic action of the pore-forming protein perforin and the serine protease granzyme B, resulting in very distinctive nuclear changes typical of apoptosis. Whereas perforin acts at the membrane, granzyme B appears to be both capable of entering the cytoplasm of target cells and accumulating in isolated nuclei. In this study we examine nuclear transport of fluoresceinated granzyme B both in vivo in intact cells in the presence of perforin and in vitro in semi-permeabilized cells using confocal laser scanning microscopy. Granzyme B alone was observed to enter the cytoplasm of intact cells but did not accumulate in nuclei. In the presence of sublytic concentrations of perforin, however, it accumulated strongly in intact cell nuclei to levels maximally about 1.5 times those in the cytoplasm after about 2.5 h. In vitro nuclear transport assays showed maximal levels of nuclear and nucleolar accumulation of granzyme B of about 2.5- and 3-fold those in the cytoplasm. In contrast to signal-dependent nuclear accumulation of SV40 large tumor antigen (T-Ag) fusion proteins in vitro, nuclear/nucleolar import of granzyme B was independent of ATP and not inhibitable by the non-hydrolyzable GTP analog GTPgammaS (guanosine 5'-O-(3-thiotriphosphate)). Similar to T-Ag fusion proteins, however, granzyme B nuclear and nucleolar accumulation was dependent on exogenously added cytosol. Specific inhibitors of granzyme B protease activity had no effect on nuclear/nucleolar accumulation, implying that proteolytic activity was not essential for nuclear targeting. The results imply that granzyme B (32 kDa) may be transported from the cytoplasm to the nucleus through passive diffusion and accumulate by binding to nuclear/nucleolar factors in a cytosolic factor-mediated process. Active and passive nuclear transport properties were normal in the presence of unlabeled granzyme B, implying that the nuclear envelope and pore complex are not granzyme B substrates.
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Affiliation(s)
- D A Jans
- Nuclear Signalling Laboratory, Division for Biochemistry and Molecular Biology, John Curtin School of Medical Research, Canberra, Australian Capital Territory 2601, Australia.
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18
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Xiao L, Eneroth PH, Qureshi GA. Nitric oxide synthase pathway may mediate human natural killer cell cytotoxicity. Scand J Immunol 1995; 42:505-11. [PMID: 7481553 DOI: 10.1111/j.1365-3083.1995.tb03687.x] [Citation(s) in RCA: 40] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/25/2023]
Abstract
The present study provides evidence that the human natural killer (NK) cell effector mechanism causing target cytolysis has a requirement for L-arginine. In a deficient medium (DM) containing only salts, buffer system and glucose, NK cell-mediated cytotoxicity was found to decrease by 70% as compared to that obtained in a complete medium (CM). However, adding L-arginine to such DM could restore the activity of NK cells to the normal level. Many other components of CM, such as serum, glutamine and vitamins did not improve NK cell-mediated killing in DM. When all amino acids except L-arginine were added to DM only a partial recovery of NK cell functional cytolysis was seen. L-arginine enhanced the NK cell activity in a dose-dependent manner. Additionally, the inhibitor of both inducible and constitutive nitric oxide synthase, N-monomethyl-L-arginine (L-NMMA) inhibited NK cytolytic activity in DM supplemented with L-arginine indicating participation of nitric oxide (NO). The results also show that the stimulatory effect of L-arginine on human NK cell-mediated cytotoxicity was accompanied by an increase in NO formation as determined by accumulation of nitrite and citrulline. L-NMMA gave a dose-dependent reduction in NO generation as well. The nitrite and citrulline production dose-dependently correlated with not only the concentration of L-arginine in the cultivation medium, but also the enhanced NK cell-mediated cytolysis. Taken together, these findings could define a L-arginine/NO-linked effector mechanism in human NK cells. Nitrite and citrulline were not formed when NK cell-mediated target cell killing took place in a L-arginine-free DM supplemented with additives. Thus, it appears as if human NK cells may cause target cell killing via both NO-dependent and -independent processes.
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Affiliation(s)
- L Xiao
- Unit for Applied Biochemistry, Novum Clinical Research Center, Huddinge Hospital, Karolinska Institute, Stockholm, Sweden
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19
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Abstract
We have discussed in the previous sections the recent progress made toward elucidating the regulatory mechanism of perforin gene transcription and the domain structure of the perforin molecule. It appears that the expression of perforin is, at least partially, controlled at the transcription level through the interaction between killer cell-specific cis- and trans- acting factors. One of such cognate pairs, NF-P motif (an EBS-homologous motif) and NF-P2 (a killer cell-specific DNA-binding protein), has been described. The regulatory mechanism of gene transcription, however, is likely to involve multiple factors which act in a coordinated fashion to bring about the most efficient expression of perforin limited strictly to activated killer lymphocytes. Through studies using synthetic peptides and recombinant perforins, it has been suggested that the N-terminal region of the perforin molecule is an important, though not the only, domain responsible for the lytic activity. Further studies are warranted to elucidate the role(s) of other potential amphiphilic structures located in the central portion of the perforin molecule in the overall pore-forming activity. The molecular basis underlying the resistance of killer lymphocytes to perforin-mediated lysis still remains an open question. Preliminary results, however, suggest that the surface protein(s) restricted to killer cells may account for their self-protection against perforin. Based on recent studies using perforin-deficient mice, the involvement of perforin in lymphocyte-mediated cytolysis both in vivo and in vitro has been confirmed. Two functional roles, a direct (lytic) and an indirect (endocytosis enhancer; conduit), both of which may contribute critically to the cell-killing event can be attributed to perforin. The fact that lymphocytes may also employ perforin-independent killing mechanism(s), e.g. Fas-dependent pathway, is beyond the scope of this review. There is, nevertheless, no doubt that these alternative cytolytic mechanisms may also play important roles in immune effector and/or regulatory responses associated with killer lymphocytes. Obviously, we are still a long way from concluding on the functional relevance of each individual cytolytic mechanism seen in different physiopathological situations. Suffice it to say, however, that a wealth of information on lymphocyte-mediated killing has already emerged through the multidisciplinary efforts conducted in our and other laboratories that promise to further dissect this complicated event in the years to come.
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Affiliation(s)
- C C Liu
- Laboratory of Molecular Immunology and Cell Biology, Rockefeller University, New York 10021, USA
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20
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Shresta S, Heusel JW, Macivor DM, Wesselschmidt RL, Russell JH, Ley TJ. Granzyme B plays a critical role in cytotoxic lymphocyte-induced apoptosis. Immunol Rev 1995; 146:211-21. [PMID: 7493755 DOI: 10.1111/j.1600-065x.1995.tb00690.x] [Citation(s) in RCA: 60] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/25/2023]
Affiliation(s)
- S Shresta
- Department of Medicine, Washington University Medical School, St. Louis, Missouri 63110, USA
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21
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Trapani J, Smyth M, Apostolidis V, Dawson M, Browne K. Granule serine proteases are normal nuclear constituents of natural killer cells. J Biol Chem 1994. [DOI: 10.1016/s0021-9258(17)32315-3] [Citation(s) in RCA: 29] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/24/2022] Open
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22
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Abstract
Zanvil Alexander Cohn, an editor of this Journal since 1973, died suddenly on June 28, 1993. Cohn is best known as the father of the current era of macrophage biology. Many of his scientific accomplishments are recounted here, beginning with seminal studies on the granules of phagocytes that were performed with his close colleague and former editor of this Journal, James Hirsch. Cohn and Hirsch identified the granules as lysosomes that discharged their contents of digestive enzymes into vacuoles containing phagocytosed microbes. These findings were part of the formative era of cell biology and initiated the modern study of endocytosis and cell-mediated resistance to infection. Cohn further explored the endocytic apparatus in pioneering studies of the mouse peritoneal macrophage in culture. He described vesicular inputs from the cell surface and Golgi apparatus and documented the thoroughness of substrate digestion within lysosomal vacuoles that would only permit the egress of monosaccharides and amino acids. These discoveries created a vigorous environment for graduate students, postdoctoral fellows, and junior and visiting faculty. Some of the major findings that emerged from Cohn's collaborations included the radioiodination of the plasma membrane for studies of composition and turnover; membrane recycling during endocytosis; the origin of the mononuclear phagocyte system in situ; the discovery of the dendritic cell system of antigen-presenting cells; the macrophage as a secretory cell, including the release of proteases and large amounts of prostaglandins and leukotrienes; several defined parameters of macrophage activation, especially the ability of T cell-derived lymphokines to enhance killing of tumor cells and intracellular protozoa; the granule discharge mechanism whereby cytotoxic lymphocytes release the pore-forming protein perforin; the signaling of macrophages via myristoylated substrates of protein kinase C; and a tissue culture model in which monocytes emigrate across tight endothelial junctions. In 1983, Cohn turned to a long-standing goal of exploring host resistance directly in humans. He studied leprosy, focusing on the disease site, the parasitized macrophages of the skin. He injected recombinant lymphokines into the skin and found that these molecules elicited several cell-mediated responses. Seeing this potential to enhance host defense in patients, Cohn was extending his clinical studies to AIDS and tuberculosis. Zanvil Cohn was a consummate physician-scientist who nurtured the relationship between cell biology and infectious disease.(ABSTRACT TRUNCATED AT 400 WORDS)
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23
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Lowin B, Krähenbühl O, Müller C, Dupuis M, Tschopp J. Perforin and its role in T lymphocyte-mediated cytolysis. EXPERIENTIA 1992; 48:911-20. [PMID: 1426142 DOI: 10.1007/bf01919138] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/27/2022]
Abstract
The killing mediated by cytotoxic T lymphocytes (CTL) represents an important mechanism in the immune defence against tumors and virus infections. The lytic mechanism has been proposed to consist of a polarized secretion of granule-stored molecules, occurring on effector-target cell contact. By electron microscopy, membrane deposited, pore-like lesions are detected on the target cell membrane during cytolysis by CTL. These structures resembled strikingly pores formed during complement attack. Granules of CTL isolated by nitrogen cavitation and Percoll gradient centrifugation were shown to retain cytotoxic activity. Further purification of proteins stored in these granules led to the discovery of a membranolytic protein named perforin which was capable of polymerizing into pore-like structures. In addition to this cytolytic protein, a set of serine esterases was found as well as lysosomal enzymes and proteoglycans, whose function are not yet clearly defined. The role of perforin in the cytotoxic process is currently being explored by ablating the active gene in mice.
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Affiliation(s)
- B Lowin
- Institute of Biochemistry, University of Lausanne, Epalinges, Switzerland
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24
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Richards SJ, Scott CS. Human NK cells in health and disease: clinical, functional, phenotypic and DNA genotypic characteristics. Leuk Lymphoma 1992; 7:377-99. [PMID: 1493440 DOI: 10.3109/10428199209049794] [Citation(s) in RCA: 26] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/27/2022]
Abstract
Natural killer (NK) cells are the subject of great current interest because of their possible (in vivo) role in tumour cell surveillance and killing, and because of the potential application of cytokine-modulated NK cells in cancer immunotherapy. In addition, clonal proliferations of NK-associated (NKa) cell populations represent a high proportion of chronic (non-B) lymphoid malignancies and abnormal (both clonal and non-clonal) NKa components are being increasingly reported in association with diverse clinical pictures such as autoimmune disease. This communication extensively reviews what is presently known regarding normal and leukaemic NKa phenotypic diversity, the mechanisms of NK-mediated cytolysis, the role of NK cells in malignancy, and the diagnostic and cellular aspects of malignant NKa proliferations.
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Affiliation(s)
- S J Richards
- Yorkshire Leukaemia Diagnostic Unit, Department of Haematology, Cookridge Hospital, Leeds, England
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25
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Hameed A, Olsen KJ, Cheng L, Fox WM, Hruban RH, Podack ER. Immunohistochemical identification of cytotoxic lymphocytes using human perforin monoclonal antibody. THE AMERICAN JOURNAL OF PATHOLOGY 1992; 140:1025-30. [PMID: 1374586 PMCID: PMC1886520] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Subscribe] [Scholar Register] [Indexed: 12/26/2022]
Abstract
Perforin is a potent cytolytic pore-forming protein expressed in cytoplasmic granules of cytotoxic T lymphocytes and natural killer cells. A new monoclonal antibody raised against human perforin was used to detect both in vitro and in vivo perforin expression in cytotoxic cells. Immunohistochemical analysis of human peripheral blood mononuclear cells cultured in recombinant interleukin-2 (rIL-2) showed strong granular cytoplasmic staining of the IL-2 activated cytotoxic cells. Fresh-frozen tissue sections from patients with heart allograft rejection were also stained. Strong granular cytoplasmic staining of the mononuclear inflammatory infiltrate characteristic for perforin in cardiac allograft rejection was observed. The detection and quantitative analysis of perforin-associated cytotoxic cells by the human anti-perforin monoclonal antibody will help to evaluate the significance of these functionally distinct cytotoxic cells in human tissue.
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Affiliation(s)
- A Hameed
- Department of Pathology, Johns Hopkins Hospital, Baltimore, Maryland 21205
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26
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Yagita H, Nakata M, Kawasaki A, Shinkai Y, Okumura K. Role of perforin in lymphocyte-mediated cytolysis. Adv Immunol 1992; 51:215-42. [PMID: 1502975 DOI: 10.1016/s0065-2776(08)60488-5] [Citation(s) in RCA: 69] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/27/2022]
Affiliation(s)
- H Yagita
- Department of Immunology, Juntendo University School of Medicine, Tokyo, Japan
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27
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Peters PJ, Borst J, Oorschot V, Fukuda M, Krähenbühl O, Tschopp J, Slot JW, Geuze HJ. Cytotoxic T lymphocyte granules are secretory lysosomes, containing both perforin and granzymes. J Exp Med 1991; 173:1099-109. [PMID: 2022921 PMCID: PMC2118839 DOI: 10.1084/jem.173.5.1099] [Citation(s) in RCA: 528] [Impact Index Per Article: 15.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/29/2022] Open
Abstract
Cytotoxic T lymphocytes (CTL) contain granules that are exocytosed during specific interaction with target cells (TC). In this process, the granule contents, including the lethal protein perforin, as well as granzymes, a family of serine esterases, are delivered to the TC. Information regarding the routing of these proteins towards the granule and their exact localization within the granule is of primary importance to resolve the mechanism of granule-mediated TC killing. In this study, the subcellular localization of perforin, granzymes, and known endosomal and lysosomal marker proteins was determined in human and murine CTL, by immunogold labeling of ultrathin cryosections followed by electron microscopy. Perforin and granzymes can be detected in rough endoplasmic reticulum, Golgi complex, trans-Golgi reticulum, and in all cytotoxic granules. Within the granules, they have a similar distribution and are localized not only in the so-called dense core but also over the region containing small internal vesicles. This finding implies that perforin and granzymes can be released in membrane-enveloped and/or -associated form into the intercellular cleft formed upon CTL-TC interaction. On the basis of the present evidence, additional release of these molecules in soluble form cannot be excluded. The lysosomal membrane glycoproteins lamp-1, lamp-2, and CD63, are abundantly present on the granule-delimiting outer membrane, which becomes incorporated into the CTL plasma membrane during lethal hit delivery. In contrast, the cation-dependent mannose 6-phosphate receptor, known to be present in endosomes and absent from lysosomes, is found only in a minority of the granules. Together with our previous findings that the granules are acidic and connected to the endocytic pathway, these observations define CTL granules as secretory lysosomes.
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Affiliation(s)
- P J Peters
- Laboratory of Cell Biology, Medical School, University of Utrecht, The Netherlands
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28
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Kato K, Agatsuma T, Tanabe T, Masuko T, Hashimoto Y. Release of esterase from murine lymphokine-activated killer cells in antibody-dependent cellular cytotoxic reaction. Jpn J Cancer Res 1991; 82:206-12. [PMID: 1900824 PMCID: PMC5918378 DOI: 10.1111/j.1349-7006.1991.tb01830.x] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/29/2022] Open
Abstract
Release of granule enzyme(s) (BLT esterase) in the antibody dependent lymphokine-activated killer (LAK) cell-mediated cytotoxic reaction (LAK ADCC) was studied using LAK cells induced from murine splenocytes and thymocytes, various human tumor cells and relevant monoclonal antibodies (mAbs) to the tumor cells. BLT esterase was not significantly released from LAK cells in direct LAK cell-mediated cytotoxic reactions (LAK CMC). However, cultures of LAK cells and IgG-coated target tumor cells resulted in release of the enzyme concomitantly with target cell lysis, although esterase release proceeded faster than target cell lysis. Anti-LFA-1 mAb showed an inhibitory effect on LAK CMC but not on either LAK ADCC or BLT esterase release in the ADCC. These results indicate that exocytosis of granule enzyme from LAK cells is triggered by stimulation of Fc receptor on LAK cells and that LAK CMC and LAK ADCC differ in their lytic mechanism in terms of the release of BLT esterase.
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Affiliation(s)
- K Kato
- Department of Hygienic Chemistry, Pharmaceutical Institute, Tohoku University, Sendai
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29
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Young JDE, Liu CC, Kwon BS, Trapani JA, Young LHY. Lymphocyte-Mediated Cytolysis Role of Granule Mediators. BLOOD CELL BIOCHEMISTRY 1991. [DOI: 10.1007/978-1-4615-3796-0_6] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.0] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/14/2023]
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30
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Rivoltini L, Colombo MP, Supino R, Ballinari D, Tsuruo T, Parmiani G. Modulation of multidrug resistance by verapamil or mdr1 anti-sense oligodeoxynucleotide does not change the high susceptibility to lymphokine-activated killers in mdr-resistant human carcinoma (LoVo) line. Int J Cancer 1990; 46:727-32. [PMID: 1976601 DOI: 10.1002/ijc.2910460429] [Citation(s) in RCA: 48] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/29/2022]
Abstract
Two sublines were derived from the colon adenocarcinoma line LoVo, the first one was sensitive (LoVo/H) and the second one was made resistant to doxorubicin (LoVo/Dx). When tested for susceptibility to lysis by different types of immune effectors, LoVo/Dx appeared more sensitive than LoVo/H to the killing of CD3+CD5+CD16-, CD3- CD16+)-enriched lymphokine activated killers (LAK) or activated macrophages. In order to check whether this effect was due to different expression of glycoprotein P170 between the two LoVo sublines (30% vs. 90% of positive cells), a pharmacological and genetic modulation of P170 was carried out in LoVo cells. Treatment of LoVo/Dx with the calcium channel blocker verpamil (VRP), strongly impaired P170 function as evaluated by reduced Dx resistance, without affecting the lysability of LoVo/Dx cells by LAKs. Moreover, the significant inhibition of P170 expression resulting from the treatment of LoVo/Dx with mdr1 anti-sense olideoxynucleotide also failed to change the high lysability of LoVo/Dx by LAKs. These results, therefore, indicate that molecules other than P170 are involved in the increased lysis of LoVo/Dx subline by immune effectors and that down-regulation of the P170 expression or function will not reduce the potential effectiveness of cancer chemo-immunotherapy.
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Affiliation(s)
- L Rivoltini
- Division of Experimental Oncology D, Istituto Nazionale Tumori, Milan, Italy
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31
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Zychlinsky A, Joag S, Liu CC, Young JD. Cytotoxic mechanisms of murine lymphokine-activated killer cells: functional and biochemical characterization of homogeneous populations of spleen LAK cells. Cell Immunol 1990; 126:377-90. [PMID: 1690083 DOI: 10.1016/0008-8749(90)90329-p] [Citation(s) in RCA: 19] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/28/2022]
Abstract
A highly purified population of murine lymphokine-activated killer (LAK) cells was obtained by selecting plastic-adherent splenocytes after incubation in high doses of recombinant IL-2. The population obtained was shown to be more than 95% positive for the cell marker asialo-GM1, and negative for both Lyt-1 (CD5) and Lyt-2 (CD8). The cells presented typical large granular lymphocyte morphology, and killed NK-susceptible target cells in an exclusively calcium-dependent fashion. A target cell DNA fragmentation activity of LAK cells could be detected even before target cell death. The presence of Hanukkah Factor/granzyme A/serine esterase 1, CTLA-1/granzyme B/serine esterase 2, and pore-forming protein (PFP/perforin) in these LAK cells was demonstrated by Northern blot analysis, suggesting that these markers are not exclusively associated with cytotoxic T lymphocytes. On immunoblots, antibodies specific for a lymphocyte PFP/perforin reacted with a 70-kDa protein of LAK cells. PFP/perforin was localized by immunofluorescence to the cell granules. A 50-kDa protein antigenically related to the macrophage cytokine tumor necrosis factor (TNF) was detected by immunoblotting and localized by immunofluorescence to both the cell granules and the cytosol. No RNA for TNF, however, could be detected using TNF-specific probes, suggesting that LAK cells may contain a cytotoxic factor which is related to, but distinct from, TNF. The work presented here demonstrates that cytotoxic mediators identified in cell lines are also present in primary cell cultures.
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Affiliation(s)
- A Zychlinsky
- Laboratory of Cellular Physiology and Immunology, Rockefeller University, New York, New York 10021
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32
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Trapani JA, Kwon BS, Kozak CA, Chintamaneni C, Young JD, Dupont B. Genomic organization of the mouse pore-forming protein (perforin) gene and localization to chromosome 10. Similarities to and differences from C9. J Exp Med 1990; 171:545-57. [PMID: 2303785 PMCID: PMC2187726 DOI: 10.1084/jem.171.2.545] [Citation(s) in RCA: 31] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/31/2022] Open
Abstract
Genomic clones encompassing the entire coding region of the mouse lymphocyte pore-forming protein gene (Pfp) have been isolated and used to determine its intron-exon organization. In contrast to C9, Pfp has a simple structure, consisting of only three exons (two of which encode polypeptide), a large 5' intron, and a single, smaller intron that is situated approximately one-third of the way through the protein-coding portions of the gene. The regions encoding the homologous domains of PFP and C9 are encoded on exons 7, 8, 9, and 10 of C9, but form only approximately half of the open reading frame of exon III in Pfp. Although encoding polypeptides with related functions, the two genes possess such sharply contrasting structures as to suggest that their analogous regions may have risen independently, by a process of convergent evolution. Using a panel of somatic cell hybrid cell lines, Pfp has been mapped to chromosome 10.
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Affiliation(s)
- J A Trapani
- Laboratory of Human Immunogenetics, Memorial Sloan-Kettering Cancer Center, New York, New York 10021
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33
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Liu CC, Rafii S, Granelli-Piperno A, Trapani JA, Young JD. Perforin and serine esterase gene expression in stimulated human T cells. Kinetics, mitogen requirements, and effects of cyclosporin A. J Exp Med 1989; 170:2105-18. [PMID: 2584937 PMCID: PMC2189526 DOI: 10.1084/jem.170.6.2105] [Citation(s) in RCA: 124] [Impact Index Per Article: 3.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/01/2023] Open
Abstract
A pore-forming protein (PFP; perforin) and various serine esterases (SE) have been identified in the cytoplasmic granules of CTL and NK cells. Perforin and several SE have recently been cloned. Northern blotting analysis was performed here using cDNA probes specific for human perforin and two SE (SE 1/HS and SE 2/GB) to monitor the levels of specific mRNAs in mitogen-stimulated primary human T cells. These mRNAs were rapidly induced by IL-2 with optimal responses at 300 U/ml. After IL-2 treatment, mRNAs for perforin, SE 1, and SE 2 peaked at 12-24 h and decreased after 48 h. The three mRNAs were also induced in T cells treated with a combination of PMA plus lectin, OKT3 mAb, or plastic-adherent accessory cells. However, the induction induced by PMA/mitogen followed a slower kinetics, peaking at 48 h. In general, we found that SE 1 mRNA was more readily induced by IL-2, while SE 2 responded better to PMA/mitogen. Similar patterns of mRNA expression were observed for both unprimed T cells and PHA-primed T blasts. After stimulation with IL-2 and PMA/mitogen, the T8+ subset was shown to be the main producer of perforin, SE 1, and SE 2. Low levels of all three mRNAs, however, were also detected in the T4+ subset. The induction of all three mRNAs by either IL-2 or PMA/mitogen was partially blocked by the immunosuppressive drug cyclosporin A (CsA), but not by the biologically inactive analogue cyclosporin H. Together, these results point to some similarities and differences with upregulation of granule mediator mRNAs relative to lymphokine mRNAs. Both sets of genes require two signals for their induction by mitogens. In contrast to lymphokines, there is a strong response of granule mRNAs to IL-2, and the induction of these transcripts is only partially blocked by CsA.
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Affiliation(s)
- C C Liu
- Laboratory of Cellular Physiology and Immunology, Rockefeller University, New York, New York 10021
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34
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Masera R, Gatti G, Sartori ML, Carignola R, Salvadori A, Magro E, Angeli A. Involvement of Ca2+-dependent pathways in the inhibition of human natural killer (NK) cell activity by cortisol. IMMUNOPHARMACOLOGY 1989; 18:11-22. [PMID: 2475455 DOI: 10.1016/0162-3109(89)90026-x] [Citation(s) in RCA: 28] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/01/2023]
Abstract
The role of Ca2+ as a second messenger of the glucocorticoid inhibition of human natural killer (NK) cell activity was evaluated using Ca2+ entry blockers (verapamil and its desmethoxy derivatives LU46973 and LU47093), calmodulin antagonists (pimozide and two naphthalensulfonamide derivatives, W-7 and W-13), the Ca2+ channel agonist BAY K 8644 and the calcium ionophore A23187. Peripheral blood mononuclear (PBM) cell preparations were incubated for 20 h with 1 x 10(-6) M cortisol and these agents in various combinations (concentration range: 1 x 10(-9) -1 x 10(-5) M) and then assayed in a direct 4-h cytolytic assay using 51Cr-labeled K 562 target cells. Exposure to cortisol led to a significant reduction of NK cell activity (about 50% vs. spontaneous activity). Ca2+ entry blockers and calmodulin antagonists were per se minimally effective, but significantly enhanced cortisol-dependent inhibition of NK cell activity. Raising extracellular Ca2+ by CaCl2 or intracellular Ca2+ by the calcium channel agonist BAY K 8644 or the ionophore A23187 resulted in an appreciable reduction of these effects. Similar results were obtained when these substances were added to monocyte-depleted or NK cell-enriched suspensions exposed to cortisol. Our data are consistent with the view that extra- and intracellular Ca2+ plays a role in the control of human NK cell activity. It is also conceivable that both calcium flux into the cell and the calcium calmodulin system are involved in the cortisol-induced inhibition of natural cytotoxicity.
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Affiliation(s)
- R Masera
- Dipartimento di Biomedicina, Università degli Studi di Torino, Ospedale San Luigi Gonzaga, Italy
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35
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Abstract
Cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells use multiple mechanisms to destroy their target cells. Pore formation resulting in osmotic lysis of the target is one mechanism; the pore-forming protein (perforin) responsible for this activity has been purified. Antigenically and functionally it resembles proteins of the membrane attack complex of complement. The other known mediators of cytotoxicity appear to be closely interrelated. Tumor necrosis factor (TNF), lymphotoxin (LT), and leukalexin are the three members of this group that have been purified, although their mechanisms of action are still unknown. CTLs fragment the DNA of target cells, as do TNF, LT, and leukalexin; this may be one of the mechanisms of action of these mediators. CTLs and NK cells do not self lyse. The basis of this phenomenon is unclear, although recent advances have shed some light on the problem.
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Affiliation(s)
- S Joag
- Laboratory of Cellular Physiology and Immunology, Rockefeller University, New York, New York 10021
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36
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Cuturi MC, Anegón I, Sherman F, Loudon R, Clark SC, Perussia B, Trinchieri G. Production of hematopoietic colony-stimulating factors by human natural killer cells. J Exp Med 1989; 169:569-83. [PMID: 2521357 PMCID: PMC2189209 DOI: 10.1084/jem.169.2.569] [Citation(s) in RCA: 131] [Impact Index Per Article: 3.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/01/2023] Open
Abstract
We have analyzed the ability of highly purified preparations of human NK cells to produce CSF. NK cells, purified by negative selection from 10-d cultures of PBMC incubated with irradiated B-lymphoblastoid cell lines, were stimulated with rIL-2, FcR(CD16) ligands (particulate immune complexes or anti-CD16 antibodies bound to Sepharose), a combination of CD16 ligands and rIL-2, or the phorbol diester phorbol dibutyrate (PDBu) together with the Ca2+ ionophore A23187. Both rIL-2 and CD16 ligands induce accumulation of GM-CSF mRNA in NK cells and the combined effect of the two stimuli is synergistic. Maximal accumulation of GM-CSF mRNA is observed after PDBu/A23187 stimulation. The participation of contaminant T cells in the observed expression of the GM-CSF gene is excluded because CD16 ligands do not stimulate T cells and CD3 ligands, powerful stimulators of T cells, are inactive on NK cells. Accumulation of CSF-1 mRNA is observed only in NK cells stimulated with both CD16 ligands and rIL-2, whereas accumulation of IL-3 mRNA is observed only in NK cells stimulated with PDBu/A23187. Transcripts of the G-CSF, IL-1 alpha, and IL-1 beta genes were never detected in NK cells in these experiments. The kinetics of accumulation of GM-CSF and CSF-1 mRNA in NK cells stimulated with CD16 ligands and rIL-2 peaked at 2-4 h and was slower than that of TNF and IFN-gamma mRNA, which peak at 1 h. GM-CSF was precipitated from the supernatant fluids of NK cells stimulated with PDBu/A23187 and its biological activity was demonstrated by the ability of the supernatants to sustain proliferation of the TALL-101 cell line or CML blasts. Biological activity of IL-3 and CSF-1 was demonstrable in supernatant fluids of NK cells stimulated with PDBu/A23187 and CD16 ligands/rIL-2, respectively.
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MESH Headings
- Antigens, Differentiation/physiology
- Antigens, Differentiation, T-Lymphocyte/physiology
- Biological Assay
- Blotting, Northern
- CD3 Complex
- Calcimycin/pharmacology
- Cells, Cultured
- Colony-Stimulating Factors/biosynthesis
- Granulocyte-Macrophage Colony-Stimulating Factor
- Growth Substances/biosynthesis
- Humans
- Interleukin-1/biosynthesis
- Interleukin-2/pharmacology
- Interleukin-3/biosynthesis
- Killer Cells, Natural/physiology
- Ligands
- Phorbol Esters/pharmacology
- Precipitin Tests
- RNA, Messenger/genetics
- Receptors, Antigen, T-Cell/physiology
- Receptors, Fc/physiology
- Receptors, IgG
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Affiliation(s)
- M C Cuturi
- Wistar Institute of Anatomy and Biology, Philadelphia, Pennsylvania 19104
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37
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Abstract
Studies of cytotoxicity by human lymphocytes revealed not only that both allogeneic and syngeneic tumor cells were lysed in a non-MHC-restricted fashion, but also that lymphocytes from normal donors were often cytotoxic. Lymphocytes from any healthy donor, as well as peripheral blood and spleen lymphocytes from several experimental animals, in the absence of known or deliberate sensitization, were found to be spontaneously cytotoxic in vitro for some normal fresh cells, most cultured cell lines, immature hematopoietic cells, and tumor cells. This type of nonadaptive, non-MHC-restricted cellmediated cytotoxicity was defined as “natural” cytotoxicity, and the effector cells mediating natural cytotoxicity were functionally defined as natural killer (NK) cells. The existence of NK cells has prompted a reinterpretation of both the studies of specific cytotoxicity against spontaneous human tumors and the theory of immune surveillance, at least in its most restrictive interpretation. Unlike cytotoxic T cells, NK cells cannot be demonstrated to have clonally distributed specificity, restriction for MHC products at the target cell surface, or immunological memory. NK cells cannot yet be formally assigned to a single lineage based on the definitive identification of a stem cell, a distinct anatomical location of maturation, or unique genotypic rearrangements.
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Affiliation(s)
- G Trinchieri
- Wistar Institute of Anatomy and Biology, Philadelphia, Pennsylvania 19104
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38
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Gambacorti-Passerini C, Rivoltini L, Radrizzani M, Supino R, Mariani M, Parmiani G. Susceptibility of human and murine drug-resistant tumor cells to the lytic activity of rIL2-activated lymphocytes (LAK). Cancer Metastasis Rev 1988; 7:335-45. [PMID: 3061677 DOI: 10.1007/bf00051374] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 01/03/2023]
Abstract
This article surveys the available data on the sensitivity of drug-resistant tumor cells to recombinant interleukin 2 (rIL2)-activated lymphocytes (LAK). In our own study, three different experimental systems were used: 1. in vitro treatment of tumor cells with an anticancer drug followed by the use of surviving cells as targets of LAK; 2. use of pairs of drug-resistant and drug-sensitive cell sublines; 3. analysis of several tumor clones obtained from the same tumor. The antitumor activity of LAK was evaluated both by the 51Cr release and the human tumor clonogenic assay (HTCA). In all the experimental systems used, drug-resistant tumor cells were found to be significantly lysed by LAK, with a consistent trend towards a higher susceptibility than their drug-sensitive counterparts. A positive correlation between the sensitivity to LAK and the ID50 for doxorubicin (Dx) was found in 44 melanoma clones analyzed, suggesting that spontaneously drug-resistant clones have a higher sensitivity to LAK than the drug-sensitive clones. Drug-resistant cells were also more sensitive to antibody and complement-mediated lysis, whereas the higher lysis of drug-resistant tumor cells exerted by LAK was maintained in a lectin dependent cytotoxicity assay. These data offer a rationale for combining chemotherapy with adoptive immunotherapy in the treatment of cancer. Moreover, studying the reasons for the higher LAK sensitivity of drug-resistant tumor cells may provide insights into the mechanisms by which tumor cells can resist LAK action.
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Jiang SB, Persechini PM, Zychlinsky A, Liu CC, Perussia B, Young JD. Resistance of cytolytic lymphocytes to perforin-mediated killing. Lack of correlation with complement-associated homologous species restriction. J Exp Med 1988; 168:2207-19. [PMID: 3199067 PMCID: PMC2189133 DOI: 10.1084/jem.168.6.2207] [Citation(s) in RCA: 48] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/04/2023] Open
Abstract
CTL and NK cells resist self-mediated killing and lysis by their own pore-forming protein (PFP; perforin). Perforin, like C, lyses RBC. Efficient C-mediated lysis of RBC occurs when both C and RBC are from different species (homologous species restriction). A protective surface protein (C8-binding protein, homologous restriction factor) has been reported to mediate both homologous species restriction in C-dependent cytolysis and protection of some target cells against perforin-induced lysis. We show here that perforin, unlike C, lyses target cells across a variety of species, including the homologous one, while the same target cell populations resist the attack by homologous C. Perforin-containing extracts of CTL and LAK/NK cells from three species (rat, mouse, and human) and purified mouse perforin were tested against RBC from 10 different species, several nucleated target cell lines, and one primary cell population (thymocytes). While resisting lysis by homologous C, most of these cell types were lysed effectively by perforin without any homologous restriction pattern. CTL and NK cells, like other nucleated targets, are resistant to lysis by homologous but not heterologous C; however, these cell types are resistant to both homologous and heterologous perforin. Together, our results suggest that the protective mechanisms associated with C- and perforin-mediated lysis are distinct.
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Affiliation(s)
- S B Jiang
- Laboratory of Cellular Physiology and Immunology, Rockefeller University, New York, New York 10021
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40
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41
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Berke G. Lymphocyte-mediated cytolysis. Effectors, lytic signals, and the mechanism whereby early membrane derangements result in target-cell death. Ann N Y Acad Sci 1988; 532:314-35. [PMID: 2460010 DOI: 10.1111/j.1749-6632.1988.tb36349.x] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/01/2023]
Affiliation(s)
- G Berke
- Department of Cell Biology, Weizmann Institute of Science, Rehovot, Israel
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42
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Zalman LS, Brothers MA, Müller-Eberhard HJ. Self-protection of cytotoxic lymphocytes: a soluble form of homologous restriction factor in cytoplasmic granules. Proc Natl Acad Sci U S A 1988; 85:4827-31. [PMID: 3260383 PMCID: PMC280529 DOI: 10.1073/pnas.85.13.4827] [Citation(s) in RCA: 14] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/04/2023] Open
Abstract
A soluble form of homologous restriction factor (HRF) has been isolated from the cytoplasmic granules of human large granular lymphocytes that were cultured in the presence of recombinant interleukin 2 for 2-3 weeks. The granule-derived protein (approximately 65 kDa) is soluble in detergent-free solution and reacts with antibody produced to membrane HRF. HRF was first described as a 65-kDa membrane protein of human erythrocytes capable of inhibiting the formation of transmembrane channels by the membrane attack complex of complement. It has also been isolated from activated human lymphocytes and shown to confer upon these cells relative resistance to lysis by the membrane attack complex and by the complement component C9-related protein of human cytotoxic lymphocytes. The soluble HRF of lymphocyte granules inhibits reactive lysis of erythrocytes by the membrane attack complex of human complement. It was also found to be a potent inhibitor of (i) the cytolytic activity of the C9-related protein of human cytotoxic lymphocytes, (ii) human large granular lymphocyte cytotoxicity, and (iii) the cytotoxic activity of human CD8+ lymphocytes obtained by cell sorting from recombinant interleukin 2-activated peripheral blood mononuclear cells. It is proposed that granule-derived soluble HRF and cell surface-membrane-bound HRF are involved in the mechanism of self-protection of killer lymphocytes.
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Affiliation(s)
- L S Zalman
- Department of Immunology, Scripps Clinic and Research Foundation, La Jolla, CA 92037
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43
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44
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Young JD, Liu CC, Persechini PM, Cohn ZA. Perforin-dependent and -independent pathways of cytotoxicity mediated by lymphocytes. Immunol Rev 1988; 103:161-202. [PMID: 3292393 DOI: 10.1111/j.1600-065x.1988.tb00755.x] [Citation(s) in RCA: 69] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/05/2023]
Abstract
There is little doubt at the present time that both perforin-dependent and -independent pathways are important in mediating the cytotoxicity associated with lymphocytes. The cell distribution of perforin, initially thought to include both CTL and NK cells, now must be viewed with caution because all previous biochemical studies on CTL have been conducted with cell lines propagated in long-term cultures in the presence of T cell growth factors (IL-2 and perhaps some still undefined factors). Under these conditions, CTL are known to assume a broader, NK-like specificity in target cell killing and may thus differ significantly from primary CTL generated in the body. Accordingly, perforin does not seem to be present in primary CTL activated directly through mixed lymphocyte reactions. It remains to be shown how primary CTL lyse target cells in vivo. Initial studies conducted in several laboratories have already provided some clues. It now seems that even in cultured, perforin-containing CTL, the perforin pathway is not an obligatory mechanism required for target cell killing. Other pathways, possibly involving TNF/lymphotoxin-like molecules, may play a direct role in this type of cytotoxicity. Other still unidentified factors now also need to be sought, including membrane polypeptides that may develop cytotoxicity directly upon cell contact and binding. Although from the studies reviewed here it is clear now that perforin has a more limited role in cell killing than originally proposed, it is still intriguing that it should share structural and functional homologies with complement proteins, drawing paradoxical analogies between two systems (the cellular and the humoral immune systems) which have evolved to become specialized to carry out separate immunological tasks. The cloning of the genes for perforin and for all the C proteins that comprise the MAC should reveal important information on how these genes originated and then diverged during evolution. The cellular distribution of other granule products, such as serine esterases, also must be viewed with caution. A serine esterase activity was initially thought to be CTL-specific. This information stimulated an intensive research activity in many laboratories that resulted in both the purification of a serine esterase family and the cloning of several serine esterase transcripts. It is becoming clear from recent evidence that this group of enzymes is not truly CTL-specific and therefore would not be expected to develop any function rendered absolutely necessary for cytolysis.(ABSTRACT TRUNCATED AT 400 WORDS)
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Affiliation(s)
- J D Young
- Laboratory of Cellular Physiology and Immunology, Rockefeller University, New York, N.Y. 10021
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45
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Müller-Eberhard HJ. The molecular basis of target cell killing by human lymphocytes and of killer cell self-protection. Immunol Rev 1988; 103:87-98. [PMID: 3292398 DOI: 10.1111/j.1600-065x.1988.tb00751.x] [Citation(s) in RCA: 34] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/05/2023]
Abstract
The cytolytic protein (C9RP) of human cytotoxic lymphocytes was isolated from large granular lymphocytes (LGL) and anti-CD3 activated cytotoxic T cells (CTL). It is immunochemically related to the channel-forming proteins of complement. Whereas LGL constitutively contain C9RP, peripheral resting CTL do not. C9RP synthesis is induced, however, in CD8+ cells upon stimulation of the T cell antigen receptor-CD3 structure. Comparison of cellular cytotoxicity and C9RP content at various times during anti-CD3 activation of CTL yielded a coefficient of correlation, r = 0.92. Isolated C9RP (Mr approximately 70,000) readily lysed a large variety of metabolically active cells tested. Certain monoclonal antibodies to C9RP inhibited target cell killing by LGL or activated CD8+ lymphocytes. Homologous restriction factor (HRF) is a normal membrane protein of blood cells that inhibits transmembrane channel formation by the membrane attack complex of complement. It has recently been found that isolated HRF (Mr approximately 65,000), bound to sheep erythrocytes, inhibited their lysis mediated by the antibody-dependent cellular cytotoxicity reaction or by isolated C9RP. Further, stimulation of resting peripheral lymphocytes with anti-CD3 resulted in increased expression of cell surface HRF. Acquisition of HRF expression conferred upon CTL relative resistance to lysis by C9RP. A soluble form of HRF (Mr approximately 65,000) was isolated from the cytoplasmic granules of LGL, which also contain C9RP, and shown to inhibit cytotoxicity of LGL and CTL. It is conceivable that HRF is opertive in self-protection of cytotoxic lymphocytes.
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Affiliation(s)
- H J Müller-Eberhard
- Bernhard Nocht Institute for Tropical Medicine, Hamburg, Federal Republic of Germany
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46
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Ferguson WS, Verret CR, Reilly EB, Iannini MJ, Eisen HN. Serine esterase and hemolytic activity in human cloned cytotoxic T lymphocytes. J Exp Med 1988; 167:528-40. [PMID: 3126252 PMCID: PMC2188843 DOI: 10.1084/jem.167.2.528] [Citation(s) in RCA: 25] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/04/2023] Open
Abstract
Target cell lysis by most murine cytotoxic T lymphocytes appears to be mediated by a complement (C9)-like protein called perforin, contained in high-density cytoplasmic granules. These granules also contain high levels of serine esterase activity, which may also play a role in cytolysis. Analysis of 17 cloned human cytotoxic T lymphocytes revealed the presence of serine esterase that is very similar to its murine counterpart in substrate and inhibitor specificities, pH optimum, and molecular mass; dot blot hybridization with synthetic oligonucleotides corresponding to the active sites of two known murine CTL esterases suggests homology to the murine enzyme HF. However, serine esterase was present at only approximately 10% of the level found in murine CTLs, and was not secreted during CTL-target cell interaction; moreover, hemolytic activity could not be detected in any of the seven cell lines tested. The results suggest that the human CTLs examined here kill their target cells by a mechanism different from that used by most cloned murine CTLs.
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Affiliation(s)
- W S Ferguson
- Department of Biology, Massachusetts Institute of Technology, Cambridge 02139
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47
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Anegón I, Cuturi MC, Trinchieri G, Perussia B. Interaction of Fc receptor (CD16) ligands induces transcription of interleukin 2 receptor (CD25) and lymphokine genes and expression of their products in human natural killer cells. J Exp Med 1988; 167:452-72. [PMID: 2831292 PMCID: PMC2188858 DOI: 10.1084/jem.167.2.452] [Citation(s) in RCA: 278] [Impact Index Per Article: 7.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/02/2023] Open
Abstract
We report evidence that FcR(CD16) on human NK cells are signal-transducing molecules that, upon ligand binding, induce transcription of genes encoding surface activation molecules [IL-2-R(CD25)] and cytokines (IFN-gamma and TNF) relevant to NK cell biology and functions. Homogeneous NK and T cell populations purified from short-term bulk cultures of PBMC with irradiated B lymphoblastoid cell lines were cultured in the presence of FcR ligands (particulate immune complexes or immobilized anti-CD16 antibodies) alone or with rIL-2. Upon 18 h of stimulation, NK cells express Tac, TfR, and 4F2 antigens and produce IFN-gamma and TNF; both effects are synergistically enhanced in the presence of rIL-2, which is itself ineffective. Treatment of NK cells with FcR(CD16) ligands induces accumulation of mRNA for IFN-gamma and TNF and, to a lesser extent, IL-2-R with fast kinetics also in the absence of de novo protein synthesis. rIL-2 and FcR(CD16) ligands synergize to induce mRNA accumulation. mRNA accumulation and transcription of TNF and IFN-gamma genes induced by FcR(CD16) ligands are greater than those induced by rIL-2, and the reverse is true for IL-2-R. The two stimuli do not synergize at the transcriptional level. These observations indicate that the mechanisms through which FcR(CD16) ligands and rIL-2 induce NK cell activation are, in part, distinct. Both operate at the transcriptional level, although other mechanisms are probably induced by the FcR ligand stimulus per se or in combination with other lymphokines and synergize at a post-transcriptional or translational level to enhance NK cell activation.
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MESH Headings
- Animals
- Antigens, Surface/biosynthesis
- Biological Products/biosynthesis
- Biological Products/metabolism
- Cell Line
- Cell Membrane/metabolism
- Cells, Cultured
- Cross-Linking Reagents
- Cytokines
- Drug Synergism
- Humans
- Interleukin-2/pharmacology
- Killer Cells, Natural/immunology
- Killer Cells, Natural/metabolism
- Kinetics
- Lymphokines/genetics
- Mice
- Protein Biosynthesis
- RNA, Messenger/metabolism
- Receptors, Fc/physiology
- Receptors, Immunologic/genetics
- Receptors, Immunologic/metabolism
- Receptors, Interleukin-2
- Recombinant Proteins/pharmacology
- Transcription, Genetic
- Tumor Necrosis Factor Receptor Superfamily, Member 7
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Affiliation(s)
- I Anegón
- Wistar Institute of Anatomy and Biology, Philadelphia, Pennsylvania 19104
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48
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Shinkai Y, Ishikawa H, Hattori M, Okumura K. Resistance of mouse cytolytic cells to pore-forming protein-mediated cytolysis. Eur J Immunol 1988; 18:29-33. [PMID: 3345794 DOI: 10.1002/eji.1830180106] [Citation(s) in RCA: 34] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/05/2023]
Abstract
Pore-forming protein (perforin, PFP) was isolated from a mouse large granular lymphocyte (LGL) [natural killer (NK-like)] cell line. Purified PFP lysed a variety of mouse tumor cell lines and helper T lymphocyte cell lines. However, LGL and cytotoxic T lymphocyte cell lines were resistant to PFP-mediated cell lysis. The presence of hemolytic activity in the granule was examined in these resistant cell lines. Four out of five of these resistant cell lines had hemolytically active granules. We determined whether NK cells freshly isolated from BALB/c nude mouse spleens were resistant to PFP-mediated cytolysis. Nylon column-passed spleen cells with an enriched content of NK cells exhibited more resistance than whole spleen cells. Moreover, when spleen cells were treated with PFP the remaining live cells showed enriched NK activity suggesting that normal peripheral cells with NK activity are resistant to PFP. These results indicate that cytolytic cells containing PFP have developed defense mechanisms to inhibit PEP-mediated cell lysis.
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MESH Headings
- Animals
- Cell Line
- Cytotoxicity Tests, Immunologic
- Cytotoxicity, Immunologic/drug effects
- Hemolysis/drug effects
- Immunity, Innate
- Killer Cells, Natural/immunology
- Membrane Glycoproteins
- Membrane Proteins/isolation & purification
- Membrane Proteins/physiology
- Mice
- Mice, Inbred BALB C
- Mice, Inbred C3H
- Mice, Inbred C57BL
- Mice, Inbred DBA
- Mice, Nude
- Perforin
- Pore Forming Cytotoxic Proteins
- Spleen/cytology
- Tumor Cells, Cultured
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Affiliation(s)
- Y Shinkai
- Department of Immunology, Juntendo University School of Medicine, Tokyo, Japan
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49
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Young JD, Liu CC, Butler G, Cohn ZA, Galli SJ. Identification, purification, and characterization of a mast cell-associated cytolytic factor related to tumor necrosis factor. Proc Natl Acad Sci U S A 1987; 84:9175-9. [PMID: 3321069 PMCID: PMC299715 DOI: 10.1073/pnas.84.24.9175] [Citation(s) in RCA: 174] [Impact Index Per Article: 4.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/05/2023] Open
Abstract
The role of mast cells and mast-cell-derived factors in natural cytotoxic reactions was investigated. Cultured and freshly isolated murine mast cells are shown to be cytotoxic to WEHI-164 and YAC-1 targets in 18-hr viability assays but not in 4-hr assays. Here, we describe a cytotoxic factor in murine mast cells that is immunologically related to tumor necrosis factor (TNF). This TNF-like factor lyses WEHI-164 cells with a slow time course requiring 16-20 hr for the lytic reaction to complete. Antibodies specific for human and murine TNF and human lymphotoxin partially block mast cell lysis of WEHI-164 cells. These antibodies react on immunoblots with one major mast cell protein band of 50 kDa. Immunoblot analysis shows this factor in cloned and uncloned cultured mouse mast cells and in mature "connective tissue-type" mast cells freshly purified from rat or mouse peritoneal cavities. The amount of this factor is greatly enhanced in cells that have been stimulated with a combination of phorbol ester/concanavalin A or bacterial lipopolysaccharide. Subcellular fractionation analysis of mast cells with Percoll gradients reveals two pools of TNF-related cytotoxic activity that are associated with free cytosolic material and granule fractions. In contrast to cytotoxic T lymphocytes and natural killer cells, granule-enriched fractions of mast cells do not contain any hemolytic activity. The localization of the TNF-like molecule in mast cell granules may play a strategical role in the rapid delivery of this mediator to the target cell membrane following cell surface stimulation and degranulation.
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Affiliation(s)
- J D Young
- Laboratory of Cellular Physiology and Immunology, Rockefeller University, New York, NY 10021
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50
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Dallegri F, Frumento G, Ballestrero A, Goretti R, Patrone F. Relationship between antibody-dependent tumour cell lysis and primary granule exocytosis by human neutrophils. Clin Exp Immunol 1987; 70:479-83. [PMID: 2827922 PMCID: PMC1542089] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/02/2023] Open
Abstract
Neutrophil-mediated antibody-dependent cellular cytotoxicity (ADCC) against Raji target cells and neutrophil degranulation during the ADCC process were evaluated in the presence and in the absence of different agents able to interfere with the neutrophil release of granule components (anion channel blockers, colchicine, isoproterenol, dimethylxanthine, cAMP). When used at concentrations incapable of preventing the target cell recognition by neutrophils, the majority of these agents inhibited both the ADCC and the release of myeloperoxidase (MPO, primary granule marker) and lysozyme (LZM, primary and secondary granule marker). The inhibition of the ADCC correlated strictly with the inhibition of the MPO release. Thus, the results are consistent with the hypothesis that neutrophil primary granules play a major role in the cytolytic process.
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Affiliation(s)
- F Dallegri
- First Medical Clinic, University of Genova Medical School, Italy
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