Retroviral Interference with Execution Pathways in Target Cells To Unravel Mechanisms of Cytotoxic T Cell-Mediated Cell Death.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1267-1267
Author(s):  
J.F. de Vries ◽  
P.A. von dem Borne ◽  
M.H.M. Heemskerk ◽  
R. Willemze ◽  
J.H.F. Falkenburg ◽  
...  
Keyword(s):  
Cell Death ◽  
T Cell ◽  
Target Cell ◽  
Death Receptor ◽  
Cytotoxic T Cell ◽  
Target Cells ◽  
Fas Receptor ◽  
Apoptotic Pathways ◽  
Kinetics Of ◽  
Effector Mechanisms

Abstract Cytotoxic T lymphocytes (CTLs) mediate target cell death by different effector mechanisms. We investigated whether a correlation exists between the kinetics of CTL-induced killing of the target cell and the different apoptotic pathways executed by the CTL. Different CTL clones were isolated using from a patient with CML after receiving donor lymphocyte infusions from an HLA-identical donor. These CTL clones recognized minor antigens expressed on the EBV-LCL cells from the patient. Since these clones were not all equally effective in killing the same target cells, we hypothesized that these T cells may induce different apoptotic pathways. In order to study the different execution pathways in the target cell, we generated retroviral constructs encoding the anti-apoptotic genes FADD-DN and FLIP, both inhibiting death receptor-mediated killing, and the granzyme B (GrB)-inhibitor PI-9. Using a retroviral delivery system, these constructs were transduced to the EBV-LCL cells. The cells highly overexpressing the gene of interest were sorted based on coexpression of a reporter gene, which was confirmed by Western Blot analysis. Both the wildtype (EBV-WT) and the transduced EBV cells (EBV-FADD-DN, EBV-FLIP and EBV-PI-9) were used as targets and the different CTL clones as effectors (E:T ratio = 1:1) in a quantitative CFSE-based cytotoxicity assay using Flow-Count Fluorospheres. To analyze the mechanism of immediate cell death caused by rapidly killing CTL clones, EBV-WT cells were exposed for 2 hours (h) to these clones, resulting in 30% lysis. This lysis could neither be blocked by FADD-DN or FLIP, nor by PI-9. Furthermore, no inhibition was obtained using the general caspase-inhibitor z-VAD-FMK, indicating that the killing was caspase-independent. In agreement with this observation, specific triggering of the Fas receptor pathway by an agonistic Fas antibody (100 ng/ml) did not induce any apoptosis within 2 h. To study CTL-induced cell death after a longer period, we analyzed CTL clones with different rates of killing and measured cytotoxicity after 5 and 24 h. Rapidly killing clones induced 60–70% cell death of the EBV-WT within 5 h, whereas slowly killing CTL clones did not show cytotoxicity after 5 h, but induced 50–60% cell death of EBV-WT after 24 h of incubation. Inhibition of CTL-mediated cell death by overexpression of the different anti-apoptotic proteins was comparable for all types of clones, showing 10–35% inhibition by either FADD-DN or FLIP and 20–55% by PI-9, indicating that both pathways are involved. As control, Fas antibody-induced apoptosis was almost completely inhibited in EBV-FADD-DN and EBV-FLIP, demonstrating highly effective block of the Fas-receptor pathway by these constructs. These results indicate that despite different kinetics of killing, all T cell clones used both the death receptor pathway and GrB release to kill its targets. In conclusion: 1. Interference with the Fas receptor or the GrB effector pathway cannot prevent CTL-mediated target cell death within the first 2 h of exposure, indicating that this lysis is probably directly perforin-mediated. 2. Both slowly and rapidly killing CTL clones use various effector mechanisms to kill their target cells, including both the death receptor and GrB pathways. In case of slower mediated cell death, not the execution pathway induced by the CTL, but affinity between TCR and MHC/peptide complex and other effector/target interactions more likely determine the kinetics of CTL-mediated cell death.

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

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

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

scholarly journals Hemagglutinin-specific cytotoxic T-cell response during influenza infection.

1977 ◽  
Vol 146 (3) ◽  
pp. 893-898 ◽  
Author(s):  
F A Ennis ◽  
W J Martin ◽  
M W Verbonitz
Keyword(s):  
T Cell ◽  
Target Cell ◽  
Cell Response ◽  
Influenza A ◽  
Influenza Infection ◽  
T Cell Response ◽  
Cytotoxic T Cell ◽  
Target Cells ◽  
Cervical Lymph Nodes ◽  
Cytotoxic Response

Specific cytotoxic thymus-derived (T) lymphocytes were detected in the cervical lymph nodes and spleen during influenza infection of mice. The cytotoxic T cells can distinguish target cells infected with different influenza A subtypes. Infection with parent viruses and their recombinant progeny possessing the hemagglutinin of one parent and the neuraminidase of the other demonstrated that significant cytotoxicity occurred only when the hemagglutinin of the immunizing viruses was the same as that of the virus used to infect the target cell. In addition to this specific cytotoxic response to the major surface antigen, a cross-reactive response could be detected when the relatively nonpermissive L cell was used as the target cell. These results indicate there is a specific cytotoxic T-cell response to the surface hemagglutinin, and a cross-reactive cytotoxic response, not directed to the hemagglutinin, during influenza infection. The cytotoxic T-cell response specific for the hemagglutinin antigen may play an important role in in vivo immunity to influenza.

New CFSE-based assay to determine susceptibility to lysis by cytotoxic T cells of leukemic precursor cells within a heterogeneous target cell population

Blood ◽  
2004 ◽  
Vol 103 (7) ◽  
pp. 2677-2682 ◽  
Author(s):  
Inge Jedema ◽  
Nicole M. van der Werff ◽  
Renée M. Y. Barge ◽  
Roel Willemze ◽  
J. H. Frederik Falkenburg
Keyword(s):  
T Cells ◽  
Cell Death ◽  
T Cell ◽  
Cell Population ◽  
Target Cell ◽  
Precursor Cells ◽  
Leukemic Cells ◽  
Cell Populations ◽  
Target Cells ◽  
Specific Cell

Abstract For the clinical evaluation of the efficacy of cellular immunotherapy it is necessary to analyze the effector functions of T cells against primary leukemic target cell populations which are usually considerably heterogeneous caused by differential maturation stages of the leukemic cells. An appropriate assay should not only allow the quantitative analysis of rapid cell death induction as measured by the conventional 51Cr release assay but also of the more slowly executing pathways of T-cell-induced apoptosis occurring within days instead of hours which cannot be measured using this method. Furthermore, it should dissect the differential susceptibility to T-cell-induced cell death of various target cell subpopulations and characterize the malignant precursor cells capable of producing malignant progeny. To fulfill these requirements we developed a new assay based on carboxyfluorescein diacetate succinimidyl ester (CFSE) labeling of the target cell population combined with antibody staining of specific cell populations and addition of fluorescent microbeads to quantitatively monitor target cell death occurring within a longer time frame up to at least 5 days. This new assay facilitates the analysis of differential recognition of distinct cell types within a heterogeneous target cell population and allows simultaneously evaluation of the proliferative status of surviving target cells in response to relevant cytokines. (Blood. 2004;103: 2677-2682)

The mechanism of T cell mediated cytotoxicity V. Morphological studies by electron microscopy

1977 ◽  
Vol 198 (1132) ◽  
pp. 315-323 ◽  
Keyword(s):  
T Cells ◽  
Cell Death ◽  
T Cell ◽  
Target Cell ◽  
Plasma Membranes ◽  
Ultrastructural Changes ◽  
Early Event ◽  
Target Cells ◽  
Cell Functions ◽  
Morphological Studies

The ultrastructural changes occurring when T cells specifically immune to antigens interact with P815 mastocytoma cells and EL4 lymphoma cells are described and related to changes previously observed by timelapse cinematography (Sanderson 1976 b ). In confirmation of work by others, pale T cells can clearly be in­criminated as cytotoxic cells. Dark T cells also form contacts with target cells, and tend to project lamellipodia over the surface of the target cell. The possibility is discussed that these represent a subset of non-cytotoxic, antigen-reactive T cells involved in other T cell functions. T cells form two types of contact: relatively small point contacts, and large areas where the two plasma membranes are in close apposition. No structures resembling specialized junctions or membrane fusions were observed in areas of contact between T cells and target cells. Close contact between pale T cells and target cells is more regular than contact with dark T cells. Many contacts were seen between morphologically normal target cells and pale T cells, and these were thought to occur in the phase between contact and target cell death. Some of these pale T cells in contact show projections towards the centre of the target cell which invaginate the cell membrane, but do not penetrate it. Remarkable T cell projections were also seen which had penetrated deeply through the membrane of one target cell. These projections appeared to have disrupted the mem­brane and had penetrated into apparently intact cytoplasm, suggesting that this penetration may be an early event in the lytic mechanism. The possibility that this phenomenon is the cause of cell death is discussed. Changes corresponding to the phase of zeiosis of the target cell are described. These commence with the formation of surface blebs, accompanied by a general mis-shapening of the cell outline and followed by vacuolation and loss of cytoplasmic organelles. Breakdown of the nucleus appears to be a later, post mortem event.

The mechanism of T cell mediated cytotoxicity I. The release of different cell components

1976 ◽  
Vol 192 (1107) ◽  
pp. 221-239 ◽  
Keyword(s):  
Cell Death ◽  
T Cell ◽  
Target Cell ◽  
Molecular Form ◽  
Molecular Size ◽  
Fixed Time ◽  
Lag Phase ◽  
Cell Contact ◽  
Target Cells ◽  
Special Cases

A comparison of the release of different markers during T cell mediated cytotoxicity of mastocytoma (P815) target cells is presented. It is shown that contrary to common assumption 51 Cr is largely held in the cell in small molecular form and only a small proportion is bound to macromolecules. Apart from this, it is shown that the release of cytoplasmic macromolecules (proteins labelled with radioactive leucine or methionine, and RNA labelled with uridine) show essentially the same kinetics (no lag phase and linear with time) as the release of phosphoryl choline, phosphorylated 3- O -methyl-glucose, sucrose and chromium, indicating that the kinetics of release of cell constituents is not related to their molecular size. Rubidium and nicotinamide are special cases in that both the spontaneous release and release in the presence of effector cells is different from the other cytoplasmic markers. Rubidium shows a rapid efflux which is complete within 15 min. This appears to be a result of T cell contact rather than the first stage of lysis. Nicotinamide shows a biphasic release suggesting that about 10% of the label effluxes as a result of T cell contact. The remaining label is released with similar kinetics to chromium. In contrast to these markers the DNA labels (thymidine and IUDR) are released with a lag of about 30 min, suggesting that the nuclear membrane is damaged later than the cytoplasmic membrane. These data are consistent with the hypothesis that while target cell death is causally related to T cell contact, there is no fixed time relation between the two events. Target cell death is accompanied by the sudden release of the cytoplasmic contents.

Lytic Polyplex Vaccines Enhance Antigen‐Specific Cytotoxic T Cell Response through Induction of Local Cell Death

2021 ◽  
pp. 2100005
Author(s):  
David J. Peeler ◽  
Albert Yen ◽  
Nicholas Luera ◽  
Patrick S. Stayton ◽  
Suzie H. Pun
Keyword(s):  
Cell Death ◽  
T Cell ◽  
Cell Response ◽  
T Cell Response ◽  
Cytotoxic T Cell ◽  
Local Cell

Beyond apoptosis: nonapoptotic cell death in physiology and disease

2005 ◽  
Vol 83 (5) ◽  
pp. 579-588 ◽  
Author(s):  
Claudio A Hetz ◽  
Vicente Torres ◽  
Andrew F.G Quest
Keyword(s):  
Cell Death ◽  
Programmed Cell Death ◽  
Death Receptor ◽  
Receptor Signaling ◽  
Family Analysis ◽  
Caspase Family ◽  
Death Receptor Signaling ◽  
Apoptotic Pathways ◽  
Nonapoptotic Cell Death ◽  
High Degree

Apoptosis is a morphologically defined form of programmed cell death (PCD) that is mediated by the activation of members of the caspase family. Analysis of death-receptor signaling in lymphocytes has revealed that caspase-dependent signaling pathways are also linked to cell death by nonapoptotic mechanisms, indicating that apoptosis is not the only form of PCD. Under physiological and pathological conditions, cells demonstrate a high degree of flexibility in cell-death responses, as is reflected in the existence of a variety of mechanisms, including necrosis-like PCD, autophagy (or type II PCD), and accidental necrosis. In this review, we discuss recent data suggesting that canonical apoptotic pathways, including death-receptor signaling, control caspase-dependent and -independent cell-death pathways.Key words: apoptosis, necrosis, nonapoptotic programmed cell death, death receptors, ceramides.

scholarly journals A novel type of cell death of lymphocytes induced by a monoclonal antibody without participation of complement.

1995 ◽  
Vol 181 (6) ◽  
pp. 2007-2015 ◽  
Author(s):  
S Matsuoka ◽  
Y Asano ◽  
K Sano ◽  
H Kishimoto ◽  
I Yamashita ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Cell Death ◽  
T Cell ◽  
B Cells ◽  
Interleukin 2 ◽  
Cytolytic Activity ◽  
Target Cells ◽  
T And B Cells ◽  
Fab Fragments ◽  
T Cell Clone

A monoclonal antibody, RE2, raised by immunizing a rat with cell lysate of a mouse T cell clone, was found to directly kill interleukin 2-dependent T cell clones without participation of serum complement. Fab fragments of RE2 had no cytolytic activity, while the cross-linking of Fab fragments with anti-rat immunoglobulin reconstituted the cytotoxicity. The cytotoxicity was temperature dependent: the antibody could kill target cells at 37 degrees C but not at 0 degrees C. Sodium azide, ethylenediaminetetraacetic acid, and forskolin did not affect the cytolytic activity of RE2, while the treatment of target cells with cytochalasin B and D completely blocked the activity. This suggested that the cell death involves a cytoskeleton-dependent active process. Giant holes on the cell membrane were formed within 5 minutes after the treatment with RE2, as observed by scanning electron microscopy. There was no indication of DNA fragmentation nor swelling of mitochondria during the cytolysis, suggesting that the cell death is neither apoptosis nor typical necrosis. The antibody also killed T cell lymphomas and T and B cell hybridomas only when these cells were preactivated with concanavalin A, lipopolysaccharide, or phorbol myristate acetate. Preactivated peripheral T and B cells were sensitive to the cytotoxicity of RE2, while resting T and B cells were insensitive. These results provide evidence for a novel pathway of cell death of activated lymphocytes by membrane excitation.

scholarly journals Absence of allogeneic restriction in human T-cell-mediated cytotoxicity to Epstein-Barr virus-infected target cells. Demonstration of an HLA-linked control at the effector level.

1979 ◽  
Vol 150 (6) ◽  
pp. 1310-1322 ◽  
Author(s):  
M Lipinski ◽  
W H Fridman ◽  
T Tursz ◽  
C Vincent ◽  
D Pious ◽  
...  
Keyword(s):  
T Cell ◽  
T Lymphocytes ◽  
Cell Surface ◽  
Target Cell ◽  
Epstein Barr Virus ◽  
Target Cells ◽  
Specific Lysis ◽  
Barr Virus ◽  
Positive Target ◽  
Epstein Barr

Peripheral T lymphocytes from patients with infectious mononucleosis (IM) are sensitized in vivo against the Epstein-Barr virus (EBV). The expression of HLA-A, B, or C molecules at the target cell surface is necessary for the cytotoxic reaction because (a) EBV-positive Daudi cells lacking HLA-A, B, and C determinants are resistant to anti-EBV T-cell lysis, (b) cytolysis of EBV-positive target cells can be consistently inhibited by anti-HLA-A, B, and C and anti-beta 2 microglobulin antibodies. However, no evidence for allogeneic restriction in this system was apparent as (a) cytotoxic T lymphocytes (CTL) from one given individual could exert a cytotoxicity of a similar magnitude on different EBV-positive target cells, regardless of the number of HLA-A or B specificities shared by the effectors and targets; (b) CTL from IM patients were able to kill target cells without any HLA-A or B antigen in common; and (c) T5-1 variants lacking one or two HLA antigens at the A, B, or D locus are killed to the same extent as the parental cells. 7 of the 9 IM patients with detectable circulating anti-EBV CTL carried the HLA-A1 antigen, whereas none of the 16 IM patients lacking detectable peripheral CTL were HLA-A1 positive (mean specific lysis of T5-1 target cells by T cells from HLA-A1 positive patients: 29.3 vs. 0.6% in HLA-A1-negative patients) (P less than 10(-9)). These data suggest an HLA-A1-linked gene control of the magnitude of the anti-EBV CTL response. Thus, the HLA region appears to act at two different level sin the T-cell-mediated lysis of EBV-infected cells by controlling first, the development of anti-EBV and second, the expression of HLA-A, B, and C molecules involved as recognition structures at the target cell surface.

scholarly journals Fas ligand mediates activation-induced cell death in human T lymphocytes.

1995 ◽  
Vol 181 (1) ◽  
pp. 71-77 ◽  
Author(s):  
M R Alderson ◽  
T W Tough ◽  
T Davis-Smith ◽  
S Braddy ◽  
B Falk ◽  
...  
Keyword(s):  
T Cells ◽  
Cell Death ◽  
T Cell ◽  
Fas Ligand ◽  
Significant Proportion ◽  
Cell Receptor ◽  
Target Cells ◽  
Activated T Cells ◽  
Activation Induced Cell Death ◽  
Fas L

A significant proportion of previously activated human T cells undergo apoptosis when triggered through the CD3/T cell receptor complex, a process termed activation-induced cell death (AICD). Ligation of Fas on activated T cells by either Fas antibodies or recombinant human Fas-ligand (Fas-L) also results in cytolysis. We demonstrate that these two pathways of apoptosis are causally related. Stimulation of previously activated T cells resulted in the expression of Fas-L mRNA and lysis of Fas-positive target cells. Fas-L antagonists inhibited AICD of T cell clones and staphylococcus enterotoxin B (SEB)-specific T cell lines. The data indicate AICD in previously stimulated T cells is mediated by Fas/Fas-L interactions.

Sign in / Sign up

Export Citation Format

Share Document