Variant antigenic peptide promotes cytotoxic T lymphocyte adhesion to target cells without cytotoxicity

Shotton, David M.; Attaran, Amir

doi:10.1073/pnas.95.26.15571

Cited by 14 publications

(16 citation statements)

References 49 publications

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“…Zeosis, which is derived from a Greek word meaning "to boil over," is characterized by rapid cytokinesis with continuous bleb extrusion and retraction (5). Zeosis has been associated with apop- tosis (9,21,22,37). As shown in supplemental video 2 (the online version of this article contains supplemental data), some cells round up and blebs are rapidly extruded and retracted on the surface of the cell, giving rise to rapid movement artifacts that cause the apparent oscillation profiles shown in the fluorescence traces.…”

Section: Effect Of Ptx On [Ca 2ϩmentioning

confidence: 99%

Palytoxin-induced cell death cascade in bovine aortic endothelial cells

Schilling¹,

Snyder²,

Sinkins³

et al. 2006

American Journal of Physiology-Cell Physiology

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ϩ -K ϩ -ATPase (NKA) pump is the receptor for the potent marine toxin palytoxin (PTX). PTX binds to the NKA and converts the pump into a monovalent cation channel that exhibits a slight permeability to Ca 2ϩ . However, the ability of PTX to directly increase cytosolic free Ca 2ϩ concentration ([Ca 2ϩ ]i) via Na ϩ pump channels and to initiate Ca 2ϩ overload-induced oncotic cell death has not been examined. Thus the purpose of this study was to determine the effect of PTX on [Ca 2ϩ ]i and the downstream events associated with cell death in bovine aortic endothelial cells. PTX (3-100 nM) produced a graded increase in [Ca 2ϩ ]i that was dependent on extracellular Ca 2ϩ . The increase in [Ca 2ϩ ]i initiated by 100 nM PTX was blocked by pretreatment with ouabain with an IC50 Ͻ 1 M. The elevation in [Ca 2ϩ ]i could be reversed by addition of ouabain at various times after PTX, but this required much higher concentrations of ouabain (0.5 mM). These results suggest that the PTX-induced rise in [Ca 2ϩ ]i occurs via the Na ϩ pump. Subsequent to the rise in [Ca 2ϩ ]i, PTX also caused a concentration-dependent increase in uptake of the vital dye ethidium bromide (EB) but not YO-PRO-1. EB uptake was also blocked by ouabain added either before or after PTX. Time-lapse video microscopy showed that PTX ultimately caused cell lysis as indicated by release of transiently expressed green fluorescent protein (molecular mass 27 kDa) and rapid uptake of propidium iodide. Cell lysis was 1) greatly delayed by removing extracellular Ca 2ϩ or by adding ouabain after PTX, 2) blocked by the cytoprotective amino acid glycine, and 3) accompanied by dramatic membrane blebbing. These results demonstrate that PTX initiates a cell death cascade characteristic of Ca 2ϩ overload.necrosis; vital dyes; membrane blebs; time-lapse video microscopy; fura-2 NATURAL TOXINS AND POISONS (e.g., cholera toxin, pertussis toxin, tetrodotoxin, conotoxin, digitalis, ryanodine, and thapsigargin) have proved useful in the identification and functional characterization of specific proteins in biochemical pathways critical for cell homeostasis and signaling. Palytoxin (PTX), originally isolated from sea corals of the genera Palythoa (24), is one of the most potent toxins known. PTX causes membrane depolarization, loss of cellular K ϩ , and a dramatic increase in cytosolic Na ϩ . PTX causes contractions of all muscles, release of neurotransmitters, hemolysis of red blood cells, and, ultimately, oncotic cell death (for review, see Ref. 38). It is now clear that the molecular receptor for PTX is the plasmalemmal Na ϩ -K ϩ -ATPase (NKA) pump. Early studies showed that the cardiac glycoside ouabain could effectively antagonize the actions of PTX, and it was suggested that PTX might convert the NKA into a channel (14, 15). Indeed, a variety of investigators showed that PTX activates a relatively nonselective cation channel with conductance in the range of 8 -14 pS (16,17,20,25,27, 28,39,43). PTX-induced cation fluxes were activated when the NKA was heterolog...

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Section: Effect Of Ptx On [Ca 2ϩmentioning

confidence: 99%

Palytoxin-induced cell death cascade in bovine aortic endothelial cells

Schilling¹,

Snyder²,

Sinkins³

et al. 2006

American Journal of Physiology-Cell Physiology

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“…To this end, we have used CD8 ϩ T cells (obtained from F5 TCR-transgenic mice) specific for the peptide NP68 (the immunodominant epitope of the influenza nucleoprotein strain A/NT/60/68) (36,37). The MHC class I anchor sites of this peptide have been previously established and a number of APLs with similar affinities to the MHC class I molecule, but different activation of T cells, have been identified (34,38,39). By using exogenous APLs (that have similar affinities to the MHC class I) as Ags, we were able to obtain a similar presentation of all APLs by the APCs.…”

mentioning

confidence: 99%

Enhanced Levels of Costimulation Lead to Reduced Effector/Memory CD8+ T Cell Functionality

Mostböck

Vidal

Schlom

et al. 2007

The Journal of Immunology

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The role of different levels of costimulation in conjunction with signal 1 in the activation of memory CD8+ T cells remains elusive. In this study, we demonstrate, in a mouse model with the influenza nucleoprotein epitope NP68, that mouse early memory (effector/memory) CD8+ T cells that were generated with high levels of costimulation have reduced CTL functionality compared with those that were generated with low levels of costimulation. This reduction is associated with increased phosphorylation of the negative regulatory site 292 on Zap70 and a decrease in granzyme B levels. Furthermore, we show that enhanced costimulation reduces proliferation and cytokine production of effector/memory CD8+ T cells in response to intermediate and weak TCR stimulation, in contrast to previously described positive effects of costimulation on naive CD8+ T cells. This effect is associated with the expression of ICAM-1 on APCs. Together, our results indicate that enhanced costimulation can lead to reduced functionality in effector/memory CD8+ T cells. This compromised effector function of effector/memory CD8+ T cells in response to high levels of costimulation can have important implications for designing immunotherapeutic strategies to enhance immune responses.

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“…Several approaches (24-33) have been described to assess immune lysis, including the classical [ 51 Cr]-release assay (26,27), the europium (Eu3+)-release assay (28)(29)(30), BLT esterase activity (31), b-galactosidase release (32), FACS assays (33), and video microscopy (34,35). Particularly relevant to the current study, a microfabricated device has been described (36) and is commercially available (xCELLigence; http://www.aceabio.com/case_info.aspx?id=249), enabling the dynamic, real-time, label-free detection of NK cell lysis by measurement of electric impedance in a microtiter format.…”

Section: Discussionmentioning

confidence: 99%

Lysis-on-Chip of Single Target Cells following Forced Interaction with CTLs or NK Cells on a Dielectrophoresis-Based Array

Abonnenc¹,

Borgatti

Fabbri

et al. 2013

The Journal of Immunology

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Guiding the interaction of single cells acting as partners in heterotypic interactions (e.g., effectors and targets of immune lysis) and monitoring the outcome of these interactions are regarded as crucial biomedical achievements. In this study, taking advantage of a dielectrophoresis (DEP)-based Laboratory-on-a-chip platform (the DEPArray), we show that it is possible to generate closed DEP cages entrapping CTLs and NK cells as either single cells or clusters; reversibly immobilize a single virus-presenting or tumor cell within the chip at a selected position; move cages and their content to predetermined spatial coordinates by software-guided routing; force a cytotoxic effector to physically interact with a putative target within a secluded area by merging their respective cages; generate cages containing effector and target cells at predetermined E:T ratios; accurately assess cytotoxicity by real-time quantitation of the release kinetics of the fluorescent dye calcein from target cells (>50 lytic events may be tested simultaneously); estimate end points of calcein release within 16 min of initial E:T cell contact; simultaneously deliver Ab-based phenotyping and on-chip lysis assessment; and identify lytic and nonlytic E:T combinations and discriminate nonlytic effector phenotypes from target refractoriness to immune lysis. The proof of principle is provided that DEPArray technology, previously used to levitate and move single cells, can be used to identify highly lytic antiviral CTLs and tumor cells that are particularly refractory to NK cell lysis. These findings are of primary interest in targeted immunotherapy.

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Variant antigenic peptide promotes cytotoxic T lymphocyte adhesion to target cells without cytotoxicity

Abstract: Timelapse video microscopy has been used to record the motility and dynamic interactions between an H-2D b -restricted murine cytotoxic T lymphocyte clone (

Cited by 14 publications

References 49 publications

Palytoxin-induced cell death cascade in bovine aortic endothelial cells

Palytoxin-induced cell death cascade in bovine aortic endothelial cells

Enhanced Levels of Costimulation Lead to Reduced Effector/Memory CD8+ T Cell Functionality

Lysis-on-Chip of Single Target Cells following Forced Interaction with CTLs or NK Cells on a Dielectrophoresis-Based Array

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