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Enhancement of FasL VP-mediated cytotoxicity depends on target FcR expression. a,Various targets were stained with biotinylated 2.4G2 anti-FcR mAb (bold line) or control IgG2b (light line) followed by PE-conjugated streptavidin (FL2). Stained cells were analyzed with a FACScan flow cytometer. The experiments were repeated twice with similar results. b, Various doses of FasL VP were used to kill FcR A20 and FcR A20 targets in the presence or absence of NOK-1 mAb (10 ng/ml). c, The ability of various doses of NOK-1 mAb to modulate FasL VP cytotoxicity is compared between the FcR A20 and FcR A20 targets.
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Bioactive Fas ligand (FasL)-expressing vesicles were generated (vesicle preparation, VP) from two cell lines overexpressing FasL. The effect of NOK-1 anti-FasL mAb (mouse IgG1) on the cytotoxicity of FasL VP against various targets was determined. At high concentrations (1-10 microg/ml), NOK-1 inhibited the cytotoxicity. By contrast, NOK-1 in the d...
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Context 1
... target FcR expres- sion was determined by fluorescent staining using the FITC-con- jugated 2.4G2 anti-FcR mAb. The results showed a specific and strong staining of LB27.4, LF , and M59 cells (target populations that exhibit "enhanceable cytotoxicity"). In contrast, staining of K3 cells, the "nonenhanceable target population," was extremely weak (Fig. 4a). To further demonstrate that the enhancement of FasL VP cytotoxicity depends on FcR expression, we compared the effect of NOK-1 mAb on the cytotoxicity of FasL VP against FcR A20 and FcR A20 target populations. As shown in Fig. 4b, FasL VP killed both targets, and the sensitivity of the two targets was comparable. In the presence of 10 ...
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... cytotoxicity"). In contrast, staining of K3 cells, the "nonenhanceable target population," was extremely weak (Fig. 4a). To further demonstrate that the enhancement of FasL VP cytotoxicity depends on FcR expression, we compared the effect of NOK-1 mAb on the cytotoxicity of FasL VP against FcR A20 and FcR A20 target populations. As shown in Fig. 4b, FasL VP killed both targets, and the sensitivity of the two targets was comparable. In the presence of 10 ng/ml NOK-1 mAb, killing of FcR A20 was significantly enhanced. The enhancement was observed over a wide range of the FasL VP concentrations exam- ined. In contrast, killing of the FcR A20 target by FasL VP was inhibited (Fig. ...
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... As shown in Fig. 4b, FasL VP killed both targets, and the sensitivity of the two targets was comparable. In the presence of 10 ng/ml NOK-1 mAb, killing of FcR A20 was significantly enhanced. The enhancement was observed over a wide range of the FasL VP concentrations exam- ined. In contrast, killing of the FcR A20 target by FasL VP was inhibited (Fig. 4b). We also determined the effect of various doses of NOK-1 mAb on FasL VP cytotoxicity. Although a dose-depen- dent inhibition was observed with the FcR A20 target, the killing of FcR A20 was enhanced by low doses of NOK-1 mAb and inhibited by high doses of NOK-1 mAb (Fig. ...
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... In contrast, killing of the FcR A20 target by FasL VP was inhibited (Fig. 4b). We also determined the effect of various doses of NOK-1 mAb on FasL VP cytotoxicity. Although a dose-depen- dent inhibition was observed with the FcR A20 target, the killing of FcR A20 was enhanced by low doses of NOK-1 mAb and inhibited by high doses of NOK-1 mAb (Fig. ...
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Citations
... These ligands belong to the TNF family and are naturally expressed by immune cells, including NK cells, granulocytes, monocytes, T cells, B cells and dendritic cells, among others (60). They are transmembrane proteins that can be proteolytically cleaved and released in a soluble form or forming part of microvesicles (61)(62)(63)(64)(65)(66)(67). In contrast to soluble FasL that does not present bioactivity, both soluble TRAIL and TNF-a mediate different biological activities including killing of cancer cells (68). ...
NK cells are key mediators of immune cell-mediated cytotoxicity toward infected and transformed cells, being one of the main executors of cell death in the immune system. NK cells recognize target cells through an array of inhibitory and activating receptors for endogenous or exogenous pathogen-derived ligands, which together with adhesion molecules form a structure known as immunological synapse that regulates NK cell effector functions. The main and best characterized mechanisms involved in NK cell-mediated cytotoxicity are the granule exocytosis pathway (perforin/granzymes) and the expression of death ligands. These pathways are recognized as activators of different cell death programmes on the target cells leading to their destruction. However, most studies analyzing these pathways have used pure recombinant or native proteins instead of intact NK cells and, thus, extrapolation of the results to NK cell-mediated cell death might be difficult. Specially, since the activation of granule exocytosis and/or death ligands during NK cell-mediated elimination of target cells might be influenced by the stimulus received from target cells and other microenvironment components, which might affect the cell death pathways activated on target cells. Here we will review and discuss the available experimental evidence on how NK cells kill target cells, with a special focus on the different cell death modalities that have been found to be activated during NK cell-mediated cytotoxicity; including apoptosis and more inflammatory pathways like necroptosis and pyroptosis. In light of this new evidence, we will develop the new concept of cell death induced by NK cells as a new regulatory mechanism linking innate immune response with the activation of tumour adaptive T cell responses, which might be the initiating stimulus that trigger the cancer-immunity cycle. The use of the different cell death pathways and the modulation of the tumour cell molecular machinery regulating them might affect not only tumour cell elimination by NK cells but, in addition, the generation of T cell responses against the tumour that would contribute to efficient tumour elimination and generate cancer immune memory preventing potential recurrences.
... Another group of EV proteins found in our N samples is potentially related to immune suppression/modulation effects. CD95, for instance, a member of the TNF family of death factors, when expressed on EVs can enhance cytotoxicity in an antibody-mediated, FcR-dependent way [44]. On the other hand, several N-EV components have been related to tumor biology or could underlie the linkage of blood transfusion with the risk of cancer recurrence and mortality in surgical cancer patients [45,46]. ...
Prestorage filtration of blood to remove contaminating donor leukocytes and platelets has substantially increased the safety level of transfusion therapy. We have previously shown that leukoreduction has a mitigating effect on the storage lesion profile by lowering the extent of hemolysis and of RBC aging and removal phenotypes, including surface signaling and microvesiculation. Even though protein composition may determine the fate of EVs in the recipient, the probable effect of leukoreduction on the EV proteome has been scarcely investigated. In the present paired study, we characterized the proteome of EVs released in prestorage leukoreduced (L) and nonleukoreduced (N) RBC units prepared from the same donors, by immunoblotting and qualitative proteomics analyses at two storage intervals. Apart from common proteofrms typically associated with the established EV biogenesis mechanisms, the comparative proteomics analyses revealed that both leukoreduction and storage duration affect the complexity of the EV proteome. Membrane and cytoskeleton-related proteins and regulators, metabolic enzymes and plasma proteins exhibited storage duration dependent variation in L- and N-EVs. Specific proteoforms prevailed in each EV group, such as transferrin in L-units or platelet glycoproteins, leukocyte surface molecules, MHC HLA, histones and tetraspanin CD9 in N-units. Of note, several unique proteins have been associated with immunomodulatory, vasoregulatory, coagulatory and anti-bacterial activities or cell adhesion events. The substantial differences between EV composition under the two RBC preparation methods shed light in the underlying EV biogenesis mechanisms and stimuli and may lead to different EV interactions and effects to target cells post transfusion.
... Fas ligand (FasL), a type II transmembrane protein and a member of the tumour necrosis factor receptors, on binding with its receptor FAS, induces apoptosis. Previous studies have reported the sorting of FasL to MVB and its release to the extracellular environment [80][81][82][83][84]. However, the sorting mechanism was not fully understood. ...
Exosomes, a type of small extracellular vesicles (sEVs), are secreted membrane vesicles that are derived from various cell types, including cancer cells, mesenchymal stem cells, and immune cells via multivesicular bodies (MVBs). These sEVs contain RNAs (mRNA, miRNA, lncRNA, and rRNA), lipids, DNA, proteins, and metabolites, all of which mediate cell-to-cell communication. This communication is known to be implicated in a diverse set of diseases such as cancers and their metastases and degenerative diseases. The molecular mechanisms, by which proteins are modified and sorted to sEVs, are not fully understood. Various cellular processes, including degradation, transcription, DNA repair, cell cycle, signal transduction, and autophagy, are known to be associated with ubiquitin and ubiquitin-like proteins (UBLs). Recent studies have revealed that ubiquitin and UBLs also regulate MVBs and protein sorting to sEVs. Ubiquitin-like 3 (UBL3)/membrane-anchored Ub-fold protein (MUB) acts as a post-translational modification (PTM) factor to regulate efficient protein sorting to sEVs. In this review, we focus on the mechanism of PTM by ubiquitin and UBLs and the pathway of protein sorting into sEVs and discuss the potential biological significance of these processes.
... The observation of a constitutive release of FasL-and TRAIL-expressing exosomes from the apical microvillous surface of the STB suggests an important role of such structures in the protection of the fetus from maternal lymphocytes (Stenqvist et al., 2013). This finding is in accordance with the first demonstrations showing that FasL is targeted to the MVB of the secretory lysosomes and is expressed on exosome-like microvesicles (Martínez-Lorenzo et al., 1999;Jodo et al., 2000;Mincheva-Nilsson et al., 2000;Monle on et al., 2001;Andreola et al., 2002;Smith et al., 2003;Fr€ angsmyr et al., 2005). ...
This review correlates and summarizes the role of the maternal-fetal interface in the immune tolerance of the fetus and the processes that lead to infection avoidance, emphasizing the participation of exosomes and other extracellular vesicles in both situations. Exosomes are released into the extracellular medium by several cell types and are excellent carriers of biomolecules. Host-derived exosomes and the transport of pathogen-derived molecules by exosomes impact infections in different ways. The interactions of exosomes with the maternal immune system are pivotal to a favorable gestational outcome. In this review, we highlight the potential role of exosomes in the establishment of an adequate milieu that enables embryo implantation and discuss the participation of exosomes released at the maternal-fetal interface during the establishment of an immune-privileged compartment for fetal development. The placenta is a component where important strategies are used to minimize the risk of infection. To present a contrast, we also discuss possible mechanisms used by pathogens to cross the maternal-fetal interface. We review the processes, mechanisms, and potential consequences of dysregulation in all of the abovementioned phenomena. Basic information about exosomes and their roles in viral immune evasion is also presented. The interactions between extracellular vesicles and bacteria, fungi, parasites and proteinaceous infectious agents are addressed. The discovery of the placental microbiota and the implications of this new microbiota are also discussed, and current proposals that explain fetal/placental colonization by both pathogenic and commensal microbes are addressed. The comprehension of such interactions will help us to understand the immune dynamics of human pregnancy and the mechanisms of immune evasion used by different pathogens.
... Furthermore, a constitutive release of FasL-and TRAIL-expressing exosomes from the apical microvillous surface of STB was demonstrated. Our results are in tune with previous investigations by others and us (22,31,32) showing that FasL is targeted to the MVB of secretory lysosomes and is expressed on exosome-like microvesicles (5,15,33,34). Two independent pathways that control the internalization of FasL on microvesicles in MVBs have been identified-phosphorylation of FasL by binding of its prolinerich domain to Fgr, Fyn, and Lyn tyrosine kinases and a direct ubiquitinylation at the lysine residues flanking the proline-rich domain (35)(36)(37). ...
... When the expression of these oligomerized ligands was lost from the exosomal membrane, the proapoptotic ability was lost as well as shown in Supplemental Fig. 1. The functional experiments are in-tune with apoptosis triggered by exosomes from other sources such as activated T cells and DC cells (5,15,34,48,49) and various cancer cells (31,33,50,51,54). To our knowledge, this is the first report of expression of preformed oligomerized FasL and TRAIL on exosomal membrane. ...
Apoptosis is crucially important in mediating immune privilege of the fetus during pregnancy. We investigated the expression and in vitro apoptotic activity of two physiologically relevant death messengers, the TNF family members Fas ligand (FasL) and TRAIL in human early and term placentas. Both molecules were intracellularly expressed, confined to the late endosomal compartment of the syncytiotrophoblast, and tightly associated to the generation and secretion of placental exosomes. Using immunoelectron microscopy, we show that FasL and TRAIL are expressed on the limiting membrane of multivesicular bodies where, by membrane invagination, intraluminal microvesicles carrying membranal bioactive FasL and TRAIL are formed and released in the extracellular space as exosomes. Analyzing exosomes secreted from placental explant cultures, to our knowledge, we demonstrate for the first time that FasL and TRAIL are clustered on the exosomal membrane as oligomerized aggregates ready to form death-inducing signaling complex. Consistently, placental FasL- and TRAIL-carrying exosomes triggered apoptosis in Jurkat T cells and activated PBMC in a dose-dependent manner. Limiting the expression of functional FasL and TRAIL to exosomes comprise a dual benefit: 1) storage of exosomal FasL and TRAIL in multivesicular bodies is protected from proteolytic cleavage and 2) upon secretion, delivery of preformed membranal death molecules by exosomes rapidly triggers apoptosis. Our results suggest that bioactive FasL- and TRAIL-carrying exosomes, able to convey apoptosis, are secreted by the placenta and tie up the immunomodulatory and protective role of human placenta to its exosome-secreting ability.
... The standard MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) (Sigma) assays were conducted 18 hours after IFN-γ treatment [11]. In some experiments, cytotoxicity assays were conducted in the presence of inhibitors such as Alf 1.2 anti-FasL, NOK-1 anti-FasL [12], cc83 anti-TAG -72 [5] or anti-L6 [6] mAb with isotype included as appropriate controls. ...
... We transfected several cell lines with the recombinant cc49scFv-FasL ext and L6scFv-FasL ext constructs and used limiting dilution technique to obtain the clones that produced the highest cytotoxicity against the A20 tumor cells, which express Fas Ag and are sensitive to sFasLmediated cytotoxicity [12]. The selected clones were then gradually adapted to growth in normal culture medium without the selection agent. ...
Targeted therapy of human cancers is an attractive approach and has been investigated with limited success. We have developed novel cytotoxic agents for targeted therapy of human cancers based on the extracellular cytotoxicity domain of CD178 (FasL) and the specificity offered by single chain antibodies (scFv) against dominant human tumor Ag TAG-72 (cc49scFv) and TAL6 (L6scFv).
The cc49scFv-FasLext is highly effective in in vitro killing of human TAG-72+ Jurkat-Ras tumor cells with a 30,000 fold greater cytotoxicity as compared to soluble FasL (sFasL). On the other hand, L6scFv-FasLext only increased cytotoxicity 500-fold as compared with sFasL against TAL6+ HeLa cells in in vitro assays. The high specificity and strong cytotoxicity of cc49scFv-FasLext made it feasible to cure IP-implanted Jurkat-Ras tumors in SCID mice.
Our study demonstrated that scFv-FasLext with a strong cytotoxicity against sensitive human tumor targets may be useful as effective chemotherapeutic agents.
... FasL can be removed from the cell surface by matrix metalloproteinase-like enzymes (21) or can be released as a membrane component of microvesicles (22). Whereas nonaggregated soluble FasL is generally accepted as a nonactive ligand (23,24), in the supernatant of stimulated T cells, the microvesicle-bound form of FasL was shown to be active in mediating cell death, indicating that AICD can be stimulated not only by direct cell-to-cell contact, but also by secreted vesicles (Vsec) called exosomes (25,26). ...
... The presence of FasL-expressing soluble granules has been observed in several culture supernatants (CSups) derived from PHA-prestimulated Jurkat cells, PBMC, or melanoma cells (25)(26)(27)(28)(29)(30)(31)(32)(33). Cytotoxic activity was detectable within 15 min after stim-ulation and could be attenuated by anti-Fas neutralizing Ab, providing evidence for functional, preformed FasL secreted upon PHA activation (26)(27)(28)(29)32). ...
... The presence of FasL-expressing soluble granules has been observed in several culture supernatants (CSups) derived from PHA-prestimulated Jurkat cells, PBMC, or melanoma cells (25)(26)(27)(28)(29)(30)(31)(32)(33). Cytotoxic activity was detectable within 15 min after stim-ulation and could be attenuated by anti-Fas neutralizing Ab, providing evidence for functional, preformed FasL secreted upon PHA activation (26)(27)(28)(29)32). Following CSup ultracentrifugation, the cytotoxic activity was retained in the pellet, and FasL could be visualized by electron microscopy in structures of 50-200 nm in diameter either in intracellular cellular stores or in the supernatant of the activated cells by Western blot (25,30,32). ...
Activated T cells secrete Fas ligand (FasL)-containing vesicles (secreted vesicles) that induce death of target cells. We provide evidence that secreted vesicles from culture supernatants (Csup) of various origins are able to generate both Fas-dependent apoptotic and Fas-independent, nonapoptotic cell death. In the absence of Fas, the nonapoptotic, Fas-independent pathway could still induce cell death. In contrast to RIP-independent classical Fas-induced cell death triggered by cross-linked or membrane-bound FasL, CSup-derived stimuli-induced apoptosis exhibited unique molecular and enzymatic characteristics. It could be partially inhibited by blocking cathepsin D enzyme activity and required the presence of RIP. Whereas stimulation with CSup, derived from both FasL-overexpressing Jurkat cells and PBMC, could induce cell death, the requirements for Fas-associated death domain protein and caspase-9 were different between the two systems. Our study highlights an important distinction between cell contact-mediated and secreted vesicle-generated activation-induced cell death and also demonstrates that the type of the secreted vesicles can also modify the cell death route. We propose that besides cell-to-cell interaction-mediated Fas triggering, stimuli induced by secreted vesicles can mediate important additional cell death signals regulating activation-induced cell death under physiological conditions.
... Once at the cell surface, FasL is cleaved via the action of metalloproteinases [13,14], liberating a soluble FasL extracellular domain, which has limited potency as an apoptosis inducer [15]. Transmembrane FasL is more effective at stimulating apoptosis, and can also be released extracellularly via secretion of microvesicles containing FasL [11,16]. ...
Fas Ligand (FasL, CD178) is a cytokine that may be secreted or expressed as a transmembrane ligand at the cell surface, and induces apoptosis by binding to the "death receptor" Fas (CD95). Here, we show that Grb2, an SH3 domain-containing adaptor protein, binds to the proline-rich domain of FasL and regulates its cell surface expression. We found that knocking down Grb2 expression decreased the amount of FasL at the cell surface and increased the abundance of intracellular vesicles containing FasL. Furthermore, we showed that Grb2 acts as an adaptor for FasL to interact with adaptin beta, a molecule known to regulate trafficking. Our data reveal that Grb2 facilitates the association of FasL with adaptin beta, and promotes sorting of FasL to the cell surface. As FasL is a potent regulator of cell death, dynamic regulation of its cell surface localization is critical for controlling local tissue remodeling and inflammation.
... FasL plays a key role in regulation within the immune system and loss of FasL gives rise to autoimmune lymphoproliferative syndrome (ALPS1B) (Wu et al., 1996). FasL is targeted to the multivesicular bodies (MVB) of secretory lysosomes (Andreola et al., 2002;Frangsmyr et al., 2005;Smith et al., 2003) and has been isolated on secreted bioactive microvesicles (Jodo et al., 2000;Martinez-Lorenzo et al., 1999;Mincheva-Nilsson et al., 2000;Monleón et al., 2001) thought to derive from these inner membrane vesicles. Small microvesicles are released into the synaptic cleft between CTL and target (Peters et al., 1989). ...
Fas ligand (FasL), a potent mediator of apoptosis expressed by CTL and NK cells, is sorted into the inner vesicles of secretory lysosomes for release via exosome-like vesicles. Previous studies identified a proline-rich domain in the cytoplasmic tail required for sorting FasL to secretory lysosomes, but the mechanisms by which this occurs have not been identified. Here we demonstrate that the PRD of FasL binds Fgr, Fyn and Lyn tyrosine kinases, leading to phosphorylation of FasL. Loss of phosphorylation reduces internalisation of FasL into multivesicular bodies. FasL is also directly mono-ubiquitylated at lysines flanking the PRD and mutation of these lysines reduces MVB localisation of FasL. Phosphorylation is not required for ubiquitylation because FasL lacking all tyrosines undergoes mono-ubiquitylation. These studies show that phosphorylation and ubiquitin signals regulate the sorting of FasL to secretory lysosomes by controlling entry into multivesicular bodies.
... Our ®ndings indicate that treatment of Jurkat T cells and peripheral blood lymphocytes with the secreted FasL, once liberated from the microvesicle, induced Fas-mediated apoptosis through activation of the caspase pathway. Other groups have reported the expression of FasL on the surface of microvesicles and found that these intact vesicles were able to induce Fas-mediated apoptosis (Martinez-Lorenzo et al., 1999;Jodo et al., 2000;Andreola et al., 2002). A possible explanation for this discrepancy may be the different cell types used for microvesicle preparation. ...
Since the invading trophoblast represents a semi-allograft, it should be rejected by the mother. It has, therefore, been postulated that during normal pregnancy the trophoblast evades the maternal immune system though the establishment of immune privilege by triggering the death of activated lymphocytes which may be sensitized to paternal alloantigens. Such peripheral tolerance may be directed through the Fas/Fas ligand (FasL) apoptotic pathway and mediated by FasL expressed by the trophoblast. However, in vivo studies show that membrane-associated expression of FasL may instead promote allograft rejection, rather than protection. The aim of this study was to determine if there is a role for FasL in trophoblast immune privilege. In this study, we demonstrate that isolated first trimester trophoblast cells lack membrane-associated FasL, but express a cytoplasmic form in association with a specialized secretory lysosomal pathway. Furthermore, this intracellular FasL is constitutively secreted by trophoblast cells via the release of microvesicles. Following disruption of these microvesicles, the whole 37 kDa secreted FasL is able to induce T-cell death by apoptosis through activation of the Fas pathway. Therefore, we propose that secretion of FasL may be one mechanism by which trophoblast cells promote a state of immune privilege and, therefore, protect themselves from maternal immune recognition.
