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Published online before print December 5, 2005
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B signaling and caspase activity
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* MediCity Research Laboratory and Department of Medical Microbiology, University of Turku, and National Public Health Institute, Turku, Finland;
Turku Centre for Biotechnology, University of Turku, and Åbo Akademi University, Finland;
Department of Biology, Åbo Akademi University, Turku, Finland;
¶ Department of Biology, Laboratory of Animal Physiology, University of Turku, Finland; and
Turku Graduate School of Biomedical Sciences, Finland
1Correspondence: MediCity Research Laboratory, University of Turku, Tykistökatu 6A, 20520 Turku, Finland. E-mail: arno.hanninen{at}utu.fi
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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(IFN-) production. To understand this costimulation, we investigated the roles of caspases and nuclear factor (NF)-
B in survival and proliferation of responding T cells. Fas ligation increased caspase-3 and -8 activities during T cell activation, irrespective of cell fate. The accelerated proliferation induced by Fas ligation could be reduced by selective inhibition of both caspases. Inhibition of NF-B simultaneously with Fas ligation inhibited the increased IFN- production and caused uniform death of all responding T cells. Thus, Fas-mediated costimulation of naïve CD4 T cells is driven by active caspases, and NF-B acts as a dominant survival-supporting factor of Fas-costimulated cells containing high levels of activated caspase-8 and -3.
Key Words: T cell activation • lymphocyte • apoptosis • dendritic cell • cell proliferation
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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B pathway (c-FLIPL) [13
]. Typically, activated T cells are sensitive to death induction via Fas signaling. Conversely, memory CD4 T cells have been reported to be costimulated to proliferate by Fas ligation [14
]. It is conceivable that T cells at different stages (naïve-proliferating-effector-memory) of their activation are phenotypically divergent, and their different intracellular signaling machineries explain how a signal from a given receptor can lead to stimulatory or inhibitory responses. It seems paradoxical that even the same machinery can be used by the cell to mediate stimulatory and inhibitory responses. In fact, many cytoplasmic proteins involved in apoptotic signaling upon ligation of death receptors are also involved in productive lymphocyte activation. Accordingly, FADD-deficient T cells have an impaired capability to proliferate [15 ], and the expression of the dominant-negative mutant protein FADD in transgenic T cells inhibits proliferation of mature T lymphocytes [16 ]. Various checkpoints have been reported to be decisive of proliferative or inhibitory signaling following ligation of Fas or other receptors of the TNFR family. Kennedy et al. [11 ] reported that stimulation of resting human T cells by CD3 cross-linking, alone or together with Fas costimulation, results in caspase-8 but not caspase-3 processing. Also, T cells in mice with a targeted caspase-8 mutation restricted to the T cell lineage proliferate weakly, and the mice fail to mount antiviral immunity [17 ]. Recently, an inherited caspase-8 mutation in humans was described that leads to immunodeficiency [18 ]. These data indicate an important role for caspase-8 in T cell activation. There is also evidence of a role for caspase-3 in T cell activation. Alam et al. [19 ] reported that T cell activation associates with selective processing of certain caspase-3 substrates and that selective inhibition of caspase-3 abrogates proliferation. This is consistent with previous studies by others showing that caspase-3 activation does not necessarily lead to apoptosis in T cells [20 , 21 ].
Caspase-8 activated by Fas ligation can also activate NF-B signaling. Procaspase-8 can form a heterodimer with c-FLIPL in the death-inducing signaling complex (DISC), where both proteins are autoproteolytically cleaved rapidly [22
]. Using cell lines, the cleaved FLIP(p43) fragment was shown to interact specifically with TNFR-associated factor (TRAF)2, which subsequently activated NF-B [23
] and extracellular signal-regulated kinase [13
] signaling pathways. Thus, activation of NF-B could explain the stimulatory effects of Fas ligation in apoptosis-resistant cells.
In the immune system, T cells are activated by dendritic cells (DC), which engage TCRs with antigen peptides complexed with major histocompatibility complex molecules. A few studies reported previously that antigen presentation by DC engineered to express FasL would induce apoptosis in antigen-specific T cells by activation of death signaling via Fas [24
25
26
]. However, more recently, Buonocore et al. [27
] reported that DC engineered to express FasL elicit stronger T helper cell type 1 and cytotoxic T cell responses than control cells in vivo. Independently of this, we have reported earlier [28
] that at the time of activation of naïve CD4 T cells with antigen-bearing DC, Fas ligation stimulates proliferation and interferon- (IFN-) production in cells surviving its apoptotic effect. The idea that Fas has a costimulatory function is supported by the facts that Fas is expressed on naïve T cells, and FasL is expressed on a highly immunogenic DC subset, activated Langerhans cells [29
, 30
].
In this work, we have elucidated the role of NF-B as a modulator of Fas-mediated costimulation in naïve CD4 T cells activated by mature antigen-bearing DC. Our results imply that Fas mediates costimulation by activating caspase-8 and -3. They also indicate that NF-B has a role as a life-supporting factor in Fas-costimulated cells containing high levels of activated caspase-8 and -3. In addition, we show that Fas ligation-induced IFN- production is dependent on NF-B signaling.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Mice were bred and maintained in the Central Animal Laboratory in Turku University (Finland) under specific pathogen-free conditions and were used at 6–10 weeks of age. The Institutional Ethical Committee of Turku University approved all animal experiments.
Preparation and activation of DC
DC were propagated from bone marrow cells according to the method of Inaba and co-workers [32
] with slight modifications. Briefly, bone marrow cells were collected from aseptically prepared (washed in 70% ethanol) femoral bones of RAG2–/– mice, and the red cells were lysed using hypotonic saline. The remaining cells were cultured for 5 days in complete medium consisting of RPMI 1640 supplemented with 10% fetal calf serum, 20 mM L-glutamine, 50 µM 2-mercaptoethanol, penicillin/streptomycin, and 20 µg/ml recombinant murine granulocyte macrophage-colony stimulating factor (PharMingen, San Diego, CA). After 5 days of culture, the cells were washed and antigen-pulsed with medium containing 2.0 mg/mL OVA (grade V, Sigma Chemical Co., St. Louis, MO). After 2 h, 10 µg/mL anti-CD40 (clone HM40-3, PharMingen) and 1 µg/mL anti-Fas (clone Jo2, PharMingen) antibodies were added to activate the DC. After overnight incubation, DC were collected, and CD11c+ cells were purified using magnetic cell sorter, anti-CD11c beads, and LS columns (Miltenyi Biotec, Bergisch Gladbach, Germany). After the purification, the cells were counted and used for OT-II cell activation.
Purification and activation of naïve CD4 T cells (OT-II T cells)
Naïve, OVA-specific OT-II T cells were purified from the spleens and lymph nodes of young (6–10 weeks of age) OT-II mice by passing the cell suspension through nylon wool columns. For studying cell division at single-cell resolution, the purified cells were counted and labeled with carboxyfluorescein diacetate succinimidyl ester (CFSE; Molecular Probes, Eugene, OR). After CFSE labeling, the cells were washed, counted, and incubated for 30 min, together with different inhibitors diluted in dimethyl sulfoxide (DMSO). Caspase-3 and caspase-8 inhibitors z-Asp-Glu-Val-Asp (zDEVD) and z-Ile-Glu-Thr-Asp (zIETD; Sigma Chemical Co.), respectively, were used at the concentration of 50 µM. NF-B signaling was blocked using BAY 11-7082 inhibitor (Calbiochem, San Diego, CA), which inhibits TNFR-induced inhibitor of B-
phosphorylation. Cells were incubated at inhibitor concentrations ranging from 2.0 to 0.067 µM or in DMSO alone. After inhibitor treatment, cells were washed twice and plated together with antigen-pulsed, activated DC using a previously optimized DC-T cell ratio of 1:10 [28
], i.e., 5 x 104 DC and 5 x 105 T cells. DC-T cell cocultures were performed in flat-bottom 96-well plates for cell division, viability, and IFN- enzyme-linked immunosorbent assay (ELISA). For CD4 expression studies and for intracellular caspase-3 and IFN- labeling, cells were cultured in 24-well plates (3x105 DC and 3x106 T cells). For fluorescence-activated cell sorting, nylon wool-purified OT-II cells were used as such for DC cocultures (2x106 DC and 2x107 T cells) in 25 cm2 culture bottles.
To deliver the Fas-mediated costimulation, anti-Fas (Jo2, PharMingen) or isotype-matched control immunoglobulin G (hamster IgG) antibodies were immediately added to the T cell-DC cocultures at the previously optimized concentration of 1 µg/mL. We confirmed previously that at this concentration, the anti-Fas antibody produces similar effects to the cross-linked recombinant FasL in our system.
Analysis of cell division, cytokine production, and cell death
After 3 or 6 days of culture, together with antigen-bearing DC, OT-II cells were recovered, and the dilution of CFSE was analyzed using FACScan flow cytometer (Becton Dickinson, San Jose, CA) and WinMDI software Version 2.8 (http://facs.scripps.edu/software.html). The effects of caspase inhibitors were determined by calculating the percentages of viable cells in each generation under each culture condition. The average number of generation of each culture condition was calculated in two steps. First, the percentage of cells in each generation was multiplied by the number of cell divisions it had completed. Then, the products of all generations were added together and divided by 100%. Cell viability was analyzed by adding 7-aminoactinomycin-D (7-AAD; Sigma Chemical Co.) to the cells prior to analysis. 7-AAD-positive cells were considered apoptotic, and 7-AAD-negative cells were viable.
IFN- was analyzed from supernatants taken at Day 6 by ELISA using anti-IFN- (R4 6A2) as the capture antibody and biotinylated anti-IFN- (XMG1.2) as the detecting antibody (both from PharMingen). Intracellular IFN- detection was performed on CFSE-labeled cells activated for 3 days using standard techniques [33
, 34
] with minor modifications. Briefly, at both time-points, cells were stimulated with phorbol 12-myristate 13-acetate (50 ng/mL) and ionomycin (500 ng/mL) for 4 h. For the last 2 h, cells were treated further with Brefeldin A (10 µg/mL). Cells were fixed in 2% paraformaldehyde (PFA) for 30 min and then permeabilized with 0.5% saponin for 10 min. Intracellular IFN- was labeled using phycoerythrin (PE)-conjugated anti-IFN- (XMG1.2) antibody or anti-rat IgG1 (R3-34) as a control antibody. Viable, proliferating cells were analyzed using flow cytometry.
To distinguish between normal and apoptotic cells, sorted OT-II cells were fixed in 3% PFA. Then, cytospin preparations were made and further mounted in Vectashield mounting medium containing 4',6-diamidino-2-phenylindole (DAPI; Vector Laboratories, Burlingame, CA). Nuclear morphology alterations were then analyzed by fluorescence microscopy.
Intracellular active caspase-3 labeling of OT-II cells
Responding OT-II cells were collected and labeled for CD4 using fluorescein isothiocyanate (FITC)-conjugated anti-CD4 (PharMingen) and for active caspase-3, using PE-conjugated monoclonal active caspase-3 antibody apoptosis kit 1 (BD Biosciences, San Jose, CA; PharMingen), according to the manufacturers’ protocol. Briefly, after 3 or 6 days of culture, the cells were labeled for CD4 and then washed twice in phosphate-buffered saline (PBS), fixed, and permeabilized using Cytoperm/Cytofix solution (Becton Dickinson) for 20 min on ice and subsequently labeled with PE-conjugated, active caspase-3 monoclonal antibody (mAb) in perm/wash solution (Becton Dickinson) for 30 min on ice. Cells were washed three times with perm/wash solution and were then analyzed on a FACScan. Only CD4hi cells were analyzed for active caspase-3. As a negative control, naïve OT-II cells were labeled for active caspase-3 using the same protocol. Where indicated, the same samples were also used for confocal microscopy analysis.
Purification of viable and apoptotic OT-II cells
Cells were collected at Days 1–3 from the small culture bottles (25 cm2) and stained with FITC-conjugated anti-Vß 5.1/2 (MR9-4; PharMingen) and PE-conjugated anti-CD4 (Caltag Laboratories, Burlingame, CA). Cells staining positively for CD4 and Vß5 (>90% of which are OT-II cells) were sorted in the presence of 7-AAD (FACSVantage, Becton Dickinson). Living cells were sorted as CD4hi and 7-AAD–, whereas apoptotic cells were sorted as CD4lo and 7-AAD+. After the sorting, cells were lysed, and poly(adenosine 5'-diphosphate-ribose) polymerase (PARP) Western blotting and ApoAlert caspase-8 and -3 activity measurements were made.
Enzymatic caspase-8 and -3 activity analysis and immunoblotting techniques
Caspase-8 and -3 activities were analyzed from 1 million naïve and fluorescence-activated cell sorter (FACS)-sorted viable OT-II cells using ApoAlert caspase-8 and caspase-3 fluorescent assay kits (BD Biosciences; Clontech, Palo Alto, CA), according to the instructions by the manufacturers. PARP cleavage was analyzed from naïve and Day 3-sorted OT-II cells by immunoblotting. Briefly, naïve and sorted cells were washed once with PBS. The cells were then lysed in Laemmli sample buffer, and the proteins were resolved on a 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The separated proteins were transferred to a nitrocellulose membrane (Schleicher and Schuell, Dassel, Germany), probed with monoclonal PARP antibody (Sigma Chemical Co.), followed by coupling to the appropriate horseradish peroxidase-conjugated secondary antibodies, and visualized with the enhanced chemiluminescence system (Amersham, Buckinghamshire, UK).
Microscopy studies
Images were collected using a Zeiss LSM 510 META laser-scanning confocal microscope (Zeiss, Jena, Germany), configured on an inverted Axiovert 200M stand (Zeiss), equipped with a Plan-Apochromat 63x/1.4 oil differential interference contrast objective. FITC fluorescence was excited at 488 nm with an argon-ion laser, and emission was recorded through a 500- to 530-nm infrared band-pass filter. PE fluorescence was excited at 543 nm with a Helium-Neon laser, and emission light was recorded through a 560-nm long-pass filter. Single z-section images were produced using Zeiss LSM 3.0 software. Alternatively, DAPI-labeled cells were viewed under a Leica DMRE epifluorescence microscope (Leica, Bannockburn, IL) equipped with a Fluotar 100x/1.3 oil PH3 objective. Images were captured using a Hamamatsu Photonics C4742-95 ORCA charged-coupled device camera (Hamamatsu, Japan).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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more vigorously [28
]. In this study, we assessed whether caspases become activated also in the surviving cells and whether activated caspases are required for the costimulatory effect of Fas ligation. For these purposes, OT-II cells were activated using OVA-pulsed, mature DC in the presence of anti-Fas or control mAb. At Days 3 and 6, the cells were collected and labeled for active caspase-3 using an antibody that specifically detects the active form of this caspase in permeabilized cells. We analyzed the active caspase-3 levels using flow cytometry in CD4hi cells, as most of them were viable (7-AAD–; Day 3 shown in Fig. 1a ). It is interesting that CD4hi (viable) cells activated under Fas costimulation contained a higher level of active caspase-3 compared with CD4hi cells activated under control conditions at Day 3. Compared with viable cells, CD4lo (dying) cells contained yet higher amounts of active caspase-3. At Day 6, Fas-costimulated cells contained still more active caspase-3 than control cells, but the overall level was lower than at Day 3 (Fig. 1b) .
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To further test the finding that caspase-3 is activated in viable cells and to test whether caspase-8 is also activated, we determined caspase-3 and caspase-8 activities in viable cells by an enzymatic assay. For this purpose, viable cells were physically separated apart from dying cells using FACS sorting with CD4 and 7-AAD staining as the sort criteria (Fig. 2a ). After sorting, viable and dead cells had characteristic light-scattering properties (Fig. 2b) . Viability of the cells was also determined by nuclear staining with DAPI, which showed intact nuclei in the viable population (left panel) but condensed and fragmented nuclei in the apoptotic cells (right panel; Fig. 2c ). To determine if substrates, which are typically processed by caspases during apoptosis, were processed in the Fas-costimulated cells, we analyzed by Western blotting the cleavage of PARP, which is normally cleaved to the p97 fragment by caspase-3 during apoptosis. PARP was not cleaved in the viable control cells, and also, viable Fas-costimulated cells mainly contained unprocessed PARP. In contrast, dying cells, sorted apart from viable, Fas-costimulated cells, contained only the processed form of PARP (Fig. 2d) , indicating that FACS sorting was effective in separating viable from dying cells.
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The enhancing effect of Fas ligation on proliferation is dependent on caspase activation
As it became evident that Fas-costimulated OT-II cells contained more active caspase-8 and -3, we tested whether the enhancing effect of Fas ligation on proliferation could be decelerated by inhibiting caspases. For this purpose, OT-II cells were CFSE-labeled, pretreated with caspase inhibitors (zDEVD or zIETD) or vehicle (DMSO) alone, and then cocultured together with mature, antigen-pulsed DC. Following each pretreatment, cells were divided into two groups, and agonistic anti-Fas mAb or control mAb was added.
After 3 days of coculture, OT-II cells responding under Fas-ligating conditions had divided significantly further than cells responding under control conditions (Fig. 3a and 3b ). At Day 6, the difference between the number of cell divisions of Fas-costimulated and control cells was even more evident. Selective inhibition of caspases reduced the proliferation of control and Fas-costimulated cells, as the percentage of cells was reduced in the more advanced and increased in the less advanced generations (Fig. 3b) . At Day 3, this phenomenon was detectable but not yet conspicuous. At Day 6, differences in proliferation induced by pretreatment with caspase inhibitors were more evident, and proliferation of Fas-costimulated cells had been inhibited more significantly than that of control cells. Thus, inhibition of caspase activity reduced the proliferation of Fas-costimulated and control cells, but the inhibitory effect was stronger on Fas-costimulated cells. Neither zIETD nor zDEVD was able to completely abolish the Fas-induced effect nor did they have an additive effect when used in combination. Also, addition of the inhibitors at Day 2 was not able to inhibit proliferation further (data not shown). These results imply that the presence of caspase inhibitors during the beginning of activation is able to inhibit the proliferation of naïve T cells for a number of forthcoming cell divisions. In Jurkat cells, both inhibitors were able to block anti-Fas-induced caspase activation completely (data not shown).
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production or cell viability. We have previously shown that by Day 6, Fas costimulation increased IFN- production by 2.5-fold compared with control cells [28
]. We therefore studied if the increase in IFN- production is dependent on initial activation of caspases. IFN- production of control and Fas-costimulated cells was unaffected by z-IETD or z-DEVD treatment (not shown).
For cells responding under Fas ligation, viability at Day 3 and at Day 6 was 
55%, irrespective of caspase inhibition, whereas the viability of control cells was approximately 85% at both time-points. Thus, caspase inhibition had no effect on the viability of the responding cells (not shown). In addition, caspase inhibition did not affect the expression of activation markers (CD4, CD44, CD25, and CD62L) of the responding T cells (data not shown).
Survival of Fas-costimulated cells depends on NF-B signaling
NF-B is known to be an important factor in mediating T cell activation and survival signals, and it has been shown recently to become activated via caspase-8 activation [23
]. We therefore studied whether control cells and Fas-costimulated cells would differ in their sensitivity to blockade of the NF-B pathway. Cell viability was analyzed using 7-AAD labeling and flow cytometry at Day 3 (not shown) and at Day 6 (Fig. 4a
). We found that 2.0 µM NF-B inhibitor BAY 11-7082 was lethal to control and Fas-costimulated cells. At 1.0 µM, BAY 11-7082 was lethal to Fas-costimulated cells, whereas control cells were not affected. At lower concentrations (0.67 µM and under), this inhibitor only modestly reduced viability of Fas-costimulated or control cells. Taken together, these results indicate that Fas-costimulated cells are more dependent on the life-supporting signals provided by NF-B than control cells.
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B inhibition on IFN- production. It is interesting that at the 0.67 µM concentration, the inhibitor had no effect on viability in either culture (Fig. 4a) but abolished the enhanced IFN- secretion (Fig. 4b)
detected by ELISA. When IFN- production was analyzed using intracellular staining from CFSE-labeled cells, it was again evident that 0.67 µM concentration of the NF-B inhibitor had no effect on control cells, whereas the percentage of IFN- producing Fas-costimulated cells was decreased to the level of control cells (Fig. 4c)
. The inhibitor did not affect proliferation of OT-II cells significantly (P>0.1). Thus, the enhancement of IFN- production after Fas costimulation is controlled by NF-B signaling, whereas the induction of caspase activity drives the accelerated proliferation. |
DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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B acts as an important survival-promoting factor during Fas-mediated costimulation of naïve CD4 T cells activated by mature, antigen-bearing DC. Although cells responding under Fas ligation proliferated vigorously, and this was mediated by caspase activation, they were particularly dependent on NF-B signaling in maintaining their viability. This was shown by their susceptibility to the NF-B inhibitor at a concentration that did not affect viability of control cells. These results suggest that inhibition of NF-B signaling converted the effect of Fas ligation from costimulation to induction of cell death. Our results clearly show that Fas ligation, at the time of activation, leads to processing of procaspase-3 and -8 in T cells that remain viable and proliferate vigorously and that this costimulatory effect is reduced by selective caspase inhibition. Although previous studies have found evidence of caspase-8 and even caspase-3 activation following TCR signaling [11 , 17 18 19 20 21 , 35 ], the importance of caspase-3 activation for Fas-mediated costimulation of naïve CD4 T cells has not been described earlier. Moreover, in direct comparison with T cell activation, simultaneous Fas ligation resulted in more pronounced and earlier activation of caspase-3 and caspase-8. These results suggest that for the responding T cells to survive Fas ligation, caspase activation need not be blocked at an early step.
Early during activation, T cells up-regulate the expression of c-FLIPS and c-FLIPL. These proteins can pair with procaspase-8 at the DISC [36
, 37
], where FLIP blocks the activation of downstream caspases and protects cells from apoptotic effects upon Fas ligation. In addition, c-FLIPL can be cleaved to c-FLIPL(p43), which in turn, leads to NF-B activation via TRAF2 [23
]. In naive CD4 T cells, c-FLIP expression is up-regulated in response to T cell activation by DC with and without Fas ligation [28
]. In addition to Fas-mediated NF-B activation, caspase-8 has recently been shown to also mediate antigen receptor-induced NF-B activation [38
].
Our results indicate that Fas costimulation-mediated induction on IFN- production is abrogated upon NF-B inhibition. In contrast to antigen receptor-induced NF-B activation, which is caspase-8-dependent, the Fas-mediated NF-B activation seems to be conducted independently of caspase activation [13
, 38
]. This is in agreement with our results, as inhibition of NF-B signaling abrogated the Fas costimulation-induced IFN- production, whereas caspase inhibition did not.
In our system, Fas ligation of responding, naïve CD4 T cells always leads to increased apoptosis and thus, diminished viability on the entire population level. It is unexpected that caspase inhibition did not increase the viability of the population subjected to Fas ligation. However, Fas ligation can also trigger caspase-independent cell death in primary T cells, which could be mediated by receptor-interacting protein kinase [39 ].
In agreement with the findings of others, showing active caspase-3 in healthy proliferating cells [19 20 21 , 40 ], we detected moderate levels of active caspase-3 in viable OT-II cells responding under control conditions. The levels were considerably higher in viable OT-II cells responding under Fas ligation, consistent with the enhanced proliferation. Yet, the highest amount of active caspase-3 was found in dying (CD4lo) cells, suggesting that only partial caspase activity is associated with costimulation. These findings were confirmed by two independent techniques: intracellular caspase-3 staining and an enzymatic assay. In preparation of viable cells for enzymatic assays, apoptotic cells were excluded on the basis of their lower levels of CD4 expression and their 7-AAD positivity [28 ]. Annexin V labeling showed similar results as 7-AAD labeling (data not shown). Intact nuclear morphology and the existence of caspase-3 substrate PARP in its unprocessed form verified that the cells purified on the basis of being CD4hi and 7-AAD– were not apoptotic. Enzymatic assays performed on these viable cells clearly showed that Fas-costimulated cells contained more active caspase-3 than control cells and also showed some caspase-8 activity already at Day 2. It is interesting that these events coincided with the time-point when T cells start to divide after establishing contact with antigen-bearing DC [41 ], suggesting that caspase activation could be linked to the cell division machinery.
An important question is what exactly determines if an individual T cell responding to its nominal antigen will die or survive after Fas ligation? Our system allowed us to address this question using homogenous T cells with similar TCR affinity to the antigen, which in turn, was efficiently presented to all responding T cells (as the DC-T cell ratio was higher than needed for an optimal T cell response). Thus, the cell fate decision in this system is not affected by differences in antigen or costimulatory receptor signaling or the prevailing cytokine environment. It is important that we found that cells responding under Fas ligation proliferated vigorously as a result of increased caspase activity but simultaneously, were more susceptible to NF-B inhibition and experienced cell death at a concentration that did not affect viability of control cells. Therefore, it appears that the decision between life and death of an individual T cell is delicately balanced and depends on the amount of active caspases and NF-B signaling available within the cell at any given time-point. Thus, NF-B appears to be able to convert the effects of active caspase-8 and -3 from death inducers to enhancers of cell division and proliferation in the surviving cells. The mechanism by which caspase-3 and caspase-8 could participate in cell division is currently unknown. One possible mechanism could be the cleavage of structural proteins, which might help or promote the actual division. Also, the mechanism by which the cells can tolerate activated effector caspase-3 is not known but similarly, long-term T cell lines have been shown to tolerate activated caspases [42
]. The notion that differential effects of activated caspases could be explained by their differential localization [21
] is not supported by our results.
In conclusion, we have shown that ligation of Fas on naïve CD4 T cells activated by DC leads to enhanced activation of caspases, which leads to accelerated proliferation and increased occurrence of cell death. Without NF-B signaling, Fas ligation will invariably lead to cell death. Thus, survival and accelerated proliferation of an individual T cell seem to depend on the amount of active caspases in combination with life-supporting NF-B signaling.
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ACKNOWLEDGEMENTS |
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Received May 4, 2005; revised October 18, 2005; accepted October 19, 2005.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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B and Erk signaling pathways Curr. Biol. 10,640-648[CrossRef][Medline]B signaling pathway Mol. Cell. Biol. 24,2627-2636- and ß-chain genes under the control of heterologous regulatory elements Immunol. Cell Biol. 76,34-40[CrossRef][Medline]B activation by antigen receptor Science 307,1465-1468This article has been cited by other articles:
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  B. Rethi, N. Vivar, S. Sammicheli, C. Fluur, N. Ruffin, A. Atlas, E. Rajnavolgyi, and F. Chiodi Priming of T cells to Fas-mediated proliferative signals by interleukin-7 Blood, August 15, 2008; 112(4): 1195 - 1204. [Abstract] [Full Text] [PDF]
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 A. Orbach, J. Rachmilewitz, M. Parnas, J.-H. Huang, M. L. Tykocinski, and M. Dranitzki-Elhalel CTLA-4 {middle dot} FasL Induces Early Apoptosis of Activated T Cells by Interfering with Anti-Apoptotic Signals J. Immunol., December 1, 2007; 179(11): 7287 - 7294. [Abstract] [Full Text] [PDF]
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) or Fas-costimulated (
) cells sorted at indicated time-points. (f, Inset) CFSE-labeled cells proliferating under control conditions at the first and the second days of culture. Results are the mean ± SEM of three individual experiments. P values were calculated using the Student’s t-test; *, P < 0.05. rfu, Relative fluorescence units.
