By
From the * Howard Hughes Medical Institute, The Rockefeller University, New York 10021
Fas(CD95) and its ligand (FasL) interaction plays a pivotal role in T cell receptor (TCR)-mediated apoptosis. However, the susceptibility of T cells to Fas-mediated apoptosis is tightly regulated during immune responses, a regulation which is thought to maintain the antigen-specificity of T cell apoptosis. Here we show that TCR stimulation enhances the induction of Fas-mediated apoptosis. In addition, using a mutant T cell hybridoma with impaired FasL expression, we show that the synergy provided by TCR stimulation can be mimicked by activators of PKC but not calcium influx. This effect cannot be inhibited by actinomycin D, suggesting that TCR stimulation leads to the alteration in preexisting signaling molecules to enhance Fas-mediated apoptosis. Our results therefore provide a mechanism of how Fas-FasL interactions lead to T cell death in an antigen-specific manner via repetitive antigen stimulation.
The T cell repertoire is determined primarily by positive and negative selection of immature thymocytes in
the thymus (1, 2). However, the establishment of antigen-specific T cell tolerance continues in mature T cell compartments to ensure that self-reactive T cells are kept inert
(2). Clonal deletion of self-reactive T cells via apoptosis is
one of the major mechanisms for establishing such tolerance (2).
Recent experiments have shown that several members of
the TNF receptor (TNFR) superfamily play critical roles
during antigen-induced T cell apoptosis. For example, the
Fas-FasL interaction participate in the induction of antigen-specific apoptosis of both immature thymocytes and mature
T cells (3), while the TNF-TNFR interaction is primarily involved in mature T cell death (8). Thus various members of the TNFR superfamily are thought to play distinct roles during T cell apoptosis depending on the developmental or activation stage of the cells or on the antigens
themselves. T cell hybridomas also mimic TCR-mediated
apoptosis via Fas-FasL interactions and have been extensively used as an in vitro model system of antigen-specific T cell deletion (11).
Some of the biochemical mechanisms that link Fas to the
apoptosis machinery has been elucidated by the identification of various signal transducing molecules which interact
with the cytoplasmic portion of Fas. Upon binding to FasL,
Fas recruits FADD/MORT1 and RIP via the interaction
of their death domains (14). Clustering of these signal
transducers leads to the further recruitment of FLICE/
MACH (15, 16) and subsequently to the activation of caspases.
Despite the direct interaction of Fas with cell death machinery, Fas-FasL interactions do not always lead to apoptosis.
For example, primary T cells have been shown to undergo
Fas-mediated apoptosis only after they are repetitively stimulated through TCR (17). Although T cells rapidly upregulate both Fas and FasL expression upon initial antigen
stimulation, apoptosis does not immediately ensue. Thus a
current consensus is that the susceptibility to Fas-mediated
apoptosis must be modified or controlled by other factors
during immune responses. In this study, we show that TCR signals, independent of de novo gene synthesis, enhance
the susceptibility of both normal T cells and T cell hybridomas to Fas-mediated apoptosis.
Transfection of Fas cDNA.
Fas cDNA (20) cloned into the retroviral vector pLXSN (21) was transiently transfected into a packaging cell line, BOSC-23, by the calcium phosphate transfection
method as described (22). The culture supernatants containing recombinant viruses were collected 2 d later, filtered, and used to
infect KIT50 as previously described (22, 23). Transfectants were
selected by their G418 (700 µg/ml) resistance (24) and the expression of Fas was confirmed by flow cytometric analysis using
the biotinylated anti-Fas Ab, Jo2 (PharMingen, San Diego, CA).
Northern Blot Analysis.
For stimulation, T cell hybridomas
were cultured in medium alone or on plates coated with anti-TCR Ab, H57-597 (10 µg/ml) (25). 6 h after stimulation, cells
were collected, washed in PBS and poly(A)+ RNA was collected
as previously described (23). 2 µg of poly(A)+ RNA was electrophoresed in 1.0% agarose-formaldehyde gel, transferred to GeneScreen membrane (DuPont-NEN, Boston, MA), and hybridized with 32P-labeled cDNA probes as described (23). Probes derived
from glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were
used to control for loading of mRNA.
Isolation and Priming of Lymph Node T cells.
Lymph node T
cells were prepared using T cell enrichment column (Biotex Laboratories Inc., Alberta, Canada) from 6-8-wk-old BALB/c mice
(Jackson ImmunoResearch Labs., Inc., West Grove, PA). Cells
were primed for apoptosis as previously described (18). In brief,
purified T cells were stimulated with concanavalin A (5 µg/ml)
and anti-CD28 Ab (1 µg/ml) for 48 h, washed three times with
10 mg/ml Stimulation of TCR or Fas by Antibody Cross-linking in the Presence of Various Biological Inhibitors and Activators.
96-well plates were
coated with various amounts of anti-TCR Ab (H57-597) (25),
anti-CD3 Ab (2C11), or anti-Fas Ab (Jo2) as indicated in the figure legends to cross-link the TCR and/or the Fas antigen, respectively. Cells were added to the plates at 2 × 104 cells/well (T
cell hybridomas) or 5 × 104 cells/well (primed LNTC). Various
inhibitors or activators were added to the wells at the final concentrations: PMA (Calbiochem-Novabiochem, La Jolla, CA) at
20 ng/ml, ionomycin (Calbiochem-Novabiochem) at 500 ng/ml,
dexamethasone (Sigma Chem. Co., St. Louis, MO) at 10 µM,
cyclosporin A (Sandoz Pharmaceuticals, East Hanover, NJ) at 50 ng/
ml, EGTA (Sigma) at 5 mM to chelate extracellular Ca2+, actinomycin D (Sigma) at 31.25 ng/ml.
Cell Death Analysis.
Percentage of apoptotic cells were measured by propidium iodide (PI; Sigma) uptake on a FACSCalibur® flow cytometer (Becton Dickinson, Mountain View, California) as previously described (8). Specific cell death was determined as follows: % specific death = [(% PI+stimulated To study the signaling pathway specific for TCR-mediated apoptosis, we
have previously generated several mutant T cell hybridoma clones which are resistant to TCR-mediated apoptosis (23,
26). One of those mutant clones, KIT50, expressed surface
TCR similar to the parental cell line, KMls-8.3.5 (data not
shown), but was resistant to the apoptosis induced by anti-TCR Ab treatment (Fig. 1 A) or by PMA plus ionomycin
(data not shown). In addition, TCR-mediated IL-2 production in KIT50 was significantly lower than that in the
parental cell line (Fig. 1 B). These results suggest that some
of the signals common to both TCR-mediated IL-2 production and apoptosis are likely to be affected in KIT50.
That these defects in apoptosis are specific for signals emanating from the TCR is suggested by the finding that dexamethasone treatment induced apoptosis in KIT50 as much
as in the parental cell line (Fig. 1 C). KIT50 thus contains
the necessary molecular machinery to carry out the apoptosis program, with the biochemical lesions likely to reside
between the TCR-proximal signaling components and the
apoptosis effector molecules (i.e., caspases) common to both TCR- and dexamethasone-mediated apoptosis.
To further study the defect(s) in KIT50, we analyzed the
expression of Nur77, Fas and FasL which were shown to
play a critical role in TCR-mediated apoptosis of T cell
hybridomas (11, 27, 28). Upon TCR-stimulation, the
induction of Nur77 was unaffected in KIT50 (Fig. 1 D).
However, both Fas and FasL mRNA induction were
markedly affected in the mutant cell line KIT50 (Fig. 1 D). The induction of IL-2 mRNA was also impaired in KIT50
(data not shown), reflecting the reduced level of IL-2 production shown in Fig. 1 B. These results suggest that some
(e.g., IL-2, FasL, and Fas expression) but not all (e.g.,
Nur77) of the signals triggered by the TCR was affected in
the mutant cell line, KIT50. In addition, the impairment in
both Fas and FasL expression appear to result in the resistance of KIT50 to TCR-mediated apoptosis.
The
susceptibility to Fas-mediated apoptosis varies among different cell types. It has been proposed that T cell proliferation is one of the requirements for susceptibility to Fas-mediated apoptosis (17). However, it is not clear whether
TCR-mediated proliferation (or cell cycle progression) is
the only requirement for T cells to become susceptible to
Fas-mediated apoptosis.
Since T cell hybridomas are continuously progressing
through the cell cycle, we tested whether additional signals
from the TCR are required for Fas-mediated apoptosis using KIT50-Fas which are derived from KIT50 by transfection of Fas (Fig. 2 A). KIT50-Fas is suitable for this study
because TCR-stimulation itself does not induce significant
apoptosis (<20% specific cell death) due to its impaired
FasL induction (Fig. 2 B). When KIT50/Fas was treated
with anti-Fas Ab alone, only a low level of apoptosis (<5%
specific cell death) was seen (Fig. 2 B). Thus cell cycle progression or continuous proliferation itself appears insufficient to render T cells susceptible to Fas-mediated cell
death. When KIT50-Fas cells were co-stimulated with
anti-TCR Ab and anti-Fas Ab, there was a synergistic effect in the induction of apoptosis (Fig. 2 B), suggesting that
TCR-mediated signals enhance Fas-mediated apoptosis in
T cell hybridomas. This enhancement is likely due to the
intracellular biochemical changes upon TCR stimulation, but not due to the residual upregulation of FasL expression
(see below).
To confirm that Fas-mediated apoptosis is enhanced by
anti-TCR Ab in primary T cells, purified T cells were
primed for anti-TCR induced apoptosis by culturing them
with ConA and a high dose of IL-2 as previously described
(18). Proliferating T cells were then restimulated with anti-CD3 Ab to induce apoptosis (18). When restimulated with
anti-CD3 Ab, a significant portion of T cells died, whereas
treatment with anti-Fas Ab alone induced only few cells
(<5%) to undergo apoptosis (Fig. 2 C). When T cells were
stimulated with both anti-CD3 and anti-Fas Ab, however,
there was a significant increase (P <0.001) in the induction
of apoptosis compared to treatment with anti-CD3 or anti-Fas Ab alone. These results suggest that TCR- and Fas-mediated signals synergize to induce apoptosis in primary T
cells (Fig. 2 C). Thus concomitant signals from TCR and
Fas appears to be a prerequisite for efficient induction of
apoptosis via Fas.
To study how TCR-mediated signals enhance Fas-mediated apoptosis, we have tested whether
cyclosporin A (CsA) can interfere with this synergy of
TCR and Fas signals. CsA treatment, which abrogates the
TCR-mediated apoptosis of T cell hybridomas by inhibiting FasL expression (data not shown) (13), did not affect the
enhancement of Fas-mediated cell death by anti-TCR Ab
(Fig. 3 A). These results suggest that calcium/calcineurin-dependent signals or FasL expression activated by the
TCR, the latter of which is also inhibited by CsA, are not
involved in the enhancement of Fas-mediated apoptosis. In
support of this, the enhancement of Fas-mediated apoptosis by TCR-stimulation was not inhibited in the presence of a
large excess EGTA (Fig. 3 A). These results are consistent
with previous reports that Fas-mediated apoptosis does not
involve calcium-regulated signals (29).
Since most of the cellular responses (e.g., IL-2 production or apoptosis) induced by TCR-stimulation can be
mimicked by treatment of a phorbol ester (PMA) and a calcium ionophore (ionomycin) (30), we tested whether these
reagents could also enhance Fas-mediated apoptosis. Similar to anti-TCR Ab, PMA plus ionomycin treatment greatly enhanced Fas-mediated apoptosis (Fig. 3 B). When
PMA or ionomycin was treated separately, PMA but not
ionomycin alone enhanced Fas-mediated apoptosis (Fig. 3
B) although it did not induce FasL expression (data not
shown) (13). Thus the enhanced Fas-mediated apoptosis
observed in these experiments is not due to increased FasL
expression. Similar synergy with PMA and anti-Fas Ab in
the induction of cell death was demonstrated in another Fas-transfected T cell hybridoma, KCIT1-8.5/Fas (23)
(Fig. 3 C). These results suggest that cellular components
activated by PMA are responsible for the enhancement of
Fas-mediated apoptosis in T cells.
Phorbol ester treatment of T cells mimics activation of
PKC- and Ras-dependent cellular responses initiated by
the TCR (30). Since Ras/MAP kinase activation can lead
to the induction of gene transcription, we tested whether
the enhancement of Fas-mediated apoptosis by PMA requires de novo macromolecular synthesis. When cells were
treated with PMA and anti-Fas Ab in the presence of actinomycin D (ActD), the level of apoptosis was comparable
to that in the absence of ActD (Fig. 3 D). Similar to PMA,
TCR signals enhanced Fas-mediated apoptosis even in the
presence of ActD (Fig. 3 D). At the same concentration of
ActD, upregulation of CD69 by TCR was efficiently
blocked (Fig. 3 E). These results suggest that TCR (or
PMA)-induced enhancement of Fas-mediated apoptosis does not depend on new gene products. Therefore, TCR-mediated activation of PKC (or other cellular signals) is
likely to modify pre-existing signaling components downstream of Fas. It is not clear at this point which cellular proteins activated by TCR are responsible to modulate the
susceptibility to Fas-mediated apoptosis. It was previously
shown that Ras, which is also activated by TCR, is activated during Fas-mediated apoptosis and that the interference of Ras activation impairs Fas-mediated apoptosis (31).
Therefore, it is possible that TCR-mediated Ras activation
may increase the susceptibility of cells to Fas-mediated apoptosis. It is also possible that Fas-associated signaling molecules can be regulated by phosphorylation events because
some of these molecules were found to be differentially
phosphorylated (32). In addition, TCR-mediated signals
may down-modulate anti-apoptotic signals which are associated with the protein phosphatase, FAP1 (33).
Antigen-specific deletion of T cells is necessary for the
control of T cell immune responses and for the maintenance of T cell homeostasis, for which Fas and FasL play
critical roles. It was previously shown that antigen (anti-TCR)-induced proliferation and cell cycle induction of
mature T cells is a prerequisite for Fas-mediated apoptosis,
thus proposing a model of how antigen specificity is attained during T cell deletion by repetitive antigen stimulation (17, 19). In this study, we provide evidence that additional TCR-mediated signals independent of cell cycle
progression and de novo gene synthesis can synergize with
Fas to trigger apoptosis. Therefore, it is likely that antigen-specificity of T cell deletion may be accomplished by coupling the TCR to Fas-mediated apoptosis via several
check-points: those which are proliferation-dependent as
well as others involving PKC activation.
-methylmannoside in balanced salt solution and then
incubated in media in the presence of 50 U/ml IL-2. After 48 h,
activated T cells (50,000 cells/well) were cultured in 96-well plates coated with anti-CD3 Ab, 2C11 (10 µg/ml), anti-Fas Ab
(Jo2, 10 µg/ml) or anti-CD3 plus anti-Fas Abs.
% PI+control)/(100%
% PI+control)] × 100%.
Isolation and Characterization of T Cell Hybridomas Which
Have Defective Fas and FasL Expression.
Fig. 1.
Isolation and characterization of a T cell hybridoma mutant
with impaired Fas and FasL expression. (A) The T cell hybridoma mutant
KIT50 is resistant to anti-TCR Ab (H57-597). Cells were incubated for
24 h on 96-well plates coated with increasing amounts of H57-597 and
then assayed for apoptosis by PI uptake and FACS® analysis. Specific cell
death was determined as described in Materials and Methods. (Squares)
The parental cell line, KMls-8.3.5; (circles) the mutant cell line, KIT50.
The representative results of at least 10 independent experiments are
shown. (B) IL-2 production upon TCR stimulation. Cells were stimulated for 18 h on plates coated with anti-TCR Ab (10 µg/ml). Supernatants were collected and their activity was assayed using HT-2 cells as previously described (23). (White bar) The parental cell line, KMls-8.3.5; (hatched bar) the mutant cell line, KIT50. The representative results of
three to seven independent experiments are shown. (C) Apoptotic cell
death induced by dexamethasone. KMls-8.3.5 and KIT50 cells were incubated in the presence of 10 µM dexamethasone or in the media alone
for 24 h and cell viability was tested by PI uptake. Spontaneous cell death
in the media alone was <2-3%. (White bar) the parental cell line, KMls-8.3.5; (hatched bar) the mutant cell line, KIT50. The representative results
of at least six independent experiments are shown. (D) Fas and FasL mRNA expression was impaired in KIT50. RNA was prepared from
control or TCR-stimulated cells and then the expression of various genes was tested by Northern blot hybridization. (c) Control, unstimulated, (a)
stimulated with anti-TCR Ab (10 µg/ml).
[View Larger Version of this Image (30K GIF file)]
Fig. 2.
TCR-mediated signals enhance Fas-mediated apoptosis. (A)
KIT50/Fas expresses relatively high levels of Fas on the cell surface. Surface Fas expression on KIT50-Fas cells were determined by FACS® analysis using biotinylated Jo2 Ab (PharMingen) and phycoerythrin (PE)-
labeled streptavidin (Becton Dickinson). (Dotted line) PE-streptavidin alone; (solid line) biotinylated Jo2 plus PE-streptavidin. (B) TCR stimulation enhances Fas-mediated cell death. KIT50-Fas cells were cultured in
96-well plates coated with increasing amounts of H57-597 ± anti-Fas Ab
(Jo2, 10 µg/ml). Cells were collected after 24 h and cell death was detected by PI uptake by FACS® analysis. The representative results of at
least three independent experiments are shown. (Filled circles) KIT50/Fas
stimulated with anti-Fas and anti-TCR Abs; (white squares) KIT50/Fas
stimulated with anti-TCR Ab alone. (C) Synergistic effect of simultaneous Fas and TCR stimulation on activated lymph node T cells. ConA-IL-2-activated LNTC were incubated on plates coated with anti-CD3
(10 µg/ml) and/or anti-Fas Abs (10 µg/ml). Cell death was determined
48 h later by PI uptake. The representative results of three independent experiments are shown. Spontaneous cell death in the media alone was
<5%. These experiments were done in the absence of cycloheximide. (White bar) anti-Fas Ab alone; (black bar) anti-CD3 Ab alone; (hatched bar)
anti-Fas Ab plus anti-CD3 Ab.
[View Larger Version of this Image (35K GIF file)]
Fig. 3.
A PKC dependent, calcium-independent pathway mediates
synergy between TCR and Fas signaling. (A) EGTA or CsA does not
block the synergy between TCR and Fas. KIT50-Fas cells were stimulated with plate-bound H57-597 (10 µg/ml) plus Jo2 (10 µg/ml) in the presence of media alone, EGTA or CsA. Cell death was measured 24 h
later by PI uptake. The representative results of three independent experiments are shown. The similar treatment of EGTA or CsA completely blocked cell death induced by anti-TCR Ab alone (data not shown). The
low level of apoptosis induced by anti-Fas Ab alone (2-10% specific cell
death) was not affected by EGTA or CsA (data not shown). Spontaneous
cell death in the media alone ± EGTA or CSA was <5%. (B) PMA can
substitute for TCR signals to enhance Fas-mediated apoptosis. KIT50-Fas
cells were cultured in 96-well plates coated with anti-Fas Ab (Jo2, 10 µg/
ml) in the presence of various stimuli. Cell death was assayed 24 h later by
PI uptake. The representative results of at least three independent experiments are shown. Cells were stimulated with Jo2 in the presence of media
alone (white bar), PMA plus ionomycin (hatched bar), PMA alone (black
bar), or ionomycin alone (gray bar). In the absence of Jo2, PMA or ionomycin alone did not induce cell death and PMA plus ionomycin induced a low level (<15%) of apoptosis in these cells (data not shown). (C) PMA
alone can enhance Fas-mediated cell death of another Fas-transfected T
cell hybridoma, KCIT1-8.5/Fas. KCIT1-8.5/Fas cells (23) were stimulated with PMA alone (white bar), plate-bound 10 µg/ml anti-Fas Ab
alone (hatched bar), or PMA plus plate-bound anti-Fas Ab (black bar). Cell
death was assayed 24 h later by PI uptake. The representative results of
three independent experiments are shown. (D) Actinomycin D does not
inhibit the synergy mediated by PMA or anti-TCR Ab. KIT50/Fas cells
were stimulated with various reagents in the presence or absence of actinomycin D. Cell death was determined 24 h later by PI uptake. The representative results of three independent experiments are shown. (White
bars) plate-bound anti-Fas Ab alone, 10 µg/ml; (hatched bars) PMA plus
plate-bound anti-Fas Ab; (black bars) plate-bound anti-TCR Ab plus anti-Fas Ab at 10 µg/ml each. In the absence of anti-Fas Ab, PMA alone did
not induce significant cell death (<5%). Anti-TCR Ab without anti-Fas
Ab induced ~10% specific cell death (data not shown). (E) Actinomycin
D inhibits CD69 induction by TCR stimulation. KIT50/Fas cells from D
were stained by FITC-labeled anti-CD69 antibody and analyzed by
FACS®.
[View Larger Version of this Image (45K GIF file)]
Address correspondence to Yongwon Choi, Ph.D., HHMI, The Rockefeller University, 1230 York Ave., Box 295, New York, NY 10021. Phone: 212-327-7441; Fax: 212-327-7319; E-mail: choi{at}rockvax.rockefeller.edu
Received for publication 31 July 1997.
Note added in proof. After this paper was accepted for publication, we found that Hornung et al. also reported that TCR signals can increase Fas-mediated apoptosis (Hornung, S., L. Zheng, and M.J. Lenardo. 1997. J. Immunol. 159:3816-3822).We would like to thank Dr. John MacMicking for his critical comments and Angela Santana and Elizabeth Robinson for their excellent technical help.
This work was supported in part by National Institutes of Health grant R01 AI/CA41082 (Y. Choi). Y. Choi is an assistant investigator of the Howard Hughes Medical Institute.
1. | von Boehmer, H.. 1994. Positive selection of lymphocytes. Cell. 76: 219-228 [Medline]. |
2. | Nossal, G.J.V.. 1994. Negative selection of lymphocytes. Cell. 76: 229-239 [Medline]. |
3. | Nagata, S., and P. Golstein. 1995. The Fas death factor. Science. 267: 1449-1456 [Medline]. |
4. | Lynch, D.H., F. Ramsdell, and M.R. Alderson. 1995. Fas and FasL in the homeostatic regulation of immune responses. Immunol. Today. 16: 569-574 [Medline]. |
5. | van Parijs, L., and A.K. Abbas. 1996. Role of Fas-mediated cell death in the regulation of immune responses. Curr. Opin. Immunol. 8: 355-361 [Medline]. |
6. | Castro, J.E., J.A. Listman, B.A. Jacobson, Y. Wang, P.A. Lopez, S. Ju, P.W. Finn, and D.L. Perkins. 1996. Fas modulation of apoptosis during negative selection of thymocytes. Immunity. 5: 617-627 [Medline]. |
7. |
Kishimoto, H., and
J. Sprent.
1997.
Negative selection in the
thymus includes semimature T cells.
J. Exp. Med.
185:
263-271
|
8. | Zheng, L., G. Fisher, R.E. Miller, J. Peschon, D.H. Lynch, and M.J. Lenardo. 1995. Induction of apoptosis in mature T cells by tumor necrosis factor. Nature. 377: 348-351 [Medline]. |
9. | Sytwu, H.-K., R.S. Libau, and H.O. McDevitt. 1996. The roles of Fas/APO-1(CD95) and TNF in antigen-induced programmed cell death in T cell receptor transgenic mice. Immunity. 5: 17-30 [Medline]. |
10. | Speiser, D.E., E. Sebzda, T. Ohteki, M.F. Bachmann, K. Pfeffer, T.W. Mak, and P.S. Ohashi. 1996. TNF receptor p55 mediates in vivo deletion of peripheral cytotoxic T lymphocytes. Eur. J. Immunol. 26: 3055-3060 [Medline]. |
11. | Brunner, T., R.J. Mogil, D. LaFace, N.J. Yoo, A. Mahboubi, F. Echeverri, S.J. Martin, W.R. Force, D.H. Lynch, C.F. Ware, and D.R. Green. 1995. Cell-autonomous Fas (CD95)/Fas-ligand interaction mediates activation-induced apoptosis in T cell hybridomas. Nature. 373: 441-444 [Medline]. |
12. | Ju, S.-T., D.J. Panka, H. Cui, R. Ettinger, M. El-Khatib, D.H. Sherr, B.Z. Stanger, and A. Marshak-Rothstein. 1995. Fas(CD95)/FasL interactions required for programmed cell death after T-cell activation. Nature. 373: 444-448 [Medline]. |
13. | Yang, Y., M. Mercep, C.F. Ware, and J.D. Ashwell. 1995. Fas and activation-induced Fas ligand mediate apoptosis of T cell hybridomas: Inhibition of Fas ligand expression by retinoic acid and glucocorticoids. J. Exp. Med. 181: 1673-1682 [Abstract]. |
14. | Chinnaiyan, A.M., and V.M. Dixit. 1997. Portrait of an executioner: the molecular mechanism of Fas/APO-1-induced apoptosis. Semin. Immunol. 9: 69-76 [Medline]. |
15. | Muzio, M., A.M. Cinnaiyan, F.C. Kischkel, K. O'Rourke, A. Shevchenko, J. Ni, C. Scaffidi, J.D. Brentz, M. Zhang, R. Gentz, et al . 1996. FLICE, a novel FADD-homologous ICE/ CED-3-like protease, is recruited to the CD95 (Fas/APO-1) death-inducing signaling complex. Cell. 85: 817-827 [Medline]. |
16. | Boldin, M.P., T.M. Goncharov, Y.V. Golstev, and D. Wallach. 1996. Involvement of MACH, a novel MORT1/ FADD-interacting protease in Fas/APO-1 and TNF receptor-induced cell death. Cell. 81: 803-815 . |
17. | Boehme, S.A., and M. Lenardo. 1993. Propriocidal apoptosis of mature T lymphocytes occurs at S phase in the cell cycle. Eur. J. Immunol. 23: 1552-1560 [Medline]. |
18. |
Lenardo, M.J..
1991.
Interleukin-2 programs mouse ![]() ![]() |
19. | Lenardo, M.J., S. Boehme, L. Chen, B. Combadiere, G. Fisher, M. Freedman, H. McFarland, C. Pelfrey, and L. Zheng. 1995. Autocrine feedback death and the regulation of mature T lymphocyte antigen receptors. Intl. Rev. Immunol. 13: 115-134 . |
20. | Ogasawara, J., R. Watanabe-Fukunaga, M. Adachi, A. Matsuzawa, T. Kasugai, Y. Kitamura, N. Itoh, T. Suda, and S. Nagata. 1993. Lethal effect of the anti-Fas antibody in mice. Nature. 364: 806-809 [Medline]. |
21. | Miller, A.D., and G.J. Rosman. 1989. Improved retroviral vectors for gene transfer and expression. Biotechniques. 7: 980-982 [Medline]. |
22. |
Pear, W.S.,
G.P. Nolan,
M.L. Scott, and
D. Baltimore.
1993.
Production of high-titer helper-free retroviruses by transient
transfection.
Proc. Natl. Acad. Sci. USA.
90:
8392-8396
|
23. | Park, C.G., S.Y. Lee, G. Kandala, S.Y. Lee, and Y. Choi. 1996. A novel gene product that couples TCR signaling to Fas(CD95) expression in activation-induced cell death. Immunity. 4: 583-591 [Medline]. |
24. | Yazdanbakhsh, K., J.-W. Choi, Y. Li, L.F. Lau, and Y. Choi. 1995. Cyclosporin A blocks apoptosis by inhibiting the DNA biding activity of the transcription factor Nur77. Proc. Natl. Acad. Sci. USA. 92: 437-441 [Abstract]. |
25. |
Kubo, R.T.,
W. Born,
J.W. Kappler,
P. Marrack, and
M. Pigeon.
1989.
Characterization of a monoclonal antibody
which detects all murine alpha beta T cell receptors.
J. Immunol.
142:
2736-2742
|
26. | Wong, B., C.G. Park, and Y. Choi. 1997. Identifying the molecular control of T cell death. Semin. Immunol. 9: 7-16 [Medline]. |
27. | Liu, Z., S.W. Smith, K.A. McLaughlin, L.M. Schwartz, and B.A. Osborne. 1994. Apoptotic signals delivered through the T-cell receptor of a T-cell hybrid require the immediate-early gene nur77. Nature. 367: 281-284 [Medline]. |
28. | Woronicz, J., B. Calnan, V. Ngo, and A. Winoto. 1994. Requirement for the orphan steroid receptor Nur77 in apoptosis of T-cell hybridomas. Nature. 367: 277-281 [Medline]. |
29. | Rouvier, E., M.-F. Luciani, and P. Golstein. 1993. Fas involvement in Ca2+-independent T cell mediated cytotoxicity. J. Exp. Med. 177: 195-200 [Abstract]. |
30. | Weiss, A., and D.R. Littman. 1994. Signal transduction by lymphocyte antigen receptors. Cell. 76: 263-274 [Medline]. |
31. | Gulbins, E., R. Bissonnette, A. Mahboubi, S. Martin, W. Nishioka, T. Brunner, G. Baier, G. Baier-Bitterlich, C. Byrd, F. Lang, et al . 1995. FAS-induced apoptosis is mediated via a ceramide-initiated RAS signaling pathway. Immunity. 2: 341-351 [Medline]. |
32. | Kischkel, F.C., S. Hellbardt, I. Behrmann, M. Germer, M. Pawlita, P.H. Krammer, and M.E. Peter. 1995. Cytotoxic-dependent APO-1(Fas/CD95)-associated proteins form a death-inducing signaling complex (DISC) with the receptor. EMBO (Eur. Mol. Biol. Organ.) J. 14: 5579-5588 [Abstract]. |
33. | Sato, T., S. Irie, S. Kitada, and J.C. Reed. 1995. FAP-1: a protein tyrosine phosphatase that associates with Fas. Science. 268: 411-415 [Medline]. |