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T cell receptor signals enhance susceptibility to Fas-mediated apoptosis.

Wong B, Arron J, Choi Y - J. Exp. Med. (1997)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Howard Hughes Medical Institute, The Rockefeller University, New York 10021, USA.

ABSTRACT
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.

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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).
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Figure 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).

Mentions: 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.


T cell receptor signals enhance susceptibility to Fas-mediated apoptosis.

Wong B, Arron J, Choi Y - J. Exp. Med. (1997)

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).
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Related In: Results  -  Collection

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Figure 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).
Mentions: 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.

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Howard Hughes Medical Institute, The Rockefeller University, New York 10021, USA.

ABSTRACT
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.

Show MeSH