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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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Related in: MedlinePlus

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®.
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Figure 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®.

Mentions: 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).


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

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

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®.
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Figure 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®.
Mentions: 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).

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
Related in: MedlinePlus