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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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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.
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Figure 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.

Mentions: 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).


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

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

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.
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Figure 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.
Mentions: 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).

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