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Membrane Fas ligand kills human peripheral blood T lymphocytes, and soluble Fas ligand blocks the killing.

Suda T, Hashimoto H, Tanaka M, Ochi T, Nagata S - J. Exp. Med. (1997)

Bottom Line: In contrast, soluble FasL kills only the latter.Soluble FasL inhibited the killing of fresh PBT by membrane FasL.These results indicate that the shedding of FasL from the membrane is a mechanism for downregulating at least part of its killing activity.

View Article: PubMed Central - PubMed

Affiliation: Osaka Bioscience Institute, Department of Molecular Biology, Suita, Osaka 565, Japan. sudat@obi.or.jp

ABSTRACT
It has been believed that the Fas expressed on human peripheral blood T cells (PBT) is nonfunctional, because these cells are insensitive to agonistic anti-Fas/Apo-1 mAbs that efficiently kill in vitro-activated T cells and many Fas-expressing cell lines. Here, we demonstrate that membrane-bound Fas ligand (FasL) kills both fresh and in vitro-activated PBT, indicating that the Fas expressed on fresh PBT is functional. In contrast, soluble FasL kills only the latter. Naive T cells in umbilical cord blood do not express Fas, but can be induced to express Fas by IFN-gamma or by a combination of IL-2 and anti-CD28 mAb, after which they acquire sensitivity to membrane but not to soluble FasL. Soluble FasL inhibited the killing of fresh PBT by membrane FasL. These results indicate that the shedding of FasL from the membrane is a mechanism for downregulating at least part of its killing activity.

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Susceptibility of fresh PBT and ConA blasts to anti-Fas mAb  (CH11), soluble human FasL (AL-1), and recombinant soluble mouse  FasL (WX1). (a–c), Fresh PBL or ConA blasts were cultured in the presence of the indicated concentrations of CH11 (a), AL-1 (b), and WX1 (c)  for 14 h. Cells were then stained with FITC-anti-Fas mAb, PE-conjugated mAb against either CD4 or CD8, and PI. Percent specific cell killing of CD4+ and CD8+ cells was determined as described in Materials  and Methods. (d–g) Fresh PBL (d and e) and ConA blasts (f and g) cultured  with (solid line) or without (dotted line) 4,000 units/ml of WX1 for 14 h  were stained as described above, and 5 × 104 total cells (including both viable  and dead cells) were analyzed in a FACScan®. Staining profiles for FITC-anti-Fas mAb of viable (PI−) CD4+ (d and f) or CD8+ cells (e and g) are  shown.
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Figure 1: Susceptibility of fresh PBT and ConA blasts to anti-Fas mAb (CH11), soluble human FasL (AL-1), and recombinant soluble mouse FasL (WX1). (a–c), Fresh PBL or ConA blasts were cultured in the presence of the indicated concentrations of CH11 (a), AL-1 (b), and WX1 (c) for 14 h. Cells were then stained with FITC-anti-Fas mAb, PE-conjugated mAb against either CD4 or CD8, and PI. Percent specific cell killing of CD4+ and CD8+ cells was determined as described in Materials and Methods. (d–g) Fresh PBL (d and e) and ConA blasts (f and g) cultured with (solid line) or without (dotted line) 4,000 units/ml of WX1 for 14 h were stained as described above, and 5 × 104 total cells (including both viable and dead cells) were analyzed in a FACScan®. Staining profiles for FITC-anti-Fas mAb of viable (PI−) CD4+ (d and f) or CD8+ cells (e and g) are shown.

Mentions: About 50% of CD4+ and CD8+ T cells in freshly isolated PBL from a healthy adult volunteer and virtually all ConA blasts generated from the PBL expressed readily detectable levels of Fas (data not shown). Consistent with previous reports, agonistic anti-Fas mAb (CH11) efficiently killed ConA blasts, whereas fresh PBT were resistant to the mAb (Fig. 1 a). We previously established a transfectant that expresses a human FasL lacking most of its cytoplasmic region. This cell line (1A12) secretes soluble FasL by a proteolytic mechanism (23). The soluble FasL was purified using an affinity column with anti-human FasL mAb. This purified soluble human FasL (named AL-1) showed similar target specificity to CH11 (Fig. 1 b). In contrast, WX1, the biologically active, recombinant soluble mouse FasL, killed both ConA blasts and fresh PBT (Fig. 1, c–g). Since WX1 specifically killed Fas+ cells in PBT, which represent ∼50% of the total PBT (Fig. 1, d and e), the dose-response curves of WX1-induced death in ConA blasts and fresh Fas+ PBT are comparable. There was no difference between CD4 and CD8 T cells in terms of susceptibility to various Fas agonists.


Membrane Fas ligand kills human peripheral blood T lymphocytes, and soluble Fas ligand blocks the killing.

Suda T, Hashimoto H, Tanaka M, Ochi T, Nagata S - J. Exp. Med. (1997)

Susceptibility of fresh PBT and ConA blasts to anti-Fas mAb  (CH11), soluble human FasL (AL-1), and recombinant soluble mouse  FasL (WX1). (a–c), Fresh PBL or ConA blasts were cultured in the presence of the indicated concentrations of CH11 (a), AL-1 (b), and WX1 (c)  for 14 h. Cells were then stained with FITC-anti-Fas mAb, PE-conjugated mAb against either CD4 or CD8, and PI. Percent specific cell killing of CD4+ and CD8+ cells was determined as described in Materials  and Methods. (d–g) Fresh PBL (d and e) and ConA blasts (f and g) cultured  with (solid line) or without (dotted line) 4,000 units/ml of WX1 for 14 h  were stained as described above, and 5 × 104 total cells (including both viable  and dead cells) were analyzed in a FACScan®. Staining profiles for FITC-anti-Fas mAb of viable (PI−) CD4+ (d and f) or CD8+ cells (e and g) are  shown.
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Related In: Results  -  Collection

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Figure 1: Susceptibility of fresh PBT and ConA blasts to anti-Fas mAb (CH11), soluble human FasL (AL-1), and recombinant soluble mouse FasL (WX1). (a–c), Fresh PBL or ConA blasts were cultured in the presence of the indicated concentrations of CH11 (a), AL-1 (b), and WX1 (c) for 14 h. Cells were then stained with FITC-anti-Fas mAb, PE-conjugated mAb against either CD4 or CD8, and PI. Percent specific cell killing of CD4+ and CD8+ cells was determined as described in Materials and Methods. (d–g) Fresh PBL (d and e) and ConA blasts (f and g) cultured with (solid line) or without (dotted line) 4,000 units/ml of WX1 for 14 h were stained as described above, and 5 × 104 total cells (including both viable and dead cells) were analyzed in a FACScan®. Staining profiles for FITC-anti-Fas mAb of viable (PI−) CD4+ (d and f) or CD8+ cells (e and g) are shown.
Mentions: About 50% of CD4+ and CD8+ T cells in freshly isolated PBL from a healthy adult volunteer and virtually all ConA blasts generated from the PBL expressed readily detectable levels of Fas (data not shown). Consistent with previous reports, agonistic anti-Fas mAb (CH11) efficiently killed ConA blasts, whereas fresh PBT were resistant to the mAb (Fig. 1 a). We previously established a transfectant that expresses a human FasL lacking most of its cytoplasmic region. This cell line (1A12) secretes soluble FasL by a proteolytic mechanism (23). The soluble FasL was purified using an affinity column with anti-human FasL mAb. This purified soluble human FasL (named AL-1) showed similar target specificity to CH11 (Fig. 1 b). In contrast, WX1, the biologically active, recombinant soluble mouse FasL, killed both ConA blasts and fresh PBT (Fig. 1, c–g). Since WX1 specifically killed Fas+ cells in PBT, which represent ∼50% of the total PBT (Fig. 1, d and e), the dose-response curves of WX1-induced death in ConA blasts and fresh Fas+ PBT are comparable. There was no difference between CD4 and CD8 T cells in terms of susceptibility to various Fas agonists.

Bottom Line: In contrast, soluble FasL kills only the latter.Soluble FasL inhibited the killing of fresh PBT by membrane FasL.These results indicate that the shedding of FasL from the membrane is a mechanism for downregulating at least part of its killing activity.

View Article: PubMed Central - PubMed

Affiliation: Osaka Bioscience Institute, Department of Molecular Biology, Suita, Osaka 565, Japan. sudat@obi.or.jp

ABSTRACT
It has been believed that the Fas expressed on human peripheral blood T cells (PBT) is nonfunctional, because these cells are insensitive to agonistic anti-Fas/Apo-1 mAbs that efficiently kill in vitro-activated T cells and many Fas-expressing cell lines. Here, we demonstrate that membrane-bound Fas ligand (FasL) kills both fresh and in vitro-activated PBT, indicating that the Fas expressed on fresh PBT is functional. In contrast, soluble FasL kills only the latter. Naive T cells in umbilical cord blood do not express Fas, but can be induced to express Fas by IFN-gamma or by a combination of IL-2 and anti-CD28 mAb, after which they acquire sensitivity to membrane but not to soluble FasL. Soluble FasL inhibited the killing of fresh PBT by membrane FasL. These results indicate that the shedding of FasL from the membrane is a mechanism for downregulating at least part of its killing activity.

Show MeSH
Related in: MedlinePlus