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Maximal proliferation of cytotoxic T lymphocytes requires reverse signaling through Fas ligand.

Suzuki I, Fink PJ - J. Exp. Med. (1998)

Bottom Line: To test this hypothesis directly, soluble FasIgG, a fusion protein of murine Fas and human IgG1, was added to FasL+ CTLs to demonstrate that blocking cell surface Fas-FasL interactions mimics the depression observed for FasL- CTLs.In contrast to these results with CD8+ T cells, alloantigen-stimulated FasL- CD4+ T cells proliferate vigorously compared to FasL+ cells.These data demonstrate that reverse signaling through FasL is required for CTLs to achieve maximal proliferation and may provide clues to differences in the homeostatic regulation of activated CD4+ and CD8+ T cells during an immune response.

View Article: PubMed Central - PubMed

Affiliation: Department of Immunology, University of Washington, Seattle, Washington 98195, USA.

ABSTRACT
Fas ligand (FasL/CD95L) is best known for its role in delivering apoptotic signals through its receptor, Fas (APO-1/CD95). In this study, we present evidence that FasL has a second role as a signaling receptor. Alloantigen-specific proliferation by multiple FasL- murine CTL lines is depressed compared to that of FasL+ CTL lines. FasL- CTLs kill efficiently on a per recovered cell basis and can achieve wild-type levels of proliferation upon stimulation by optimal doses of anti-CD3, suggesting the lack of a costimulatory signal during antigen stimulation. To test this hypothesis directly, soluble FasIgG, a fusion protein of murine Fas and human IgG1, was added to FasL+ CTLs to demonstrate that blocking cell surface Fas-FasL interactions mimics the depression observed for FasL- CTLs. In addition, plate-bound FasIgG in conjunction with suboptimal anti-CD3 stimulation augments proliferative signals in FasL+ but not FasL- CTLs. In contrast to these results with CD8+ T cells, alloantigen-stimulated FasL- CD4+ T cells proliferate vigorously compared to FasL+ cells. These data demonstrate that reverse signaling through FasL is required for CTLs to achieve maximal proliferation and may provide clues to differences in the homeostatic regulation of activated CD4+ and CD8+ T cells during an immune response.

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Depressed proliferation and cell recovery of FasL-deficient  CTLs relative to FasL+ CTLs over the course of an MLC. (a and b)  [3H]TdR uptake and viable cell counts over days 1–5 from long-term CTL  lines from B6, B6.lpr, and B6.gld mice cultured with allogeneic H-2k splenocytes. APCs were removed before counting by antibody depletion. (c)  CTL responders were purified CD8+ T cells derived from B6 and B6.gld  mice cultured with H-2bm1 splenocytes. (d) [3H]TdR uptake by CTL responders used in a, cultured with C3H.gld splenocytes. Experiments were  repeated two to seven times. All data are averages of triplicate wells and  error bars represent the SD of the means. Percentages appearing to the  right of the figures indicate normalized comparison to B6 wild-type.
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Figure 1: Depressed proliferation and cell recovery of FasL-deficient CTLs relative to FasL+ CTLs over the course of an MLC. (a and b) [3H]TdR uptake and viable cell counts over days 1–5 from long-term CTL lines from B6, B6.lpr, and B6.gld mice cultured with allogeneic H-2k splenocytes. APCs were removed before counting by antibody depletion. (c) CTL responders were purified CD8+ T cells derived from B6 and B6.gld mice cultured with H-2bm1 splenocytes. (d) [3H]TdR uptake by CTL responders used in a, cultured with C3H.gld splenocytes. Experiments were repeated two to seven times. All data are averages of triplicate wells and error bars represent the SD of the means. Percentages appearing to the right of the figures indicate normalized comparison to B6 wild-type.

Mentions: Proliferation assays using CTL lines derived from wild-type B6, B6.lpr, and B6.gld mice revealed that FasL+ CTLs proliferate better in response to allogeneic stimulation than do FasL− CTLs (Fig. 1, a and c). APC titration from 5 × 104 to 1 × 106 cells cocultured per well with CTLs did not alter the relative depression in proliferation of B6.gld CTL lines (data not shown). In addition, kinetic analysis of viable cell counts revealed that the lack of FasL function also influences the ability of CTLs to increase in number over the course of an MLC (Fig. 1 b), and that this expansion coincides with FasL expression by the responding T cells. As determined by flow cytometry, FasL expression on the CTL lines is upregulated by 3 h after antigenic stimulation, peaks on day 3, and begins to decrease by day 4 (data not shown). This pattern of depressed proliferative capacity and number of recovered cells was reproducible for three independent CTL lines and was not reversed by the addition of exogenous IL-2. The depressed proliferation and recovered cell counts were also seen upon coculture of B6.gld CTLs with APCs from C3H.gld mice, demonstrating that this phenomenon is not caused by Fas-mediated killing of gld CTLs by FasL expressed on the APCs (Fig. 1 d). Autocrine Fas-mediated suicide (10–12) can also be ruled out as a mechanism for depressed proliferation because gld CTLs do not express functional cell surface FasL. Analysis by flow cytometry (data not shown) also ensured that there is not an accumulation of Thy1+CD8−CD4− cells analogous to those found in vivo in gld and lpr mice (1).


Maximal proliferation of cytotoxic T lymphocytes requires reverse signaling through Fas ligand.

Suzuki I, Fink PJ - J. Exp. Med. (1998)

Depressed proliferation and cell recovery of FasL-deficient  CTLs relative to FasL+ CTLs over the course of an MLC. (a and b)  [3H]TdR uptake and viable cell counts over days 1–5 from long-term CTL  lines from B6, B6.lpr, and B6.gld mice cultured with allogeneic H-2k splenocytes. APCs were removed before counting by antibody depletion. (c)  CTL responders were purified CD8+ T cells derived from B6 and B6.gld  mice cultured with H-2bm1 splenocytes. (d) [3H]TdR uptake by CTL responders used in a, cultured with C3H.gld splenocytes. Experiments were  repeated two to seven times. All data are averages of triplicate wells and  error bars represent the SD of the means. Percentages appearing to the  right of the figures indicate normalized comparison to B6 wild-type.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2199194&req=5

Figure 1: Depressed proliferation and cell recovery of FasL-deficient CTLs relative to FasL+ CTLs over the course of an MLC. (a and b) [3H]TdR uptake and viable cell counts over days 1–5 from long-term CTL lines from B6, B6.lpr, and B6.gld mice cultured with allogeneic H-2k splenocytes. APCs were removed before counting by antibody depletion. (c) CTL responders were purified CD8+ T cells derived from B6 and B6.gld mice cultured with H-2bm1 splenocytes. (d) [3H]TdR uptake by CTL responders used in a, cultured with C3H.gld splenocytes. Experiments were repeated two to seven times. All data are averages of triplicate wells and error bars represent the SD of the means. Percentages appearing to the right of the figures indicate normalized comparison to B6 wild-type.
Mentions: Proliferation assays using CTL lines derived from wild-type B6, B6.lpr, and B6.gld mice revealed that FasL+ CTLs proliferate better in response to allogeneic stimulation than do FasL− CTLs (Fig. 1, a and c). APC titration from 5 × 104 to 1 × 106 cells cocultured per well with CTLs did not alter the relative depression in proliferation of B6.gld CTL lines (data not shown). In addition, kinetic analysis of viable cell counts revealed that the lack of FasL function also influences the ability of CTLs to increase in number over the course of an MLC (Fig. 1 b), and that this expansion coincides with FasL expression by the responding T cells. As determined by flow cytometry, FasL expression on the CTL lines is upregulated by 3 h after antigenic stimulation, peaks on day 3, and begins to decrease by day 4 (data not shown). This pattern of depressed proliferative capacity and number of recovered cells was reproducible for three independent CTL lines and was not reversed by the addition of exogenous IL-2. The depressed proliferation and recovered cell counts were also seen upon coculture of B6.gld CTLs with APCs from C3H.gld mice, demonstrating that this phenomenon is not caused by Fas-mediated killing of gld CTLs by FasL expressed on the APCs (Fig. 1 d). Autocrine Fas-mediated suicide (10–12) can also be ruled out as a mechanism for depressed proliferation because gld CTLs do not express functional cell surface FasL. Analysis by flow cytometry (data not shown) also ensured that there is not an accumulation of Thy1+CD8−CD4− cells analogous to those found in vivo in gld and lpr mice (1).

Bottom Line: To test this hypothesis directly, soluble FasIgG, a fusion protein of murine Fas and human IgG1, was added to FasL+ CTLs to demonstrate that blocking cell surface Fas-FasL interactions mimics the depression observed for FasL- CTLs.In contrast to these results with CD8+ T cells, alloantigen-stimulated FasL- CD4+ T cells proliferate vigorously compared to FasL+ cells.These data demonstrate that reverse signaling through FasL is required for CTLs to achieve maximal proliferation and may provide clues to differences in the homeostatic regulation of activated CD4+ and CD8+ T cells during an immune response.

View Article: PubMed Central - PubMed

Affiliation: Department of Immunology, University of Washington, Seattle, Washington 98195, USA.

ABSTRACT
Fas ligand (FasL/CD95L) is best known for its role in delivering apoptotic signals through its receptor, Fas (APO-1/CD95). In this study, we present evidence that FasL has a second role as a signaling receptor. Alloantigen-specific proliferation by multiple FasL- murine CTL lines is depressed compared to that of FasL+ CTL lines. FasL- CTLs kill efficiently on a per recovered cell basis and can achieve wild-type levels of proliferation upon stimulation by optimal doses of anti-CD3, suggesting the lack of a costimulatory signal during antigen stimulation. To test this hypothesis directly, soluble FasIgG, a fusion protein of murine Fas and human IgG1, was added to FasL+ CTLs to demonstrate that blocking cell surface Fas-FasL interactions mimics the depression observed for FasL- CTLs. In addition, plate-bound FasIgG in conjunction with suboptimal anti-CD3 stimulation augments proliferative signals in FasL+ but not FasL- CTLs. In contrast to these results with CD8+ T cells, alloantigen-stimulated FasL- CD4+ T cells proliferate vigorously compared to FasL+ cells. These data demonstrate that reverse signaling through FasL is required for CTLs to achieve maximal proliferation and may provide clues to differences in the homeostatic regulation of activated CD4+ and CD8+ T cells during an immune response.

Show MeSH
Related in: MedlinePlus