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.

costimulation ͉ homeostasis ͉ peripheral T cell

Fas ligand (FasL/CD95L/APO-1L) is one of a growing number of TNF family members whose triggering costimulates maximal proliferation of activated T cells. In this study we show that maximal Ag-dependent accumulation of transferred TCR-transgenic CD8+ T cells requires Fas (CD95/APO-1) expression by the adoptive hosts. Additionally, adoptively transferred FasL+ CD8+ T cells demonstrate a 2-fold advantage in Ag-driven expansion over their FasL−counterparts. This study illustrates the in vivo role of TCR-dependent FasL costimulation in the Ag-specific proliferation of both heterogeneous and homogeneous populations of primary CD8+ T cells and long-term CTL lines. Thus, cross-linking FasL on naive and Ag-experienced CD8+ T cells whose Ag-specific TCRs are engaged is required to drive maximal cellular proliferation in vivo.

Productive T cell activation generally requires costimulation in addition to a signal delivered through the TCR. Although FasL is well-characterized for its capacity to deliver a death signal through Fas, this TNF family member can also transmit a reverse signal to enhance Ag-driven T cell proliferation. In this study, we define this reverse signal through FasL as costimulation by showing it requires TCR coengagement and is CD28 independent. We demonstrate that FasL-mediated costimulation drives FasL recruitment into lipid rafts and association with select Src homology 3 (SH3)-containing proteins. We further show that the proline-rich intracellular domain of FasL is sufficient to costimulate by enhancing the phosphorylation of Akt, ERK1/2, JNK, and FasL itself, by activating the transcription factors NFAT and AP-1, and by enhancing IFN-γ production. These results elucidate the pathway of costimulation through the death inducer FasL, and comprise the first mechanistic analysis of a newly emerging group of costimulators, the TNF family.

No abstract