Maximal Proliferation of Cytotoxic T Lymphocytes Requires Reverse Signaling through Fas Ligand

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.

The Fas ligand intracellular domain is released by ADAM10 and SPPL2a cleavage in T-cells

“…33 Much less is known about the molecular mechanisms underlying its capacity to transduce nonapoptotic signals into the ligand-bearing cell. FasL reverse signaling has been shown to enhance T-cell proliferation, [11][12][13] and a recent publication has demonstrated that expression of the cytoplasmic FasL domain alone is sufficient to costimulate T-cell receptor signals. 11 Nevertheless, it remains unclear exactly how the FasL ICD is engaged in the transmission of such costimulatory signals.…”

“…[10][11][12][13] For example, crosslinking cell surface FasL during T-cell receptor activation led to maximal CD8 þ T-cell proliferation, indicating an ancillary role of FasL signaling in T-cell activation. [11][12][13] This phenomenon, known as 'reverse' or 'retrograde' signaling, has also been associated with other members of the TNF superfamily. Its common outcomes are changes in proliferation and cytokine production profiles of the affected cells.…”

Critical role of lipid rafts in CD154‐mediated T cell signaling

“…However, the precise mechanism by which CD154-triggered signaling is accomplished remains largely unknown since the cytoplasmic tail does not contain any signaling motif. Reverse signaling described for other members of the TNF family such as FasL [24], TNF-a [25], and Ox40L, results in modulating the cell's functions and survival [26][27][28].…”

“…However, this activation of the PKC is involved only in the downstream signaling implicating p38 but not ERK1/2 since their inhibition by the total inhibitor chelerythrine did not affect the phosphorylation of the ERK1/2 kinase. Therefore, it seems that the signal provided through CD154 into lipid raft involves activation of PKCa/d which in return will mediate phosphorylation of p38.Similar to the requirement of lipid rafts integrity for translocation of FasL, other member of the TNF superfamily into lipid rafts [24], membrane integrity is also essential for relocation of CD154 into lipid rafts and consequent phosphorylation of PKC and p38, including production of IL-2 in the Jurkat D1.1 cells. Cholesterol depletion that prevented the association of hypercross linked CD20 with detergent-insoluble rafts, and resulted in attenuation of both calcium mobilization and rituximab-induced apoptosis also support our conclusions regarding the requirement of the integrity of lipid rafts [45].…”

“…However, the precise mechanism by which CD154-triggered signaling is accomplished remains largely unknown since the cytoplasmic tail does not contain any signaling motif. Reverse signaling described for other members of the TNF family such as FasL [24], , and Ox40L, results in modulating the cell's functions and survival [26][27][28].Lipid rafts are membrane microdomains rich in cholesterol and sphingolipids known to contain many adaptor proteins involved in signalization. Lipid rafts are the highly organized microdomains of the plasma membrane that facilitate intermolecular association and the propagation of signal transduction pathways including Ca2 1 and PKC.…”