Cutting Edge: FADD Is Not Required for Antigen Receptor-Mediated NF-κB Activation

Abstract

Recently, it has been demonstrated that stimulated T cells bearing defects in caspase-8 fail to promote nuclear shuttling of NF-κB complexes. Such cells display strikingly similar proliferative and survival defects as T cells lacking Fas-associated death domain protein (FADD) function. We characterized NF-κB signaling in T cells bearing a dominant-negative FADD transgene (FADDdd). Whereas FADDdd T cells displayed proliferative defects following activation, these were not a consequence of aberrant NF-κB signaling, as measured by IKK/IκB phosphorylation and IκB degradation. There were no appreciable defects in nuclear translocation of p65/Rel using ImageStream, a flow-based imaging cytometer. Pretreatment with benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone, a potent caspase inhibitor, also failed to impede canonical NF-κB signaling. Secretion of IL-2 and up-regulation of various activation markers occurred normally. Thus, FADD does not play an essential role in NF-κB activation, suggesting an alternative route by which this adaptor promotes the clonal expansion of T cells.

FADD and caspase-8 control the outcome of autophagic signaling in proliferating T cells

“…Consistent with this, we recently reported defects in regulation of S6 kinase in FADDdd transgenic T cells (16), a key intermediate in autophagy regulation (30). Further, bromodeoxyuridine (BrdU) incorporation assays revealed diminished entry of FADDdd transgenic and casp8 Ϫ/Ϫ T cells into S-phase and enhanced proportions of cells bearing subdiploid DNA content, a defect particularly acute in FADDdd-expressing CD8 ϩ T cells bearing an ovalbumin (OVA)-specific OT-I TCR transgene (14,15). To investigate the morphology of activated FADDdd T cells, naïve and 3-day activated OT1 and OT1-FADDdd (OT1-FD) T cells were subjected to transmission electron microscopy (TEM) analysis (Fig.…”

“…Indeed, T cells expressing a dominantly interfering form of FADD (FADDdd) or those lacking FADD, casp8, or c-FLIP fail to efficiently proliferate following antigen receptor stimulation (4,(8)(9)(10)(11)(12)(13). These defects were not rescued by provision of interleukin-2 (IL-2), a potent T cell mitogenic cytokine, and the proliferative defects observed in these mutant T cells were not due to defective IL-2 production (4,14,15). Curiously, the most profound requirement for FADD and casp8 exists in CD8 ϩ T cells (14,16), potentially as a consequence of their rapid proliferative capacity (17).…”

Antigen-mediated T cell expansion regulated by parallel pathways of death

“…Similarly, B cells deficient for Fadd or caspase-8 exhibit defects in Tlr3,4-mediated proliferation and mitogen-dependent antibody responses (19)(20)(21). To explain this effect, reports have supported a role for caspase-8 and Fadd in NF B activation (20,(22)(23)(24), whereas other studies show their absence has no effect (18,19,21,25).…”

Sensitivity measurement and compensation in spectral imaging

“…After compensation, the imagery is analyzed with the IDEAS software package where over two hundred photometric and morphological features are automatically calculated for each cell for the determination and identification of biologically relevant events or populations. Amongst possible examples are quantitation of nuclear translocation, image-based determination of stages of apoptosis, organelle identification, and colocalization of signals (25,26).…”