Bryan D. Bell

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

Fas-associated death domain protein (FADD) and caspase-8 (casp8) are vital intermediaries in apoptotic signaling induced by tumor necrosis factor family ligands. Paradoxically, lymphocytes lacking FADD or casp8 fail to undergo normal clonal expansion following antigen receptor cross-linking and succumb to caspase-independent cell death upon activation. Here we show that T cells lacking FADD or casp8 activity are subject to hyperactive autophagic signaling and subvert a cellular survival mechanism into a potent death process. T cell autophagy, enhanced by mitogenic signaling, recruits casp8 through interaction with FADD:Atg5-Atg12 complexes. Inhibition of autophagic signaling with 3-methyladenine, dominant-negative Vps34, or Atg7 shRNA rescued T cells expressing a dominant-negative FADD protein. The necroptosis inhibitor Nec-1, which blocks receptor interacting protein kinase 1 (RIP kinase 1), also completely rescued T cells lacking FADD or casp8 activity. Thus, while autophagy is necessary for rapid T cell proliferation, our findings suggest that FADD and casp8 form a feedback loop to limit autophagy and prevent this salvage pathway from inducing RIPK1-dependent necroptotic cell death. Thus, linkage of FADD and casp8 to autophagic signaling intermediates is essential for rapid T cell clonal expansion and may normally serve to promote caspase-dependent apoptosis under hyperautophagic conditions, thereby averting necrosis and inflammation in vivo.

Complementary roles of Fas-associated death domain (FADD) and receptor interacting protein kinase-3 (RIPK3) in T-cell homeostasis and antiviral immunity

Caspase-8 (casp8) is required for extrinsic apoptosis, and mice deficient in casp8 fail to develop and die in utero while ultimately failing to maintain the proliferation of T cells, B cells, and a host of other cell types. Paradoxically, these failures are not caused by a defect in apoptosis, but by a presumed proliferative function of this protease. Indeed, following mitogenic stimulation, T cells lacking casp8 or its adaptor protein FADD (Fas-associated death domain protein) develop a hyperautophagic morphology, and die a programmed necrosis-like death process termed necroptosis. Recent studies have demonstrated that receptor-interacting protein kinases (RIPKs) RIPK1 and RIPK3 together facilitate TNF-induced necroptosis, but the precise role of RIPKs in the demise of T cells lacking FADD or casp8 activity is unknown. Here we demonstrate that RIPK3 and FADD have opposing and complementary roles in promoting T-cell clonal expansion and homeostasis. We show that the defective proliferation of T cells bearing an interfering form of FADD (FADDdd) is rescued by crossing with RIPK3−/− mice, although such rescue ultimately leads to lymphadenopathy. Enhanced recovery of these double-mutant T cells following stimulation demonstrates that FADD, casp8, and RIPK3 are all essential for clonal expansion, contraction, and antiviral responses. Finally, we demonstrate that caspase-mediated cleavage of RIPK1-containing necrosis inducing complexes (necrosomes) is sufficient to prevent necroptosis in the face of death receptor signaling. These studies highlight the “two-faced” nature of casp8 activity, promoting clonal expansion in some situations and apoptotic demise in others.

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

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.

A Fas-Associated Death Domain Protein/Caspase-8-Signaling Axis Promotes S-Phase Entry and Maintains S6 Kinase Activity in T Cells Responding to IL-2

Fas-associated death domain protein (FADD) constitutes an essential component of TNFR-induced apoptotic signaling. Paradoxically, FADD has also been shown to be crucial for lymphocyte development and activation. In this study, we report that FADD is necessary for long-term maintenance of S6 kinase (S6K) activity. S6 phosphorylation at serines 240 and 244 was only observed after long-term stimulation of wild-type cells, roughly corresponding to the time before S-phase entry, and was poorly induced in T cells expressing a dominantly interfering form of FADD (FADDdd), viral FLIP, or possessing a deficiency in caspase-8. Defects in S6K1 phosphorylation were also observed. However, defective S6K1 phosphorylation was not a consequence of a wholesale defect in mammalian target of rapamycin function, because 4E-BP1 phosphorylation following T cell activation was unaffected by FADDdd expression. Although cyclin D3 up-regulation and retinoblastoma hypophosphorylation occurred normally in FADDdd T cells, cyclin E expression and cyclin-dependent kinase 2 activation were markedly impaired in FADDdd T cells. These results demonstrate that a FADD/caspase-8-signaling axis promotes T cell cycle progression and sustained S6K activity.

Coordinate regulation of autophagy and apoptosis in T cells by death effectors: FADD or foundation

During an immune response, specific recognition of microbial and tumor antigens leads to the rapid proliferation of lymphocytes. Once the immunological challenge is eliminated, the vast majority of these lymphocytes must be removed via apoptosis. Cell death is also vital for the deletion of autoreactive or chronically activated lymphocytes to prevent the development of autoimmunity in the host. Such processes are highly dependent on death receptors (DRs), molecules of the TNF receptor family. While these DRs promote apoptosis, interference with DR signaling paradoxically interferes with rapid lymphocyte proliferation. Recently, we discovered that T cells lacking Fas-Associated protein with Death Domain (FADD) or caspase-8 (casp8) function, both essential for DR-induced apoptosis, succumb to hyperactivation of autophagy and die through a non-apoptotic form of cell death rather than proliferating following mitogen stimulation. We observed recruitment of FADD, casp8 and serine/threonine kinase RIPK1 to complexes containing Atg5, Atg12 and Atg16L, suggesting that the generation of early autophagosomes leads to the assembly of complexes that activate casp8. Since blockade of RIPK1 or interference with autophagic signaling inhibited this alternative death process, we propose that hyperactive autophagy induced in the absence of caspase activity leads to a necrosis-like form of death that depends on RIPK1 enzymatic function. Herein, we summarize these findings and speculate on the significance and means by which autophagy is normally activated in proliferating lymphocytes.

T cell intrinsic roles of autophagy in promoting adaptive immunity

Autophagy, an ancient cellular response where autophagic vacuoles are formed within the cytosol, is induced in response to a variety of cellular insults, including growth factor or nutrient withdrawal, organelle damage, and misfolded proteins. Autophagy is rapidly induced in T lymphocytes following antigenic stimulation and blockade of autophagic signaling greatly reduces T cell clonal expansion, suggesting that autophagy is primarily involved in promoting T cell survival. In contrast, a recently identified negative feedback loop involving FADD and caspase-8 limits the level of autophagy in T cells. Failure to activate caspase-8 during T cell mitogenesis leads to hyperactive autophagy and cellular death through a programmed necrotic mechanism. These findings suggest that crosstalk between these cellular processes is essential for T cell activation and homeostasis.