Craig M. Walsh

A Role for FADD in T Cell Activation and Development

FADD is a cytoplasmic adapter molecule that links the family of death receptors to the activation of caspases during apoptosis. We have produced transgenic mice expressing a dominantly interfering mutant of FADD, lacking the caspase-dimerizing death effector domain, as well as mice overexpressing the poxvirus serpin, CrmA, an inhibitor of caspases downstream of FADD. While thymocytes from either line of mice were completely protected from CD95-dependent cytotoxicity, neither transgene afforded protection from apoptosis induced during thymocyte selection and neither led to the lymphoproliferative disorders associated with deficiencies in CD95. However, in FADD dominant negative (FADDdd) mice, early thymocyte development was retarded and peripheral lymphocyte pools were devoid of normal populations of T cells. We show that thymocytes and peripheral T cells from FADDdd display signaling anomalies, implying that FADD plays a previously uncharacterized role in T cell development and activation.

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.

The complex interplay between autophagy, apoptosis, and necrotic signals promotes T-cell homeostasis.

Summary:  Intense research efforts over the last two decades have focused on establishing the significance of apoptotic signaling in adaptive immunity. Without doubt, caspase‐dependent apoptosis plays vital roles in many immune processes, including lymphocyte development, positive and negative selection, homeostasis, and self‐tolerance. Cell biologists have developed new insights into cell death, establishing that other modes of cell death exist, such as programmed necrosis and type II/autophagic cell death. Additionally, immunologists have identified a number of immunological processes that are highly dependent upon cellular autophagy, including antigen presentation, lymphocyte development and function, pathogen recognition and destruction, and inflammatory regulation. In this review, we provide detailed mechanistic descriptions of cellular autophagy and programmed necrosis induced in response to death receptor ligation, including methods to identify them, and compare and contrast these processes with apoptosis. The crosstalk between these three processes is emphasized as newly formulated evidence suggests that this interplay is vital for efficient T‐cell clonal expansion. This new evidence indicates that in addition to apoptosis, autophagy and programmed necrosis play significant roles in the termination of T‐cell‐dependent immune responses.

The Requirements for Fas-Associated Death Domain Signaling in Mature T Cell Activation and Survival

Fas-associated death domain (FADD) is a death domain containing cytoplasmic adapter molecule required for the induction of apoptosis by death receptors. Paradoxically, FADD also plays a crucial role in the development and proliferation of T cells. Using T cells from mice expressing a dominant negative form of FADD (FADDdd), activation with anti-TCR Ab and costimulation or exogenous cytokines is profoundly diminished. This is also seen in wild-type primary T cells transduced with the same transgene, demonstrating that FADD signaling is required in normally differentiated T cells. The defective proliferation does not appear to be related to the early events associated with TCR stimulation. Rather, with a block in FADD signaling, stimulated T cells exhibit a high rate of cell death corresponding to the initiation of cell division. Although CD4 T cells exhibit a moderate deficiency, this effect is most profound in CD8 T cells. In vivo, the extent of this defective accumulation is most apparent; lymphocytic choriomenigitis virus-infected FADDdd-expressing mice completely fail to mount an Ag-specific response. These results show that, in a highly regulated fashion, FADD, and most likely caspases, can transduce either a signal for survival or one that leads directly to apoptosis and that the balance between these opposing outcomes is crucial to adaptive immunity.

Functions of Caspase 8: the Identified and the Mysterious

Initially discovered as an initiator protease in apoptosis mediated by death receptors, caspase-8 is now known to have an apparently confounding opposing effect in securing cell survival. It is required to allow mouse embryo survival, and the survival of hematopoietic cells during their development and activation. Classic models in which caspase-8 is depleted or inhibited frequently result in inhibition of apoptosis, and conversion to death through a necrotic pathway. This bewildering switch is now known to be driven by activation of a pathway dependent on protein kinases of the RIP family, which engage a pathway known as necroptosis. If caspase-8 does not control this pathway, necrotic death results. The pro-apoptotic and pro-survival functions of caspase-8 are regulated by a specific interaction with the pseudo-caspase cFLIP, and it is thought that the heterocomplex between these two partners alters the substrate specificity of caspase-8 in favor of inactivating components of the RIP kinase pathway. The description of how caspase-8 and cFLIP coordinate the switch between apoptosis and survival is just beginning. The mechanism is not known, the differential targets are not known, and the reason of why an apoptotic initiator has been co-opted as a critical survival factor is only guessed at. Elucidating these unknowns will be important in understanding mechanisms and possible therapeutic targets in autoimmune, inflammatory, and metastatic diseases.

Programmed necrosis and autophagy in immune function

It has long been known that apoptosis is vital to the generation and maintenance of proper adaptive immune function. An example is the essential requirement for apoptotic signaling during the generation of self‐tolerant lymphocytes: the apoptotic death of B and T cells with overt autoreactivity is essential to central tolerance. More recently, the contributions of additional processes including cellular autophagy and programmed necrosis have been implicated in controlling both innate and adaptive immune functions. Evidence has been provided to demonstrate that the death of cells following ligation of death receptors (DRs), a subfamily of cell surface molecules related to tumor necrosis factor receptor 1, is not exclusively the domain of caspase‐dependent apoptosis. In cells lacking the capacity to activate caspase‐8 following DR ligation, cell death instead occurs via programmed necrosis, or as it has been recently termed, ‘necroptosis’. This death process depends on RIP1 and RIP3, serine/threonine kinases that are recruited by DRs, and likely by other cellular signals including DNA damage and antigen receptor ligation. The generation of RIP1/RIP3 containing ‘necrosomes’ activates downstream necroptotic signaling that ultimately targets cellular energetic metabolism. Also related to cellular metabolic regulation, cellular autophagy has also been found to play unique and important roles in immunity. In this review, we describe the roles of necroptosis and autophagy in innate and adaptive immunity and speculate on the intriguing interplay between these two cellular processes.

FADD self-association is required for stable interaction with an activated death receptor

Receptor-mediated programmed cell death proceeds through an activated receptor to which the death adaptor FADD and the initiator procaspases 8 and/or 10 are recruited following receptor stimulation. The adaptor FADD is responsible for both receptor binding and recruitment of the procaspases into the death-inducing signaling complex. Biochemical dissection of the FADD death effector domain and functional replacement with a coiled-coil motif demonstrates that there is an obligatory FADD self-association via the DED during assembly of the death-inducing signaling complex. Using engineered oligomerization motifs with defined stoichiometries, the requirement for FADD self-association through the DED can be separated from the caspase-recruitment function of the domain. Disruption of FADD self-association precludes formation of a competent signaling complex. On this basis, we propose an alternative architecture for the FADD signaling complex in which FADD acts as a molecular bridge to stitch together an array of activated death receptors.

Apoptotic signal transduction and T cell tolerance.

The healthy immune system makes use of a variety of surveillance mechanisms at different stages of lymphoid development to prevent the occurrence and expansion of potentially harmful autoreactive T cell clones. Disruption of these mechanisms may lead to inappropriate activation of T cells and the development of autoimmune and lymphoproliferative diseases [such as multiple sclerosis, rheumatoid arthritis, lupus erythematosus, diabetes and autoimmune lymphoproliferative syndrome (ALPS)]. Clonal deletion of T cells with high affinities for self-peptide-MHC via programmed cell death (apoptosis) is an essential mechanism leading to self-tolerance. Referred to as negative selection, central tolerance in the thymus serves as the first checkpoint for the developing T cell repertoire and involves the apoptotic elimination of potentially autoreactive T cells clones bearing high affinity T cell receptors (TCR) that recognize autoantigens presented by thymic epithelial cells. Autoreactive T cells that escape negative selection are held in check in the periphery by either functional inactivation (“anergy”) or extrathymic clonal deletion, both of which are dependent on the strength and frequency of the TCR signal and the costimulatory context, or by regulatory T cells. This review provides an overview of the different molecular executioners of cell death programs that are vital to intrathymic or extrathymic clonal deletion of T cells. Further, the potential involvement of various apoptotic signaling paradigms are discussed with respect to the genesis and pathophysiology of autoimmune disease.

Complement protein C1q bound to apoptotic cells suppresses human macrophage and dendritic cell-mediated Th17 and Th1 T cell subset proliferation

A complete genetic deficiency of the complement protein C1q results in SLE with nearly 100% penetrance in humans, but the molecular mechanisms responsible for this association have not yet been fully determined. C1q opsonizes ACs for enhanced ingestion by phagocytes, such as Mφ and iDCs, avoiding the extracellular release of inflammatory DAMPs upon loss of the membrane integrity of the dying cell. We previously showed that human monocyte‐derived Mφ and DCs ingesting autologous, C1q‐bound LALs (C1q‐polarized Mφ and C1q‐polarized DCs), enhance the production of anti‐inflammatory cytokines, and reduce proinflammatory cytokines relative to Mφ or DC ingesting LAL alone. Here, we show that C1q‐polarized Mφ have elevated PD‐L1 and PD‐L2 and suppressed surface CD40, and C1q‐polarized DCs have higher surface PD‐L2 and less CD86 relative to Mφ or DC ingesting LAL alone, respectively. In an MLR, C1q‐polarized Mφ reduced allogeneic and autologous Th17 and Th1 subset proliferation and demonstrated a trend toward increased Treg proliferation relative to Mφ ingesting LAL alone. Moreover, relative to DC ingesting AC in the absence of C1q, C1q‐polarized DCs decreased autologous Th17 and Th1 proliferation. These data demonstrate that a functional consequence of C1q‐polarized Mφ and DC is the regulation of Teff activation, thereby “sculpting” the adaptive immune system to avoid autoimmunity, while clearing dying cells. It is noteworthy that these studies identify novel target pathways for therapeutic intervention in SLE and other autoimmune diseases.