Fas (CD95, Apo-1, or TNFRSF6) is a prototypical apoptosis-inducing death receptor in the tumor necrosis factor receptor (TNFR) superfamily. While the extracellular domains of TNFRs form trimeric ...complexes with their ligands and the intracellular domains engage in higher-order oligomerization, the role of the transmembrane (TM) domains is unknown. We determined the NMR structures of mouse and human Fas TM domains in bicelles that mimic lipid bilayers. Surprisingly, these domains use proline motifs to create optimal packing in homotrimer assembly distinct from classical trimeric coiled-coils in solution. Cancer-associated and structure-based mutations in Fas TM disrupt trimerization in vitro and reduce apoptosis induction in vivo, indicating the essential role of intramembrane trimerization in receptor activity. Our data suggest that the structures represent the signaling-active conformation of Fas TM, which appears to be different from the pre-ligand conformation. Analysis of other TNFR sequences suggests proline-containing sequences as common motifs for receptor TM trimerization.
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•Fas transmembrane domain structures show proline-containing motif for trimerization•The trimer conformation of the transmembrane domain is important for Fas signaling•The trimer conformation represents signaling-active, but not pre-liganded, state of Fas•Structural and functional data explain cancer mutations in transmembrane domain
Fas/CD95 is an apoptosis-inducing death receptor. Fu et al. determined the transmembrane domain structure of Fas and showed that the trimer assembly, which is mediated by a proline-containing motif, is essential for Fas signaling. The study provides structural explanation for several known cancer mutations in the transmembrane domain of Fas.
Caspase-8 activation can be triggered by death receptor-mediated formation of the death-inducing signaling complex (DISC) and by the inflammasome adaptor ASC. Caspase-8 assembles with FADD at the ...DISC and with ASC at the inflammasome through its tandem death effector domain (tDED), which is regulated by the tDED-containing cellular inhibitor cFLIP and the viral inhibitor MC159. Here we present the caspase-8 tDED filament structure determined by cryoelectron microscopy. Extensive assembly interfaces not predicted by the previously proposed linear DED chain model were uncovered, and were further confirmed by structure-based mutagenesis in filament formation in vitro and Fas-induced apoptosis and ASC-mediated caspase-8 recruitment in cells. Structurally, the two DEDs in caspase-8 use quasi-equivalent contacts to enable assembly. Using the tDED filament structure as a template, structural analyses reveal the interaction surfaces between FADD and caspase-8 and the distinct mechanisms of regulation by cFLIP and MC159 through comingling and capping, respectively.
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•Caspase-8 tDED assembles into filaments through quasi-equivalent contacts•The assembly of caspase-8 filaments is nucleated by the upstream Fas/FADD complex•cFLIP tDED also forms filaments, which interact with caspase-8 by comingling•MC159 inhibits caspase-8 filament assembly by a unique capping mechanism
How caspase-8 is activated has been a long-standing question. Fu et al. show that its tDED forms filaments using quasi-equivalent interactions. Cryo-EM structure of the filament reveals mechanisms of caspase-8 activation and its regulation by cFLIP and MC159.
Mutations affecting the apoptosis-inducing function of the Fas/CD95 TNF-family receptor result in autoimmune and lymphoproliferative disease. However, Fas can also costimulate T-cell activation and ...promote tumour cell growth and metastasis. Palmitoylation at a membrane proximal cysteine residue enables Fas to localize to lipid raft microdomains and induce apoptosis in cell lines. Here, we show that a palmitoylation-defective Fas C194V mutant is defective in inducing apoptosis in primary mouse T cells, B cells and dendritic cells, while retaining the ability to enhance naive T-cell differentiation. Despite inability to efficiently induce cell death, the Fas C194V receptor prevents the lymphoaccumulation and autoimmunity that develops in Fas-deficient mice. These findings indicate that induction of apoptosis through Fas is dependent on receptor palmitoylation in primary immune cells, and Fas may prevent autoimmunity by mechanisms other than inducing apoptosis.
TNF, acting through p55 tumor necrosis factor receptor 1 (TNFR1), contributes to the pathogenesis of many inflammatory diseases. TNFR-associated periodic syndrome (TRAPS, OMIM 142680) is an autosomal ...dominant autoinflammatory disorder characterized by prolonged attacks of fevers, peritonitis, and soft tissue inflammation. TRAPS is caused by missense mutations in the extracellular domain of TNFR1 that affect receptor folding and trafficking. These mutations lead to loss of normal function rather than gain of function, and thus the pathogenesis of TRAPS is an enigma. Here we show that mutant TNFR1 accumulates intracellularly in peripheral blood mononuclear cells of TRAPS patients and in multiple cell types from two independent lines of knockin mice harboring TRAPS-associated TNFR1 mutations. Mutant TNFR1 did not function as a surface receptor for TNF but rather enhanced activation of MAPKs and secretion of proinflammatory cytokines upon stimulation with LPS. Enhanced inflammation depended on autocrine TNF secretion and WT TNFR1 in mouse and human myeloid cells but not in fibroblasts. Heterozygous TNFR1-mutant mice were hypersensitive to LPS-induced septic shock, whereas homozygous TNFR1-mutant mice resembled TNFR1-deficient mice and were resistant to septic shock. Thus WT and mutant TNFR1 act in concert from distinct cellular locations to potentiate inflammation in TRAPS. These findings establish a mechanism of pathogenesis in autosomal dominant diseases where full expression of the disease phenotype depends on functional cooperation between WT and mutant proteins and also may explain partial responses of TRAPS patients to TNF blockade.
Background The B-cell receptor transmembrane activator and calcium modulator ligand interactor (TACI) is important for T-independent antibody responses. One in 200 blood donors are heterozygous for ...the TACI A181E mutation. Objective We sought to investigate the effect on B-cell function of TACI A181E heterozygosity in reportedly healthy subjects and of the corresponding TACI A144E mutation in mice. Methods Nuclear factor κB (NF-κB) activation was measured by using the luciferase assay in 293T cells cotransfected with wild-type and mutant TACI. TACI-driven proliferation, isotype switching, and antibody responses were measured in B cells from heterozygous TACI A144E knock-in mice. Mouse mortality was monitored after intranasal pneumococcal challenge. Results Levels of natural antibodies to the pneumococcal polysaccharide component phosphocholine were significantly lower in A181E-heterozygous than TACI-sufficient Swedish blood donors never immunized with pneumococcal antigens. Although overexpressed hTACI A181E and mTACI A144E acted as dominant-negative mutations in transfectants, homozygosity for A144E in mice resulted in absent TACI expression in B cells, indicating that the mutant protein is unstable when naturally expressed. A144E heterozygous mice, such as TACI+/− mice, expressed half the normal level of TACI on their B cells and exhibited similar defects in a proliferation-inducing ligand–driven B-cell activation, antibody responses to TNP-Ficoll, production of natural antibodies to phosphocholine, and survival after intranasal pneumococcal challenge. Conclusion These results suggest that TACI A181E heterozygosity results in TACI haploinsufficiency with increased susceptibility to pneumococcal infection. This has important implications for asymptomatic TACI A181E carriers.
Originally discovered as an inducer of apoptosis, the TNF-family receptor Fas (CD95, APO-1, TNFRSF6) has more recently been found to have functions beyond cell death, including T cell co-stimulation ...and promoting terminal differentiation of CD4+ and CD8+ T cells. Other TNF family members also discovered as apoptosis inducers, such as TRAIL (APO-2L, TNFSF10), can promote inflammation through caspase-8. Surprisingly, non-apoptotic signaling through Fas can protect from the autoimmunity seen in Fas deficiency independently from the cell death inducing functions of the receptor. Non-apoptotic Fas signaling can induce tumor cell growth and migration, and impair the efficacy of T cell adoptive immunotherapy. Blocking of non-apoptotic functions of these receptors may be a novel strategy to regulate autoimmunity and inflammation, and enhance antitumor immunity.
Tumor necrosis factor (TNF) family cytokine receptors containing a death domain, known informally as ‘death receptors’, trigger a range of functions beyond inducing programmed cell death, including cell growth and proliferation.
The TNFR Fas, or CD95, can co-stimulate CD4+ and CD8+ T cell activation and accelerate T cell differentiation into short-lived effector memory T cells.
Fas-induced effector memory T cell differentiation limits the efficacy of adoptive T cell immunotherapy and is also important for preventing the autoimmune syndrome that arises in the absence of a functional Fas receptor.
TRAIL receptors can also trigger inflammatory responses through a scaffolding function of caspase-8 and activation of caspase-1 and interleukin 1β activation and release.
These novel functions of TNF receptors may constitute targets for approaches to improve cancer immunotherapy and treat autoimmune diseases.
Signaling through the TNF-family receptor Fas/CD95 can trigger apoptosis or non-apoptotic cellular responses and is essential for protection from autoimmunity. Receptor clustering has been observed ...following interaction with Fas ligand (FasL), but the stoichiometry of Fas, particularly when triggered by membrane-bound FasL, the only form of FasL competent at inducing programmed cell death, is not known. Here we used super-resolution microscopy to study the behavior of single molecules of Fas/CD95 on the plasma membrane after interaction of Fas with FasL on planar lipid bilayers. We observed rapid formation of Fas protein superclusters containing more than 20 receptors after interactions with membrane-bound FasL. Fluorescence correlation imaging demonstrated recruitment of FADD dependent on an intact Fas death domain, with lipid raft association playing a secondary role. Flow-cytometric FRET analysis confirmed these results, and also showed that some Fas clustering can occur in the absence of FADD and caspase-8. Point mutations in the Fas death domain associated with autoimmune lymphoproliferative syndrome (ALPS) completely disrupted Fas reorganization and FADD recruitment, confirming structure-based predictions of the critical role that these residues play in Fas–Fas and Fas–FADD interactions. Finally, we showed that induction of apoptosis correlated with the ability to form superclusters and recruit FADD.
Across clinical trials, T cell expansion and persistence following adoptive cell transfer (ACT) have correlated with superior patient outcomes. Herein, we undertook a pan-cancer analysis to identify ...actionable ligand-receptor pairs capable of compromising T cell durability following ACT. We discovered that FASLG, the gene encoding the apoptosis-inducing ligand FasL, is overexpressed within the majority of human tumor microenvironments (TMEs). Further, we uncovered that Fas, the receptor for FasL, is highly expressed on patient-derived T cells used for clinical ACT. We hypothesized that a cognate Fas-FasL interaction within the TME might limit both T cell persistence and antitumor efficacy. We discovered that genetic engineering of Fas variants impaired in the ability to bind FADD functioned as dominant negative receptors (DNRs), preventing FasL-induced apoptosis in Fas-competent T cells. T cells coengineered with a Fas DNR and either a T cell receptor or chimeric antigen receptor exhibited enhanced persistence following ACT, resulting in superior antitumor efficacy against established solid and hematologic cancers. Despite increased longevity, Fas DNR-engineered T cells did not undergo aberrant expansion or mediate autoimmunity. Thus, T cell-intrinsic disruption of Fas signaling through genetic engineering represents a potentially universal strategy to enhance ACT efficacy across a broad range of human malignancies.
Adoptive cell transfer (ACT) of purified naive, stem cell memory, and central memory T cell subsets results in superior persistence and antitumor immunity compared with ACT of populations containing ...more-differentiated effector memory and effector T cells. Despite a clear advantage of the less-differentiated populations, the majority of ACT trials utilize unfractionated T cell subsets. Here, we have challenged the notion that the mere presence of less-differentiated T cells in starting populations used to generate therapeutic T cells is sufficient to convey their desirable attributes. Using both mouse and human cells, we identified a T cell-T cell interaction whereby antigen-experienced subsets directly promote the phenotypic, functional, and metabolic differentiation of naive T cells. This process led to the loss of less-differentiated T cell subsets and resulted in impaired cellular persistence and tumor regression in mouse models following ACT. The T memory-induced conversion of naive T cells was mediated by a nonapoptotic Fas signal, resulting in Akt-driven cellular differentiation. Thus, induction of Fas signaling enhanced T cell differentiation and impaired antitumor immunity, while Fas signaling blockade preserved the antitumor efficacy of naive cells within mixed populations. These findings reveal that T cell subsets can synchronize their differentiation state in a process similar to quorum sensing in unicellular organisms and suggest that disruption of this quorum-like behavior among T cells has potential to enhance T cell-based immunotherapies.
The death-inducing signaling complex (DISC) formed by the death receptor Fas, the adaptor protein FADD and caspase-8 mediates the extrinsic apoptotic program. Mutations in Fas that disrupt the DISC ...cause autoimmune lymphoproliferative syndrome (ALPS). Here we show that the Fas-FADD death domain (DD) complex forms an asymmetric oligomeric structure composed of 5-7 Fas DD and 5 FADD DD, whose interfaces harbor ALPS-associated mutations. Structure-based mutations disrupt the Fas-FADD interaction in vitro and in living cells; the severity of a mutation correlates with the number of occurrences of a particular interaction in the structure. The highly oligomeric structure explains the requirement for hexameric or membrane-bound FasL in Fas signaling. It also predicts strong dominant negative effects from Fas mutations, which are confirmed by signaling assays. The structure optimally positions the FADD death effector domain (DED) to interact with the caspase-8 DED for caspase recruitment and higher-order aggregation.
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