T cells genetically engineered to overcome death signaling enhance adoptive cancer immunotherapy.

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.

Engagement of Fas differentially regulates the production of LPS-induced proinflammatory cytokines and type I interferons.

Fas (CD95) signalling is best known for its role in apoptosis, however, recent reports have shown it to be involved in other cellular responses as well, including inflammation. Fas and its adaptor protein FADD are known to negatively regulate LPS-induced proinflammatory responses, but their role in LPS-induced type I interferon production is unknown. Here, we demonstrate that Fas engagement on macrophages, using an agonistic Fas antibody CH11, augments LPS-induced NF-κB responses, causing increased production of TNFα, IL-8, IL-6 and IL-12. Conversely, costimulation with both LPS and CH11 causes a significant reduction in the level of interferon-beta (IFNß) production. This differential effect involves the Fas adaptor FADD because while LPS-induced IL-6 production increased in FADD-/- murine embryonic fibroblasts, LPS-induced IFNß production was significantly reduced in these cells. Overexpression of a dominant negative form of FADD (FADD-DD) inhibits LPS-induced IFNß luciferase but not LPS-induced NF-κB luciferase. In contrast, overexpression of full-length FADD inhibited LPS-induced NF-κB luciferase activation but was seen to augment LPS-induced IFNß luciferase. Moreover, FADD-DD inhibits TRIF-, TRAM-, IKKε-, TBK-1- and TRAF3-induced IFNß luciferase production, with coimmunoprecipitation experiments demonstrating an interaction between FADD and TRIF. These data identify FADD as a novel component of the noncanonical Toll-like receptor 4/IFNß signalling pathway and demonstrate that both Fas and its adaptor FADD can differentially regulate the production of LPS-induced proinflammatory cytokines and type I interferons.

FADD at the Crossroads between Cancer and Inflammation.

Initially described as an adaptor molecule for death receptor (DR)-mediated apoptosis, Fas-associated death domain (FADD) was later implicated in nonapoptotic cellular processes. During the last decade, FADD has been shown to participate and regulate most of the signalosome complexes, including necrosome, FADDosome, innateosome, and inflammasome. Given the role of these signaling complexes, FADD has emerged as a new actor in innate immunity, inflammation, and cancer development. Concomitant to these new roles, a surprising number of mechanisms deemed to regulate FADD functions have been identified, including post-translational modifications of FADD protein and FADD secretion. This review focuses on recent knowledge of the biological roles of FADD, a pleiotropic molecule having multiple partners, and its impact in cancer, innate immunity, and inflammation.

Molecular cloning and characterization of FADD from the orange-spotted grouper (Epinephelus coioides).

Fas-associated protein with death domain (FADD) is the key adaptor protein that transmits apoptotic signals mediated by the main death receptors. Besides being an essential instrument in cell death, FADD is also implicated in proliferation, cell cycle progression, tumor development, inflammation, innate immunity, and autophagy. In the present study, a FADD homologue (EcFADD) from the orange-spotted grouper (Epinephelus coioides) was cloned and its possible role in fish immunity was analyzed. The full length cDNA of EcFADD contains 808 base pairs (bp), including a 573 bp open reading frame that encodes a 190 amino acid protein with a predicted molecular mass of 21.81 kDa. Quantitative real-time polymerase chain reaction analysis indicated that EcFADD was distributed in all examined tissues. The expression of EcFADD in the spleen of E. coioides was differentially up-regulated when challenged with Singapore grouper iridovirus (SGIV) or polyinosine-polycytidylic acid(poly[I:C]). EcFADD was abundantly distributed in both the cytoplasm and nucleus in grouper spleen (GS) and fathead minnow (FHM) epithelial cells. Over-expression of EcFADD inhibited SGIV infection and replication and SGIV-induced apoptosis. To achieve antiviral and anti-apoptosis activities, FADD promoted the activation of interferon-stimulated response element (ISRE) and type I interferon (IFN) genes in the antiviral IFN signaling pathway and inhibited activation of apoptosis-related transcription factors p53. Our results not only characterize FADD but also reveal new immune functions and the molecular mechanisms by which FADD responds to virus infection and virus-induced apoptosis.

Decreased linear ubiquitination of NEMO and FADD on apoptosis with caspase-mediated cleavage of HOIP.

NF-κB is crucial to regulate immune and inflammatory responses and cell survival. LUBAC generates a linear ubiquitin chain and activates NF-κB through ubiquitin ligase (E3) activity in the HOIP subunit. Here, we show that HOIP is predominantly cleaved by caspase at Asp390 upon apoptosis, and that is subjected to proteasomal degradation. We identified that FADD, as well as NEMO, is a substrate for LUBAC. Although the C-terminal fragment of HOIP retains NF-κB activity, linear ubiquitination of NEMO and FADD decreases upon apoptosis. Moreover, the N-terminal fragment of HOIP binds with deubiquitinases, such as OTULIN and CYLD-SPATA2. These results indicate that caspase-mediated cleavage of HOIP divides critical functional regions of HOIP, and that this regulates linear (de)ubiquitination of substrates upon apoptosis.

MLKL and FADD Are Critical for Suppressing Progressive Lymphoproliferative Disease and Activating the NLRP3 Inflammasome.

MLKL, a key component downstream of RIPK3, is suggested to be a terminal executor of necroptosis. Genetic studies have revealed that Ripk3 ablation rescues embryonic lethality in Fadd- or Caspase-8-deficient mice. Given that RIPK3 has also been implicated in non-necroptotic pathways including apoptosis and inflammatory signaling, it remains unclear whether the lethality in Fadd(-/-) mice is indeed caused by necropotosis. Here, we show that genetic deletion of Mlkl rescues the developmental defect in Fadd-deficient mice and that Fadd(-/-)Mlkl(-/-) mice are viable and fertile. Mlkl(-/-)Fadd(-/-) mice display significantly accelerated lymphoproliferative disease characterized by lymphadenopathy and splenomegaly when compared to Ripk3(-/-)Fadd(-/-) mice. Mlkl(-/-)Fadd(-/-) bone-marrow-derived macrophages and dendritic cells have impaired NLRP3 inflammasome activation associated with defects in ASC speck formation and NF-κB-dependent NLRP3 transcription. Our findings reveal that MLKL and FADD play critical roles in preventing lymphoproliferative disease and activating the NLRP3 inflammasome.

The Mitochondrial Phosphatase PGAM5 Is Dispensable for Necroptosis but Promotes Inflammasome Activation in Macrophages.

The cytokine IL-1ß is intimately linked to many pathological inflammatory conditions. Mature IL-1ß secretion requires cleavage by the inflammasome. Recent evidence indicates that many cell death signal adaptors have regulatory roles in inflammasome activity. These include the apoptosis inducers FADD and caspase 8, and the necroptosis kinases receptor interacting protein kinase 1 (RIPK1) and RIPK3. PGAM5 is a mitochondrial phosphatase that has been reported to function downstream of RIPK3 to promote necroptosis and IL-1ß secretion. To interrogate the biological function of PGAM5, we generated Pgam5(-/-) mice. We found that Pgam5(-/-) mice were smaller compared with wild type littermates, and male Pgam5(-/-) mice were born at sub-Mendelian ratio. Despite these growth and survival defects, Pgam5(-/-) cells responded normally to multiple inducers of apoptosis and necroptosis. Rather, we found that PGAM5 is critical for IL-1ß secretion in response to NLRP3 and AIM2 inflammasome agonists. Moreover, vesicular stomatosis virus-induced IL-1ß secretion was impaired in Pgam5(-/-) bone marrow-derived macrophages, but not in Ripk3(-/-) bone marrow-derived dendritic cells, indicating that PGAM5 functions independent of RIPK3 to promote inflammasome activation. Mechanistically, PGAM5 promotes ASC polymerization, maintenance of mitochondrial integrity, and optimal reactive oxygen species production in response to inflammasome signals. Hence PGAM5 is a novel regulator of inflammasome and caspase 1 activity that functions independently of RIPK3.

The expanding spectrum of the autoimmune lymphoproliferative syndromes.

PURPOSE OF REVIEW: Several autoimmune lymphoproliferative syndromes have been described lately. We review here the main clinical and laboratory findings of these new disorders. RECENT FINDINGS: The prototypical autoimmune lymphoproliferative syndrome (ALPS) has had its diagnostic criteria modified, somatic mutations in RAS genes were found to cause an ALPS-like syndrome in humans, and mutations in a gene encoding a protein kinase C (PRKCD) were discovered to cause a syndrome of lymphoproliferation, autoimmunity and natural killer cell defect. SUMMARY: The recent discoveries shed light on the molecular pathways governing lymphocyte death, proliferation and immune tolerance in humans.

Signal-transducing adaptor protein-2 modulates Fas-mediated T cell apoptosis by interacting with caspase-8.

We found that an adaptor protein, signal-transducing adaptor protein (STAP)-2, is a new member of the Fas-death-inducing signaling complex and participates in activation-induced cell death in T cells. STAP-2 enhanced Fas-mediated apoptosis and caspase-8 aggregation and activation in Jurkat T cells. Importantly, STAP-2 directly interacted with caspase-8 and Fas, resulting in enhanced interactions between caspase-8 and FADD in the Fas-death-inducing signaling complex. Moreover, STAP-2 protein has a consensus caspase-8 cleavage sequence, VEAD, in its C-terminal domain, and processing of STAP-2 by caspase-8 was crucial for Fas-induced apoptosis. Physiologic roles of STAP-2 were confirmed by observations that STAP-2-deficient mice displayed impaired activation-induced cell death and superantigen-induced T cell depletion. Therefore, STAP-2 is a novel participant in the regulation of T cell apoptosis after stimulation.

Fas-associated death domain protein and adenosine partnership: fad in RA.

Inflammation is the principal hallmark of RA. Different pathways are implicated in the production of pro-inflammatory cytokines, the bona fide mediators of this inflammation. Among them are the TNF pathway and the IL-1 receptor/Toll-like receptor (IL-1R/TLR4) pathway. One of the potential negative regulators of IL-1R/TLR4 signalling is the Fas-associated death domain protein (FADD), which is the pivotal adaptor of the apoptotic signal mediated by death receptors of the TNF family. FADD can sequester myeloid differentiation primary response gene 88 (MyD88), the common adaptor of most TLRs, and hence hinder the activation of nuclear factor κB (NF-κB), the downstream transcription factor. We recently described a new regulatory mechanism of FADD expression, via the shedding of microvesicles, mediated by adenosine receptors. Interestingly, adenosine is found in high concentrations in the joints of RA patients and has been largely reported as a regulator of inflammation. This review discusses the possible link that could exist between the adenosine-dependent regulation of FADD in the inflammatory context of RA and the potential role of FADD as a therapeutic target in the treatment of RA. We will see that the modulation of FADD expression may be a double-edged sword by increasing apoptosis and at the same time limiting NF-κB activation.

Common variation in genes related to immune response and risk of childhood leukemia.

An abnormal immune response to common infection(s) may be a plausible etiological mechanism in childhood leukemia. We investigated whether 931 tagging single nucleotide polymorphisms (SNPs) selected in gene regions related to immune response are associated with childhood leukemia susceptibility in a hospital-based case-control study (63 cases and 148 controls) conducted among Korean children. The AT or TT genotype of rs7939734 in Fas-associated protein with death domain (FADD) was associated with increased risk of childhood leukemia compared with the AA genotype (odds ratio [OR] = 2.26, 95% confidence interval [95% CI] = 1.20-4.25, p(trend) = 0.0007, min p = 0.002, false discovery rate [FDR] p = 0.17). The CG or GG genotype of rs2301696 in TRPM5 was associated with decreased risk of childhood leukemia compared with the CC genotype (OR = 0.30, 95% CI = 0.14-0.63, p(trend) = 0.002, min p = 0.004, FDR p = 0.17). Our findings suggest that genetic polymorphisms in immune response genes might play a role in childhood leukemia development with limited biologic evidence.

RIP1-mediated regulation of lymphocyte survival and death responses.

RIP1 is an adaptor serine/threonine kinase associated with the signaling complex of death receptors (DRs) including Fas, TNFR1, and TRAIL-Rs which can initiate apoptosis. While DRs are dispensable throughout development, RIP1 deletion results in perinatal lethality. The developmental defect caused by absence of RIP1 remains unexplained. In previous studies, RIP1-deficient hematopoietic progenitors failed to reconstitute the T cell compartment and our recent data indicate a new role for RIP1 in TCR-induced activation of the pro-survival NF-κB pathway. Here, we show that RIP1 is also critical for B cell development. In addition, RIP1(-/-) B cells stimulated through LPS/TLR4 are impaired in NF-κB activation but have no major defect in the Akt pathway. Recently, RIP1 has also emerged as a critical player in necrosis-like death, necroptosis, in various cell lines. We have demonstrated that RIP1 deficiency can reverse the embryonic and T cell proliferation defects in mice lacking FADD, a caspase adaptor protein, which indicates a potential role for RIP1 in mediating in vivo necroptosis. We provide an overview and discussion of the accumulating data revealing insights into the diverse functions of RIP1 in survival and death signaling in lymphocytes.

The adaptor protein FADD protects epidermal keratinocytes from necroptosis in vivo and prevents skin inflammation.

Epidermal keratinocytes provide an essential structural and immunological barrier forming the first line of defense against potentially pathogenic microorganisms. Mechanisms regulating barrier integrity and innate immune responses in the epidermis are important for the maintenance of skin immune homeostasis and the pathogenesis of inflammatory skin diseases. Here, we show that epidermal keratinocyte-restricted deficiency of the adaptor protein FADD (FADD(E-KO)) induced severe inflammatory skin lesions in mice. The development of skin inflammation in FADD(E-KO) mice was triggered by RIP kinase 3 (RIP3)-mediated programmed necrosis (termed necroptosis) of FADD-deficient keratinocytes, which was partly dependent on the deubiquitinating enzyme CYLD and tumor necrosis factor (TNF)-TNF receptor 1 signaling. Collectively, our findings provide an in vivo experimental paradigm that regulation of necroptosis in keratinocytes is important for the maintenance of immune homeostasis and the prevention of chronic inflammation in the skin.

Differential expression of factors involved in the intrinsic and extrinsic apoptotic pathways in juvenile systemic lupus erythematosus.

Dysregulated neutrophil apoptosis may result in the development of autoimmune disease by contributing to nuclear autoantigen exposure, leading to autoantibody generation and a breakdown in immune tolerance. It has previously been shown that neutrophil apoptosis is increased in juvenile-onset systemic lupus erythematosus (JSLE). This study aims to investigate the pathways involved in JSLE serum-induced apoptosis. Caspases 3, 7-9, IAP1/2, XIAP and FADD mRNA levels and TRAIL R2, BID/tBID, caspase 8 and 9 protein expression were measured in neutrophils from JSLE patients (n = 14) and controls (n = 10). The mRNA levels of caspases 7-9 were significantly higher in JSLE neutrophils than in controls, whereas the mRNA levels of IAP1, IAP2 and XIAP were decreased (p < 0.05). A decrease in neutrophil apoptosis induced by JSLE serum was observed in the presence of caspase 8 and 9 inhibitors (p < 0.05), and the activity of caspases 8 and 9 increased over time. tBID protein expression increased following incubation with JSLE serum. These data focus specifically on the expression and activity of the main caspases in the intrinsic and extrinsic apoptotic pathways. Increased expression of factors involved in the downstream signalling of the extrinsic apoptotic pathway indicates a prominent involvement of this pathway in JSLE serum-induced apoptosis.

Oligodendrocyte-specific FADD deletion protects mice from autoimmune-mediated demyelination.

Apoptosis of oligodendrocytes (ODCs), the myelin-producing glial cells in the CNS, plays a central role in demyelinating diseases such as multiple sclerosis and experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis. To investigate the mechanism behind ODC apoptosis in EAE, we made use of conditional knockout mice lacking the adaptor protein FADD specifically in ODCs (FADD(ODC-KO)). FADD mediates apoptosis by coupling death receptors with downstream caspase activation. In line with this, ODCs from FADD(ODC-KO) mice were completely resistant to death receptor-induced apoptosis in vitro. In the EAE model, FADD(ODC-KO) mice followed an ameliorated clinical disease course in comparison with control littermates. Lymphocyte and macrophage infiltration into the spinal cord parenchyma was significantly reduced, as was the extent of demyelination and proinflammatory gene expression. Collectively, our data show that FADD is critical for ODC apoptosis and the development of autoimmune demyelinating disease.

FADD: a regulator of life and death.

FAS-associated protein with death domain (FADD) is the key adaptor protein transmitting apoptotic signals mediated by the main death receptors (DRs). Besides being an essential instrument in cell death, FADD is also implicated in proliferation, cell cycle progression, tumor development, inflammation, innate immunity, and autophagy. Recently, many of these new functions of FADD were shown to be independent of DRs. Moreover, FADD function is dictated by protein localization and phosphorylation state. Thus, FADD is a crucial and unique controller of many essential cellular processes. The full understanding of the networks dictating the ultimate function of FADD may provide a new paradigm for other multifaceted proteins.

An emerging player in the adaptive immune response: microRNA-146a is a modulator of IL-2 expression and activation-induced cell death in T lymphocytes.

Activation of the T cell-mediated immune response has been associated with changes in the expression of specific microRNAs (miRNAs). However, the role of miRNAs in the development of an effective immune response is just beginning to be explored. This study focuses on the functional role of miR-146a in T lymphocyte-mediated immune response and provides interesting clues on the transcriptional regulation of miR-146a during T-cell activation. We show that miR-146a is low in human naive T cells and is abundantly expressed in human memory T cells; consistently, miR-146a is induced in human primary T lymphocytes upon T-cell receptor (TCR) stimulation. Moreover, we identified NF-kB and c-ETS binding sites as required for the induction of miR-146a transcription upon TCR engagement. Our results demonstrate that several signaling pathways, other than inflammation, are influenced by miR-146a. In particular, we provide experimental evidence that miR-146a modulates activation-induced cell death (AICD), acting as an antiapoptotic factor, and that Fas-associated death domain (FADD) is a target of miR-146a. Furthermore, miR-146a enforced expression impairs both activator protein 1 (AP-1) activity and interleukin-2 (IL-2) production induced by TCR engagement, thus suggesting a role of this miRNA in the modulation of adaptive immunity.

Death receptor signal transducers: nodes of coordination in immune signaling networks.

Death receptors (DRs) are members of the tumor necrosis factor receptor superfamily that possess a cytoplasmic death domain (DD). DRs regulate important operational and homeostatic aspects of the immune system. They transmit signals through apical protein complexes, which are nucleated by the DD adaptors FADD and TRADD, to control cellular outcomes that range from apoptosis to gene activation. FADD and TRADD also nucleate several distal signaling complexes, which mediate cross-talk between distinct DR signaling pathways. Moreover, together with other DR signal transducers, FADD and TRADD participate in functional complexes assembled by certain non-DR immune cell receptors, such as pattern-recognition receptors. Thus, DR signal transducers may provide important nodes of coordination in immune signaling networks.

A monoclonal-antibody-based ELISA for the detection of human FADD (Fas-associated death domain).

FADD (Fas-associated death domain) has been widely expressed in various tissues and its expression has been recently demonstrated to correlate with tumour progression and prognosis. Currently, measurement of FADD expression mainly depends on Western-blot or immunohistochemical approaches. To develop a conventional sandwich ELISA avenue for the detection of FADD protein to supplement Western blotting or immunohistochemistry, a series of mAbs (monoclonal antibodies) specific for FADD protein, designated 3A3, 3F9, 3G4, 4B9, 4G1, 7A8, 7B8 and 7F4, were produced by fusing mouse s/p20 myeloma cells with the spleen cells of a mouse immunized with the Escherichia coli-expressed recombinant His(6)-FADD protein. On the basis of the characterization of these mAbs, purified 3F9 was selected as the capture antibody and the biotin-conjugated 3A3 was selected as the detection antibody in sandwich ELISA. The limit of detection for the ELISA was 0.3 ng of purified His(6)-FADD (FADD tagged with hexahistidine), and it could detect both recombinant and native human FADD protein. Furthermore, the positive reaction of the ELISA could be blocked by rabbit anti-FADD sera. All of these results indicated that the ELISA developed in the present paper could be a promising tool for detection of FADD protein.