Members of the tumor necrosis factor (TNF) receptor family and their corresponding ligands are critical regulators of apoptosis and various other cellular processes. Some of the receptors (Fas, TNF-R1, TRAIL-R1, TRAIL-R2, TRAMP/ DR3, DR6, and EDA-R) contain a cytoplasmic region, called the death domain (DD), which is essential for cell death signaling (18,22). Signals emanating from Fas and TNF-R1 have been intensively studied (14). Upon receptor activation, the DD of Fas undergoes direct homotypic interaction with a DD in the adapter protein FADD, while FADD recruitment is indirect (via TRADD) in the case of TNF-R1 (4). The death effector domain (DED) at the amino terminus of FADD then recruits pro-caspase 8 via homotypic interaction with its two DEDs. The high local concentration of caspase 8 zymogens facilitates self-processing and assembly of the mature enzyme. Activated caspase 8 initiates apoptosis by subsequent cleavage of downstream caspases (caspase-3, -6, and -7).Death induced by death receptors is tightly regulated by genes that are activated by the transcription factor NF-B (25). Modulation of the response in favor of NF-B protects cells from apoptosis; failure to do so results in increased cell death. At least six NF-B-responsive genes are involved in this survival amplification loop (26), i.e., those that encode IAP-1 and IAP-2, which block caspase activity (7); that which encodes the Bcl-2 family member A1 (25); and those that encode TRAF-1, TRAF-2 (1), and A20 (21), which are themselves implicated in the NF-B signaling pathway. However, overexpression of all of these genes affords, at best, partial protection, in particular from death triggered by TNF (25). The only known potent inhibitor of death receptor signals is c-FLIP. Two c-FLIPs have been characterized (23, 24). The full-length 55-kDa-long form of FLIP (FLIP L ) exhibits overall structural homology to caspase 8, containing two DEDs that interact with FADD and an inactive caspase-like domain. An alternatively spliced short form of FLIP (FLIP S ) contains only the two DEDs and displays reduced antiapoptotic capacity.We undertook a series of experiments to investigate whether c-FLIP is implicated in the antiapoptotic NF-B response. Here we provide evidence that FLIP expression is upregulated upon the stimulation of several signaling pathways that are known to trigger activation of the transcription factor NF-B. Moreover, we found that cells that were rendered highly sensitive to death ligand-induced apoptosis by blocking NF-B activation could be rescued by expressing FLIP. FLIP may therefore play a key role in the NF-B-mediated control of death signals. MATERIALS AND METHODSAntibodies and materials. Rabbit anti-TRAF2 polyclonal antibody C20, rabbit anti-cIAP1 polyclonal antibody H-85, and mouse anti-TRAF1 monoclonal antibody H3 were purchased from Santa Cruz Biotechnology (Santa Cruz, Calif.). Rat anti-cFLIP monoclonal antibody Dave II was from Alexis, Lausen Switzerland, and anti-Phospho-IBa antibody was from Biolab. Anti-tubulin and antiFl...

The paracaspase MALT1 is pivotal in antigen receptor-mediated lymphocyte activation and lymphomagenesis. MALT1 contains a caspase-like domain, but it is unknown whether this domain is proteolytically active. Here we report that MALT1 had arginine-directed proteolytic activity that was activated after T cell stimulation, and we identify the signaling protein Bcl-10 as a MALT1 substrate. Processing of Bcl-10 after Arg228 was required for T cell receptor-induced cell adhesion to fibronectin. In contrast, MALT1 activity but not Bcl-10 cleavage was essential for optimal activation of transcription factor NF-kappaB and production of interleukin 2. Thus, the proteolytic activity of MALT1 is central to T cell activation, which suggests a possible target for the development of immunomodulatory or anticancer drugs.

Spontaneous CD8 T-cell responses occur in growing tumors but are usually poorly effective. Understanding the molecular and cellular mechanisms that drive these responses is of major interest as they could be exploited to generate a more efficacious antitumor immunity. As such, stimulator of IFN genes (STING), an adaptor molecule involved in cytosolic DNA sensing, is required for the induction of antitumor CD8 T responses in mouse models of cancer. Here, we find that enforced activation of STING by intratumoral injection of cyclic dinucleotide GMP-AMP (cGAMP), potently enhanced antitumor CD8 T responses leading to growth control of injected and contralateral tumors in mouse models of melanoma and colon cancer. The ability of cGAMP to trigger antitumor immunity was further enhanced by the blockade of both PD1 and CTLA4. The STING-dependent antitumor immunity, either induced spontaneously in growing tumors or induced by intratumoral cGAMP injection was dependent on type I IFNs produced in the tumor microenvironment. In response to cGAMP injection, both in the mouse melanoma model and an ex vivo model of cultured human melanoma explants, the principal source of type I IFN was not dendritic cells, but instead endothelial cells. Similarly, endothelial cells but not dendritic cells were found to be the principal source of spontaneously induced type I IFNs in growing tumors. These data identify an unexpected role of the tumor vasculature in the initiation of CD8 T-cell antitumor immunity and demonstrate that tumor endothelial cells can be targeted for immunotherapy of melanoma.M etastatic melanoma is a highly aggressive cancer with a fast increasing incidence worldwide. Unless diagnosed early and surgically resected, the disease becomes metastatic and life threatening. Both chemotherapy and irradiation are ineffective. Novel therapies that target oncogenic drivers have brought some improvements, but tumor cells escape regularly (1). Melanoma is a prototypical immunogenic tumor, as shown by the occurrence of spontaneous CD8 T-cell responses that drive tumor regressions and by the identification of CD8 T cells that recognize melanoma antigens (2, 3). Although many immunotherapeutic strategies have been developed to induce such responses, clinical efficacies have been poor. More recently, "checkpoint blockade" therapies that target T-cell-inhibitory pathways mediated by CTLA4 (4) and PD1 (5) have yielded encouraging clinical results and demonstrated that spontaneous CD8 T-cell responses in tumors can be boosted to treat melanoma.The mechanisms that drive spontaneous antitumor immune responses are poorly understood. Type I IFNs (IFN-α and IFN-β) may play a role as the expression type I IFN-related genes in primary melanoma has been associated with spontaneous tumor regressions (6) and correlated to the tumor infiltration by specific CD8+ T cells (6, 7). Furthermore, the lack of type I IFN signaling or IFN-β expression inhibited the generation of tumor-specific CD8 T cells and accelerated tumor growth in a murine melanoma mo...

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The inflammasome is a cytosolic protein complex regulating the activation of caspase-1, which cleaves the pro-inflammatory cytokines IL-1beta and IL-18 into their active form. The inflammasome is composed of a NACHT-, LRR- and pyrin (NALP) family member that acts as a sensor for danger signals and the adaptor protein apoptosis-associated speck-like protein containing a CARD domain (ASC), which allows the recruitment of caspase-1 in the complex. In the skin, exposure to contact sensitizers (CS) such as trinitro-chlorobenzene causes an immune response called contact hypersensitivity (CHS) or eczema. In this delayed-type hypersensitivity response, efficient priming of the adaptive immunity depends on the concomitant activation of the innate immune system, including IL-1beta/IL-18 activation in the skin. To determine if the inflammasome contributes to CHS, we have analyzed its capacity to react to CS in vitro and in vivo. We show here that key components of the inflammasome are present in human keratinocytes and that CS like trinitro-chlorobenzene induce caspase-1/ASC dependent IL-1beta and IL-18 processing and secretion. We also show that ASC- and NALP3-deficient mice display an impaired response to CS. These findings suggest that CS act as danger signals that activate the inflammasome in the skin, and reveal a new role of NALP3 and ASC as regulators of innate immunity in CHS.

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CARMA1 is a lymphocyte-specific member of the membrane-associated guanylate kinase (MAGUK) family of scaffolding proteins, which coordinate signaling pathways emanating from the plasma membrane. CARMA1 interacts with Bcl10 via its caspase-recruitment domain (CARD). Here we investigated the role of CARMA1 in T cell activation and found that T cell receptor (TCR) stimulation induced a physical association of CARMA1 with the TCR and Bcl10. We found that CARMA1 was constitutively associated with lipid rafts, whereas cytoplasmic Bcl10 translocated into lipid rafts upon TCR engagement. A CARMA1 mutant, defective for Bcl10 binding, had a dominant-negative (DN) effect on TCR-induced NF-kappa B activation and IL-2 production and on the c-Jun NH(2)-terminal kinase (Jnk) pathway when the TCR was coengaged with CD28. Together, our data show that CARMA1 is a critical lipid raft-associated regulator of TCR-induced NF-kappa B activation and CD28 costimulation-dependent Jnk activation.