Over-expression of B7-H1 (PD-L1) molecule in the tumor microenvironment (TME) is a major immune evasion mechanism in some cancer patients and antibody blockade of the B7-H1/PD-1 interaction can normalize compromised immunity without excessive side-effects. Using a genomescale T-cell activity array, we identified Siglec-15 as a critical immune suppressor. While only expressed on some myeloid cells normally, Siglec-15 is broadly upregulated on human cancer cells and tumor-infiltrating myeloid cells, and its expression is mutually exclusive to B7-H1, partially due to its induction by M-CSF and downregulation by IFN-γ. We demonstrate that Siglec-15 suppresses antigen-specific T-cell responses in vitro and in vivo. Genetic ablation or antibody blockade of Siglec-15 amplifies anti-tumor immunity in the TME and inhibits tumor Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use:

The immune system must discriminate between pathogenic and nonpathogenic microbes in order to initiate an appropriate response. Toll-like receptors (TLRs) detect microbial components common to both pathogenic and nonpathogenic bacteria, whereas Nod-like receptors (NLRs) sense microbial components introduced into the host cytosol by the specialized secretion systems or pore-forming toxins of bacterial pathogens. The host signaling pathways that respond to bacterial secretion systems remain poorly understood. Infection with the pathogen Legionella pneumophila, which utilizes a type IV secretion system (T4SS), induced an increased proinflammatory cytokine response compared to avirulent bacteria in which the T4SS was inactivated. This enhanced response involved NF-κB activation by TLR signaling as well as Nod1 and Nod2 detection of type IV secretion. Furthermore, a TLR- and RIP2-independent pathway leading to p38 and SAPK/JNK MAPK activation was found to play an equally important role in the host response to virulent L. pneumophila. Activation of this MAPK pathway was T4SS-dependent and coordinated with TLR signaling to mount a robust proinflammatory cytokine response to virulent L. pneumophila. These findings define a previously uncharacterized host response to bacterial type IV secretion that activates MAPK signaling and demonstrate that coincident detection of multiple bacterial components enables immune discrimination between virulent and avirulent bacteria.

Inflammasomes are intracellular multiprotein signaling complexes that activate Caspase-1, leading to the cleavage and secretion of IL-1β and IL-18, and ultimately host cell death. Inflammasome activation is a common cellular response to infection; however, the consequences of inflammasome activation during acute infection and in the development of long-term protective immunity is not well understood. To investigate the role of the inflammasome in vivo, we engineered a strain of Listeria monocytogenes that ectopically expresses Legionella pneumophila flagellin, a potent activator of the Nlrc4 inflammasome. Compared with wild-type L. monocytogenes , strains that ectopically secreted flagellin induced robust host cell death and IL-1β secretion. These strains were highly attenuated both in bone marrow-derived macrophages and in vivo compared with wild-type L. monocytogenes . Attenuation in vivo was dependent on Nlrc4, but independent of IL-1β/IL-18 or neutrophil activity. L. monocytogenes strains that activated the inflammasome generated significantly less protective immunity, a phenotype that correlated with decreased induction of antigen-specific T cells. Our data suggest that avoidance of inflammasome activation is a critical virulence strategy for intracellular pathogens, and that activation of the inflammasome leads to decreased long-term protective immunity and diminished T-cell responses.

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is a cytokine with potential therapeutic value against cancers because of its selective cytotoxicity to many transformed, but not normal, cells. The ''decoy receptors'' TRAIL-R3 (TR3) and TRAIL-R4 (TR4) were believed to negatively regulate TRAIL-induced cytotoxicity by competing for ligand binding with TRAIL-R1 (TR1) and TRAIL-R2 (TR2). Here, we show that inhibition of TRAILinduced apoptosis by TR4 critically depends on its association with TR2 via the NH 2-terminal preligand assembly domain overlapping the first partial cysteine-rich domain of both receptors. By contrast, ligand binding by TR4 is dispensable for its apoptosis inhibitory function, thereby excluding the possibility that TR4 was a ''decoy'' to inhibit apoptosis by binding up TRAIL. In primary CD8 ؉ T cells, which express only TR2 and TR4 and are resistant to TRAIL-induced apoptosis, stimulation with phorbol myristate acetate abrogated the ligand-independent interaction between TR2 and TR4 and enhanced their sensitivity to TRAIL-induced apoptosis. Hence, whereas most TNF receptors normally form only homotrimeric complexes, the preligand assembly domains in TR2 and TR4 permit mixed complex formation as a means to regulate apoptosis induction. We propose that TR4 is a ''regulatory'' rather than ''decoy'' receptor that inhibits apoptosis signaling by TRAIL through this previously uncharacterized ligand-independent mechanism. decoy receptors T he regulation of cell death by members of the TNF family plays a critical role in immune function and homeostasis (1). TRAIL is a TNF-like cytokine that selectively induces apoptosis in many tumor cells, but not in normal cells. Administration of recombinant TRAIL or antibodies against TR2 in several experimental tumor models exhibited potent antitumor activity with minimal hepatic toxicity (2-6). Moreover, recombinant TRAIL or agonist TRAIL receptor antibody often synergizes with chemotherapy or radiation to induce tumor-cell apoptosis (7-10). This unique property of TRAIL has prompted many to vaunt it as a potential therapeutic agent against malignant diseases. Despite its potency against tumor cells, the physiological function of TRAIL is largely unknown, although some reports have implicated TRAIL to be involved in tumor surveillance (11), target cell killing by various immune effector cells (12,13), and the regulation of innate immune responses (14).TRAIL binds to five distinct TNF receptor (TNFR)-like receptors, TR1 (TRAIL-R1͞DR4), TR2 (TRAIL-R2͞DR5͞ Killer͞Trick), TR3 (TRAIL-R3͞DcR1͞LIT͞TRID), TR4 (TRAIL-R4͞DcR2͞TRUNDD), and the soluble receptor osteoprotegerin (OPG). OPG is a soluble receptor that also binds another TNF-like cytokine called TRANCE͞RANK-L and may have a more prominent role in bone and myeloid cell development than in regulating TRAIL-induced apoptosis. The four membrane-anchored TRAIL receptors contain two complete cysteine-rich domains (CRDs) for ligand binding that are preceded at the NH 2 termini by a highly conserved...

Legionella pneumophila is a gram-negative facultative intracellular parasite of macrophages. Although L. pneumophila is the causative agent of a severe pneumonia known as Legionnaires' disease, it is likely that most infections caused by this organism are cleared by the host innate immune system. It is predicted that host pattern recognition proteins belonging to the Toll-like receptor (TLR) family are involved in the protective innate immune responses. We examined the role of TLR-mediated responses in L. pneumophila detection and clearance using genetically altered mouse hosts in which the macrophages are permissive for L. pneumophila intracellular replication. Our data demonstrate that cytokine production by bone marrow-derived macrophages (BMMs) in response to L. pneumophila infection requires the TLR adapter protein MyD88 and is reduced in the absence of TLR2 but not in the absence of TLR4. Bacterial growth ex vivo in BMMs from MyD88-deficient mice was not enhanced compared to bacterial growth ex vivo in BMMs from heterozygous littermate controls. Wild-type mice were able to clear L. pneumophila from the lung, whereas respiratory infection of MyD88-deficient mice caused death that resulted from robust bacterial replication and dissemination. In contrast to an infection with virulent L. pneumophila, MyD88-deficient mice were able to clear infections with L. pneumophila dotA mutants, indicating that MyD88-independent responses in the lung are sufficient to clear bacteria that are unable to replicate intracellularly. In vivo growth of L. pneumophila was enhanced in the lungs of TLR2-deficient mice, which resulted in a delay in bacterial clearance. No significant differences were observed in the growth and clearance of L. pneumophila in the lungs of TLR4-deficient mice and heterozygous littermate control mice. Our data indicate that MyD88 is crucial for eliciting a protective innate immune response against virulent L. pneumophila and that TLR2 is one of the pattern recognition receptors involved in initiating this MyD88-dependent response.