Boosting Natural Killer Cell-Mediated Targeting of Sarcoma Through DNAM-1 and NKG2D.

Sarcomas are malignancies of mesenchymal origin that occur in bone and soft tissues. Many are chemo- and radiotherapy resistant, thus conventional treatments fail to increase overall survival. Natural Killer (NK) cells exert anti-tumor activity upon detection of a complex array of tumor ligands, but this has not been thoroughly explored in the context of sarcoma immunotherapy. In this study, we investigated the NK cell receptor/ligand immune profile of primary human sarcoma explants. Analysis of tumors from 32 sarcoma patients identified the proliferative marker PCNA and DNAM-1 ligands CD112 and/or CD155 as commonly expressed antigens that could be efficiently targeted by genetically modified (GM) NK cells. Despite the strong expression of CD112 and CD155 on sarcoma cells, characterization of freshly dissociated sarcomas revealed a general decrease in tumor-infiltrating NK cells compared to the periphery, suggesting a defect in the endogenous NK cell response. We also applied a functional screening approach to identify relevant NK cell receptor/ligand interactions that induce efficient anti-tumor responses using a panel NK-92 cell lines GM to over-express 12 different activating receptors. Using GM NK-92 cells against primary sarcoma explants (n = 12) revealed that DNAM-1 over-expression on NK-92 cells led to efficient degranulation against all tested explants (n = 12). Additionally, NKG2D over-expression showed enhanced responses against 10 out of 12 explants. These results show that DNAM-1+ or NKG2D+ GM NK-92 cells may be an efficient approach in targeting sarcomas. The degranulation capacity of GM NK-92 cell lines was also tested against various established tumor cell lines, including neuroblastoma, Schwannoma, melanoma, myeloma, leukemia, prostate, pancreatic, colon, and lung cancer. Enhanced degranulation of DNAM-1+ or NKG2D+ GM NK-92 cells was observed against the majority of tumor cell lines tested. In conclusion, DNAM-1 or NKG2D over-expression elicited a dynamic increase in NK cell degranulation against all sarcoma explants and cancer cell lines tested, including those that failed to induce a notable response in WT NK-92 cells. These results support the broad therapeutic potential of DNAM-1+ or NKG2D+ GM NK-92 cells and GM human NK cells for the treatment of sarcomas and other malignancies.

Interactions between naïve and infected macrophages reduce Mycobacterium tuberculosis viability.

A high intracellular bacillary load of Mycobacterium tuberculosis in macrophages induces an atypical lysosomal cell death with early features of apoptosis that progress to necrosis within hours. Unlike classical apoptosis, this cell death mode does not appear to diminish M. tuberculosis viability. We previously reported that culturing heavily infected macrophages with naïve macrophages produced an antimicrobial effect, but only if naïve macrophages were added during the pre-necrotic phase of M. tuberculosis-induced cell death. In the present study we investigated the mechanism of antimicrobial activity in co-cultures, anticipating that efferocytosis of bacilli in apoptotic bodies would be required. Confocal microscopy revealed frustrated phagocytosis of M. tuberculosis-infected macrophages with no evidence that significant numbers of bacilli were transferred to the naïve macrophages. The antimicrobial effect of naïve macrophages was retained when they were separated from infected macrophages in transwells, and conditioned co-culture supernatants transferred antimicrobial activity to cultures of infected macrophages alone. Antimicrobial activity in macrophage co-cultures was abrogated when the naïve population was deficient in IL-1 receptor or when the infected population was deficient in inducible nitric oxide synthase. The participation of nitric oxide suggested a conventional antimicrobial mechanism requiring delivery of bacilli to a late endosomal compartment. Using macrophages expressing GFP-LC3 we observed the induction of autophagy specifically by a high intracellular load of M. tuberculosis. Bacilli were identified in LC3-positive compartments and LC3-positive compartments were confirmed to be acidified and LAMP1 positive. Thus, the antimicrobial effect of naïve macrophages acting on M. tuberculosis in heavily-infected macrophages is contact-independent. Interleukin-1 provides an afferent signal that induces an as yet unidentified small molecule which promotes nitric oxide-dependent antimicrobial activity against bacilli in autolysosomes of heavily infected macrophages. This cooperative, innate antimicrobial interaction may limit the maximal growth rate of M. tuberculosis prior to the expression of adaptive immunity in pulmonary tuberculosis.

Autophagy controls IL-1beta secretion by targeting pro-IL-1beta for degradation.

Autophagy is a key regulator of cellular homeostasis that can be activated by pathogen-associated molecules and recently has been shown to influence IL-1ß secretion by macrophages. However, the mechanisms behind this are unclear. Here, we describe a novel role for autophagy in regulating the production of IL-1ß in antigen-presenting cells. After treatment of macrophages with Toll-like receptor ligands, pro-IL-1ß was specifically sequestered into autophagosomes, whereas further activation of autophagy with rapamycin induced the degradation of pro-IL-1ß and blocked secretion of the mature cytokine. Inhibition of autophagy promoted the processing and secretion of IL-1ß by antigen-presenting cells in an NLRP3- and TRIF-dependent manner. This effect was reduced by inhibition of reactive oxygen species but was independent of NOX2. Induction of autophagy in mice in vivo with rapamycin reduced serum levels of IL-1ß in response to challenge with LPS. These data demonstrate that autophagy controls the production of IL-1ß through at least two separate mechanisms: by targeting pro-IL-1ß for lysosomal degradation and by regulating activation of the NLRP3 inflammasome.

Macrophage apoptosis in tuberculosis.

Mycobacterium tuberculosis (Mtb) is an intracellular pathogen that infects alveolar macrophages following aerosol transmission. Lung macrophages provide a critical intracellular niche that is required for Mtb to establish infection in the human host. This parasitic relationship is made possible by the capacity of Mtb to block phagosome maturation following entry into the host macrophage, creating an environment that supports bacillary replication. Apoptosis is increasingly understood to play a role in host defense against intracellular pathogens including viruses, fungi, protozoa and bacteria. In the last 15 years an understanding of the role that macrophage apoptosis plays in TB has begun to emerge. Here we review the history and current state of the art of this topic and we offer a model of the macrophage-pathogen interaction that takes into the account the complexities of programmed cell death and the relationship between various death signaling pathways and host defense in TB.

High-affinity ligand binding by wild-type/mutant heteromeric complexes of the mannose 6-phosphate/insulin-like growth factor II receptor.

The mannose 6-phosphate/insulin-like growth factor II receptor has diverse ligand-binding properties contributing to its roles in lysosome biogenesis and growth suppression. Optimal receptor binding and internalization of mannose 6-phosphate (Man-6-P)-bearing ligands requires a dimeric structure leading to bivalent high-affinity binding, presumably mediated by cooperation between sites on both subunits. Insulin-like growth factor II (IGF-II) binds to a single site on each monomer. It is hypothesized that IGF-II binding to cognate sites on each monomer occurs independently, but bivalent Man-6-P ligand binding requires cooperative contributions from sites on both monomers. To test this hypothesis, we co-immunoprecipitated differentially epitope-tagged soluble mini-receptors and assessed ligand binding. Pairing of wild-type and point-mutated IGF-II binding sites between two dimerized mini-receptors had no effect on the function of the contralateral binding site, indicating IGF-II binding to each side of the dimer is independent and manifests no intersubunit effects. As expected, heterodimeric receptors composed of a wild-type monomer and a mutant bearing two Man-6-P-binding knockout mutations form functional IGF-II binding sites. By contrast to prediction, such heterodimeric receptors also bind Man-6-P-based ligands with high affinity, and the amount of binding can be attributed entirely to the immunoprecipitated wild-type receptors. Anchoring of both C-terminal ends of the heterodimer produces optimal binding of both IGF-II and Man-6-P ligands. Thus, IGF-II binds independently to both subunits of the dimeric mannose 6-phosphate/insulin-like growth factor II receptor. Although wild-type/mutant hetero-oligomers form readily when mixed, it appears that multivalent Man-6-P ligands bind preferentially to wild-type sites, possibly by cross-bridging receptors within clusters of immobilized receptors.

A functional gene array for detection of bacterial virulence elements.

Emerging known and unknown pathogens create profound threats to public health. Platforms for rapid detection and characterization of microbial agents are critically needed to prevent and respond to disease outbreaks. Available detection technologies cannot provide broad functional information about known or novel organisms. As a step toward developing such a system, we have produced and tested a series of high-density functional gene arrays to detect elements of virulence and antibiotic resistance mechanisms. Our first generation array targets genes from Escherichia coli strains K12 and CFT073, Enterococcus faecalis and Staphylococcus aureus. We determined optimal probe design parameters for gene family detection and discrimination. When tested with organisms at varying phylogenetic distances from the four target strains, the array detected orthologs for the majority of targeted gene families present in bacteria belonging to the same taxonomic family. In combination with whole-genome amplification, the array detects femtogram concentrations of purified DNA, either spiked in to an aerosol sample background, or in combinations from one or more of the four target organisms. This is the first report of a high density NimbleGen microarray system targeting microbial antibiotic resistance and virulence mechanisms. By targeting virulence gene families as well as genes unique to specific biothreat agents, these arrays will provide important data about the pathogenic potential and drug resistance profiles of unknown organisms in environmental samples.

Statistical challenges in the analysis of two-dimensional difference gel electrophoresis experiments using DeCyder.

MOTIVATION: The DeCyder software (GE Healthcare) is the current state-of-the-art commercial product for the analysis of two-dimensional difference gel electrophoresis (2D DIGE) experiments. Analyses complementing DeCyder are suggested by incorporating recent advances from the microarray data analysis literature. A case study on the effect of smallpox vaccination is used to compare the results obtained from DeCyder with the results obtained by applying moderated t-tests adjusted for multiple comparisons to DeCyder output data that was additionally normalized. RESULTS: Application of the more stringent statistical tests applied to the normalized 2D DIGE data decreased the number of potentially differentially expressed proteins from the number obtained from DeCyder and increased the confidence in detecting differential expression in human clinical studies.

Loss of compartmentalization causes misregulation of lysine biosynthesis in peroxisome-deficient yeast cells.

To characterize the metabolic role of peroxisomes in yeast cells under physiological conditions, we performed a comprehensive meta-analysis of published microarray data. Previous studies of yeast peroxisomes have mainly been focused on the function of peroxisomes under extreme conditions, such as growth on oleate or methanol as the sole carbon source, and may therefore not be representative of the normal physiological role of yeast peroxisomes. Surprisingly, our analysis of the microarray data reveals that the only pathway responding to peroxisome deficiency in mid-log phase is lysine biosynthesis, whereas classical peroxisomal pathways such as beta-oxidation are unaffected. We show that the upregulation of lysine biosynthesis genes in peroxisome-deficient yeasts shares many characteristics with the physiological response to lysine starvation. We provide data that suggest that this is the result of a "pathological" stimulation of the Lys14p transcriptional activator by the pathway intermediate aminoadipate semialdehyde. Mistargeting of the peroxisomal lysine pathway to the cytosol increases the active concentration of aminoadipate semialdehyde, which is no longer contained in the peroxisome and can now activate Lys14p at much lower levels than in wild-type yeasts. This is the first well-documented example of pathway misregulation in response to peroxisome deficiency and will be useful in understanding the phenotypic details of human peroxisome-deficient patients (Zellweger syndrome).

The relationship between simian immunodeficiency virus RNA levels and the mRNA levels of alpha/beta interferons (IFN-alpha/beta) and IFN-alpha/beta-inducible Mx in lymphoid tissues of rhesus macaques during acute and chronic infection.

To define the role of alpha/beta interferons (IFN-alpha/beta) in simian immunodeficiency virus (SIV) infection, IFN-alpha and IFN-beta mRNA levels and mRNA levels of Mx, an antiviral effector molecule, were determined in lymphoid tissues of rhesus macaques infected with pathogenic SIV. IFN-alpha/beta responses were induced during the acute phase and persisted in various lymphoid tissues throughout the chronic phase of infection. IFN-alpha/beta responses were most consistent in tissues with high viral RNA levels; thus, IFN-alpha/beta responses were not generally associated with effective control of SIV replication. IFN-alpha/beta responses were differentially regulated in different lymphoid tissues and at different stages of infection. The most consistent IFN-alpha/beta responses in acute and chronic SIV infection were observed in peripheral lymph nodes. In the spleen, only a transient increase in IFN-alpha/beta mRNA levels during acute SIV infection was observed. Further, IFN-alpha and IFN-beta mRNA levels showed a tissue-specific expression pattern during the chronic, but not the acute, phase of infection. In the acute phase of infection, SIV RNA levels in lymphoid tissues of rhesus macaques correlated with mRNA levels of both IFN-alpha and IFN-beta, whereas during chronic SIV infection only increased IFN-alpha mRNA levels correlated with the level of virus replication in the same tissues. In lymphoid tissues of all SIV-infected monkeys, higher viral RNA levels were associated with increased Mx mRNA levels. We found no evidence that monkeys with increased Mx mRNA levels in lymphoid tissues had enhanced control of virus replication. In fact, Mx mRNA levels were associated with high viral RNA levels in lymphoid tissues of chronically infected animals.