The immune microenvironment of tumors can play a critical role in promoting or inhibiting tumor progression depending on the context. We present evidence that tumor-associated macrophages/microglia ...(TAMs) can promote tumor progression in the sonic hedgehog subgroup of medulloblastoma (SHH-MB). By combining longitudinal manganese-enhanced magnetic resonance imaging (MEMRI) and immune profiling of a sporadic mouse model of SHH-MB, we found the density of TAMs is higher in the ~50% of tumors that progress to lethal disease. Furthermore, reducing regulatory T cells or eliminating B and T cells in Rag1 mutants does not alter SHH-MB tumor progression. As TAMs are a dominant immune component in tumors and are normally dependent on colony-stimulating factor 1 receptor (CSF1R), we treated mice with a CSF1R inhibitor, PLX5622. Significantly, PLX5622 reduces a subset of TAMs, prolongs mouse survival, and reduces the volume of most tumors within 4 weeks of treatment. Moreover, concomitant with a reduction in TAMs the percentage of infiltrating cytotoxic T cells is increased, indicating a change in the tumor environment. Our studies in an immunocompetent preclinical mouse model demonstrate TAMs can have a functional role in promoting SHH-MB progression. Thus, CSF1R inhibition could have therapeutic potential for a subset of SHH-MB patients.
Phagocytosis is a fundamental cellular process that is pivotal for immunity as it coordinates microbial killing, innate immune activation and antigen presentation. An essential step in this process ...is phagosome acidification, which regulates many functions of these organelles that allow phagosomes to participate in processes that are essential to both innate and adaptive immunity. Here we report that acidification of phagosomes containing Gram-positive bacteria is regulated by the NLRP3 inflammasome and caspase-1. Active caspase-1 accumulates on phagosomes and acts locally to control the pH by modulating buffering by the NADPH oxidase NOX2. These data provide insight into a mechanism by which innate immune signals can modify cellular defenses and establish a new function for the NLRP3 inflammasome and caspase-1 in host defense.
Recent studies have demonstrated abundant transcription of a set of noncoding RNAs (ncRNAs) preferentially within tumors as opposed to normal tissue. Using an approach from statistical physics, we ...quantify global transcriptome-wide motif use for the first time, to our knowledge, in human and murine ncRNAs, determining that most have motif use consistent with the coding genome. However, an outlier subset of tumor-associated ncRNAs, typically of recent evolutionary origin, has motif use that is often indicative of pathogenassociated RNA. For instance, we show that the tumor-associated human repeat human satellite repeat II (HSATII) is enriched in motifs containing CpG dinucleotides in AU-rich contexts that most of the human genome and human adapted viruses have evolved to avoid. We demonstrate that a key subset of these ncRNAs functions as immunostimulatory “self-agonists” and directly activates cells of the mononuclear phagocytic system to produce proinflammatory cytokines. These ncRNAs arise from endogenous repetitive elements that are normally silenced, yet are often very highly expressed in cancers. We propose that the innate response in tumors may partially originate from direct interaction of immunogenic ncRNAs expressed in cancer cells with innate pattern recognition receptors, and thereby assign a previously unidentified danger-associated function to a set of dark matter repetitive elements. These findings potentially reconcile several observations concerning the role of ncRNA expression in cancers and their relationship to the tumor microenvironment.
Mononuclear phagocytes (MP) comprise monocytes, macrophages (MΦ) and dendritic cells (DC), including their lineage-committed progenitors, which together have an eminent role in health and disease. ...Lipid-based siRNA-mediated gene inactivation is an established approach to investigate gene function in MP cells. However, although there are few protocols dedicated for siRNA-mediated gene inactivation in primary human DC and MΦ, there are none available for primary human monocytes. Moreover, there is no available method to perform comparative studies of a siRNA-mediated gene silencing in primary monocytes and other MP cells. Here, we describe a protocol optimized for the lipid-based delivery of siRNA to perform gene silencing in primary human blood monocytes, which is applicable to DCs, and differs from the classical route of siRNA delivery into MΦs. Along with this protocol, we provide a comparative analysis of how monocytes, DC and MΦ are efficiently transfected with the target siRNA without affecting cell viability, resulting in strong gene knockdown efficiency, including the simultaneous inactivation of two genes. Moreover, siRNA delivery does not affect classical functions in MP such as differentiation, phagocytosis and migration, demonstrating that this protocol does not induce non-specific major alterations in these cells. As a proof-of-principle, a functional analysis of hematopoietic cell kinase (Hck) shows for the first time that this kinase regulates the protease-dependent migration mode in human monocytes. Collectively, this protocol enables efficient gene inactivation in primary MP, suggesting a wide spectrum of applications such as siRNA-based high-throughput screening, which could ultimately improve our knowledge about MP biology.
Transcriptional profiling using microarrays provides a unique opportunity to decipher host pathogen cross-talk on the global level. Here, for the first time, we have been able to investigate gene ...expression changes in both Mycobacterium tuberculosis, a major human pathogen, and its human host cells, macrophages and dendritic cells.
In addition to common responses, we could identify eukaryotic and microbial transcriptional signatures that are specific to the cell type involved in the infection process. In particular M. tuberculosis shows a marked stress response when inside dendritic cells, which is in accordance with the low permissivity of these specialized phagocytes to the tubercle bacillus and to other pathogens. In contrast, the mycobacterial transcriptome inside macrophages reflects that of replicating bacteria. On the host cell side, differential responses to infection in macrophages and dendritic cells were identified in genes involved in oxidative stress, intracellular vesicle trafficking and phagosome acidification.
This study provides the proof of principle that probing the host and the microbe transcriptomes simultaneously is a valuable means to accessing unique information on host pathogen interactions. Our results also underline the extraordinary plasticity of host cell and pathogen responses to infection, and provide a solid framework to further understand the complex mechanisms involved in immunity to M. tuberculosis and in mycobacterial adaptation to different intracellular environments.
BackgroundagenT-797 is an allogeneic, native invariant natural killer T (iNKT) cell therapy product currently in phase I clinical trials for cancer (heme and solid). iNKT cells are a distinct ...population of T cells that can recognize tumors via direct recognition of CD1d (an MHC-I like molecule presenting glycolipids) through the TCR or recognition of NK cell receptor ligands via various NK receptors. We developed agenT-797 from isolated and ex-vivo expanded peripheral blood iNKT cells. Here we describe in vivo xenograft models to demonstrate the overall tissue distribution, tumor infiltration and efficacy of agenT-797 in liquid as well as solid tumors.MethodsWe utilized NOG-hIL15 (human IL-15) transgenic mice to ensure persistence/maintenance of ex-vivo expanded human iNKT cells throughout the studies. For studying efficacy in liquid tumors, we used NALM6, an acute lymphoblastic leukemia (ALL) cell line and for solid tumors selected A375, a melanoma cell line. Both cell lines were engineered to overexpress CD1d. Upon injection of iNKT cells, tumor growth and iNKT cell tissue/tumor infiltration as well as phenotype were studied.ResultsInjection of iNKT cells in NALM6- engrafted NOG-hIL15 mice resulted in an overall reduction in leukemic burden as measured by luminescence-based imaging. Flow cytometric analysis revealed infiltration of iNKT cells at the site of leukemic expansion, namely blood, spleen, bone marrow and liver. Cells were activated when reaching the site of the tumor. In addition, iNKT cells produced IFNγ and TNFα and low levels of IL-13/IL-4, consistent with a Th1 response. When iNKT cells were injected into A375 engrafted mice we observed infiltration of iNKT cells into the tumor, where they become activated and proliferate overtime. We observed an overall reduction in tumor size when iNKT cells were injected compared to control group, demonstrating the impact of iNKT cells on tumor growth.ConclusionsWe established xenograft mouse models to address various biological questions around human iNKT cells as a cell therapy product. We have demonstrated homing and infiltration of iNKT cells at the site of tumor and relative proliferation and expansion. These models provide a suitable platform for in-vivo preclinical studies on agenT -797 in cancer.Ethics ApprovalAll procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards.
The C-type lectin dendritic cell-specific intercellular adhesion molecule-3 grabbing nonintegrin (DC-SIGN) mediates the innate immune recognition of microbial carbohydrates. We investigated the ...function of this molecule in the host response to pathogens in vivo, by generating mouse lines lacking the DC-SIGN homologues SIGNR1, SIGNR3, and SIGNR5. Resistance to Mycobacterium tuberculosis was impaired only in SIGNR3-deficient animals. SIGNR3 was expressed in lung phagocytes during infection, and interacted with M. tuberculosis bacilli and mycobacterial surface glycoconjugates to induce secretion of critical host defense inflammatory cytokines, including tumor necrosis factor (TNF). SIGNR3 signaling was dependent on an intracellular tyrosine-based motif and the tyrosine kinase Syk. Thus, the mouse DC-SIGN homologue SIGNR3 makes a unique contribution to protection of the host against a pulmonary bacterial pathogen.
Many vaccines, including those using recombinant antigen subunits, rely on adjuvant(s) to enhance the efficacy of the host immune responses. Among the few adjuvants clinically approved, QS-21, a ...saponin-based immunomodulatory molecule isolated from the tree bark of Quillaja saponaria (QS) is used in complex formulations in approved effective vaccines. High demand of the QS raw material as well as manufacturing scalability limitation has been barriers here. We report for the first-time successful plant cell culture production of QS-21 having structural, chemical, and biologic, properties similar to the bark extracted product. These data ensure QS-21 and related saponins are broadly available and accessible to drug developers.
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•Transformative method for manufacturing QS-21 vaccine adjuvant•Enhancing the sustainability of QS-21 vaccine adjuvant production•Bioequivalence of plant cell culture and conventional bark extract QS-21 vaccine adjuvants•Revolutionary in vitro platform to develop plant saponin-derived vaccine adjuvants
Natural product synthesis; Medical biochemistry; Cell; Phytochemistry; Bioactive plant product
Host defense against pathogens involves various receptors expressed in cells of the immune system. Upon pathogen recognition, these proteins mediate a plethora of effector functions, such as the ...secretion of key protective cytokines and other immune mediators. These receptors include C-type lectins (CTLs), which are increasingly being recognized as major players in the host response to microbes. One particular CTL, DCSIGN/CD209, recognizes conserved sugar motifs in a number of viruses, parasites and bacteria. In particular, we and others have shown that DC-SIGN plays an important part in the recognition by dendritic cells and macrophages of Mycobacterium tuberculosis, the causal agent of tuberculosis in humans. Using the mouse as a model
host for M. tuberculosis, we recently showed that the DC-SIGN homologue SIGNR3 mediates protection against the tubercle bacillus, possibly through secretion of the key cytokines interleukin 6 and tumor necrosis factor. Here, we summarize and discuss these findings and their implications for the design of future studies aiming to improve our understanding of the role of DC-SIGN and other C-type lectins in immunity to mycobacteria and other pathogens.
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