Atopic dermatitis (AD) is a chronic itch and inflammatory disorder of the skin that affects one in ten people. Patients suffering from severe AD eventually progress to develop asthma and allergic ...rhinitis, in a process known as the “atopic march.” Signaling between epithelial cells and innate immune cells via the cytokine thymic stromal lymphopoietin (TSLP) is thought to drive AD and the atopic march. Here, we report that epithelial cells directly communicate to cutaneous sensory neurons via TSLP to promote itch. We identify the ORAI1/NFAT calcium signaling pathway as an essential regulator of TSLP release from keratinocytes, the primary epithelial cells of the skin. TSLP then acts directly on a subset of TRPA1-positive sensory neurons to trigger robust itch behaviors. Our results support a model whereby calcium-dependent TSLP release by keratinocytes activates both primary afferent neurons and immune cells to promote inflammatory responses in the skin and airways.
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•Epithelial cells communicate to sensory neurons via TSLP to promote itch•ORAI1/NFAT calcium signaling regulates release of TSLP from keratinocytes•TSLP is a robust pruritogen that promotes itch-evoked scratching•TSLP-evoked itch behaviors require TRPA1 ion channels that promote inflammation
The proinflammatory cytokine TSLP is a potent driver of dermatitis and is primarily thought to act through modulation of immune cells. TSLP is now found to directly activate sensory neurons via signaling involving ORAI1 and TRPA1 channels to trigger itch behaviors.
Spontaneous tumor-initiated T cell priming is dependent on IFN-β production by tumor-resident dendritic cells. On the basis of recent observations indicating that IFN-β expression was dependent upon ...activation of the host STING pathway, we hypothesized that direct engagement of STING through intratumoral (IT) administration of specific agonists would result in effective anti-tumor therapy. After proof-of-principle studies using the mouse STING agonist DMXAA showed a potent therapeutic effect, we generated synthetic cyclic dinucleotide (CDN) derivatives that activated all human STING alleles as well as murine STING. IT injection of STING agonists induced profound regression of established tumors in mice and generated substantial systemic immune responses capable of rejecting distant metastases and providing long-lived immunologic memory. Synthetic CDNs have high translational potential as a cancer therapeutic.
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•STING activation in the TME leads to a potent anti-tumor response in mouse tumor models•Modified CDNs that activate all hSTING alleles and mSTING have been generated•In mouse tumor models, ML RR-S2 CDA shows potent anti-tumor immune efficacy•Injection of one tumor induces regression of distant tumors and T cell memory
Endogenous STING pathway activation within the tumor induces spontaneous T cell priming. Corrales et al. generated synthetic cyclic dinucleotides that activate murine and all human STING alleles as a therapeutic. Intratumoral injection induced regression of established tumors and generated systemic immune responses, mediating rejection of distant metastases and providing immunologic memory.
The accumulation of DNA in the cytosol serves as a key immunostimulatory signal associated with infections, cancer and genomic damage
. Cytosolic DNA triggers immune responses by activating the ...cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) pathway
. The binding of DNA to cGAS activates its enzymatic activity, leading to the synthesis of a second messenger, cyclic guanosine monophosphate-adenosine monophosphate (2'3'-cGAMP)
. This cyclic dinucleotide (CDN) activates STING
, which in turn activates the transcription factors interferon regulatory factor 3 (IRF3) and nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB), promoting the transcription of genes encoding type I interferons and other cytokines and mediators that stimulate a broader immune response. Exogenous 2'3'-cGAMP produced by malignant cells
and other CDNs, including those produced by bacteria
and synthetic CDNs used in cancer immunotherapy
, must traverse the cell membrane to activate STING in target cells. How these charged CDNs pass through the lipid bilayer is unknown. Here we used a genome-wide CRISPR-interference screen to identify the reduced folate carrier SLC19A1, a folate-organic phosphate antiporter, as the major transporter of CDNs. Depleting SLC19A1 in human cells inhibits CDN uptake and functional responses, and overexpressing SLC19A1 increases both uptake and functional responses. In human cell lines and primary cells ex vivo, CDN uptake is inhibited by folates as well as two medications approved for treatment of inflammatory diseases, sulfasalazine and the antifolate methotrexate. The identification of SLC19A1 as the major transporter of CDNs into cells has implications for the immunotherapeutic treatment of cancer
, host responsiveness to CDN-producing pathogenic microorganisms
and-potentially-for some inflammatory diseases.
Interferon-induced proteins, including the largely uncharacterized interferon-induced tetratricopeptide repeat (IFIT) protein family, provide defenses against pathogens. Differing from expectations ...for tetratricopeptide repeat (TPR) proteins and from human IFIT1, IFIT2, and IFIT3, we show that human IFIT5 recognizes cellular RNA instead of protein partners. In vivo and in vitro, IFIT5 bound to endogenous 5′-phosphate-capped RNAs, including transfer RNAs. The crystal structure of IFIT5 revealed a convoluted intramolecular packing of eight TPRs as a fold that we name the TPR eddy. Additional, non-TPR structural elements contribute to an RNA binding cleft. Instead of general cytoplasmic distribution, IFIT5 concentrated in actin-rich protrusions from the apical cell surface colocalized with the RNA-binding retinoic acid-inducible gene-I (RIG-I). These findings establish compartmentalized cellular RNA binding activity as a mechanism for IFIT5 function and reveal the TPR eddy as a scaffold for RNA recognition.
► Human IFIT5 specifically and directly binds cellular RNAs, including transfer RNAs ► IFIT5 binding requires a 5′ monophosphate or polyphosphate cap ► IFIT5 folds as an intramolecular TPR eddy that scaffolds unique additional helices ► IFIT5 colocalizes with RIG-I to actin structures at the apical cell surface
Intratumoral (IT) STING activation results in tumor regression in preclinical models, yet factors dictating the balance between innate and adaptive anti-tumor immunity are unclear. Here, clinical ...candidate STING agonist ADU-S100 (S100) is used in an IT dosing regimen optimized for adaptive immunity to uncover requirements for a T cell-driven response compatible with checkpoint inhibitors (CPIs). In contrast to high-dose tumor ablative regimens that result in systemic S100 distribution, low-dose immunogenic regimens induce local activation of tumor-specific CD8+ effector T cells that are responsible for durable anti-tumor immunity and can be enhanced with CPIs. Both hematopoietic cell STING expression and signaling through IFNAR are required for tumor-specific T cell activation, and in the context of optimized T cell responses, TNFα is dispensable for tumor control. In a poorly immunogenic model, S100 combined with CPIs generates a survival benefit and durable protection. These results provide fundamental mechanistic insights into STING-induced anti-tumor immunity.
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•Dose of intratumoral STING agonist dictates expansion of tumor-specific T cells•Lower immunogenic dosing regimens elicit durable tumor control via CD8+ T cells•Higher tumor ablative dosing regimens compromise durable anti-tumor immunity•Immunogenic dosing is ideal for checkpoint inhibitor combination therapy
Intratumoral STING pathway activation is a promising therapeutic approach to treat cancer. While high doses of STING agonist are effective at clearing injected tumors, Sivick et al. find that lower doses of STING agonist are optimal for generating robust systemic tumor-specific T cell responses and durable anti-tumor immunity.
Realizing the full potential of human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs) requires efficient methods for genetic modification. However, techniques to generate ...cell type-specific lineage reporters, as well as reliable tools to disrupt, repair or overexpress genes by gene targeting, are inefficient at best and thus are not routinely used. Here we report the highly efficient targeting of three genes in human pluripotent cells using zinc-finger nuclease (ZFN)-mediated genome editing. First, using ZFNs specific for the OCT4 (POU5F1) locus, we generated OCT4-eGFP reporter cells to monitor the pluripotent state of hESCs. Second, we inserted a transgene into the AAVS1 locus to generate a robust drug-inducible overexpression system in hESCs. Finally, we targeted the PITX3 gene, demonstrating that ZFNs can be used to generate reporter cells by targeting non-expressed genes in hESCs and hiPSCs.
The development of zinc finger nucleases for targeted gene modification can benefit from rapid functional assays that directly quantify activity at the endogenous target. Here we describe a simple ...procedure for quantifying mutations that result from DNA double-strand break repair via non-homologous end joining. The assay is based on the ability of the Surveyor nuclease to selectively cleave distorted duplex DNA formed via cross-annealing of mutated and wild-type sequence.
Interferon (IFN) responses play key roles in cellular defense against pathogens. Highly expressed IFN-induced proteins with tetratricopeptide repeats (IFITs) are proposed to function as RNA binding ...proteins, but the RNA binding and discrimination specificities of IFIT proteins remain unclear. Here we show that human IFIT5 has comparable affinity for RNAs with diverse phosphate-containing 5′-ends, excluding the higher eukaryotic mRNA cap. Systematic mutagenesis revealed that sequence substitutions in IFIT5 can alternatively expand or introduce bias in protein binding to RNAs with 5′ monophosphate, triphosphate, cap0 (triphosphate-bridged N7-methylguanosine), or cap1 (cap0 with RNA 2′- O -methylation). We defined the breadth of cellular ligands for IFIT5 by using a thermostable group II intron reverse transcriptase for RNA sequencing. We show that IFIT5 binds precursor and processed tRNAs, as well as other RNA polymerase III transcripts. Our findings establish the RNA recognition specificity of the human innate immune response protein IFIT5.
Chromosomal translocations are signatures of numerous cancers and lead to expression of fusion genes that act as oncogenes. The wealth of genomic aberrations found in cancer, however, makes it ...challenging to assign a specific phenotypic change to a specific aberration. In this study, we set out to use genome editing with zinc finger (ZFN) and transcription activator-like effector (TALEN) nucleases to engineer, de novo, translocation-associated oncogenes at cognate endogenous loci in human cells. Using ZFNs and TALENs designed to cut precisely at relevant translocation breakpoints, we induced cancer-relevant t(11;22)(q24;q12) and t(2;5)(p23;q35) translocations found in Ewing sarcoma and anaplastic large cell lymphoma (ALCL), respectively. We recovered both translocations with high efficiency, resulting in the expression of the EWSR1-FLI1 and NPM1-ALK fusions. Breakpoint junctions recovered after ZFN cleavage in human embryonic stem (ES) cell-derived mesenchymal precursor cells fully recapitulated the genomic characteristics found in tumor cells from Ewing sarcoma patients. This approach with tailored nucleases demonstrates that expression of fusion genes found in cancer cells can be induced from the native promoter, allowing interrogation of both the underlying mechanisms and oncogenic consequences of tumor-related translocations in human cells. With an analogous strategy, the ALCL translocation was reverted in a patient cell line to restore the integrity of the two participating chromosomes, further expanding the repertoire of genomic rearrangements that can be engineered by tailored nucleases.
Agricultural biotechnology is limited by the inefficiencies of conventional random mutagenesis and transgenesis. Because targeted genome modification in plants has been intractable, plant trait ...engineering remains a laborious, time-consuming and unpredictable undertaking. Here we report a broadly applicable, versatile solution to this problem: the use of designed zinc-finger nucleases (ZFNs) that induce a double-stranded break at their target locus. We describe the use of ZFNs to modify endogenous loci in plants of the crop species Zea mays. We show that simultaneous expression of ZFNs and delivery of a simple heterologous donor molecule leads to precise targeted addition of an herbicide-tolerance gene at the intended locus in a significant number of isolated events. ZFN-modified maize plants faithfully transmit these genetic changes to the next generation. Insertional disruption of one target locus, IPK1, results in both herbicide tolerance and the expected alteration of the inositol phosphate profile in developing seeds. ZFNs can be used in any plant species amenable to DNA delivery; our results therefore establish a new strategy for plant genetic manipulation in basic science and agricultural applications.
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