DPP9 holds all the CARD8s for inflammasome regulation.

CARD8 senses pathogen-associated protease activity and assembles a pyroptosis-inducing inflammasome, but detailed regulatory mechanisms have remained elusive. In this issue of Immunity, Sharif et al. use cryo-EM and biochemical assays to unveil how DPP9 sequesters the inflammasome-forming C-terminal fragment of CARD8 to suppress its activation.

Immune Sensing of Synthetic, Bacterial, and Protozoan RNA by Toll-like Receptor 8 Requires Coordinated Processing by RNase T2 and RNase 2.

Human toll-like receptor 8 (TLR8) activation induces a potent T helper-1 (Th1) cell response critical for defense against intracellular pathogens, including protozoa. The receptor harbors two distinct binding sites, uridine and di- and/or trinucleotides, but the RNases upstream of TLR8 remain poorly characterized. We identified two endolysosomal endoribonucleases, RNase T2 and RNase 2, that act synergistically to release uridine from oligoribonucleotides. RNase T2 cleaves preferentially before, and RNase 2 after, uridines. Live bacteria, P. falciparum-infected red blood cells, purified pathogen RNA, and synthetic oligoribonucleotides all required RNase 2 and T2 processing to activate TLR8. Uridine supplementation restored RNA recognition in RNASE2-/- or RNASET2-/- but not RNASE2-/-RNASET2-/- cells. Primary immune cells from RNase T2-hypomorphic patients lacked a response to bacterial RNA but responded robustly to small-molecule TLR8 ligands. Our data identify an essential function of RNase T2 and RNase 2 upstream of TLR8 and provide insight into TLR8 activation.

Influenza A Infection Stimulates RIG-I and Enhances Effector Function of Primary Human NK Cells.

Immune surveillance by natural killer (NK) cells and their recruitment to sites of inflammation renders them susceptible to viral infection, potentially modulating their effector function. Here, we analyzed innate RNA receptor signaling in NK cells downstream of direct Influenza A virus (IAV) infection and its impact on NK cell effector function. Infection of NK cells with IAV resulted in the activation of TBK1, NF-Ï°B and subsequent type-I IFN secretion. CRISPR-generated knockouts in primary human NK cells revealed that this effect depended on the antiviral cytosolic RNA receptor RIG-I. Transfection of NK cells with synthetic 3p-dsRNA, a strong RIG-I agonist that mimics viral RNA, resulted in a similar phenotype and rendered NK cells resistant to subsequent IAV infection. Strikingly, both IAV infection and 3p-dsRNA transfection enhanced degranulation and cytokine production by NK cells when exposed to target cells. Thus, RIG-I activation in NK cells both supports their cell intrinsic viral defense and enhances their cytotoxic effector function against target cells.

Quantitative analysis of zero-field splitting parameter distributions in Gd(iii) complexes.

The magnetic properties of paramagnetic species with spin S > 1/2 are parameterized by the familiar g tensor as well as "zero-field splitting" (ZFS) terms that break the degeneracy between spin states even in the absence of a magnetic field. In this work, we determine the mean values and distributions of the ZFS parameters D and E for six Gd(iii) complexes (S = 7/2) and critically discuss the accuracy of such determination. EPR spectra of the Gd(iii) complexes were recorded in glassy frozen solutions at 10 K or below at Q-band (∼34 GHz), W-band (∼94 GHz) and G-band (240 GHz) frequencies, and simulated with two widely used models for the form of the distributions of the ZFS parameters D and E. We find that the form of the distribution of the ZFS parameter D is bimodal, consisting roughly of two Gaussians centered at D and -D with unequal amplitudes. The extracted values of D (σD) for the six complexes are, in MHz: Gd-NO3Pic, 485 ± 20 (155 ± 37); Gd-DOTA/Gd-maleimide-DOTA, -714 ± 43 (328 ± 99); iodo-(Gd-PyMTA)/MOMethynyl-(Gd-PyMTA), 1213 ± 60 (418 ± 141); Gd-TAHA, 1361 ± 69 (457 ± 178); iodo-Gd-PCTA-[12], 1861 ± 135 (467 ± 292); and Gd-PyDTTA, 1830 ± 105 (390 ± 242). The sign of D was adjusted based on the Gaussian component with larger amplitude. We relate the extracted P(D) distributions to the structure of the individual Gd(iii) complexes by fitting them to a model that superposes the contribution to the D tensor from each coordinating atom of the ligand. Using this model, we predict D, σD, and E values for several additional Gd(iii) complexes that were not measured in this work. The results of this paper may be useful as benchmarks for the verification of quantum chemical calculations of ZFS parameters, and point the way to designing Gd(iii) complexes for particular applications and estimating their magnetic properties a priori.

High-density preculture of PBMCs restores defective sensitivity of circulating CD8 T cells to virus- and tumor-derived antigens.

Peripheral blood mononuclear cells (PBMCs) are the only source of human lymphoid cells routinely available for immunomonitoring of T-cell responses to microbial and tumor-associated antigens. However, previous work in mice and humans had indicated that CD4 T cells transiently lose antigen sensitivity when cellular contacts are lost (eg, by entering the circulation). Using the simple and robust protocol for resetting T cells to original reactivity (RESTORE; ie, preculturing PBMCs for 2 days at a high cell density before initiation of antigenic stimulation), we show that CD8 T-cell responses to viral and tumor-associated antigens are greatly underestimated in blood, and sometimes even remain undetected, if conventional, unprocessed PBMC cultures are used. The latter finding is particularly striking with regard to the appearance of Wilms tumor 1 protein-specific CD8 T-cell responses in leukemia patients after allogeneic bone marrow transplantation. The dramatic increase in antigen sensitivity of "restored" CD8 T cells is associated with phosphorylation of proximal T-cell receptor signaling components, and with the upregulation of genes involved in aerobic glycolysis, thereby increasing T-cell functionality. The RESTORE protocol permits a more meaningful monitoring of CD8 memory T-cell responses to viral infections and tumors and vaccination success. Furthermore, when generating T-cell lines for adoptive T-cell therapy, it avoids the loss of those clones, which strictly depend on the primed status conferred by cellular interactions in the tissue context for their initial reactivation by antigen. The data reported in this article have been deposited in the Gene Expression Omnibus database (accession number GSE63430).

Minicircle-Based Engineering of Chimeric Antigen Receptor (CAR) T Cells.

Plasmid DNA is being used as a pharmaceutical agent in vaccination, as well as a basic substance and starting material in gene and cell therapy, and viral vector production. Since the uncontrolled expression of backbone sequences present in such plasmids and the dissemination of antibiotic resistance genes may have profound detrimental effects, an important goal in vector development was to produce supercoiled DNA lacking bacterial backbone sequences: Minicircle (MC) DNA. The Sleeping Beauty (SB) transposon system is a non-viral gene delivery platform enabling a close-to-random profile of genomic integration. In combination, the MC platform greatly enhances SB transposition and transgene integration resulting in higher numbers of stably modified target cells. We have recently developed a strategy for MC-based SB transposition of chimeric antigen receptor (CAR) transgenes that enable improved transposition rates compared to conventional plasmids and rapid manufacturing of therapeutic CAR T cell doses (Monjezi et al. 2016). This advance enables manufacturing CAR T cells in a virus-free process that relies on SB-mediated transposition from MC DNA to accomplish gene-transfer. Advantages of this approach include a strong safety profile due to the nature of the MC itself and the genomic insertion pattern of MC-derived CAR transposons. In addition, stable transposition and high-level CAR transgene expression, as well as easy and reproducible handling, make MCs a preferred vector source for gene-transfer in advanced cellular and gene therapy. In this chapter, we will review our experience in MC-based CAR T cell engineering and discuss our recent advances in MC manufacturing to accelerate both pre-clinical and clinical implementation.

EPR characterization of Mn(ii) complexes for distance determination with pulsed dipolar spectroscopy.

The four Mn(ii) complexes Mn-DOTA, Mn-TAHA, Mn-PyMTA, and Mn-NO3Py were characterized by electron paramagnetic resonance (EPR), electron-nuclear double resonance (ENDOR), and relaxation measurements, to predict their relative performance in the EPR pulse dipolar spectroscopy (PDS) experiments. High spin density localization on the metal ions was proven by ENDOR on 1H, D, 14N, and 55Mn nuclei. The transverse relaxation of the Mn(ii) complexes appears to be slow enough for PDS-based spin-spin distance determination. Rather advantageous ratios of T1/Tm were measured allowing for good relaxation induced dipolar modulation enhancement (RIDME) performance and, in general, fast shot repetitions in any PDS experiment. Relaxation properties of the Mn(ii) complexes correlate with the strengths of their zero field splitting (ZFS). Further, a comparison of Mn(ii)-DOTA and Gd(iii)-DOTA based spin labels is presented. The RIDME technique to measure nanometer-range Mn(ii)-Mn(ii) distances in biomolecules is discussed as an alternative to the well-known DEER technique that often appears challenging in cases of metal-metal distance measurements. The use of a modified kernel function that includes dipolar harmonic overtones allows model-free computation of the Mn(ii)-Mn(ii) distance distributions. Mn(ii)-Mn(ii) distances are computed from RIDME data of Mn-rulers consisting of two Mn-PyMTA complexes connected by a rodlike spacer of defined length. Level crossing effects seem to have only a weak influence on the distance distributions computed from this set of Mn(ii)-Mn(ii) RIDME data.

Increased IKKϵ protein stability ensures efficient type I interferon responses in conditions of TBK1 deficiency.

TBK1 and IKKϵ are related, crucial kinases in antiviral immune signaling pathways downstream of cytosolic nucleic acid receptors such as cGAS and RIG-I-like receptors. Upon activation, they phosphorylate the transcription factors IRF3 and IRF7 and thereby initiate the expression of type I interferons and antiviral effectors. While point mutation-induced loss of TBK1 kinase activity results in clinical hyper-susceptibility to viral infections, a complete lack of TBK1 expression in humans is unexpectedly not associated with diminished antiviral responses. Here, we provide a mechanistic explanation for these so-far unexplained observations by showing that TBK1 controls the protein expression of its related kinase IKKϵ in human myeloid cells. Mechanistically, TBK1 constitutively diminishes the protein stability of IKKϵ independent of TBK1 kinase activity but dependent on its interaction with the scaffold protein TANK. In consequence, depletion of TBK1 protein but not mutation-induced kinase deficiency induces the upregulation of IKKϵ. Due to the functional redundancy of the kinases in cGAS-STING and RIG-I-like receptor signaling in human myeloid cells, enhanced IKKϵ expression can compensate for the loss of TBK1. We show that IKKϵ upregulation is crucial to ensure unmitigated type I interferon production in conditions of TBK1 deficiency: While the type I interferon response to Listeria monocytogenes infection is maintained upon TBK1 loss, it is strongly diminished in cells harboring a kinase-deficient TBK1 variant, in which IKKϵ is not upregulated. Many pathogens induce TBK1 degradation, suggesting that loss of TBK1-mediated destabilization of IKKϵ is a critical backup mechanism to prevent diminished interferon responses upon TBK1 depletion.

3D Immuno-DNA Fluorescence In Situ Hybridization (FISH) for Detection of HIV-1 and Cellular Genes in Primary CD4+ T Cells.

Fluorescence in situ hybridization (FISH) is a powerful, broadly used microscopy-based technique that leverages fluorescently labeled nucleic acid probes to detect parts of the genome inside metaphase or interphase cell nuclei. In recent years, different methodologies developed to visualize genome topology and spatial relationships between genes have gained much attention as instruments to decode the relationship between chromatin structure and function. In addition to chromosome conformation capture-based techniques, highly multiplexed forms of FISH combined with high-throughput and super-resolution microscopy are used to map and spatially define contact frequencies between different genomic regions. All these approaches have strongly contributed to our knowledge of how the human genome is packed in the cell nucleus.In this chapter, we describe detailed step-by-step protocols for 3D immuno-DNA FISH detection of genes and Human immunodeficiency virus 1 (HIV-1) provirus in primary CD4+ T cells from healthy donors, or cells infected in vitro with the virus. Our multicolor 3D-FISH technique allows, by using up to three fluorophores, visualization of spatial positioning of loci inside a 3D cell nucleus.

An epigenetic GPI anchor defect impairs TLR4 signaling in the B cell transdifferentiation model for primary human monocytes BLaER1.

Antigen-presenting myeloid cells like monocytes detect invading pathogens via pattern recognition receptors (PRRs) and initiate adaptive and innate immune responses. As analysis of PRR signaling in primary human monocytes is hampered by their restricted expandability, human monocyte models like THP-1 cells are commonly used for loss-of-function studies, such as with CRISPR-Cas9 editing. A recently developed transdifferentiation cell culture system, BLaER1, enables lineage conversion from malignant B cells to monocytes and was found superior to THP-1 in mimicking PRR signaling, thus being the first model allowing TLR4 and inflammasome pathway analysis. Here, we identified an important caveat when investigating TLR4-driven signaling in BLaER1 cells. We show that this model contains glycosylphosphatidylinositol (GPI) anchor-deficient cells, which lack CD14 surface expression when differentiated to monocytes, resulting in diminished LPS/TLR4 but not TLR7/TLR8 responsiveness. This GPI anchor defect is caused by epigenetic silencing of PIGH, leading to a random distribution of intact and PIGH-deficient clones after single-cell cloning. Overexpressing PIGH restored GPI-anchored protein (including CD14) expression and LPS responsiveness. When studying CD14- or other GPI-anchored protein-dependent pathways, researchers should consider this anomaly and ensure equal GPI-anchored protein expression when comparing cells that have undergone single-cell cloning, e. g. after CRISPR-Cas9 editing.

MAPK-pathway inhibition mediates inflammatory reprogramming and sensitizes tumors to targeted activation of innate immunity sensor RIG-I.

Kinase inhibitors suppress the growth of oncogene driven cancer but also enforce the selection of treatment resistant cells that are thought to promote tumor relapse in patients. Here, we report transcriptomic and functional genomics analyses of cells and tumors within their microenvironment across different genotypes that persist during kinase inhibitor treatment. We uncover a conserved, MAPK/IRF1-mediated inflammatory response in tumors that undergo stemness- and senescence-associated reprogramming. In these tumor cells, activation of the innate immunity sensor RIG-I via its agonist IVT4, triggers an interferon and a pro-apoptotic response that synergize with concomitant kinase inhibition. In humanized lung cancer xenografts and a syngeneic Egfr-driven lung cancer model these effects translate into reduction of exhausted CD8+ T cells and robust tumor shrinkage. Overall, the mechanistic understanding of MAPK/IRF1-mediated intratumoral reprogramming may ultimately prolong the efficacy of targeted drugs in genetically defined cancer patients.

Spatially clustered loci with multiple enhancers are frequent targets of HIV-1 integration.

HIV-1 recurrently targets active genes and integrates in the proximity of the nuclear pore compartment in CD4+ T cells. However, the genomic features of these genes and the relevance of their transcriptional activity for HIV-1 integration have so far remained unclear. Here we show that recurrently targeted genes are proximal to super-enhancer genomic elements and that they cluster in specific spatial compartments of the T cell nucleus. We further show that these gene clusters acquire their location during the activation of T cells. The clustering of these genes along with their transcriptional activity are the major determinants of HIV-1 integration in T cells. Our results provide evidence of the relevance of the spatial compartmentalization of the genome for HIV-1 integration, thus further strengthening the role of nuclear architecture in viral infection.