Cohesin-mediated 3D contacts tune enhancer-promoter regulation.

Enhancers are key drivers of gene regulation thought to act via 3D physical interactions with the promoters of their target genes. However, genome-wide depletions of architectural proteins such as cohesin result in only limited changes in gene expression, despite a loss of contact domains and loops. Consequently, the role of cohesin and 3D contacts in enhancer function remains debated. Here, we developed CRISPRi of regulatory elements upon degron operation (CRUDO), a novel approach to measure how changes in contact frequency impact enhancer effects on target genes by perturbing enhancers with CRISPRi and measuring gene expression in the presence or absence of cohesin. We systematically perturbed all 1,039 candidate enhancers near five cohesin-dependent genes and identified 34 enhancer-gene regulatory interactions. Of 26 regulatory interactions with sufficient statistical power to evaluate cohesin dependence, 18 show cohesin-dependent effects. A decrease in enhancer-promoter contact frequency upon removal of cohesin is frequently accompanied by a decrease in the regulatory effect of the enhancer on gene expression, consistent with a contact-based model for enhancer function. However, changes in contact frequency and regulatory effects on gene expression vary as a function of distance, with distal enhancers (e.g., >50Kb) experiencing much larger changes than proximal ones (e.g., <50Kb). Because most enhancers are located close to their target genes, these observations can explain how only a small subset of genes - those with strong distal enhancers - are sensitive to cohesin. Together, our results illuminate how 3D contacts, influenced by both cohesin and genomic distance, tune enhancer effects on gene expression.

Highlights from the 2023 International Meeting on the Molecular Biology of Hepatitis B virus.

Since its discovery in 1965, our understanding of the hepatitis B virus (HBV) replication cycle and host immune responses has increased markedly. In contrast, our knowledge of the molecular biology of hepatitis delta virus (HDV), which is associated with more severe liver disease, is less well understood. Despite the progress made, critical gaps remain in our knowledge of HBV and HDV replication and the mechanisms underlying viral persistence and evasion of host immunity. The International HBV Meeting is the leading annual scientific meeting for presenting the latest advances in HBV and HDV molecular virology, immunology, and epidemiology. In 2023, the annual scientific meeting was held in Kobe, Japan and this review summarises some of the advances presented at the Meeting and lists gaps in our knowledge that may facilitate the development of new therapies.

Revealing emergent magnetic charge in an antiferromagnet with diamond quantum magnetometry.

Whirling topological textures play a key role in exotic phases of magnetic materials and are promising for logic and memory applications. In antiferromagnets, these textures exhibit enhanced stability and faster dynamics with respect to their ferromagnetic counterparts, but they are also difficult to study due to their vanishing net magnetic moment. One technique that meets the demand of highly sensitive vectorial magnetic field sensing with negligible backaction is diamond quantum magnetometry. Here we show that an archetypal antiferromagnet-haematite-hosts a rich tapestry of monopolar, dipolar and quadrupolar emergent magnetic charge distributions. The direct read-out of the previously inaccessible vorticity of an antiferromagnetic spin texture provides the crucial connection to its magnetic charge through a duality relation. Our work defines a paradigmatic class of magnetic systems to explore two-dimensional monopolar physics, and highlights the transformative role that diamond quantum magnetometry could play in exploring emergent phenomena in quantum materials.

An encyclopedia of enhancer-gene regulatory interactions in the human genome.

Identifying transcriptional enhancers and their target genes is essential for understanding gene regulation and the impact of human genetic variation on disease1-6. Here we create and evaluate a resource of >13 million enhancer-gene regulatory interactions across 352 cell types and tissues, by integrating predictive models, measurements of chromatin state and 3D contacts, and largescale genetic perturbations generated by the ENCODE Consortium7. We first create a systematic benchmarking pipeline to compare predictive models, assembling a dataset of 10,411 elementgene pairs measured in CRISPR perturbation experiments, >30,000 fine-mapped eQTLs, and 569 fine-mapped GWAS variants linked to a likely causal gene. Using this framework, we develop a new predictive model, ENCODE-rE2G, that achieves state-of-the-art performance across multiple prediction tasks, demonstrating a strategy involving iterative perturbations and supervised machine learning to build increasingly accurate predictive models of enhancer regulation. Using the ENCODE-rE2G model, we build an encyclopedia of enhancer-gene regulatory interactions in the human genome, which reveals global properties of enhancer networks, identifies differences in the functions of genes that have more or less complex regulatory landscapes, and improves analyses to link noncoding variants to target genes and cell types for common, complex diseases. By interpreting the model, we find evidence that, beyond enhancer activity and 3D enhancer-promoter contacts, additional features guide enhancerpromoter communication including promoter class and enhancer-enhancer synergy. Altogether, these genome-wide maps of enhancer-gene regulatory interactions, benchmarking software, predictive models, and insights about enhancer function provide a valuable resource for future studies of gene regulation and human genetics.

Engineering immunosuppressive drug-resistant armored (IDRA) SARS-CoV-2 T cells for cell therapy.

Solid organ transplant (SOT) recipients receive immunosuppressive drugs (ISDs) and are susceptible to developing severe COVID-19. Here, we analyze the Spike-specific T-cell response after 3 doses of mRNA vaccine in a group of SOT patients (n = 136) treated with different ISDs. We demonstrate that a combination of a calcineurin inhibitor (CNI), mycophenolate mofetil (MMF), and prednisone (Pred) treatment regimen strongly suppressed the mRNA vaccine-induced Spike-specific cellular response. Such defects have clinical consequences because the magnitude of vaccine-induced Spike-specific T cells was directly proportional to the ability of SOT patients to rapidly clear SARS-CoV-2 after breakthrough infection. To then compensate for the T-cell defects induced by immunosuppressive treatment and to develop an alternative therapeutic strategy for SOT patients, we describe production of 6 distinct SARS-CoV-2 epitope-specific ISD-resistant T-cell receptor (TCR)-T cells engineered using the mRNA electroporation method with reactivity minimally affected by mutations occurring in Beta, Delta, Gamma, and Omicron variants. This strategy with transient expression characteristics marks an improvement in the immunotherapeutic field and provides an attractive and novel therapeutic possibility for immunosuppressed COVID-19 patients.

Imaging Nucleation and Propagation of Pinned Domains in Few-Layer Fe5-xGeTe2.

Engineering nontrivial spin textures in magnetic van der Waals materials is highly desirable for spintronic applications based on hybrid heterostructures. The recent observation of labyrinth and bubble domains in the near room-temperature ferromagnet Fe5-xGeTe2 down to a bilayer thickness was thus a significant advancement toward van der Waals-based many-body physics. However, the physical mechanism responsible for stabilizing these domains remains unclear and requires further investigation. Here, we combine cryogenic scanning diamond quantum magnetometry and field reversal techniques to elucidate the high-field propagation and nucleation of bubble domains in trilayer Fe5-xGeTe2. We provide evidence of pinning-induced nucleation of magnetic bubbles and further show an unexpectedly high layer-dependent coercive field. These measurements can be easily extended to a wide range of magnetic materials to provide valuable nanoscale insight into domain processes critical for spintronic applications.

Lytic efficiency of immunosuppressive drug-resistant armoured T cells against circulating HBV-related HCC in whole blood.

Recurrence of hepatitis B virus-related hepatocellular carcinoma (HBV-HCC) after liver transplant (LT) is mediated by circulating tumour cells (CTCs) and exacerbated by the immunosuppressants required to prevent graft rejection. To circumvent the effects of immunosuppressants, we developed immunosuppressive drug-resistant armoured HBV-specific T-cell receptor-redirected T cells (IDRA HBV-TCR). However, their ability to eliminate HBV-HCC circulating in the whole blood has never been tested, and whether their lytic efficacy is compatible with the number of adoptively transferred T cells in vivo has never been measured. Hence, we developed a microscopy-based assay to quantify CTCs in whole blood. The assay was then used to quantify the efficacy of IDRA HBV-TCRs to lyse free-floating HBV-HCC cells in the presence of Tacrolimus and Mycophenolate Mofetil (MMF). We demonstrated that a panel of antibodies (AFP, GPC3, Vimentin, pan-Cytokeratin, and CD45) specific for HCC tumour antigens and immune cells can effectively differentiate HCC-CTCs in whole blood. Through dose-titration experiments, we observed that in the presence of immunosuppressive drugs, a minimum of 20 000 IDRA HBV-TCR T cells/ml of whole blood is necessary to lyse ~63.5% of free-floating HBV-HCC cells within 16 hours. In conclusion, IDRA HBV-TCR T cells can lyse free-floating HBV-HCC cells in whole blood in the presence of Tacrolimus and MMF. The quantity of IDRA-HBV TCR T cells required can be achieved by the adoptive transfer of 5 × 106 IDRA-HBV TCR-T cells/kg, supporting the utilisation of IDRA HBV-TCR T cells to eliminate CTCs as prophylaxis against recurrence after LT.

Intrinsic giant magnetoresistance due to exchange-bias-type effects at the surface of single-crystalline NiS2 nanoflakes.

The coexistence of different properties in the same material often results in exciting physical effects. At low temperatures, the pyrite transition-metal disulphide NiS2 hosts both antiferromagnetic and weak ferromagnetic orders, along with surface metallicity dominating its electronic transport. The interplay between such a complex magnetic structure and surface-dominated conduction in NiS2, however, is still not understood. A possible reason for this limited understanding is that NiS2 has been available primarily in bulk single-crystal form, which makes it difficult to perform studies combining magnetometry and transport measurements with high spatial resolution. Here, NiS2 nanoflakes are produced via mechanical cleaving and exfoliation of NiS2 single crystals and their properties are studied on a local (micron-size) scale. Strongly field-asymmetric magnetotransport features are found at low temperatures, which resemble those of more complex magnetic thin film heterostructures. Using nitrogen vacancy magnetometry, these magnetotransport features are related to exchange-bias-type effects between ferromagnetic and antiferromagnetic regions forming near step edges at the nanoflake surface. Nanoflakes with bigger steps exhibit giant magnetoresistance, which suggests a strong influence of magnetic spin textures at the NiS2 surface on its electronic transport. These findings pave the way for the application of NiS2 nanoflakes in van der Waals heterostructures for low-temperature spintronics and superconducting spintronics.

Protocol to detect antigen-specific nasal-resident T cells in humans.

Specialized T cells are located in the nasal cavity and act as the first line of defense against respiratory viral infection. Here, we present a protocol for the detection and characterization of antigen-specific nasal-resident T cells. We detail steps for localized nasal swabbing to collect the nasal samples. We then describe IFN-γ ELISpot and an activation-induced marker assay to detect and characterize antigen-specific nasal-resident T cells. For complete details on the use and execution of this protocol, please refer to Lim et al. (2022).1.

A comparative characterization of SARS-CoV-2-specific T cells induced by mRNA or inactive virus COVID-19 vaccines.

Unlike mRNA vaccines based only on the spike protein, inactivated severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) vaccines should induce a diversified T cell response recognizing distinct structural proteins. Here, we perform a comparative analysis of SARS-CoV-2-specific T cells in healthy individuals following vaccination with inactivated SARS-CoV-2 or mRNA vaccines. Relative to spike mRNA vaccination, inactivated vaccines elicit a lower magnitude of spike-specific T cells, but the combination of membrane, nucleoprotein, and spike-specific T cell response is quantitatively comparable with the sole spike T cell response induced by mRNA vaccine, and they efficiently tolerate the mutations characterizing the Omicron lineage. However, this multi-protein-specific T cell response is not mediated by a coordinated CD4 and CD8 T cell expansion but by selective priming of CD4 T cells. These findings can help in understanding the role of CD4 and CD8 T cells in the efficacy of the different vaccines to control severe COVID-19 after Omicron infection.

SARS-CoV-2-specific T cells in the changing landscape of the COVID-19 pandemic.

Since the onset of the coronavirus disease 2019 (COVID-19) pandemic, multiple severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants with increasing ability to evade neutralizing antibodies have emerged. Thus, earlier interest in defining the correlates of protection from infection, mainly mediated by humoral immunity, has shifted to correlates of protection from disease, which require a more comprehensive analysis of both humoral and cellular immunity. In this review, we summarized the evidence that supports the role of SARS-CoV-2-specific T cells induced by infection, by vaccination or by their combination (defined as hybrid immunity) in disease protection. We then analyzed the different epidemiological and virological variables that can modify the magnitude, function, and anatomical localization of SARS-CoV-2-specific T cells and their influence in the possible ability of T cells to protect the host from severe COVID-19 development.

T cell immunity is key to the pandemic endgame: How to measure and monitor it.

As vaccine deployment improves the healthcare emergency status caused by the SARS-CoV-2 pandemic, we need reliable tools to evaluate the duration of protective immunity at a global scale. Seminal studies have demonstrated that while neutralizing antibodies can protect us from viral infection, T cell-mediated cellular immunity provides long-term protection from severe COVID-19, even in the case of emerging new variants of concern (VOC). Indeed, the emergence of VOCs, able to substantially escape antibodies generated by current vaccines, has made the analysis of correlates of humoral protection against infection obsolete. The focus should now shift towards immunological correlates of protection from disease based on quantification of cellular immunity. Despite this evidence, an assessment of T cell responses is still overlooked. This is largely due to technical challenges and lack of validated diagnostic tests. Here, we review the current state of the art of available tests to distinguish between SARS-CoV-2 antigen-specific Tcells and non-antigen specific T-cells. These assays range from the analysis of the T cell-receptor (TCR) diversity (i.e. Immunoseq and MHC tetramer staining) to the detection of functional T cell activation (i.e. ICS, AIM, Elispot, ELLA, dqTACT, etc.) either from purified Peripheral Blood Mononuclear Cells (PBMCs) or whole blood. We discuss advantages and disadvantages of each assay, proposing their ideal use for different scopes. Finally, we argue how it is paramount to deploy cheap, standardized, and scalable assays to measure T cell functionality to fill this critical diagnostic gap and manage these next years of the pandemic.

Act Early and at the Right Location: SARS-CoV-2 T Cell Kinetics and Tissue Localization.

The emergence of new SARS-CoV-2 lineages able to escape antibodies elicited by infection or vaccination based on the Spike protein of the Wuhan isolates has reduced the ability of Spike-specific antibodies to protect previously infected or vaccinated individuals from infection. Therefore, the role played by T cells in the containment of viral replication and spread after infection has taken a more central stage. In this brief review, we will discuss the role played by T cells in the protection from COVID-19, with a particular emphasis on the kinetics of the T cell response and its localization at the site of primary infection.

HBV-HCC treatment with mRNA electroporated HBV-TCR T cells.

Hepatocellular carcinoma is a significant global health challenge with steadily increasing incidence in the East Asia region. While both Hepatitis C and B virus infections account for the majority of HCC cases, the advent of potent antivirals against HCV infection has biased the aetiology towards chronic HBV infection that at the moment remains without an effective cure. For this reason, HBV-HCC remains a persistent global problem. Treatment options for intermediate to advanced stages of HBV-HCC remain limited, hence novel therapeutic strategies are required to fulfil this medical need. Following the considerable success of adoptive T-cell immunotherapy against B-cell malignancies, it is conceivable to envision whether the same could be achieved against HBV-HCC. In this review, we describe the development of T-cell therapy strategies for HBV-HCC and discuss the safety and the efficacy of the strategies in terms of the direct killing of tumour cells and the other alterations possibly induced by the action of the T cells.

SARS-CoV-2 breakthrough infection in vaccinees induces virus-specific nasal-resident CD8+ and CD4+ T cells of broad specificity.

Rapid recognition of SARS-CoV-2-infected cells by resident T cells in the upper airway might provide an important layer of protection against COVID-19. Whether parenteral SARS-CoV-2 vaccination or infection induces nasal-resident T cells specific for distinct SARS-CoV-2 proteins is unknown. We isolated T cells from the nasal mucosa of COVID-19 vaccinees who either experienced SARS-CoV-2 infection after vaccination (n = 34) or not (n = 16) and analyzed their phenotype, SARS-CoV-2 specificity, function, and persistence. Nasal-resident SARS-CoV-2-specific CD8+ and CD4+ T cells were detected almost exclusively in vaccinees who experienced SARS-CoV-2 breakthrough infection. Importantly, the Spike-specific T cells primed by vaccination did not suppress the induction of T cells specific for other SARS-CoV-2 proteins. The nasal-resident T cell responses persisted for ≥140 d, with minimal sign of waning. These data highlight the importance of viral nasal challenge in the formation of SARS-CoV-2-specific antiviral immunity at the site of primary infection and further define the immunological features of SARS-CoV-2 hybrid immunity.

Rapid, scalable assessment of SARS-CoV-2 cellular immunity by whole-blood PCR.

Fast, high-throughput methods for measuring the level and duration of protective immune responses to SARS-CoV-2 are needed to anticipate the risk of breakthrough infections. Here we report the development of two quantitative PCR assays for SARS-CoV-2-specific T cell activation. The assays are rapid, internally normalized and probe-based: qTACT requires RNA extraction and dqTACT avoids sample preparation steps. Both assays rely on the quantification of CXCL10 messenger RNA, a chemokine whose expression is strongly correlated with activation of antigen-specific T cells. On restimulation of whole-blood cells with SARS-CoV-2 viral antigens, viral-specific T cells secrete IFN-γ, which stimulates monocytes to produce CXCL10. CXCL10 mRNA can thus serve as a proxy to quantify cellular immunity. Our assays may allow large-scale monitoring of the magnitude and duration of functional T cell immunity to SARS-CoV-2, thus helping to prioritize revaccination strategies in vulnerable populations.

Unveiling the Emergent Traits of Chiral Spin Textures in Magnetic Multilayers.

Magnetic skyrmions are topologically wound nanoscale textures of spins whose ambient stability and electrical manipulation in multilayer films have led to an explosion of research activities. While past efforts focused predominantly on isolated skyrmions, recently ensembles of chiral spin textures, consisting of skyrmions and magnetic stripes, are shown to possess rich interactions with potential for device applications. However, several fundamental aspects of chiral spin texture phenomenology remain to be elucidated, including their domain wall (DW) structure, thermodynamic stability, and morphological transitions. Here the evolution of these textural characteristics are unveiled on a tunable multilayer platform-wherein chiral interactions governing spin texture energetics can be widely varied-using a combination of full-field electron and soft X-ray microscopies with numerical simulations. With increasing chiral interactions, the emergence of Néel helicity, followed by a marked reduction in domain compressibility, and finally a transformation in the skyrmion formation mechanism are demonstrated. Together with an analytical model, these experiments establish a comprehensive microscopic framework for investigating and tailoring chiral spin texture character in multilayer films.