bims-nimamd Biomed News
on Neuroimmunity and neuroinflammation in ageing and metabolic disease
Issue of 2022‒04‒03
forty-four papers selected by
Fawaz Alzaïd
Sorbonne Université

  1. Nat Commun. 2022 Mar 29. 13(1): 1669
      Prime editor (PE), which is developed by combining Cas9 nickase and an engineered reverse transcriptase, can mediate all twelve types of base substitutions and small insertions or deletions in living cells but its efficiency remains low. Here, we develop spegRNA by introducing same-sense mutations at proper positions in the reverse-transcription template of pegRNA to increase PE's base-editing efficiency up-to 4,976-fold (on-average 353-fold). We also develop apegRNA by altering the pegRNA secondary structure to increase PE's indel-editing efficiency up-to 10.6-fold (on-average 2.77-fold). The spegRNA and apegRNA can be combined to further enhance editing efficiency. When spegRNA and apegRNA are used in PE3 and PE5 systems, the efficiencies of sPE3, aPE3, sPE5 and aPE5 systems are all enhanced significantly. The strategies developed in this study realize highly efficient prime editing at certain previously uneditable sites.
  2. Nature. 2022 Mar 30.
      Polycomb repressive complexes 1 and 2 (PRC1 and PRC2) are histone-modifying and -binding complexes that mediate the formation of facultative heterochromatin and are required for silencing of developmental genes and maintenance of cell fate1-3. Multiple pathways of RNA decay work together to establish and maintain heterochromatin in fission yeast, including a recently identified role for a conserved RNA-degradation complex known as the rixosome or RIX1 complex4-6. Whether RNA degradation also has a role in the stability of mammalian heterochromatin remains unknown. Here we show that the rixosome contributes to silencing of many Polycomb targets in human cells. The rixosome associates with human PRC complexes and is enriched at promoters of Polycomb target genes. Depletion of either the rixosome or Polycomb results in accumulation of paused and elongating RNA polymerase at Polycomb target genes. We identify point mutations in the RING1B subunit of PRC1 that disrupt the interaction between PRC1 and the rixosome and result in diminished silencing, suggesting that direct recruitment of the rixosome to chromatin is required for silencing. Finally, we show that the RNA endonuclease and kinase activities of the rixosome and the downstream XRN2 exoribonuclease, which degrades RNAs with 5' monophosphate groups generated by the rixosome, are required for silencing. Our findings suggest that rixosomal degradation of nascent RNA is conserved from fission yeast to human, with a primary role in RNA degradation at facultative heterochromatin in human cells.
  3. Nat Commun. 2022 Mar 30. 13(1): 1702
      Global Genomic Repair (GGR) and Transcription-Coupled Repair (TCR) have been viewed, respectively, as major and minor sub-pathways of the nucleotide excision repair (NER) process that removes bulky lesions from the genome. Here we applied a next generation sequencing assay, CPD-seq, in E. coli to measure the levels of cyclobutane pyrimidine dimer (CPD) lesions before, during, and after UV-induced genotoxic stress, and, therefore, to determine the rate of genomic recovery by NER at a single nucleotide resolution. We find that active transcription is necessary for the repair of not only the template strand (TS), but also the non-template strand (NTS), and that the bulk of TCR is independent of Mfd - a DNA translocase that is thought to be necessary and sufficient for TCR in bacteria. We further show that repair of both TS and NTS is enhanced by increased readthrough past Rho-dependent terminators. We demonstrate that UV-induced genotoxic stress promotes global antitermination so that TCR is more accessible to the antisense, intergenic, and other low transcribed regions. Overall, our data suggest that GGR and TCR are essentially the same process required for complete repair of the bacterial genome.
  4. Nat Commun. 2022 03 28. 13(1): 1638
      COVID-19 pathogen SARS-CoV-2 has infected hundreds of millions and caused over 5 million deaths to date. Although multiple vaccines are available, breakthrough infections occur especially by emerging variants. Effective therapeutic options such as monoclonal antibodies (mAbs) are still critical. Here, we report the development, cryo-EM structures, and functional analyses of mAbs that potently neutralize SARS-CoV-2 variants of concern. By high-throughput single cell sequencing of B cells from spike receptor binding domain (RBD) immunized animals, we identify two highly potent SARS-CoV-2 neutralizing mAb clones that have single-digit nanomolar affinity and low-picomolar avidity, and generate a bispecific antibody. Lead antibodies show strong inhibitory activity against historical SARS-CoV-2 and several emerging variants of concern. We solve several cryo-EM structures at ~3 Å resolution of these neutralizing antibodies in complex with prefusion spike trimer ectodomain, and reveal distinct epitopes, binding patterns, and conformations. The lead clones also show potent efficacy in vivo against authentic SARS-CoV-2 in both prophylactic and therapeutic settings. We also generate and characterize a humanized antibody to facilitate translation and drug development. The humanized clone also has strong potency against both the original virus and the B.1.617.2 Delta variant. These mAbs expand the repertoire of therapeutics against SARS-CoV-2 and emerging variants.
  5. Nat Commun. 2022 Apr 01. 13(1): 1732
      Clathrin polymerization and changes in plasma membrane architecture are necessary steps in forming vesicles to internalize cargo during clathrin-mediated endocytosis (CME). Simultaneous analysis of clathrin dynamics and membrane structure is challenging due to the limited axial resolution of fluorescence microscopes and the heterogeneity of CME. This has fueled conflicting models of vesicle assembly and obscured the roles of flat clathrin assemblies. Here, using Simultaneous Two-wavelength Axial Ratiometry (STAR) microscopy, we bridge this critical knowledge gap by quantifying the nanoscale dynamics of clathrin-coat shape change during vesicle assembly. We find that de novo clathrin accumulations generate both flat and curved structures. High-throughput analysis reveals that the initiation of vesicle curvature does not directly correlate with clathrin accumulation. We show clathrin accumulation is preferentially simultaneous with curvature formation at shorter-lived clathrin-coated vesicles (CCVs), but favors a flat-to-curved transition at longer-lived CCVs. The broad spectrum of curvature initiation dynamics revealed by STAR microscopy supports multiple productive mechanisms of vesicle formation and advocates for the flexible model of CME.
  6. Nature. 2022 Mar 30.
    Keywords:  Epigenetics; Genetics; Molecular biology
  7. Nat Commun. 2022 Apr 01. 13(1): 1742
      Colorectal cancer (CRC) is among the most common malignancies with limited treatments other than surgery. The tumor microenvironment (TME) profiling enables the discovery of potential therapeutic targets. Here, we profile 54,103 cells from tumor and adjacent tissues to characterize cellular composition and elucidate the potential origin and regulation of tumor-enriched cell types in CRC. We demonstrate that the tumor-specific FAP+ fibroblasts and SPP1+ macrophages were positively correlated in 14 independent CRC cohorts containing 2550 samples and validate their close localization by immuno-fluorescent staining and spatial transcriptomics. This interaction might be regulated by chemerin, TGF-β, and interleukin-1, which would stimulate the formation of immune-excluded desmoplasic structure and limit the T cell infiltration. Furthermore, we find patients with high FAP or SPP1 expression achieved less therapeutic benefit from an anti-PD-L1 therapy cohort. Our results provide a potential therapeutic strategy by disrupting FAP+ fibroblasts and SPP1+ macrophages interaction to improve immunotherapy.
  8. Nat Commun. 2022 Mar 30. 13(1): 1680
      Categorical neural responses underlie various forms of selection and decision-making. Such binary-like responses promote robust signaling of the winner in the presence of input ambiguity and neural noise. Here, we show that a 'donut-like' inhibitory mechanism in which each competing option suppresses all options except itself, is highly effective at generating categorical neural responses. It surpasses motifs of feedback inhibition, recurrent excitation, and divisive normalization invoked frequently in decision-making models. We demonstrate experimentally not only that this mechanism operates in the midbrain spatial selection network in barn owls, but also that it is necessary for categorical signaling by it. The functional pattern of neural inhibition in the midbrain forms an exquisitely structured 'multi-holed' donut consistent with this network's combinatorial inhibitory function for stimulus selection. Additionally, modeling reveals a generalizable neural implementation of the donut-like motif for categorical selection. Self-sparing inhibition may, therefore, be a powerful circuit module central to categorization.
  9. Nat Commun. 2022 Mar 31. 13(1): 1698
      Combining single-cell cytometry datasets increases the analytical flexibility and the statistical power of data analyses. However, in many cases the full potential of co-analyses is not reached due to technical variance between data from different experimental batches. Here, we present cyCombine, a method to robustly integrate cytometry data from different batches, experiments, or even different experimental techniques, such as CITE-seq, flow cytometry, and mass cytometry. We demonstrate that cyCombine maintains the biological variance and the structure of the data, while minimizing the technical variance between datasets. cyCombine does not require technical replicates across datasets, and computation time scales linearly with the number of cells, allowing for integration of massive datasets. Robust, accurate, and scalable integration of cytometry data enables integration of multiple datasets for primary data analyses and the validation of results using public datasets.
  10. Nat Commun. 2022 Apr 01. 13(1): 1752
      Human Immunodeficiency Virus (HIV) relies on host molecular machinery for replication. Systematic attempts to genetically or biochemically define these host factors have yielded hundreds of candidates, but few have been functionally validated in primary cells. Here, we target 426 genes previously implicated in the HIV lifecycle through protein interaction studies for CRISPR-Cas9-mediated knock-out in primary human CD4+ T cells in order to systematically assess their functional roles in HIV replication. We achieve efficient knockout (>50% of alleles) in 364 of the targeted genes and identify 86 candidate host factors that alter HIV infection. 47 of these factors validate by multiplex gene editing in independent donors, including 23 factors with restrictive activity. Both gene editing efficiencies and HIV-1 phenotypes are highly concordant among independent donors. Importantly, over half of these factors have not been previously described to play a functional role in HIV replication, providing numerous novel avenues for understanding HIV biology. These data further suggest that host-pathogen protein-protein interaction datasets offer an enriched source of candidates for functional host factor discovery and provide an improved understanding of the mechanics of HIV replication in primary T cells.
  11. iScience. 2022 Apr 15. 25(4): 103907
      While microbial communities inhabit a wide variety of complex natural environments, in vitro culturing enables highly controlled conditions and high-throughput interrogation for generating mechanistic insights. In vitro assemblies of gut commensals have recently been introduced as models for the intestinal microbiota, which plays fundamental roles in host health. However, a protocol for 16S rRNA sequencing and analysis of in vitro samples that optimizes financial cost, time/effort, and accuracy/reproducibility has yet to be established. Here, we systematically identify protocol elements that have significant impact, introduce bias, and/or can be simplified. Our results indicate that community diversity and composition are generally unaffected by substantial protocol streamlining. Additionally, we demonstrate that a strictly aerobic halophile is an effective spike-in for estimating absolute abundances in communities of anaerobic gut commensals. This time- and money-saving protocol should accelerate discovery by increasing 16S rRNA data reliability and comparability and through the incorporation of absolute abundance estimates.
    Keywords:  Methodology in biological sciences; Microbiology
  12. Nat Commun. 2022 Mar 30. 13(1): 1676
      Running profoundly alters stimulus-response properties in mouse primary visual cortex (V1), but its effect in higher-order visual cortex is under-explored. Here we systematically investigate how visual responses vary with locomotive state across six visual areas and three cortical layers using a massive dataset from the Allen Brain Institute. Although previous work has shown running speed to be positively correlated with neural activity in V1, here we show that the sign of correlations between speed and neural activity varies across extra-striate cortex, and is even negative in anterior extra-striate cortex. Nevertheless, across all visual cortices, neural responses can be decoded more accurately during running than during stationary periods. We show that this effect is not attributable to changes in population activity structure, and propose that it instead arises from an increase in reliability of single-neuron responses during locomotion.
  13. Eur J Immunol. 2022 Mar 27.
      Progressive loss of effector functions, especially IFN-γ secreting capability, in effector memory CD8+ T (CD8+ TEM ) cells plays a crucial role in asthma worsening. However, the mechanisms of CD8+ TEM cell dysfunction remain elusive. Here, we report that S100A4 drives CD8+ TEM cell dysfunction, impairing their protective memory response and promoting asthma worsening in an ovalbumin (OVA)-induced asthmatic murine model. We find that CD8+ TEM cells contain two subsets based on S100A4 expression. S100A4+ subsets exhibit dysfunctional effector phenotypes with increased proliferative capability, whereas S100A4- subsets retain effector function but are more inclined to apoptosis, giving rise a dysfunctional CD8+ TEM cell pool. Mechanistically, S100A4 upregulation of mitochondrial metabolism results in a decrease of acetyl-CoA levels, which impair the transcription of effector genes, especially ifn-γ, facilitating cell survival, tolerance and memory potential. Our findings thus reveal general insights into how S100A4 CD8+ TEM cells reprogram into dysfunctional and less protective phenotypes to aggravate asthma. This article is protected by copyright. All rights reserved.
    Keywords:  IFN-γ; S100A4; allergic asthma; dysfunction; effector memory CD8+ T cells
  14. Nature. 2022 Mar;603(7903): S54-S56
    Keywords:  Diseases; Health care; Public health; Society
  15. Methods Mol Biol. 2022 ;2463 53-66
      Humanized mice, which we define as immunodeficient mice that have been reconstituted with a human immune system, represent promising preclinical models for translational research and precision medicine as they allow modeling and therapy of human diseases in vivo. The first generation of humanized mice showed insufficient development, diversity and function of human immune cells, in particular human natural killer (NK) cells and type 1 innate lymphoid cells (ILC1). This limited the applicability of humanized mice for studying ILC1 and NK cells in the context of human cancers and immunotherapeutic manipulation. However, since 2014, several next-generation humanized mouse models have been developed that express human IL-15 either as a transgene or knock-in (NOG-IL15, NSG-IL15, NSG-IL7-IL15, SRG-15) or show improved development of human myeloid cells, which express human IL-15 and thereby promote human NK cell development (NSG-SGM3, MISTRG, BRGSF). Here we compare the various next-generation humanized mouse models and describe the methodological procedures for creating mice with a functioning human immune system and how they can be used to study and manipulate human NK cells in health and disease.
    Keywords:  Cancer immunotherapy; Hematopoietic stem cells; Humanized mice; Immuno-oncology; Innate lymphoid cells; NK cells; Precision medicine; Preclinical model; Translational research
  16. J Immunol. 2022 Mar 28. pii: ji2100948. [Epub ahead of print]
      B cell differentiation is associated with substantial transcriptional, metabolic, and epigenetic remodeling, including redistribution of histone 3 lysine 27 trimethylation (H3K27me3), which is associated with a repressive chromatin state and gene silencing. Although the role of the methyltransferase EZH2 (Enhancer of zeste homolog 2) in B cell fate decisions has been well established, it is not known whether H3K27me3 demethylation is equally important. In this study, we showed that simultaneous genetic deletion of the two H3K27 demethylases UTX and JMJD3 (double-knockout [Utx fl/fl Jmjd3 fl/fl Cd19 cre/+] [dKO]) led to a significant increase in plasma cell (PC) formation after stimulation with the T cell-independent Ags LPS and NP-Ficoll. This phenotype occurred in a UTX-dependent manner as UTX single-knockout mice, but not JMJD3 single-knockout mice, mimicked the dKO. Although UTX- and JMJD3-deficient marginal zone B cells showed increased proliferation, dKO follicular B cells also showed increased PC formation. PCs from dKO mice upregulated genes associated with oxidative phosphorylation and exhibited increased spare respiratory capacity. Mechanistically, deletion of Utx and Jmjd3 resulted in higher levels of H3K27me3 at proapoptotic genes and resulted in reduced apoptosis of dKO PCs in vivo. Furthermore, UTX regulated chromatin accessibility at regions containing ETS and IFN regulatory factor (IRF) transcription factor family motifs, including motifs of known repressors of PC fate. Taken together, these data demonstrate that the H3K27me3 demethylases restrain B cell differentiation.
  17. Nature. 2022 Mar 31.
    Keywords:  Diabetes; Infection; SARS-CoV-2
  18. Nature. 2022 Mar 30.
      Decades of work have elucidated cytokine signalling and transcriptional pathways that control T cell differentiation and have led the way to targeted biologic therapies that are effective in a range of autoimmune, allergic and inflammatory diseases. Recent evidence indicates that obesity and metabolic disease can also influence the immune system1-7, although the mechanisms and effects on immunotherapy outcomes remain largely unknown. Here, using two models of atopic dermatitis, we show that lean and obese mice mount markedly different immune responses. Obesity converted the classical type 2 T helper (TH2)-predominant disease associated with atopic dermatitis to a more severe disease with prominent TH17 inflammation. We also observed divergent responses to biologic therapies targeting TH2 cytokines, which robustly protected lean mice but exacerbated disease in obese mice. Single-cell RNA sequencing coupled with genome-wide binding analyses revealed decreased activity of nuclear receptor peroxisome proliferator-activated receptor-γ (PPARγ) in TH2 cells from obese mice relative to lean mice. Conditional ablation of PPARγ in T cells revealed that PPARγ is required to focus the in vivo TH response towards a TH2-predominant state and prevent aberrant non-TH2 inflammation. Treatment of obese mice with a small-molecule PPARγ agonist limited development of TH17 pathology and unlocked therapeutic responsiveness to targeted anti-TH2 biologic therapies. These studies reveal the effects of obesity on immunological disease and suggest a precision medicine approach to target the immune dysregulation caused by obesity.
  19. Nat Commun. 2022 Mar 29. 13(1): 1671
      The pulmonary immune system consists of a network of tissue-resident cells as well as immune cells that are recruited to the lungs during infection and/or inflammation. How these immune components function during an acute poxvirus infection is not well understood. Intranasal infection of mice with vaccinia virus causes lethal pneumonia and systemic dissemination. Here we report that vaccinia C7 is a crucial virulence factor that blocks activation of the transcription factor IRF3. We provide evidence that type II alveolar epithelial cells (AECIIs) respond to pulmonary infection of vaccinia virus by inducing IFN-β and IFN-stimulated genes via the activation of the MDA5 and STING-mediated nucleic acid-sensing pathways and the type I IFN positive feedback loop. This leads to the recruitment and activation of CCR2+ inflammatory monocytes in the infected lungs and subsequent differentiation into Lyve1- interstitial macrophages (Lyve1- IMs), which efficiently engulf viral particles and block viral replication. Our results provide insights into how innate immune sensing of viral infection by lung AECIIs influences the activation and differentiation of CCR2+ inflammatory monocytes to defend against pulmonary poxvirus infection.
  20. J Immunol. 2022 Apr 01. pii: ji2101071. [Epub ahead of print]
      The germinal center (GC) response is essential for generating memory B and long-lived Ab-secreting plasma cells during the T cell-dependent immune response. In the GC, signals via the BCR and CD40 collaboratively promote the proliferation and positive selection of GC B cells expressing BCRs with high affinities for specific Ags. Although a complex gene transcriptional regulatory network is known to control the GC response, it remains elusive how the positive selection of GC B cells is modulated posttranscriptionally. In this study, we show that methyltransferase like 14 (Mettl14)-mediated methylation of adenosines at the position N 6 of mRNA (N 6-methyladenosine [m6A]) is essential for the GC B cell response in mice. Ablation of Mettl14 in B cells leads to compromised GC B cell proliferation and a defective Ab response. Interestingly, we unravel that Mettl14-mediated m6A regulates the expression of genes critical for positive selection and cell cycle regulation of GC B cells in a Ythdf2-dependent but Myc-independent manner. Furthermore, our study reveals that Mettl14-mediated m6A modification promotes mRNA decay of negative immune regulators, such as Lax1 and Tipe2, to upregulate genes requisite for GC B cell positive selection and proliferation. Thus, our findings suggest that Mettl14-mediated m6A modification plays an essential role in the GC B cell response.
  21. Nature. 2022 Mar 30.
      Mapping the spatial distribution and molecular identity of constituent cells is essential for understanding tissue dynamics in health and disease. We lack a comprehensive map of human distal airways, including the terminal and respiratory bronchioles (TRBs), which are implicated in respiratory diseases1-4. Here, using spatial transcriptomics and single-cell profiling of microdissected distal airways, we identify molecularly distinct TRB cell types that have not-to our knowledge-been previously characterized. These include airway-associated LGR5+ fibroblasts and TRB-specific alveolar type-0 (AT0) cells and TRB secretory cells (TRB-SCs). Connectome maps and organoid-based co-cultures reveal that LGR5+ fibroblasts form a signalling hub in the airway niche. AT0 cells and TRB-SCs are conserved in primates and emerge dynamically during human lung development. Using a non-human primate model of lung injury, together with human organoids and tissue specimens, we show that alveolar type-2 cells in regenerating lungs transiently acquire an AT0 state from which they can differentiate into either alveolar type-1 cells or TRB-SCs. This differentiation programme is distinct from that identified in the mouse lung5-7. Our study also reveals mechanisms that drive the differentiation of the bipotent AT0 cell state into normal or pathological states. In sum, our findings revise human lung cell maps and lineage trajectories, and implicate an epithelial transitional state in primate lung regeneration and disease.
  22. Nat Commun. 2022 Apr 01. 13(1): 1751
      The interaction between tumor suppressor BRCA2 and DSS1 is essential for RAD51 recruitment and repair of DNA double stand breaks (DSBs) by homologous recombination (HR). We have generated mice with a leucine to proline substitution at position 2431 of BRCA2, which disrupts this interaction. Although a significant number of mutant mice die during embryogenesis, some homozygous and hemizygous mutant mice undergo normal postnatal development. Despite lack of radiation induced RAD51 foci formation and a severe HR defect in somatic cells, mutant mice are fertile and exhibit normal RAD51 recruitment during meiosis. We hypothesize that the presence of homologous chromosomes in close proximity during early prophase I may compensate for the defect in BRCA2-DSS1 interaction. We show the restoration of RAD51 foci in mutant cells when Topoisomerase I inhibitor-induced single strand breaks are converted into DSBs during DNA replication. We also partially rescue the HR defect by tethering the donor DNA to the site of DSBs using streptavidin-fused Cas9. Our findings demonstrate that the BRCA2-DSS1 complex is dispensable for RAD51 loading when the homologous DNA is close to the DSB.
  23. Nature. 2022 Mar 30.
    Keywords:  Immunology; Medical research; Public health; SARS-CoV-2
  24. Nat Commun. 2022 Mar 29. 13(1): 1652
      Obesity is the major driver of the global epidemic in type 2 diabetes (T2D). In individuals with obesity, impaired insulin action leads to increased lipolysis in adipocytes, resulting in elevated plasma free fatty acid (FFA) levels that promote peripheral insulin resistance, a hallmark of T2D. Here we show, by using a combined genetic/biochemical/pharmacologic approach, that increased adipocyte lipolysis can be prevented by selective activation of adipocyte Gq signaling in vitro and in vivo (in mice). Activation of this pathway by a Gq-coupled designer receptor or by an agonist acting on an endogenous adipocyte Gq-coupled receptor (CysLT2 receptor) greatly improved glucose and lipid homeostasis in obese mice or in mice with adipocyte insulin receptor deficiency. Our findings identify adipocyte Gq signaling as an essential regulator of whole-body glucose and lipid homeostasis and should inform the development of novel classes of GPCR-based antidiabetic drugs.
  25. Nat Commun. 2022 Mar 29. 13(1): 1665
      Receptor dimerization of urokinase-type plasminogen activator receptor (uPAR) was previously identified at protein level and on cell surface. Recently, a dimeric form of mouse uPAR isoform 2 was proposed to induce kidney disease. Here, we report the crystal structure of human uPAR dimer at 2.96 Å. The structure reveals enormous conformational changes of the dimer compared to the monomeric structure: D1 of uPAR opens up into a large expanded ring that captures a β-hairpin loop of a neighboring uPAR to form an expanded β-sheet, leading to an elongated, highly intertwined dimeric uPAR. Based on the structure, we identify E49P as a mutation promoting dimer formation. The mutation increases receptor binding to the amino terminal fragment of its primary ligand uPA, induces the receptor to distribute to the basal membrane, promotes cell proliferation, and alters cell morphology via β1 integrin signaling. These results reveal the structural basis for uPAR dimerization, its effect on cellular functions, and provide a basis to further study this multifunctional receptor.
  26. Nature. 2022 Mar;603(7903): 795-796
    Keywords:  Biophysics; Fluid dynamics; Microbiology
  27. Nat Biomed Eng. 2022 Mar 28.
      The human immunodeficiency virus (HIV) integrates its genome into that of infected cells and may enter an inactive state of reversible latency that cannot be targeted using antiretroviral therapy. Sequencing such a provirus and the adjacent host junctions in individual cells may elucidate the mechanisms of the persistence of infected cells, but this is difficult owing to the 150-million-fold higher amount of background human DNA. Here we show that full-length proviruses connected to their contiguous HIV-host DNA junctions can be assembled via a high-throughput microfluidic assay where droplet-based whole-genome amplification of HIV DNA in its native context is followed by a polymerase chain reaction (PCR) to tag droplets containing proviruses for sequencing. We assayed infected cells from people with HIV receiving suppressive antiretroviral therapy, resulting in the detection and sequencing of paired proviral genomes and integration sites, 90% of which were not recovered by commonly used nested-PCR methods. The sequencing of individual proviral genomes with their integration sites could improve the genetic analysis of persistent HIV-infected cell reservoirs.
  28. Nature. 2022 Mar 29.
    Keywords:  Immunology; Medical research; SARS-CoV-2; Vaccines
  29. Nat Commun. 2022 Mar 29. 13(1): 1658
      The process of epigenetic silencing, while fundamentally important, is not yet completely understood. Here we report a replenishable female mouse embryonic stem cell (mESC) system, Xmas, that allows rapid assessment of X chromosome inactivation (XCI), the epigenetic silencing mechanism of one of the two X chromosomes that enables dosage compensation in female mammals. Through a targeted genetic screen in differentiating Xmas mESCs, we reveal that the BAF complex is required to create nucleosome-depleted regions at promoters on the inactive X chromosome during the earliest stages of establishment of XCI. Without this action gene silencing fails. Xmas mESCs provide a tractable model for screen-based approaches that enable the discovery of unknown facets of the female-specific process of XCI and epigenetic silencing more broadly.
  30. Nature. 2022 Mar 30.
      Transcription-coupled DNA repair (TCR) is presumed to be a minor sub-pathway of nucleotide excision repair (NER) in bacteria. Global genomic repair is thought to perform the bulk of repair independently of transcription. TCR is also believed to be mediated exclusively by Mfd-a DNA translocase of a marginal NER phenotype1-3. Here we combined in cellulo cross-linking mass spectrometry with structural, biochemical and genetic approaches to map the interactions within the TCR complex (TCRC) and to determine the actual sequence of events that leads to NER in vivo. We show that RNA polymerase (RNAP) serves as the primary sensor of DNA damage and acts as a platform for the recruitment of NER enzymes. UvrA and UvrD associate with RNAP continuously, forming a surveillance pre-TCRC. In response to DNA damage, pre-TCRC recruits a second UvrD monomer to form a helicase-competent UvrD dimer that promotes backtracking of the TCRC. The weakening of UvrD-RNAP interactions renders cells sensitive to genotoxic stress. TCRC then recruits a second UvrA molecule and UvrB to initiate the repair process. Contrary to the conventional view, we show that TCR accounts for the vast majority of chromosomal repair events; that is, TCR thoroughly dominates over global genomic repair. We also show that TCR is largely independent of Mfd. We propose that Mfd has an indirect role in this process: it participates in removing obstructive RNAPs in front of TCRCs and also in recovering TCRCs from backtracking after repair has been completed.
  31. Cancer Immunol Res. 2022 Apr 01. 10(4): 372-383
      Immune-checkpoint inhibitors (ICI), although revolutionary in improving long-term survival outcomes, are mostly effective in patients with immune-responsive tumors. Most patients with cancer either do not respond to ICIs at all or experience disease progression after an initial period of response. Treatment resistance to ICIs remains a major challenge and defines the biggest unmet medical need in oncology worldwide. In a collaborative workshop, thought leaders from academic, biopharma, and nonprofit sectors convened to outline a resistance framework to support and guide future immune-resistance research. Here, we explore the initial part of our effort by collating seminal discoveries through the lens of known biological processes. We highlight eight biological processes and refer to them as immune resistance nodes. We examine the seminal discoveries that define each immune resistance node and pose critical questions, which, if answered, would greatly expand our notion of immune resistance. Ultimately, the expansion and application of this work calls for the integration of multiomic high-dimensional analyses from patient-level data to produce a map of resistance phenotypes that can be utilized to guide effective drug development and improved patient outcomes.
  32. Nat Commun. 2022 Mar 29. 13(1): 1663
      Rho is a general transcription termination factor in bacteria, but many aspects of its mechanism of action are unclear. Diverse models have been proposed for the initial interaction between the RNA polymerase (RNAP) and Rho (catch-up and stand-by pre-terminational models); for the terminational release of the RNA transcript (RNA shearing, RNAP hyper-translocation or displacing, and allosteric models); and for the post-terminational outcome (whether the RNAP dissociates or remains bound to the DNA). Here, we use single-molecule fluorescence assays to study those three steps in transcription termination mediated by E. coli Rho. We find that different mechanisms previously proposed for each step co-exist, but apparently occur on various timescales and tend to lead to specific outcomes. Our results indicate that three kinetically distinct routes take place: (1) the catch-up mode leads first to RNA shearing for RNAP recycling on DNA, and (2) later to RNAP displacement for decomposition of the transcriptional complex; (3) the last termination usually follows the stand-by mode with displacing for decomposing. This three-route model would help reconcile current controversies on the mechanisms.
  33. J Clin Invest. 2022 Mar 29. pii: e154334. [Epub ahead of print]
      BACKGROUND: Responses to conventional donor lymphocyte infusion (DLI) for post-allogeneic hematopoietic cell transplantation (HCT) relapse are typically poor. Natural killer (NK) cell-based therapy is a promising modality to treat post-HCT relapse.METHODS: We initiated this ongoing phase I trial of adoptively transferred cytokine induced memory-like (CIML) NK cells in patients with myeloid malignancies relapsed after haploidentical HCT. All patients received a donor-derived NK cell dose of 5-10 million cells/kg after lymphodepleting chemotherapy, followed by systemic IL-2 for 7 doses. High resolution profiling with mass cytometry and single cell RNA sequencing characterized the expanding and persistent NK cell subpopulations in a longitudinal manner after infusion.
    RESULTS: In the first 6 enrolled patients on the trial, infusion of CIML NK cells led to a rapid 10 to 50-fold in vivo expansion that was sustained over months. The infusion was well-tolerated, with fever and pancytopenia as the most common adverse events. Expansion of NK cells was distinct from IL-2 effects on endogenous post-HCT NK cells, and not dependent on CMV viremia. Immunophenotypic and transcriptional profiling revealed a dynamic evolution of the activated CIML NK cell phenotype, superimposed on the natural variation in donor NK cell repertoires.
    CONCLUSION: Given their rapid expansion and long-term persistence in an immune compatible environment, CIML NK cells serve as a promising platform for the treatment of post-transplant relapse of myeloid disease. Further characterization of their unique in vivo biology and interaction with both T cells and tumor targets will lead to improvements in cell-based immunotherapies.
    TRIAL REGISTRATION: NCT04024761FUNDING. Supported by Dunkin Donuts Breakthrough Award, the NIH/National Cancer Institute R21 CA245413, the Leukemia and Lymphoma Society Scholar and TRP awards.
    Keywords:  Immunotherapy; NK cells; Stem cell transplantation; Stem cells; Transplantation
  34. Nat Commun. 2022 Mar 28. 13(1): 1643
      Disruption of mental functions in Alzheimer's disease (AD) and related disorders is accompanied by selective degeneration of brain regions. These regions comprise large-scale ensembles of cells organized into systems for mental functioning, however the relationship between clinical symptoms of dementia, patterns of neurodegeneration, and functional systems is not clear. Here we present a model of the association between dementia symptoms and degenerative brain anatomy using F18-fluorodeoxyglucose PET and dimensionality reduction techniques in two cohorts of patients with AD. This reflected a simple information processing-based functional description of macroscale brain anatomy which we link to AD physiology, functional networks, and mental abilities. We further apply the model to normal aging and seven degenerative diseases of mental functions. We propose a global information processing model for mental functions that links neuroanatomy, cognitive neuroscience and clinical neurology.
  35. Methods Mol Biol. 2022 ;2463 103-116
      Natural killer (NK) cells are innate lymphocytes that control tumors and microbial infections. Human NK cells are transcriptomically and phenotypically heterogeneous. The site where NK cells develop and reside determines their phenotype and effector functions. Our current knowledge about human NK cells is primarily from blood- and bone marrow-derived NK cells. The major limitation in formulating organ-specific clinical therapy is the knowledge gap on how tissue-resident NK cells develop, home, and function. Thus, it is crucial to define the transcriptomic profiles and the transcriptional regulation of tissue-resident NK cells. The major challenges in studying tissue-resident NK cells include their total number and the complexity of the tissue. Additionally, during isolation, keeping them viable and naïve without activation are challenging tasks. Here, we provide methods for isolating and performing transcriptomic analyses of NK cells at the individual cell level. Single-cell RNA sequencing provides a higher resolution of cellular heterogeneity and a better understanding of cell-cell interactions within the microenvironment. Using these methods, we can efficiently identify distinct populations of NK cells in tissues and define their unique transcriptomic profiles.
    Keywords:  Cell dissociation; NK cell development; Seurat/Single-R; Single-cell transcriptome; Tissue-resident NK cells
  36. Sci Immunol. 2022 Apr;7(70): eabq1730
      Platelet- and mast cell-derived serotonin metabolite 5-HIAA binding to GPR35 mediates neutrophil recruitment to acute inflammatory sites.
  37. Nat Commun. 2022 Mar 31. 13(1): 1704
      In Vaccinia virus (VACV), the prototype poxvirus, scaffold protein D13 forms a honeycomb-like lattice on the viral membrane that results in formation of the pleomorphic immature virion (IV). The structure of D13 is similar to those of major capsid proteins that readily form icosahedral capsids in nucleocytoplasmic large DNA viruses (NCLDVs). However, the detailed assembly mechanism of the nonicosahedral poxvirus scaffold has never been understood. Here we show the cryo-EM structures of the D13 trimer and scaffold intermediates produced in vitro. The structures reveal that the displacement of the short N-terminal α-helix is critical for initiation of D13 self-assembly. The continuous curvature of the IV is mediated by electrostatic interactions that induce torsion between trimers. The assembly mechanism explains the semiordered capsid-like arrangement of D13 that is distinct from icosahedral NCLDVs. Our structures explain how a single protein can self-assemble into different capsid morphologies and represent a local exception to the universal Caspar-Klug theory of quasi-equivalence.
  38. Immunity. 2022 Mar 25. pii: S1074-7613(22)00125-X. [Epub ahead of print]
      Lymph node (LN) stromal cells play a crucial role in LN development and in supporting adaptive immune responses. However, their origin, differentiation pathways, and transcriptional programs are still elusive. Here, we used lineage-tracing approaches and single-cell transcriptome analyses to determine origin, transcriptional profile, and composition of LN stromal and endothelial progenitors. Our results showed that all major stromal cell subsets and a large proportion of blood endothelial cells originate from embryonic Hoxb6+ progenitors of the lateral plate mesoderm (LPM), whereas lymphatic endothelial cells arise from Pax3+ progenitors of the paraxial mesoderm (PXM). Single-cell RNA sequencing revealed the existence of different Cd34+ and Cxcl13+ stromal cell subsets and showed that embryonic LNs contain proliferating progenitors possibly representing the amplifying populations for terminally differentiated cells. Taken together, our work identifies the earliest embryonic sources of LN stromal and endothelial cells and demonstrates that stromal diversity begins already during LN development.
    Keywords:  fibroblastic reticular cell; follicular dendritic cell; lineage tracing; lymph node development; marginal reticular cell; perivascular cell; single cell RNA-seq
  39. Nat Commun. 2022 Apr 01. 13(1): 1779
      Common variable immunodeficiency (CVID), the most prevalent symptomatic primary immunodeficiency, displays impaired terminal B-cell differentiation and defective antibody responses. Incomplete genetic penetrance and ample phenotypic expressivity in CVID suggest the participation of additional pathogenic mechanisms. Monozygotic (MZ) twins discordant for CVID are uniquely valuable for studying the contribution of epigenetics to the disease. Here, we generate a single-cell epigenomics and transcriptomics census of naïve-to-memory B cell differentiation in a CVID-discordant MZ twin pair. Our analysis identifies DNA methylation, chromatin accessibility and transcriptional defects in memory B-cells mirroring defective cell-cell communication upon activation. These findings are validated in a cohort of CVID patients and healthy donors. Our findings provide a comprehensive multi-omics map of alterations in naïve-to-memory B-cell transition in CVID and indicate links between the epigenome and immune cell cross-talk. Our resource, publicly available at the Human Cell Atlas, gives insight into future diagnosis and treatments of CVID patients.
  40. Science. 2022 Apr;376(6588): eabk3112
      Mobile elements and repetitive genomic regions are sources of lineage-specific genomic innovation and uniquely fingerprint individual genomes. Comprehensive analyses of such repeat elements, including those found in more complex regions of the genome, require a complete, linear genome assembly. We present a de novo repeat discovery and annotation of the T2T-CHM13 human reference genome. We identified previously unknown satellite arrays, expanded the catalog of variants and families for repeats and mobile elements, characterized classes of complex composite repeats, and located retroelement transduction events. We detected nascent transcription and delineated CpG methylation profiles to define the structure of transcriptionally active retroelements in humans, including those in centromeres. These data expand our insight into the diversity, distribution, and evolution of repetitive regions that have shaped the human genome.
  41. J Immunol. 2022 Apr 01. pii: ji2100794. [Epub ahead of print]
      Regulatory T cells (Tregs) are critical for regulating immunopathogenic responses in a variety of infections, including infection of mice with JHM strain of mouse hepatitis virus (JHMV), a neurotropic coronavirus that causes immune-mediated demyelinating disease. Although virus-specific Tregs are known to mitigate disease in this infection by suppressing pathogenic effector T cell responses of the same specificity, it is unclear whether these virus-specific Tregs form memory populations and persist similar to their conventional T cell counterparts of the same epitope specificity. Using congenically labeled JHMV-specific Tregs, we found that virus-specific Tregs persist long-term after murine infection, through at least 180 d postinfection and stably maintain Foxp3 expression. We additionally demonstrate that these cells are better able to proliferate and inhibit virus-specific T cell responses postinfection than naive Tregs of the same specificity, further suggesting that these cells differentiate into memory Tregs upon encountering cognate Ag. Taken together, these data suggest that virus-specific Tregs are able to persist long-term in the absence of viral Ag as memory Tregs.
  42. Cell Rep. 2022 Mar 29. pii: S2211-1247(22)00309-6. [Epub ahead of print]38(13): 110565
      Interleukin (IL)-10 is considered a prototypical anti-inflammatory cytokine, significantly contributing to the maintenance and reestablishment of immune homeostasis. Accordingly, it has been shown in the intestine that IL-10 produced by Tregs can act on effector T cells, thereby limiting inflammation. Herein, we investigate whether this role also applies to IL-10 produced by T cells during central nervous system (CNS) inflammation. During neuroinflammation, both CNS-resident and -infiltrating cells produce IL-10; yet, as IL-10 has a pleotropic function, the exact contribution of the different cellular sources is not fully understood. We find that T-cell-derived IL-10, but not other relevant IL-10 sources, can promote inflammation in experimental autoimmune encephalomyelitis. Furthermore, in the CNS, T-cell-derived IL-10 acts on effector T cells, promoting their survival and thereby enhancing inflammation and CNS autoimmunity. Our data indicate a pro-inflammatory role of T-cell-derived IL-10 in the CNS.
    Keywords:  CP: Immunology; CP: Neuroscience; T cells; autoimmunity; cell death; experimental autoimmune encephalomyelitis, EAE; inflammation; interleukin-10
  43. Nat Commun. 2022 Apr 01. 13(1): 1744
      Accurate descriptions of protein-protein interactions are essential for understanding biological systems. Remarkably accurate atomic structures have been recently computed for individual proteins by AlphaFold2 (AF2). Here, we demonstrate that the same neural network models from AF2 developed for single protein sequences can be adapted to predict the structures of multimeric protein complexes without retraining. In contrast to common approaches, our method, AF2Complex, does not require paired multiple sequence alignments. It achieves higher accuracy than some complex protein-protein docking strategies and provides a significant improvement over AF-Multimer, a development of AlphaFold for multimeric proteins. Moreover, we introduce metrics for predicting direct protein-protein interactions between arbitrary protein pairs and validate AF2Complex on some challenging benchmark sets and the E. coli proteome. Lastly, using the cytochrome c biogenesis system I as an example, we present high-confidence models of three sought-after assemblies formed by eight members of this system.
  44. Nat Commun. 2022 Mar 30. 13(1): 1675
      The epidemiology of infectious causes of meningitis in sub-Saharan Africa is not well understood, and a common cause of meningitis in this region, Mycobacterium tuberculosis (TB), is notoriously hard to diagnose. Here we show that integrating cerebrospinal fluid (CSF) metagenomic next-generation sequencing (mNGS) with a host gene expression-based machine learning classifier (MLC) enhances diagnostic accuracy for TB meningitis (TBM) and its mimics. 368 HIV-infected Ugandan adults with subacute meningitis were prospectively enrolled. Total RNA and DNA CSF mNGS libraries were sequenced to identify meningitis pathogens. In parallel, a CSF host transcriptomic MLC to distinguish between TBM and other infections was trained and then evaluated in a blinded fashion on an independent dataset. mNGS identifies an array of infectious TBM mimics (and co-infections), including emerging, treatable, and vaccine-preventable pathogens including Wesselsbron virus, Toxoplasma gondii, Streptococcus pneumoniae, Nocardia brasiliensis, measles virus and cytomegalovirus. By leveraging the specificity of mNGS and the sensitivity of an MLC created from CSF host transcriptomes, the combined assay has high sensitivity (88.9%) and specificity (86.7%) for the detection of TBM and its many mimics. Furthermore, we achieve comparable combined assay performance at sequencing depths more amenable to performing diagnostic mNGS in low resource settings.