Targeted reconstruction of T cell receptor sequence from single cell RNA-seq links CDR3 length to T cell differentiation state

Abstract The T cell compartment must contain diversity in both T cell receptor (TCR) repertoire and cell state to provide effective immunity against pathogens. However, it remains unclear how differences in the TCR contribute to heterogeneity in T cell state. Single cell RNA-sequencing (scRNA-seq) can allow simultaneous measurement of TCR sequence and global transcriptional profile from single cells. However, current methods for TCR inference from scRNA-seq are limited in their sensitivity and require long sequencing reads, thus increasing the cost and decreasing the number of cells that can be feasibly analyzed. Here we present TRAPeS, a publicly available tool that can efficiently extract TCR sequence information from short-read scRNA-seq libraries. We apply it to investigate heterogeneity in the CD8+ T cell response in humans and mice, and show that it is accurate and more sensitive than existing approaches. Coupling TRAPeS with transcriptome analysis of CD8+ T cells specific for a single epitope from Yellow Fever Virus (YFV), we show that the recently described ‘naive-like’ memory population have significantly longer CDR3 regions and greater divergence from germline sequence than do effector-memory phenotype cells. This suggests that TCR usage is associated with the differentiation state of the CD8+ T cell response to YFV

Antigen presentation deficiency, mesenchymal differentiation, and resistance to immunotherapy in the murine syngeneic CT2A tumor model

BackgroundThe GL261 and CT2A syngeneic tumor lines are frequently used as immunocompetent orthotopic mouse models of human glioblastoma (huGBM) but demonstrate distinct differences in their responses to immunotherapy.MethodsTo decipher the cell-intrinsic mechanisms that drive immunotherapy resistance in CT2A-luc and to define the aspects of human cancer biology that these lines can best model, we systematically compared their characteristics using whole exome and transcriptome sequencing, and protein analysis through immunohistochemistry, Western blot, flow cytometry, immunopeptidomics, and phosphopeptidomics.ResultsThe transcriptional profiles of GL261-luc2 and CT2A-luc tumors resembled those of some huGBMs, despite neither line sharing the essential genetic or histologic features of huGBM. Both models exhibited striking hypermutation, with clonal hotspot mutations in RAS genes (Kras p.G12C in GL261-luc2 and Nras p.Q61L in CT2A-luc). CT2A-luc distinctly displayed mesenchymal differentiation, upregulated angiogenesis, and multiple defects in antigen presentation machinery (e.g. Tap1 p.Y488C and Psmb8 p.A275P mutations) and interferon response pathways (e.g. copy number losses of loci including IFN genes and reduced phosphorylation of JAK/STAT pathway members). The defect in MHC class I expression could be overcome in CT2A-luc by interferon-γ treatment, which may underlie the modest efficacy of some immunotherapy combinations. Additionally, CT2A-luc demonstrated substantial baseline secretion of the CCL-2, CCL-5, and CCL-22 chemokines, which play important roles as myeloid chemoattractants.ConclusionAlthough the clinical contexts that can be modeled by GL261 and CT2A for huGBM are limited, CT2A may be an informative model of immunotherapy resistance due to its deficits in antigen presentation machinery and interferon response pathways

The epigenetic landscape of T cell exhaustion.

Exhausted T cells in cancer and chronic viral infection express distinctive patterns of genes, including sustained expression of programmed cell death protein 1 (PD-1). However, the regulation of gene expression in exhausted T cells is poorly understood. Here, we define the accessible chromatin landscape in exhausted CD8+ T cells and show that it is distinct from functional memory CD8+ T cells. Exhausted CD8+ T cells in humans and a mouse model of chronic viral infection acquire a state-specific epigenetic landscape organized into functional modules of enhancers. Genome editing shows that PD-1 expression is regulated in part by an exhaustion-specific enhancer that contains essential RAR, T-bet, and Sox3 motifs. Functional enhancer maps may offer targets for genome editing that alter gene expression preferentially in exhausted CD8+ T cells

Molecular definition of group 1 innate lymphoid cells in the mouse uterus

Determining the function of uterine lymphocytes is challenging because of the rapidly changing nature of the organ in response to sex hormones and, during pregnancy, to the invading fetal trophoblast cells. Here we provide the first genome-wide transcriptome atlas of mouse uterine group 1 innate lymphoid cells (g1 ILCs) at mid-gestation. The composition of g1 ILCs fluctuates throughout reproductive life, with Eomes-veCD49a+ ILC1s dominating before puberty and specifically expanding in second pregnancies, when the expression of CXCR6, a marker of memory cells, is upregulated. Tissue-resident Eomes+CD49a+ NK cells (trNK), which resemble human uterine NK cells, are most abundant during early pregnancy, and showcase gene signatures of responsiveness to TGF-β, connections with trophoblast, epithelial, endothelial and smooth muscle cells, leucocytes, as well as extracellular matrix. Unexpectedly, trNK cells express genes involved in anaerobic glycolysis, lipid metabolism, iron transport, protein ubiquitination, and recognition of microbial molecular patterns. Conventional NK cells expand late in gestation and may engage in crosstalk with trNK cells involving IL-18 and IFN-γ. These results identify trNK cells as the cellular hub of uterine g1 ILCs at mid-gestation and mark CXCR6+ ILC1s as potential memory cells of pregnancy.This work was funded by a Wellcome Trust Investigator Award 200841/Z/16/Z, the Centre for Trophoblast Research (CTR), and the Cambridge NIHR BRC Cell Phenotyping Hub to FC, the Associazione Italiana Ricerca per la Ricerca sul Cancro (AIRC) - Special Project 5x1000 no. 9962, AIRC IG 2017 Id.19920 and AIRC 2014 Id. 15283 to LM, and Ministero della Salute RF-2013, GR-2013-02356568 to PV. IF was funded by a CTR PhD fellowship

Young and vulnerable: Spatial-temporal trends and risk factors for infant mortality in rural South Africa (Agincourt), 1992-2007

<p>Abstract</p> <p>Background</p> <p>Infant mortality is an important indicator of population health in a country. It is associated with several health determinants, such as maternal health, access to high-quality health care, socioeconomic conditions, and public health policy and practices.</p> <p>Methods</p> <p>A spatial-temporal analysis was performed to assess changes in infant mortality patterns between 1992-2007 and to identify factors associated with infant mortality risk in the Agincourt sub-district, rural northeast South Africa. Period, sex, refugee status, maternal and fertility-related factors, household mortality experience, distance to nearest primary health care facility, and socio-economic status were examined as possible risk factors. All-cause and cause-specific mortality maps were developed to identify high risk areas within the study site. The analysis was carried out by fitting Bayesian hierarchical geostatistical negative binomial autoregressive models using Markov chain Monte Carlo simulation. Simulation-based Bayesian kriging was used to produce maps of all-cause and cause-specific mortality risk.</p> <p>Results</p> <p>Infant mortality increased significantly over the study period, largely due to the impact of the HIV epidemic. There was a high burden of neonatal mortality (especially perinatal) with several hot spots observed in close proximity to health facilities. Significant risk factors for all-cause infant mortality were mother's death in first year (most commonly due to HIV), death of previous sibling and increasing number of household deaths. Being born to a Mozambican mother posed a significant risk for infectious and parasitic deaths, particularly acute diarrhoea and malnutrition.</p> <p>Conclusions</p> <p>This study demonstrates the use of Bayesian geostatistical models in assessing risk factors and producing smooth maps of infant mortality risk in a health and socio-demographic surveillance system. Results showed marked geographical differences in mortality risk across a relatively small area. Prevention of vertical transmission of HIV and survival of mothers during the infants' first year in high prevalence villages needs to be urgently addressed, including expanded antenatal testing, prevention of mother-to-child transmission, and improved access to antiretroviral therapy. There is also need to assess and improve the capacity of district hospitals for emergency obstetric and newborn care. Persisting risk factors, including inadequate provision of clean water and sanitation, are yet to be fully addressed.</p

A Global Census of Fission Yeast Deubiquitinating Enzyme Localization and Interaction Networks Reveals Distinct Compartmentalization Profiles and Overlapping Functions in Endocytosis and Polarity

Proteomic, localization, and enzymatic activity screens in fission yeast reveal how deubiquitinating enzyme localization and function are tuned

The James Webb Space Telescope Mission

Twenty-six years ago a small committee report, building on earlier studies, expounded a compelling and poetic vision for the future of astronomy, calling for an infrared-optimized space telescope with an aperture of at least $4m$. With the support of their governments in the US, Europe, and Canada, 20,000 people realized that vision as the $6.5m$ James Webb Space Telescope. A generation of astronomers will celebrate their accomplishments for the life of the mission, potentially as long as 20 years, and beyond. This report and the scientific discoveries that follow are extended thank-you notes to the 20,000 team members. The telescope is working perfectly, with much better image quality than expected. In this and accompanying papers, we give a brief history, describe the observatory, outline its objectives and current observing program, and discuss the inventions and people who made it possible. We cite detailed reports on the design and the measured performance on orbit.Comment: Accepted by PASP for the special issue on The James Webb Space Telescope Overview, 29 pages, 4 figure

GWAS meta-analysis of intrahepatic cholestasis of pregnancy implicates multiple hepatic genes and regulatory elements

Intrahepatic cholestasis of pregnancy (ICP) is a pregnancy-specific liver disorder affecting 0.5–2% of pregnancies. The majority of cases present in the third trimester with pruritus, elevated serum bile acids and abnormal serum liver tests. ICP is associated with an increased risk of adverse outcomes, including spontaneous preterm birth and stillbirth. Whilst rare mutations affecting hepatobiliary transporters contribute to the aetiology of ICP, the role of common genetic variation in ICP has not been systematically characterised to date. Here, we perform genome-wide association studies (GWAS) and meta-analyses for ICP across three studies including 1138 cases and 153,642 controls. Eleven loci achieve genome-wide significance and have been further investigated and fine-mapped using functional genomics approaches. Our results pinpoint common sequence variation in liver-enriched genes and liver-specific cis-regulatory elements as contributing mechanisms to ICP susceptibility

Bi-allelic Loss-of-Function CACNA1B Mutations in Progressive Epilepsy-Dyskinesia.

The occurrence of non-epileptic hyperkinetic movements in the context of developmental epileptic encephalopathies is an increasingly recognized phenomenon. Identification of causative mutations provides an important insight into common pathogenic mechanisms that cause both seizures and abnormal motor control. We report bi-allelic loss-of-function CACNA1B variants in six children from three unrelated families whose affected members present with a complex and progressive neurological syndrome. All affected individuals presented with epileptic encephalopathy, severe neurodevelopmental delay (often with regression), and a hyperkinetic movement disorder. Additional neurological features included postnatal microcephaly and hypotonia. Five children died in childhood or adolescence (mean age of death: 9 years), mainly as a result of secondary respiratory complications. CACNA1B encodes the pore-forming subunit of the pre-synaptic neuronal voltage-gated calcium channel Cav2.2/N-type, crucial for SNARE-mediated neurotransmission, particularly in the early postnatal period. Bi-allelic loss-of-function variants in CACNA1B are predicted to cause disruption of Ca2+ influx, leading to impaired synaptic neurotransmission. The resultant effect on neuronal function is likely to be important in the development of involuntary movements and epilepsy. Overall, our findings provide further evidence for the key role of Cav2.2 in normal human neurodevelopment.MAK is funded by an NIHR Research Professorship and receives funding from the Wellcome Trust, Great Ormond Street Children's Hospital Charity, and Rosetrees Trust. E.M. received funding from the Rosetrees Trust (CD-A53) and Great Ormond Street Hospital Children's Charity. K.G. received funding from Temple Street Foundation. A.M. is funded by Great Ormond Street Hospital, the National Institute for Health Research (NIHR), and Biomedical Research Centre. F.L.R. and D.G. are funded by Cambridge Biomedical Research Centre. K.C. and A.S.J. are funded by NIHR Bioresource for Rare Diseases. The DDD Study presents independent research commissioned by the Health Innovation Challenge Fund (grant number HICF-1009-003), a parallel funding partnership between the Wellcome Trust and the Department of Health, and the Wellcome Trust Sanger Institute (grant number WT098051). We acknowledge support from the UK Department of Health via the NIHR comprehensive Biomedical Research Centre award to Guy's and St. Thomas' National Health Service (NHS) Foundation Trust in partnership with King's College London. This research was also supported by the NIHR Great Ormond Street Hospital Biomedical Research Centre. J.H.C. is in receipt of an NIHR Senior Investigator Award. The research team acknowledges the support of the NIHR through the Comprehensive Clinical Research Network. The views expressed are those of the author(s) and not necessarily those of the NHS, the NIHR, Department of Health, or Wellcome Trust. E.R.M. acknowledges support from NIHR Cambridge Biomedical Research Centre, an NIHR Senior Investigator Award, and the University of Cambridge has received salary support in respect of E.R.M. from the NHS in the East of England through the Clinical Academic Reserve. I.E.S. is supported by the National Health and Medical Research Council of Australia (Program Grant and Practitioner Fellowship)

Retrospective evaluation of whole exome and genome mutation calls in 746 cancer samples

Funder: NCI U24CA211006Abstract: The Cancer Genome Atlas (TCGA) and International Cancer Genome Consortium (ICGC) curated consensus somatic mutation calls using whole exome sequencing (WES) and whole genome sequencing (WGS), respectively. Here, as part of the ICGC/TCGA Pan-Cancer Analysis of Whole Genomes (PCAWG) Consortium, which aggregated whole genome sequencing data from 2,658 cancers across 38 tumour types, we compare WES and WGS side-by-side from 746 TCGA samples, finding that ~80% of mutations overlap in covered exonic regions. We estimate that low variant allele fraction (VAF < 15%) and clonal heterogeneity contribute up to 68% of private WGS mutations and 71% of private WES mutations. We observe that ~30% of private WGS mutations trace to mutations identified by a single variant caller in WES consensus efforts. WGS captures both ~50% more variation in exonic regions and un-observed mutations in loci with variable GC-content. Together, our analysis highlights technological divergences between two reproducible somatic variant detection efforts