Publications by authors named "Alex K Shalek"

95 Publications

Leukocyte dynamics after intracerebral hemorrhage in a living patient reveal rapid adaptations to tissue milieu.

JCI Insight 2021 Mar 22;6(6). Epub 2021 Mar 22.

Department of Neurology, Yale University School of Medicine, New Haven, Connecticut, USA.

Intracerebral hemorrhage (ICH) is a devastating form of stroke with a high mortality rate and few treatment options. Discovery of therapeutic interventions has been slow given the challenges associated with studying acute injury in the human brain. Inflammation induced by exposure of brain tissue to blood appears to be a major part of brain tissue injury. Here, we longitudinally profiled blood and cerebral hematoma effluent from a patient enrolled in the Minimally Invasive Surgery with Thrombolysis in Intracerebral Hemorrhage Evacuation trial, offering a rare window into the local and systemic immune responses to acute brain injury. Using single-cell RNA-Seq (scRNA-Seq), this is the first report to our knowledge that characterized the local cellular response during ICH in the brain of a living patient at single-cell resolution. Our analysis revealed shifts in the activation states of myeloid and T cells in the brain over time, suggesting that leukocyte responses are dynamically reshaped by the hematoma microenvironment. Interestingly, the patient had an asymptomatic rebleed that our transcriptional data indicated occurred prior to detection by CT scan. This case highlights the rapid immune dynamics in the brain after ICH and suggests that sensitive methods such as scRNA-Seq would enable greater understanding of complex intracerebral events.
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http://dx.doi.org/10.1172/jci.insight.145857DOI Listing
March 2021

A single-cell and spatial atlas of autopsy tissues reveals pathology and cellular targets of SARS-CoV-2.

Authors:
Toni M Delorey Carly G K Ziegler Graham Heimberg Rachelly Normand Yiming Yang Asa Segerstolpe Domenic Abbondanza Stephen J Fleming Ayshwarya Subramanian Daniel T Montoro Karthik A Jagadeesh Kushal K Dey Pritha Sen Michal Slyper Yered H Pita-Juárez Devan Phillips Zohar Bloom-Ackerman Nick Barkas Andrea Ganna James Gomez Erica Normandin Pourya Naderi Yury V Popov Siddharth S Raju Sebastian Niezen Linus T-Y Tsai Katherine J Siddle Malika Sud Victoria M Tran Shamsudheen K Vellarikkal Liat Amir-Zilberstein Deepak S Atri Joseph Beechem Olga R Brook Jonathan Chen Prajan Divakar Phylicia Dorceus Jesse M Engreitz Adam Essene Donna M Fitzgerald Robin Fropf Steven Gazal Joshua Gould John Grzyb Tyler Harvey Jonathan Hecht Tyler Hether Judit Jane-Valbuena Michael Leney-Greene Hui Ma Cristin McCabe Daniel E McLoughlin Eric M Miller Christoph Muus Mari Niemi Robert Padera Liuliu Pan Deepti Pant Carmel Pe'er Jenna Pfiffner-Borges Christopher J Pinto Jacob Plaisted Jason Reeves Marty Ross Melissa Rudy Erroll H Rueckert Michelle Siciliano Alexander Sturm Ellen Todres Avinash Waghray Sarah Warren Shuting Zhang Daniel R Zollinger Lisa Cosimi Rajat M Gupta Nir Hacohen Winston Hide Alkes L Price Jayaraj Rajagopal Purushothama Rao Tata Stefan Riedel Gyongyi Szabo Timothy L Tickle Deborah Hung Pardis C Sabeti Richard Novak Robert Rogers Donald E Ingber Z Gordon Jiang Dejan Juric Mehrtash Babadi Samouil L Farhi James R Stone Ioannis S Vlachos Isaac H Solomon Orr Ashenberg Caroline B M Porter Bo Li Alex K Shalek Alexandra-Chloé Villani Orit Rozenblatt-Rosen Aviv Regev

bioRxiv 2021 Feb 25. Epub 2021 Feb 25.

The SARS-CoV-2 pandemic has caused over 1 million deaths globally, mostly due to acute lung injury and acute respiratory distress syndrome, or direct complications resulting in multiple-organ failures. Little is known about the host tissue immune and cellular responses associated with COVID-19 infection, symptoms, and lethality. To address this, we collected tissues from 11 organs during the clinical autopsy of 17 individuals who succumbed to COVID-19, resulting in a tissue bank of approximately 420 specimens. We generated comprehensive cellular maps capturing COVID-19 biology related to patients' demise through single-cell and single-nucleus RNA-Seq of lung, kidney, liver and heart tissues, and further contextualized our findings through spatial RNA profiling of distinct lung regions. We developed a computational framework that incorporates removal of ambient RNA and automated cell type annotation to facilitate comparison with other healthy and diseased tissue atlases. In the lung, we uncovered significantly altered transcriptional programs within the epithelial, immune, and stromal compartments and cell intrinsic changes in multiple cell types relative to lung tissue from healthy controls. We observed evidence of: alveolar type 2 (AT2) differentiation replacing depleted alveolar type 1 (AT1) lung epithelial cells, as previously seen in fibrosis; a concomitant increase in myofibroblasts reflective of defective tissue repair; and, putative TP63 intrapulmonary basal-like progenitor (IPBLP) cells, similar to cells identified in H1N1 influenza, that may serve as an emergency cellular reserve for severely damaged alveoli. Together, these findings suggest the activation and failure of multiple avenues for regeneration of the epithelium in these terminal lungs. SARS-CoV-2 RNA reads were enriched in lung mononuclear phagocytic cells and endothelial cells, and these cells expressed distinct host response transcriptional programs. We corroborated the compositional and transcriptional changes in lung tissue through spatial analysis of RNA profiles and distinguished unique tissue host responses between regions with and without viral RNA, and in COVID-19 donor tissues relative to healthy lung. Finally, we analyzed genetic regions implicated in COVID-19 GWAS with transcriptomic data to implicate specific cell types and genes associated with disease severity. Overall, our COVID-19 cell atlas is a foundational dataset to better understand the biological impact of SARS-CoV-2 infection across the human body and empowers the identification of new therapeutic interventions and prevention strategies.
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http://dx.doi.org/10.1101/2021.02.25.430130DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7924267PMC
February 2021

Human airway mast cells proliferate and acquire distinct inflammation-driven phenotypes during type 2 inflammation.

Sci Immunol 2021 Feb;6(56)

Jeff and Penny Vinik Immunology Center, Division of Allergy and Clinical Immunology, Brigham and Women's Hospital, Boston, MA, USA.

Mast cells (MCs) play a pathobiologic role in type 2 (T2) allergic inflammatory diseases of the airway, including asthma and chronic rhinosinusitis with nasal polyposis (CRSwNP). Distinct MC subsets infiltrate the airway mucosa in T2 disease, including subepithelial MCs expressing the proteases tryptase and chymase (MC) and epithelial MCs expressing tryptase without chymase (MC). However, mechanisms underlying MC expansion and the transcriptional programs underlying their heterogeneity are poorly understood. Here, we use flow cytometry and single-cell RNA-sequencing (scRNA-seq) to conduct a comprehensive analysis of human MC hyperplasia in CRSwNP, a T2 cytokine-mediated inflammatory disease. We link discrete cell surface phenotypes to the distinct transcriptomes of CRSwNP MC and MC, which represent polarized ends of a transcriptional gradient of nasal polyp MCs. We find a subepithelial population of CD38CD117 MCs that is markedly expanded during T2 inflammation. These CD38CD117 MCs exhibit an intermediate phenotype relative to the expanded MC and MC subsets. CD38CD117 MCs are distinct from circulating MC progenitors and are enriched for proliferation, which is markedly increased in CRSwNP patients with aspirin-exacerbated respiratory disease, a severe disease subset characterized by increased MC burden and elevated MC activation. We observe that MCs expressing a polyp MC-like effector program are also found within the lung during fibrotic diseases and asthma, and further identify marked differences between MC in nasal polyps and skin. These results indicate that MCs display distinct inflammation-associated effector programs and suggest that in situ MC proliferation is a major component of MC hyperplasia in human T2 inflammation.
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http://dx.doi.org/10.1126/sciimmunol.abb7221DOI Listing
February 2021

Impaired local intrinsic immunity to SARS-CoV-2 infection in severe COVID-19.

bioRxiv 2021 Feb 20. Epub 2021 Feb 20.

Infection with SARS-CoV-2, the virus that causes COVID-19, can lead to severe lower respiratory illness including pneumonia and acute respiratory distress syndrome, which can result in profound morbidity and mortality. However, many infected individuals are either asymptomatic or have isolated upper respiratory symptoms, which suggests that the upper airways represent the initial site of viral infection, and that some individuals are able to largely constrain viral pathology to the nasal and oropharyngeal tissues. Which cell types in the human nasopharynx are the primary targets of SARS-CoV-2 infection, and how infection influences the cellular organization of the respiratory epithelium remains incompletely understood. Here, we present nasopharyngeal samples from a cohort of 35 individuals with COVID-19, representing a wide spectrum of disease states from ambulatory to critically ill, as well as 23 healthy and intubated patients without COVID-19. Using standard nasopharyngeal swabs, we collected viable cells and performed single-cell RNA-sequencing (scRNA-seq), simultaneously profiling both host and viral RNA. We find that following infection with SARS-CoV-2, the upper respiratory epithelium undergoes massive reorganization: secretory cells diversify and expand, and mature epithelial cells are preferentially lost. Further, we observe evidence for deuterosomal cell and immature ciliated cell expansion, potentially representing active repopulation of lost ciliated cells through coupled secretory cell differentiation. Epithelial cells from participants with mild/moderate COVID-19 show extensive induction of genes associated with anti-viral and type I interferon responses. In contrast, cells from participants with severe lower respiratory symptoms appear globally muted in their anti-viral capacity, despite substantially higher local inflammatory myeloid populations and equivalent nasal viral loads. This suggests an essential role for intrinsic, local epithelial immunity in curbing and constraining viral-induced pathology. Using a custom computational pipeline, we characterized cell-associated SARS-CoV-2 RNA and identified rare cells with RNA intermediates strongly suggestive of active replication. Both within and across individuals, we find remarkable diversity and heterogeneity among SARS-CoV-2 RNA+ host cells, including developing/immature and interferon-responsive ciliated cells, "hillock"-like cells, and unique subsets of secretory, goblet, and squamous cells. Finally, SARS-CoV-2 RNA+ cells, as compared to uninfected bystanders, are enriched for genes involved in susceptibility (e.g., , ) or response (e.g., , , ) to infection. Together, this work defines both protective and detrimental host responses to SARS-CoV-2, determines the direct viral targets of infection, and suggests that failed anti-viral epithelial immunity in the nasal mucosa may underlie the progression to severe COVID-19.
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http://dx.doi.org/10.1101/2021.02.20.431155DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7899452PMC
February 2021

Spatiotemporal single-cell profiling reveals that invasive and tissue-resident memory donor CD8 T cells drive gastrointestinal acute graft-versus-host disease.

Sci Transl Med 2021 Jan;13(576)

Division of Pediatric Hematology/Oncology, Boston Children's Hospital, Department of Pediatric Oncology, Dana-Farber Cancer Institute, Department of Pediatrics, Harvard Medical School, Boston, MA 02115, USA.

Organ infiltration by donor T cells is critical to the development of acute graft-versus-host disease (aGVHD) in recipients after allogeneic hematopoietic stem cell transplant (allo-HCT). However, deconvoluting the transcriptional programs of newly recruited donor T cells from those of tissue-resident T cells in aGVHD target organs remains a challenge. Here, we combined the serial intravascular staining technique with single-cell RNA sequencing to dissect the tightly connected processes by which donor T cells initially infiltrate tissues and then establish a pathogenic tissue residency program in a rhesus macaque allo-HCT model that develops aGVHD. Our results enabled creation of a spatiotemporal map of the transcriptional programs controlling donor CD8 T cell infiltration into the primary aGVHD target organ, the gastrointestinal (GI) tract. We identified the large and small intestines as the only two sites demonstrating allo-specific, rather than lymphodepletion-driven, T cell infiltration. GI-infiltrating donor CD8 T cells demonstrated a highly activated, cytotoxic phenotype while simultaneously developing a canonical tissue-resident memory T cell (T) transcriptional signature driven by interleukin-15 (IL-15)/IL-21 signaling. We found expression of a cluster of genes directly associated with tissue invasiveness, including those encoding adhesion molecules (), specific chemokines ( and ) and chemokine receptors (), as well as multiple cytoskeletal proteins. This tissue invasion transcriptional signature was validated by its ability to discriminate the CD8 T cell transcriptome of patients with GI aGVHD from those of GVHD-free patients. These results provide insights into the mechanisms controlling tissue occupancy of target organs by pathogenic donor CD8 T cells during aGVHD in primate transplant recipients.
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http://dx.doi.org/10.1126/scitranslmed.abc0227DOI Listing
January 2021

Lymph nodes are innervated by a unique population of sensory neurons with immunomodulatory potential.

Cell 2021 Jan 16;184(2):441-459.e25. Epub 2020 Dec 16.

The Ragon Institute of MGH, MIT and Harvard, Cambridge, MA 02139, USA; Department of Immunology & HMS Center for Immune Imaging, Harvard Medical School, Boston, MA 02115, USA. Electronic address:

Barrier tissue immune responses are regulated in part by nociceptors. Nociceptor ablation alters local immune responses at peripheral sites and within draining lymph nodes (LNs). The mechanisms and significance of nociceptor-dependent modulation of LN function are unknown. Using high-resolution imaging, viral tracing, single-cell transcriptomics, and optogenetics, we identified and functionally tested a sensory neuro-immune circuit that is responsive to lymph-borne inflammatory signals. Transcriptomics profiling revealed that multiple sensory neuron subsets, predominantly peptidergic nociceptors, innervate LNs, distinct from those innervating surrounding skin. To uncover LN-resident cells that may interact with LN-innervating sensory neurons, we generated a LN single-cell transcriptomics atlas and nominated nociceptor target populations and interaction modalities. Optogenetic stimulation of LN-innervating sensory fibers triggered rapid transcriptional changes in the predicted interacting cell types, particularly endothelium, stromal cells, and innate leukocytes. Thus, a unique population of sensory neurons monitors peripheral LNs and may locally regulate gene expression.
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http://dx.doi.org/10.1016/j.cell.2020.11.028DOI Listing
January 2021

Cyclin D3 drives inertial cell cycling in dark zone germinal center B cells.

J Exp Med 2021 Apr;218(4)

Laboratory of Lymphocyte Dynamics, The Rockefeller University, New York, NY.

During affinity maturation, germinal center (GC) B cells alternate between proliferation and somatic hypermutation in the dark zone (DZ) and affinity-dependent selection in the light zone (LZ). This anatomical segregation imposes that the vigorous proliferation that allows clonal expansion of positively selected GC B cells takes place ostensibly in the absence of the signals that triggered selection in the LZ, as if by "inertia." We find that such inertial cycles specifically require the cell cycle regulator cyclin D3. Cyclin D3 dose-dependently controls the extent to which B cells proliferate in the DZ and is essential for effective clonal expansion of GC B cells in response to strong T follicular helper (Tfh) cell help. Introduction into the Ccnd3 gene of a Burkitt lymphoma-associated gain-of-function mutation (T283A) leads to larger GCs with increased DZ proliferation and, in older mice, clonal B cell lymphoproliferation, suggesting that the DZ inertial cell cycle program can be coopted by B cells undergoing malignant transformation.
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http://dx.doi.org/10.1084/jem.20201699DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7754672PMC
April 2021

Functional compensation precedes recovery of tissue mass following acute liver injury.

Nat Commun 2020 11 19;11(1):5785. Epub 2020 Nov 19.

Genetics Division, Brigham and Women's Hospital, Harvard Medical School, Boston, MA, 02115, USA.

The liver plays a central role in metabolism, protein synthesis and detoxification. It possesses unique regenerative capacity upon injury. While many factors regulating cellular proliferation during liver repair have been identified, the mechanisms by which the injured liver maintains vital functions prior to tissue recovery are unknown. Here, we identify a new phase of functional compensation following acute liver injury that occurs prior to cellular proliferation. By coupling single-cell RNA-seq with in situ transcriptional analyses in two independent murine liver injury models, we discover adaptive reprogramming to ensure expression of both injury response and core liver function genes dependent on macrophage-derived WNT/β-catenin signaling. Interestingly, transcriptional compensation is most prominent in non-proliferating cells, clearly delineating two temporally distinct phases of liver recovery. Overall, our work describes a mechanism by which the liver maintains essential physiological functions prior to cellular reconstitution and characterizes macrophage-derived WNT signals required for this compensation.
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http://dx.doi.org/10.1038/s41467-020-19558-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7677389PMC
November 2020

Evolution and Diversity of Immune Responses during Acute HIV Infection.

Immunity 2020 Nov;53(5):908-924

Ragon Institute of MGH, MIT, and Harvard, Cambridge, MA, USA; Institute for Medical Engineering and Science (IMES), Massachusetts Institute of Technology, Cambridge, MA, USA; Department of Chemistry, Massachusetts Institute of Technology, Cambridge, MA, USA; Broad Institute of MIT and Harvard, Cambridge, MA, USA; Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA, USA. Electronic address:

Understanding the earliest immune responses following HIV infection is critical to inform future vaccines and therapeutics. Here, we review recent prospective human studies in at-risk populations that have provided insight into immune responses during acute infection, including additional relevant data from non-human primate (NHP) studies. We discuss the timing, nature, and function of the diverse immune responses induced, the onset of immune dysfunction, and the effects of early anti-retroviral therapy administration. Treatment at onset of viremia mitigates peripheral T and B cell dysfunction, limits seroconversion, and enhances cellular antiviral immunity despite persistence of infection in lymphoid tissues. We highlight pertinent areas for future investigation, and how application of high-throughput technologies, alongside targeted NHP studies, may elucidate immune response features to target in novel preventions and cures.
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http://dx.doi.org/10.1016/j.immuni.2020.10.015DOI Listing
November 2020

Inflammasomes within Hyperactive Murine Dendritic Cells Stimulate Long-Lived T Cell-Mediated Anti-tumor Immunity.

Cell Rep 2020 Nov;33(7):108381

Harvard Medical School and Division of Gastroenterology, Boston Children's Hospital, Boston, MA, USA. Electronic address:

Central to anti-tumor immunity are dendritic cells (DCs), which stimulate long-lived protective T cell responses. Recent studies have demonstrated that DCs can achieve a state of hyperactivation, which is associated with inflammasome activities within living cells. Herein, we report that hyperactive DCs have an enhanced ability to migrate to draining lymph nodes and stimulate potent cytotoxic T lymphocyte (CTL) responses. This enhanced migratory activity is dependent on the chemokine receptor CCR7 and is associated with a unique transcriptional program that is not observed in conventionally activated or pyroptotic DCs. We show that hyperactivating stimuli are uniquely capable of inducing durable CTL-mediated anti-tumor immunity against tumors that are sensitive or resistant to PD-1 inhibition. These protective responses are intrinsic to the cDC1 subset of DCs, depend on the inflammasome-dependent cytokine IL-1β, and enable tumor lysates to serve as immunogens. If these activities are verified in humans, hyperactive DCs may impact immunotherapy.
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http://dx.doi.org/10.1016/j.celrep.2020.108381DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7727444PMC
November 2020

Single-Cell Profiling of Ebola Virus Disease In Vivo Reveals Viral and Host Dynamics.

Cell 2020 Nov 6;183(5):1383-1401.e19. Epub 2020 Nov 6.

Integrated Research Facility, Division of Clinical Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Frederick, MD 21702, USA.

Ebola virus (EBOV) causes epidemics with high mortality yet remains understudied due to the challenge of experimentation in high-containment and outbreak settings. Here, we used single-cell transcriptomics and CyTOF-based single-cell protein quantification to characterize peripheral immune cells during EBOV infection in rhesus monkeys. We obtained 100,000 transcriptomes and 15,000,000 protein profiles, finding that immature, proliferative monocyte-lineage cells with reduced antigen-presentation capacity replace conventional monocyte subsets, while lymphocytes upregulate apoptosis genes and decline in abundance. By quantifying intracellular viral RNA, we identify molecular determinants of tropism among circulating immune cells and examine temporal dynamics in viral and host gene expression. Within infected cells, EBOV downregulates STAT1 mRNA and interferon signaling, and it upregulates putative pro-viral genes (e.g., DYNLL1 and HSPA5), nominating pathways the virus manipulates for its replication. This study sheds light on EBOV tropism, replication dynamics, and elicited immune response and provides a framework for characterizing host-virus interactions under maximum containment.
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http://dx.doi.org/10.1016/j.cell.2020.10.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7707107PMC
November 2020

Second-Strand Synthesis-Based Massively Parallel scRNA-Seq Reveals Cellular States and Molecular Features of Human Inflammatory Skin Pathologies.

Immunity 2020 10;53(4):878-894.e7

Institute for Medical Engineering & Science (IMES), MIT, Cambridge, Massachusetts, USA; Department of Immunology, Harvard Medical School, Boston, MA, USA; Broad Institute of MIT and Harvard, Cambridge, MA, USA; Ragon Institute of MGH, MIT and Harvard, Cambridge, MA, USA; Koch Institute for Integrative Cancer Research, MIT, Cambridge, MA, USA; Harvard Stem Cell Institute, Cambridge, MA, USA. Electronic address:

High-throughput single-cell RNA-sequencing (scRNA-seq) methodologies enable characterization of complex biological samples by increasing the number of cells that can be profiled contemporaneously. Nevertheless, these approaches recover less information per cell than low-throughput strategies. To accurately report the expression of key phenotypic features of cells, scRNA-seq platforms are needed that are both high fidelity and high throughput. To address this need, we created Seq-Well S ("Second-Strand Synthesis"), a massively parallel scRNA-seq protocol that uses a randomly primed second-strand synthesis to recover complementary DNA (cDNA) molecules that were successfully reverse transcribed but to which a second oligonucleotide handle, necessary for subsequent whole transcriptome amplification, was not appended due to inefficient template switching. Seq-Well S increased the efficiency of transcript capture and gene detection compared with that of previous iterations by up to 10- and 5-fold, respectively. We used Seq-Well S to chart the transcriptional landscape of five human inflammatory skin diseases, thus providing a resource for the further study of human skin inflammation.
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http://dx.doi.org/10.1016/j.immuni.2020.09.015DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7562821PMC
October 2020

The Human Cell Atlas and equity: lessons learned.

Nat Med 2020 10;26(10):1509-1511

Institute for Medical Engineering & Science, Department of Chemistry, and Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA, USA.

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http://dx.doi.org/10.1038/s41591-020-1100-4DOI Listing
October 2020

Improved haplotype inference by exploiting long-range linking and allelic imbalance in RNA-seq datasets.

Nat Commun 2020 09 16;11(1):4662. Epub 2020 Sep 16.

Computer Science & Artificial Intelligence Laboratory, Massachusetts Institute of Technology, Cambridge, MA, 02139, USA.

Haplotype reconstruction of distant genetic variants remains an unsolved problem due to the short-read length of common sequencing data. Here, we introduce HapTree-X, a probabilistic framework that utilizes latent long-range information to reconstruct unspecified haplotypes in diploid and polyploid organisms. It introduces the observation that differential allele-specific expression can link genetic variants from the same physical chromosome, thus even enabling using reads that cover only individual variants. We demonstrate HapTree-X's feasibility on in-house sequenced Genome in a Bottle RNA-seq and various whole exome, genome, and 10X Genomics datasets. HapTree-X produces more complete phases (up to 25%), even in clinically important genes, and phases more variants than other methods while maintaining similar or higher accuracy and being up to 10×  faster than other tools. The advantage of HapTree-X's ability to use multiple lines of evidence, as well as to phase polyploid genomes in a single integrative framework, substantially grows as the amount of diverse data increases.
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http://dx.doi.org/10.1038/s41467-020-18320-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7494856PMC
September 2020

Innate Lymphoid Cell Activation and Sustained Depletion in Blood and Tissue of Children Infected with HIV from Birth Despite Antiretroviral Therapy.

Cell Rep 2020 09;32(11):108153

Africa Health Research Institute (AHRI), Durban 4001, South Africa; Department of Immunology and Microbiology, University of Copenhagen, Copenhagen 2200N, Denmark; University College London, Division of Infection and Immunity, London WC1E 6AE, UK; School of Laboratory Medicine and Medical Sciences, University of KwaZulu-Natal, Durban 4001, South Africa. Electronic address:

Innate lymphoid cells (ILCs) are important for response to infection and for immune development in early life. HIV infection in adults depletes circulating ILCs, but the impact on children infected from birth remains unknown. We study vertically HIV-infected children from birth to adulthood and find severe and persistent depletion of all circulating ILCs that, unlike CD4 T cells, are not restored by long-term antiretroviral therapy unless initiated at birth. Remaining ILCs upregulate genes associated with cellular activation and metabolic perturbation. Unlike HIV-infected adults, ILCs are also profoundly depleted in tonsils of vertically infected children. Transcriptional profiling of remaining ILCs reveals ongoing cell-type-specific activity despite antiretroviral therapy. Collectively, these data suggest an important and ongoing role for ILCs in lymphoid tissue of HIV-infected children from birth, where persistent depletion and sustained transcriptional activity are likely to have long-term immune consequences that merit further investigation.
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http://dx.doi.org/10.1016/j.celrep.2020.108153DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7495043PMC
September 2020

A Single Human V-gene Allows for a Broad-Spectrum Antibody Response Targeting Bacterial Lipopolysaccharides in the Blood.

Cell Rep 2020 08;32(8):108065

The Ragon Institute of Massachusetts General Hospital, Massachusetts Institute of Technology and Harvard University, 400 Technology Square, Cambridge, MA 02139, USA. Electronic address:

B cell receptors (BCRs) display a combination of variable (V)-gene-encoded complementarity determining regions (CDRs) and adaptive/hypervariable CDR3 loops to engage antigens. It has long been proposed that the former tune for recognition of pathogens or groups of pathogens. To experimentally evaluate this within the human antibody repertoire, we perform immune challenges in transgenic mice that bear diverse human CDR3 and light chains but are constrained to different human Vgenes. We find that, of six commonly deployed V sequences, only those CDRs encoded by IGHV1-202 enable polyclonal antibody responses against bacterial lipopolysaccharide (LPS) when introduced to the bloodstream. The LPS is from diverse strains of gram-negative bacteria, and the V-gene-dependent responses are directed against the non-variable and universal saccrolipid substructure of this antigen. This reveals a broad-spectrum anti-LPS response in which germline-encoded CDRs naturally hardwire the human antibody repertoire for recognition of a conserved microbial target.
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http://dx.doi.org/10.1016/j.celrep.2020.108065DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7446668PMC
August 2020

Single-Cell Analyses of Colon and Blood Reveal Distinct Immune Cell Signatures of Ulcerative Colitis and Crohn's Disease.

Gastroenterology 2020 08 16;159(2):591-608.e10. Epub 2020 May 16.

Division of Newborn Medicine, Boston Children's Hospital, Boston, Massachusetts; Department of Pediatrics, University of Pittsburgh Medical Center Children's Hospital, Pittsburgh, Pennsylvania; Department of Immunology, University of Pittsburgh, Pittsburgh, Pennsylvania; Department of Developmental Biology University of Pittsburgh, Pittsburgh, Pennsylvania; Department of Pediatrics, Yale University School of Medicine, New Haven, Connecticut. Electronic address:

Background & Aims: Studies are needed to determine the mechanisms of mucosal dysregulation in patients with inflammatory bowel diseases (IBDs) and differences in inflammatory responses of patients with ulcerative colitis (UC) vs Crohn's disease (CD). We used mass cytometry (CyTOF) to characterize and compare immune cell populations in the mucosa and blood from patients with IBD and without IBD (controls) at single-cell resolution.

Methods: We performed CyTOF analysis of colonic mucosa samples (n = 87) and peripheral blood mononuclear cells (n = 85) from patients with active or inactive UC or CD and controls. We also performed single-cell RNA sequencing, flow cytometry, and RNA in situ hybridization analyses to validate key findings. We used random forest modeling to identify differences in signatures across subject groups.

Results: Compared with controls, colonic mucosa samples from patients with IBD had increased abundances of HLA-DR+CD38+ T cells, including T-regulatory cells that produce inflammatory cytokines; CXCR3+ plasmablasts; and IL1B+ macrophages and monocytes. Colonic mucosa samples from patients with UC were characterized by expansion of IL17A+ CD161+ effector memory T cells and IL17A+ T-regulatory cells; expansion of HLA-DR+CD56+ granulocytes; and reductions in type 3 innate lymphoid cells. Mucosal samples from patients with active CD were characterized by IL1B+HLA-DR+CD38+ T cells, IL1B+TNF+IFNG naïve B cells, IL1B+ dendritic cells (DCs), and IL1B+ plasmacytoid DCs. Peripheral blood mononuclear cells from patients with active CD differed from those of active UC in that the peripheral blood mononuclear cells from patients with CD had increased IL1B+ T-regulatory cells, IL1B+ DCs and IL1B+ plasmacytoid DCs, IL1B+ monocytes, and fewer group 1 innate lymphoid cells. Random forest modeling differentiated active UC from active CD in colonic mucosa and blood samples; top discriminating features included many of the cellular populations identified above.

Conclusions: We used single-cell technologies to identify immune cell populations specific to mucosa and blood samples from patients with active or inactive CD and UC and controls. This information might be used to develop therapies that target specific cell populations in patients with different types of IBD.
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http://dx.doi.org/10.1053/j.gastro.2020.04.074DOI Listing
August 2020

SARS-CoV-2 Receptor ACE2 Is an Interferon-Stimulated Gene in Human Airway Epithelial Cells and Is Detected in Specific Cell Subsets across Tissues.

Cell 2020 05 27;181(5):1016-1035.e19. Epub 2020 Apr 27.

Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA; Program in Immunology, Harvard Medical School, Boston, MA 02115, USA; Division of Gastroenterology, Hepatology, and Nutrition, Boston Children's Hospital, Boston, MA 02115, USA; Harvard Stem Cell Institute, Cambridge, MA 02138, USA. Electronic address:

There is pressing urgency to understand the pathogenesis of the severe acute respiratory syndrome coronavirus clade 2 (SARS-CoV-2), which causes the disease COVID-19. SARS-CoV-2 spike (S) protein binds angiotensin-converting enzyme 2 (ACE2), and in concert with host proteases, principally transmembrane serine protease 2 (TMPRSS2), promotes cellular entry. The cell subsets targeted by SARS-CoV-2 in host tissues and the factors that regulate ACE2 expression remain unknown. Here, we leverage human, non-human primate, and mouse single-cell RNA-sequencing (scRNA-seq) datasets across health and disease to uncover putative targets of SARS-CoV-2 among tissue-resident cell subsets. We identify ACE2 and TMPRSS2 co-expressing cells within lung type II pneumocytes, ileal absorptive enterocytes, and nasal goblet secretory cells. Strikingly, we discovered that ACE2 is a human interferon-stimulated gene (ISG) in vitro using airway epithelial cells and extend our findings to in vivo viral infections. Our data suggest that SARS-CoV-2 could exploit species-specific interferon-driven upregulation of ACE2, a tissue-protective mediator during lung injury, to enhance infection.
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http://dx.doi.org/10.1016/j.cell.2020.04.035DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7252096PMC
May 2020

Systematic comparison of single-cell and single-nucleus RNA-sequencing methods.

Nat Biotechnol 2020 06 6;38(6):737-746. Epub 2020 Apr 6.

Broad Institute of MIT and Harvard, Cambridge, MA, USA.

The scale and capabilities of single-cell RNA-sequencing methods have expanded rapidly in recent years, enabling major discoveries and large-scale cell mapping efforts. However, these methods have not been systematically and comprehensively benchmarked. Here, we directly compare seven methods for single-cell and/or single-nucleus profiling-selecting representative methods based on their usage and our expertise and resources to prepare libraries-including two low-throughput and five high-throughput methods. We tested the methods on three types of samples: cell lines, peripheral blood mononuclear cells and brain tissue, generating 36 libraries in six separate experiments in a single center. To directly compare the methods and avoid processing differences introduced by the existing pipelines, we developed scumi, a flexible computational pipeline that can be used with any single-cell RNA-sequencing method. We evaluated the methods for both basic performance, such as the structure and alignment of reads, sensitivity and extent of multiplets, and for their ability to recover known biological information in the samples.
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http://dx.doi.org/10.1038/s41587-020-0465-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7289686PMC
June 2020

The Human Tumor Atlas Network: Charting Tumor Transitions across Space and Time at Single-Cell Resolution.

Cell 2020 04;181(2):236-249

Department of Data Sciences, Dana-Farber Cancer Institute, Boston, MA 02215, USA; Present address: Foundation Medicine, Cambridge, MA 02141, USA.

Crucial transitions in cancer-including tumor initiation, local expansion, metastasis, and therapeutic resistance-involve complex interactions between cells within the dynamic tumor ecosystem. Transformative single-cell genomics technologies and spatial multiplex in situ methods now provide an opportunity to interrogate this complexity at unprecedented resolution. The Human Tumor Atlas Network (HTAN), part of the National Cancer Institute (NCI) Cancer Moonshot Initiative, will establish a clinical, experimental, computational, and organizational framework to generate informative and accessible three-dimensional atlases of cancer transitions for a diverse set of tumor types. This effort complements both ongoing efforts to map healthy organs and previous large-scale cancer genomics approaches focused on bulk sequencing at a single point in time. Generating single-cell, multiparametric, longitudinal atlases and integrating them with clinical outcomes should help identify novel predictive biomarkers and features as well as therapeutically relevant cell types, cell states, and cellular interactions across transitions. The resulting tumor atlases should have a profound impact on our understanding of cancer biology and have the potential to improve cancer detection, prevention, and therapeutic discovery for better precision-medicine treatments of cancer patients and those at risk for cancer.
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http://dx.doi.org/10.1016/j.cell.2020.03.053DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7376497PMC
April 2020

Integrated single-cell analysis of multicellular immune dynamics during hyperacute HIV-1 infection.

Nat Med 2020 04 23;26(4):511-518. Epub 2020 Mar 23.

Ragon Institute of MGH, MIT and Harvard, Cambridge, MA, USA.

Cellular immunity is critical for controlling intracellular pathogens, but individual cellular dynamics and cell-cell cooperativity in evolving human immune responses remain poorly understood. Single-cell RNA-sequencing (scRNA-seq) represents a powerful tool for dissecting complex multicellular behaviors in health and disease and nominating testable therapeutic targets. Its application to longitudinal samples could afford an opportunity to uncover cellular factors associated with the evolution of disease progression without potentially confounding inter-individual variability. Here, we present an experimental and computational methodology that uses scRNA-seq to characterize dynamic cellular programs and their molecular drivers, and apply it to HIV infection. By performing scRNA-seq on peripheral blood mononuclear cells from four untreated individuals before and longitudinally during acute infection, we were powered within each to discover gene response modules that vary by time and cell subset. Beyond previously unappreciated individual- and cell-type-specific interferon-stimulated gene upregulation, we describe temporally aligned gene expression responses obscured in bulk analyses, including those involved in proinflammatory T cell differentiation, prolonged monocyte major histocompatibility complex II upregulation and persistent natural killer (NK) cell cytolytic killing. We further identify response features arising in the first weeks of infection, for example proliferating natural killer cells, which potentially may associate with future viral control. Overall, our approach provides a unified framework for characterizing multiple dynamic cellular responses and their coordination.
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http://dx.doi.org/10.1038/s41591-020-0799-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7237067PMC
April 2020

IL-5Rα marks nasal polyp IgG4- and IgE-expressing cells in aspirin-exacerbated respiratory disease.

J Allergy Clin Immunol 2020 06 19;145(6):1574-1584. Epub 2020 Mar 19.

Department of Medicine, Harvard Medical School, Boston, Mass; Division of Allergy and Clinical Immunology, Brigham and Women's Hospital, Boston, Mass. Electronic address:

Background: The cause of severe nasal polyposis in aspirin-exacerbated respiratory disease (AERD) is unknown. Elevated antibody levels have been associated with disease severity in nasal polyps, but upstream drivers of local antibody production in nasal polyps are undetermined.

Objective: We sought to identify upstream drivers and phenotypic properties of local antibody-expressing cells in nasal polyps from subjects with AERD.

Methods: Sinus tissue was obtained from subjects with AERD, chronic rhinosinusitis (CRS) with nasal polyps (CRSwNP), CRS without nasal polyps, and controls without CRS. Tissue antibody levels were quantified via ELISA and immunohistochemistry and were correlated with disease severity. Antibody-expressing cells were profiled with single-cell RNA sequencing, flow cytometry, and immunofluorescence, with IL-5Rα function determined through IL-5 stimulation and subsequent RNA sequencing and quantitative PCR.

Results: Tissue IgE and IgG4 levels were elevated in AERD compared with in controls (P < .01 for IgE and P < .001 for IgG4 vs CRSwNP). Subjects with AERD whose nasal polyps recurred rapidly had higher IgE levels than did subjects with AERD, with slower regrowth (P = .005). Single-cell RNA sequencing revealed increased IL5RA, IGHG4, and IGHE in antibody-expressing cells from patients with AERD compared with antibody-expressing cells from patients with CRSwNP. There were more IL-5Rα plasma cells in the polyp tissue from those with AERD than in polyp tissue from those with CRSwNP (P = .026). IL-5 stimulation of plasma cells in vitro induced changes in a distinct set of transcripts.

Conclusions: Our study identifies an increase in antibody-expressing cells in AERD defined by transcript enrichment of IL5RA and IGHG4 or IGHE, with confirmed surface expression of IL-5Rα and functional IL-5 signaling. Tissue IgE and IgG4 levels are elevated in AERD, and higher IgE levels are associated with faster nasal polyp regrowth. Our findings suggest a role for IL-5Rα antibody-expressing cells in facilitating local antibody production and severe nasal polyps in AERD.
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http://dx.doi.org/10.1016/j.jaci.2020.02.035DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7282948PMC
June 2020

Distribution and storage of inflammatory memory in barrier tissues.

Nat Rev Immunol 2020 05 3;20(5):308-320. Epub 2020 Feb 3.

Institute for Medical Engineering and Science (IMES), MIT, Cambridge, MA, USA.

Memories of previous immune events enable barrier tissues to rapidly recall distinct environmental exposures. To effectively inform future responses, these past experiences can be stored in cell types that are long-term residents or essential constituents of tissues. There is an emerging understanding that, in addition to antigen-specific immune cells, diverse haematopoietic, stromal, parenchymal and neuronal cell types can store inflammatory memory. Here, we explore the impact of previous immune activity on various cell lineages with the goal of presenting a unified view of inflammatory memory to environmental exposures (such as allergens, antigens, noxious agents and microorganisms) at barrier tissues. We propose that inflammatory memory is distributed across diverse cell types and stored through shifts in cell states, and we provide a framework to guide future experiments. This distribution and storage may promote adaptation or maladaptation in homeostatic, maintenance and disease settings - especially if the distribution of memory favours cellular cooperation during storage or recall.
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http://dx.doi.org/10.1038/s41577-019-0263-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7547402PMC
May 2020

Immunological Fingerprints of Controllers Developing Neutralizing HIV-1 Antibodies.

Cell Rep 2020 01;30(4):984-996.e4

Ragon Institute of MGH, MIT, and Harvard, Boston, MA, USA; Broad Institute of MIT and Harvard, Cambridge, MA, USA; Infectious Disease Division, Brigham and Women's Hospital, Boston, MA, USA; Infectious Disease Division, Massachusetts General Hospital, Boston, MA, USA. Electronic address:

The induction of broadly neutralizing antibodies (bnAbs) is highly desired for an effective vaccine against HIV-1. Typically, bnAbs develop in patients with high viremia, but they can also evolve in some untreated HIV-1 controllers with low viral loads. Here, we identify a subgroup of neutralizer-controllers characterized by myeloid DCs (mDCs) with a distinct inflammatory signature and a superior ability to prime T follicular helper (Tfh)-like cells in an STAT4-dependent fashion. This distinct immune profile is associated with a higher frequency of Tfh-like cells in peripheral blood (pTfh) and an enrichment for Tfh-defining genes in circulating CD4 T cells. Correspondingly, monocytes from this neutralizer controller subgroup upregulate genes encoding for chemotaxis and inflammation, and they secrete high levels of IL-12 in response to TLR stimulation. Our results suggest the existence of multi-compartment immune networks between mDCs, Tfh, and monocytes that may facilitate the development of bnAbs in a subgroup of HIV-1 controllers.
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http://dx.doi.org/10.1016/j.celrep.2019.12.087DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6990401PMC
January 2020

Identification of a Master Regulator of Differentiation in Toxoplasma.

Cell 2020 01 16;180(2):359-372.e16. Epub 2020 Jan 16.

Whitehead Institute for Biomedical Research, Cambridge, MA 02142, USA; Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02142, USA. Electronic address:

Toxoplasma gondii chronically infects a quarter of the world's population, and its recrudescence can cause life-threatening disease in immunocompromised individuals and recurrent ocular lesions in the immunocompetent. Acute-stage tachyzoites differentiate into chronic-stage bradyzoites, which form intracellular cysts resistant to immune clearance and existing therapies. The molecular basis of this differentiation is unknown, despite being efficiently triggered by stresses in culture. Through Cas9-mediated screening and single-cell profiling, we identify a Myb-like transcription factor (BFD1) necessary for differentiation in cell culture and in mice. BFD1 accumulates during stress and its synthetic expression is sufficient to drive differentiation. Consistent with its function as a transcription factor, BFD1 binds the promoters of many stage-specific genes and represents a counterpoint to the ApiAP2 factors that dominate our current view of parasite gene regulation. BFD1 provides a genetic switch to study and control Toxoplasma differentiation and will inform prevention and treatment of chronic infections.
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http://dx.doi.org/10.1016/j.cell.2019.12.013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6978799PMC
January 2020

Prevention of tuberculosis in macaques after intravenous BCG immunization.

Nature 2020 01 1;577(7788):95-102. Epub 2020 Jan 1.

Vaccine Research Center, National Institute of Allergy and Infectious Diseases (NIAID), National Institutes of Health (NIH), Bethesda, MD, USA.

Mycobacterium tuberculosis (Mtb) is the leading cause of death from infection worldwide. The only available vaccine, BCG (Bacillus Calmette-Guérin), is given intradermally and has variable efficacy against pulmonary tuberculosis, the major cause of mortality and disease transmission. Here we show that intravenous administration of BCG profoundly alters the protective outcome of Mtb challenge in non-human primates (Macaca mulatta). Compared with intradermal or aerosol delivery, intravenous immunization induced substantially more antigen-responsive CD4 and CD8 T cell responses in blood, spleen, bronchoalveolar lavage and lung lymph nodes. Moreover, intravenous immunization induced a high frequency of antigen-responsive T cells across all lung parenchymal tissues. Six months after BCG vaccination, macaques were challenged with virulent Mtb. Notably, nine out of ten macaques that received intravenous BCG vaccination were highly protected, with six macaques showing no detectable levels of infection, as determined by positron emission tomography-computed tomography imaging, mycobacterial growth, pathology and granuloma formation. The finding that intravenous BCG prevents or substantially limits Mtb infection in highly susceptible rhesus macaques has important implications for vaccine delivery and clinical development, and provides a model for defining immune correlates and mechanisms of vaccine-elicited protection against tuberculosis.
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http://dx.doi.org/10.1038/s41586-019-1817-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7015856PMC
January 2020

TCR sequencing paired with massively parallel 3' RNA-seq reveals clonotypic T cell signatures.

Nat Immunol 2019 12 19;20(12):1692-1699. Epub 2019 Nov 19.

Koch Institute for Integrative Cancer Research, MIT, Cambridge, MA, USA.

High-throughput 3' single-cell RNA-sequencing (scRNA-seq) allows cost-effective, detailed characterization of individual immune cells from tissues. Current techniques, however, are limited in their ability to elucidate essential immune cell features, including variable sequences of T cell antigen receptors (TCRs) that confer antigen specificity. Here, we present a strategy that enables simultaneous analysis of TCR sequences and corresponding full transcriptomes from 3'-barcoded scRNA-seq samples. This approach is compatible with common 3' scRNA-seq methods, and adaptable to processed samples post hoc. We applied the technique to identify transcriptional signatures associated with T cells sharing common TCRs from immunized mice and from patients with food allergy. We observed preferential phenotypes among subsets of expanded clonotypes, including type 2 helper CD4 T cell (T2) states associated with food allergy. These results demonstrate the utility of our method when studying diseases in which clonotype-driven responses are critical to understanding the underlying biology.
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http://dx.doi.org/10.1038/s41590-019-0544-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7528220PMC
December 2019

Single-cell transcriptomic atlas of the human retina identifies cell types associated with age-related macular degeneration.

Nat Commun 2019 10 25;10(1):4902. Epub 2019 Oct 25.

Broad Institute of MIT and Harvard, Cambridge, MA, 02142, USA.

Genome-wide association studies (GWAS) have identified genetic variants associated with age-related macular degeneration (AMD), one of the leading causes of blindness in the elderly. However, it has been challenging to identify the cell types associated with AMD given the genetic complexity of the disease. Here we perform massively parallel single-cell RNA sequencing (scRNA-seq) of human retinas using two independent platforms, and report the first single-cell transcriptomic atlas of the human retina. Using a multi-resolution network-based analysis, we identify all major retinal cell types, and their corresponding gene expression signatures. Heterogeneity is observed within macroglia, suggesting that human retinal glia are more diverse than previously thought. Finally, GWAS-based enrichment analysis identifies glia, vascular cells, and cone photoreceptors to be associated with the risk of AMD. These data provide a detailed analysis of the human retina, and show how scRNA-seq can provide insight into cell types involved in complex, inflammatory genetic diseases.
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http://dx.doi.org/10.1038/s41467-019-12780-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6814749PMC
October 2019