Publications by authors named "Pradyot Dash"

30 Publications

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A population of proinflammatory T cells coexpresses αβ and γδ T cell receptors in mice and humans.

J Exp Med 2020 05;217(5)

Immune Regulation Research Group, School of Biochemistry and Immunology, Trinity Biomedical Sciences Institute, Trinity College Dublin, Dublin, Ireland.

T cells are classically recognized as distinct subsets that express αβ or γδ TCRs. We identify a novel population of T cells that coexpress αβ and γδ TCRs in mice and humans. These hybrid αβ-γδ T cells arose in the murine fetal thymus by day 16 of ontogeny, underwent αβ TCR-mediated positive selection into CD4+ or CD8+ thymocytes, and constituted up to 10% of TCRδ+ cells in lymphoid organs. They expressed high levels of IL-1R1 and IL-23R and secreted IFN-γ, IL-17, and GM-CSF in response to canonically restricted peptide antigens or stimulation with IL-1β and IL-23. Hybrid αβ-γδ T cells were transcriptomically distinct from conventional γδ T cells and displayed a hyperinflammatory phenotype enriched for chemokine receptors and homing molecules that facilitate migration to sites of inflammation. These proinflammatory T cells promoted bacterial clearance after infection with Staphylococcus aureus and, by licensing encephalitogenic Th17 cells, played a key role in the development of autoimmune disease in the central nervous system.
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http://dx.doi.org/10.1084/jem.20190834DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7201916PMC
May 2020

Human γδ T-cell receptor repertoire is shaped by influenza viruses, age and tissue compartmentalisation.

Clin Transl Immunology 2019 23;8(9):e1079. Epub 2019 Sep 23.

Department of Microbiology and Immunology University of Melbourne at The Peter Doherty Institute for Infection and Immunity Melbourne VIC Australia.

Background: Although γδ T cells comprise up to 10% of human peripheral blood T cells, questions remain regarding their role in disease states and T-cell receptor (TCR) clonal expansions. We dissected anti-viral functions of human γδ T cells towards influenza viruses and defined influenza-reactive γδ TCRs in the context of γδ-TCRs across the human lifespan.

Methods: We performed Cr-killing assay and single-cell time-lapse live video microscopy to define mechanisms underlying γδ T-cell-mediated killing of influenza-infected targets. We assessed cytotoxic profiles of γδ T cells in influenza-infected patients and IFN-γ production towards influenza-infected lung epithelial cells. Using single-cell RT-PCR, we characterised paired TCRγδ clonotypes for influenza-reactive γδ T cells in comparison with TCRs from healthy neonates, adults, elderly donors and tissues.

Results: We provide the first visual evidence of γδ T-cell-mediated killing of influenza-infected targets and show distinct features to those reported for CD8 T cells. γδ T cells displayed poly-cytotoxic profiles in influenza-infected patients and produced IFN-γ towards influenza-infected cells. These IFN-γ-producing γδ T cells were skewed towards the γ9δ2 TCRs, particularly expressing the public GV9-TCRγ, capable of pairing with numerous TCR-δ chains, suggesting their significant role in γδ T-cell immunity. Neonatal γδ T cells displayed extensive non-overlapping TCRγδ repertoires, while adults had enriched γ9δ2-pairings with diverse CDR3γδ regions. Conversely, the elderly showed distinct γδ-pairings characterised by large clonal expansions, a profile also prominent in adult tissues.

Conclusion: Human TCRγδ repertoire is shaped by age, tissue compartmentalisation and the individual's history of infection, suggesting that these somewhat enigmatic γδ T cells indeed respond to antigen challenge.
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http://dx.doi.org/10.1002/cti2.1079DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6756999PMC
September 2019

Human CD8 T cell cross-reactivity across influenza A, B and C viruses.

Nat Immunol 2019 05 18;20(5):613-625. Epub 2019 Feb 18.

Department of Microbiology and Immunology, University of Melbourne, at the Peter Doherty Institute for Infection and Immunity, Parkville, Victoria, Australia.

Influenza A, B and C viruses (IAV, IBV and ICV, respectively) circulate globally and infect humans, with IAV and IBV causing the most severe disease. CD8 T cells confer cross-protection against IAV strains, however the responses of CD8 T cells to IBV and ICV are understudied. We investigated the breadth of CD8 T cell cross-recognition and provide evidence of CD8 T cell cross-reactivity across IAV, IBV and ICV. We identified immunodominant CD8 T cell epitopes from IBVs that were protective in mice and found memory CD8 T cells directed against universal and influenza-virus-type-specific epitopes in the blood and lungs of healthy humans. Lung-derived CD8 T cells displayed tissue-resident memory phenotypes. Notably, CD38Ki67CD8 effector T cells directed against novel epitopes were readily detected in IAV- or IBV-infected pediatric and adult subjects. Our study introduces a new paradigm whereby CD8 T cells confer unprecedented cross-reactivity across all influenza viruses, a key finding for the design of universal vaccines.
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http://dx.doi.org/10.1038/s41590-019-0320-6DOI Listing
May 2019

Activity of enisamium, an isonicotinic acid derivative, against influenza viruses in differentiated normal human bronchial epithelial cells.

Antivir Chem Chemother 2018 Jan-Dec;26:2040206618811416

3 Farmak Public Joint Stock Company, Kiev, Ukraine.

Aims: New therapeutics for the control of influenza virus infections are needed to alleviate the burden caused by seasonal epidemics and occasional pandemics, and to overcome the potential risk of drug-resistance emergence. Enisamium iodide (Amizon®, Farmak) is currently approved for clinical use for the treatment of influenza in 11 countries which includes Ukraine, Russia, Belarus, Kazakhstan, and Uzbekistan. However, experimental evidence of the antiviral activity of enisamium has not been reported.

Methods: Antiviral activity of enisamium was assessed by virus yield reduction assays using differentiated normal human bronchial epithelial cells. Permeability of enisamium into differentiated normal human bronchial epithelial cells and its cytotoxicity were also assessed, and comparisons with other cell lines were made.

Results: Enisamium inhibited replication of multiple subtypes of influenza A viruses, including seasonal H1N1, 2009 pandemic H1N1, seasonal H3N2, the zoonotic H5N1 and H7N9, neuraminidase inhibitor-resistant variant carrying the H275Y NA substitution (N1 numbering), and influenza B virus at doses 23- to 64-fold lower than cytotoxic concentrations. The permeability of enisamium in Madin-Darby canine kidney cells (where no antiviral activity was found) was less than 0.08%, while higher permeability was observed in differentiated normal human bronchial epithelial cells (1.9%). The kinetics of enisamium intracellular uptake in differentiated normal human bronchial epithelial cells was concentration dependent. In time-of-addition experiments in differentiated normal human bronchial epithelial cells, enisamium treatment within 4 h after A(H1N1) virus inoculation resulted in 100-fold or greater reductions in virus titers, suggesting that it affects an early stage of the virus life cycle.

Conclusions: Enisamium exhibits antiviral activity against influenza viruses in vitro, supporting the reported clinical efficacy against influenza virus infections.
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http://dx.doi.org/10.1177/2040206618811416DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6961345PMC
June 2019

Lung γδ T Cells Mediate Protective Responses during Neonatal Influenza Infection that Are Associated with Type 2 Immunity.

Immunity 2018 09 28;49(3):531-544.e6. Epub 2018 Aug 28.

Department of Immunology, St. Jude Children's Research Hospital, Memphis, TN 38105, USA; Integrated Biomedical Sciences Program, University of Tennessee Health Science Center, Memphis, TN 38163, USA. Electronic address:

Compared to adults, infants suffer higher rates of hospitalization, severe clinical complications, and mortality due to influenza infection. We found that γδ T cells protected neonatal mice against mortality during influenza infection. γδ T cell deficiency did not alter viral clearance or interferon-γ production. Instead, neonatal influenza infection induced the accumulation of interleukin-17A (IL-17A)-producing γδ T cells, which was associated with IL-33 production by lung epithelial cells. Neonates lacking IL-17A-expressing γδ T cells or Il33 had higher mortality upon influenza infection. γδ T cells and IL-33 promoted lung infiltration of group 2 innate lymphoid cells and regulatory T cells, resulting in increased amphiregulin secretion and tissue repair. In influenza-infected children, IL-17A, IL-33, and amphiregulin expression were correlated, and increased IL-17A levels in nasal aspirates were associated with better clinical outcomes. Our results indicate that γδ T cells are required in influenza-infected neonates to initiate protective immunity and mediate lung homeostasis.
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http://dx.doi.org/10.1016/j.immuni.2018.07.011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6345262PMC
September 2018

Metabolic signaling directs the reciprocal lineage decisions of αβ and γδ T cells.

Sci Immunol 2018 07;3(25)

Department of Immunology, St. Jude Children's Research Hospital, Memphis, TN 38105, USA.

The interaction between extrinsic factors and intrinsic signal strength governs thymocyte development, but the mechanisms linking them remain elusive. We report that mechanistic target of rapamycin complex 1 (mTORC1) couples microenvironmental cues with metabolic programs to orchestrate the reciprocal development of two fundamentally distinct T cell lineages, the αβ and γδ T cells. Developing thymocytes dynamically engage metabolic programs including glycolysis and oxidative phosphorylation, as well as mTORC1 signaling. Loss of RAPTOR-mediated mTORC1 activity impairs the development of αβ T cells but promotes γδ T cell generation, associated with disrupted metabolic remodeling of oxidative and glycolytic metabolism. Mechanistically, we identify mTORC1-dependent control of reactive oxygen species production as a key metabolic signal in mediating αβ and γδ T cell development, and perturbation of redox homeostasis impinges upon thymocyte fate decisions and mTORC1-associated phenotypes. Furthermore, single-cell RNA sequencing and genetic dissection reveal that mTORC1 links developmental signals from T cell receptors and NOTCH to coordinate metabolic activity and signal strength. Our results establish mTORC1-driven metabolic signaling as a decisive factor for reciprocal αβ and γδ T cell development and provide insight into metabolic control of cell signaling and fate decisions.
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http://dx.doi.org/10.1126/sciimmunol.aas9818DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6230375PMC
July 2018

VDJdb: a curated database of T-cell receptor sequences with known antigen specificity.

Nucleic Acids Res 2018 01;46(D1):D419-D427

Pirogov Russian National Research Medical University, Moscow 117997, Russia.

The ability to decode antigen specificities encapsulated in the sequences of rearranged T-cell receptor (TCR) genes is critical for our understanding of the adaptive immune system and promises significant advances in the field of translational medicine. Recent developments in high-throughput sequencing methods (immune repertoire sequencing technology, or RepSeq) and single-cell RNA sequencing technology have allowed us to obtain huge numbers of TCR sequences from donor samples and link them to T-cell phenotypes. However, our ability to annotate these TCR sequences still lags behind, owing to the enormous diversity of the TCR repertoire and the scarcity of available data on T-cell specificities. In this paper, we present VDJdb, a database that stores and aggregates the results of published T-cell specificity assays and provides a universal platform that couples antigen specificities with TCR sequences. We demonstrate that VDJdb is a versatile instrument for the annotation of TCR repertoire data, enabling a concatenated view of antigen-specific TCR sequence motifs. VDJdb can be accessed at https://vdjdb.cdr3.net and https://github.com/antigenomics/vdjdb-db.
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http://dx.doi.org/10.1093/nar/gkx760DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5753233PMC
January 2018

De Novo Epigenetic Programs Inhibit PD-1 Blockade-Mediated T Cell Rejuvenation.

Cell 2017 Jun 22;170(1):142-157.e19. Epub 2017 Jun 22.

Department of Immunology, St. Jude Children's Research Hospital, Memphis, TN 38105, USA. Electronic address:

Immune-checkpoint-blockade (ICB)-mediated rejuvenation of exhausted T cells has emerged as a promising approach for treating various cancers and chronic infections. However, T cells that become fully exhausted during prolonged antigen exposure remain refractory to ICB-mediated rejuvenation. We report that blocking de novo DNA methylation in activated CD8 T cells allows them to retain their effector functions despite chronic stimulation during a persistent viral infection. Whole-genome bisulfite sequencing of antigen-specific murine CD8 T cells at the effector and exhaustion stages of an immune response identified progressively acquired heritable de novo methylation programs that restrict T cell expansion and clonal diversity during PD-1 blockade treatment. Moreover, these exhaustion-associated DNA-methylation programs were acquired in tumor-infiltrating PD-1hi CD8 T cells, and approaches to reverse these programs improved T cell responses and tumor control during ICB. These data establish de novo DNA-methylation programming as a regulator of T cell exhaustion and barrier of ICB-mediated T cell rejuvenation.
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http://dx.doi.org/10.1016/j.cell.2017.06.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5568784PMC
June 2017

Quantifiable predictive features define epitope-specific T cell receptor repertoires.

Nature 2017 07 21;547(7661):89-93. Epub 2017 Jun 21.

Department of Immunology, St Jude Children's Research Hospital, Memphis, Tennessee 38105, USA.

T cells are defined by a heterodimeric surface receptor, the T cell receptor (TCR), that mediates recognition of pathogen-associated epitopes through interactions with peptide and major histocompatibility complexes (pMHCs). TCRs are generated by genomic rearrangement of the germline TCR locus, a process termed V(D)J recombination, that has the potential to generate marked diversity of TCRs (estimated to range from 10 (ref. 1) to as high as 10 (ref. 2) possible receptors). Despite this potential diversity, TCRs from T cells that recognize the same pMHC epitope often share conserved sequence features, suggesting that it may be possible to predictively model epitope specificity. Here we report the in-depth characterization of ten epitope-specific TCR repertoires of CD8 T cells from mice and humans, representing over 4,600 in-frame single-cell-derived TCRαβ sequence pairs from 110 subjects. We developed analytical tools to characterize these epitope-specific repertoires: a distance measure on the space of TCRs that permits clustering and visualization, a robust repertoire diversity metric that accommodates the low number of paired public receptors observed when compared to single-chain analyses, and a distance-based classifier that can assign previously unobserved TCRs to characterized repertoires with robust sensitivity and specificity. Our analyses demonstrate that each epitope-specific repertoire contains a clustered group of receptors that share core sequence similarities, together with a dispersed set of diverse 'outlier' sequences. By identifying shared motifs in core sequences, we were able to highlight key conserved residues driving essential elements of TCR recognition. These analyses provide insights into the generalizable, underlying features of epitope-specific repertoires and adaptive immune recognition.
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http://dx.doi.org/10.1038/nature22383DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5616171PMC
July 2017

Surveillance states.

Nat Struct Mol Biol 2017 04;24(4):339-341

Department of Immunology, St. Jude Children's Research Hospital, Memphis, Tennessee, USA.

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http://dx.doi.org/10.1038/nsmb.3398DOI Listing
April 2017

Rapid cloning, expression, and functional characterization of paired αβ and γδ T-cell receptor chains from single-cell analysis.

Mol Ther Methods Clin Dev 2016 27;3:15054. Epub 2016 Jan 27.

Department of Immunology, St. Jude Children's Research Hospital, Memphis, Tennessee, USA; Integrated Biomedical Sciences Program, Department of Microbiology, Immunology and Biochemistry, University of Tennessee Health Science Center, Memphis, Tennessee, USA.

Transgenic expression of antigen-specific T-cell receptor (TCR) genes is a promising approach for immunotherapy against infectious diseases and cancers. A key to the efficient application of this approach is the rapid and specific isolation and cloning of TCRs. Current methods are often labor-intensive, nonspecific, and/or relatively slow. Here, we describe an efficient system for antigen-specific αβTCR cloning and CDR3 substitution. We demonstrate the capability of cloning influenza-specific TCRs within 10 days using single-cell polymerase chain reaction (PCR) and Gibson Assembly techniques. This process can be accelerated to 5 days by generating receptor libraries, requiring only the exchange of the antigen-specific CDR3 region into an existing backbone. We describe the construction of this library for human γδ TCRs and report the cloning and expression of a TRGV9/TRDV2 receptor that is activated by zoledronic acid. The functional activity of these αβ and γδ TCRs can be characterized in a novel reporter cell line (Nur77-GFP Jurkat 76 TCRα(-)β(-)) for screening of TCR specificity and avidity. In summary, we provide a rapid method for the cloning, expression, and functional characterization of human and mouse TCRs that can assist in the development of TCR-mediated therapeutics.
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http://dx.doi.org/10.1038/mtm.2015.54DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4729322PMC
February 2016

Single-Cell Analysis of T-Cell Receptor αβ Repertoire.

Methods Mol Biol 2015 ;1343:181-97

Department of Immunology, St. Jude Children's Research Hospital, 262 Danny Thomas Place/MS 351‬, Memphis, TN, 38105, USA.

The unbiased, paired analysis of T-cell receptor (TCR) α- and β-chain usage at the single-cell level provides a valuable window of understanding into the TCR repertoire and the nature of the immune response. Earlier technologies for TCR repertoire analysis were often limited to examining TCR complementarity-determining region 3 (CDR3) β expression or required in vitro cloning procedures that can artificially skew the TCR repertoire from its in vivo state. We describe here a direct ex vivo, single-cell-based strategy for the clonotypic analysis of TCRαβ repertoires that utilizes multiplexed panels of TCRα and TCRβ-specific primers in a nested PCR to amplify expressed transcripts from individual, epitope-specific T cells. This strategy yields the paired TCRαβ sequences of any given population of αβ T cells of interest.
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http://dx.doi.org/10.1007/978-1-4939-2963-4_15DOI Listing
June 2016

Paired TCRαβ analysis of virus-specific CD8(+) T cells exposes diversity in a previously defined 'narrow' repertoire.

Immunol Cell Biol 2015 Oct 25;93(9):804-14. Epub 2015 Mar 25.

Department of Microbiology and Immunology, University of Melbourne, Peter Doherty Institute of Infection and Immunity, Melbourne, VIC, Australia.

T-cell receptor (TCR) usage has an important role in determining the outcome of CD8(+) cytotoxic T-lymphocyte responses to viruses and other pathogens. However, the characterization of TCR usage from which such conclusions are drawn is based on exclusive analysis of either the TCRα chain or, more commonly, the TCRβ chain. Here, we have used a multiplexed reverse transcription-PCR protocol to analyse the CDR3 regions of both TCRα and β chains from single naive or immune epitope-specific cells to provide a comprehensive picture of epitope-specific TCR usage and selection into the immune response. Analysis of TCR repertoires specific for three influenza-derived epitopes (D(b)NP(366), D(b)PA(224) and D(b)PB1-F2(62)) showed preferential usage of particular TCRαβ proteins in the immune repertoire relative to the naive repertoire, in some cases, resulting in a complete shift in TRBV preference or CDR3 length, and restricted repertoire diversity. The NP(366)-specific TCRαβ repertoire, previously defined as clonally restricted based on TCRβ analysis, was similarly diverse as the PA(224)- and PB1-F2(62)-specific repertoires. Intriguingly, preferred TCR characteristics (variable gene usage, CDR3 length and junctional gene usage) appeared to be able to confer specificity either independently or in concert with one another, depending on the epitope specificity. These data have implications for established correlations between the nature of the TCR repertoire and response outcomes after infection, and suggest that analysis of a subset of cells or a single TCR chain does not accurately depict the nature of the antigen-specific TCRαβ repertoire.
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http://dx.doi.org/10.1038/icb.2015.44DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4618100PMC
October 2015

Host detection and the stealthy phenotype in influenza virus infection.

Curr Top Microbiol Immunol 2015 ;386:121-47

Department of Immunology, St. Jude Children's Research Hospital, Memphis, TN, 38105, USA.

The innate host response to influenza virus infection plays a critical role in determining the subsequent course of infection and the clinical outcome of disease. The host has a diverse array of detection and effector mechanisms that are able to recognize and initiate effective antiviral responses. In opposition, the virus utilizes a number of distinct mechanisms to evade host detection and effector activity in order to remain "stealthy" throughout its replication cycle. In this review, we describe these host and viral mechanisms, including the major pattern recognition receptor families (the TLRs, NLRs, and RLRs) in the host and the specific viral proteins such as NS1 that are key players in this interaction. Additionally, we explore nonreductive mechanisms of viral immune evasion and propose areas important for future inquiry.
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http://dx.doi.org/10.1007/82_2014_412DOI Listing
March 2015

Membrane association of the CD3ε signaling domain is required for optimal T cell development and function.

J Immunol 2014 Jul 4;193(1):258-67. Epub 2014 Jun 4.

Department of Immunology, St. Jude Children's Research Hospital, Memphis, TN, 38105;

The TCR:CD3 complex transduces signals that are critical for optimal T cell development and adaptive immunity. In resting T cells, the CD3ε cytoplasmic tail associates with the plasma membrane via a proximal basic-rich stretch (BRS). In this study, we show that mice lacking a functional CD3ε-BRS exhibited substantial reductions in thymic cellularity and limited CD4- CD8- double-negative (DN) 3 to DN4 thymocyte transition, because of enhanced DN4 TCR signaling resulting in increased cell death and TCR downregulation in all subsequent populations. Furthermore, positive, but not negative, T cell selection was affected in mice lacking a functional CD3ε-BRS, which led to limited peripheral T cell function and substantially reduced responsiveness to influenza infection. Collectively, these results indicate that membrane association of the CD3ε signaling domain is required for optimal thymocyte development and peripheral T cell function.
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http://dx.doi.org/10.4049/jimmunol.1400322DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4065803PMC
July 2014

A comprehensive collection of systems biology data characterizing the host response to viral infection.

Sci Data 2014 14;1:140033. Epub 2014 Oct 14.

Seattle Biomedical Research Institute , Seattle, WA 98109, USA.

The Systems Biology for Infectious Diseases Research program was established by the U.S. National Institute of Allergy and Infectious Diseases to investigate host-pathogen interactions at a systems level. This program generated 47 transcriptomic and proteomic datasets from 30 studies that investigate in vivo and in vitro host responses to viral infections. Human pathogens in the Orthomyxoviridae and Coronaviridae families, especially pandemic H1N1 and avian H5N1 influenza A viruses and severe acute respiratory syndrome coronavirus (SARS-CoV), were investigated. Study validation was demonstrated via experimental quality control measures and meta-analysis of independent experiments performed under similar conditions. Primary assay results are archived at the GEO and PeptideAtlas public repositories, while processed statistical results together with standardized metadata are publically available at the Influenza Research Database (www.fludb.org) and the Virus Pathogen Resource (www.viprbrc.org). By comparing data from mutant versus wild-type virus and host strains, RNA versus protein differential expression, and infection with genetically similar strains, these data can be used to further investigate genetic and physiological determinants of host responses to viral infection.
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http://dx.doi.org/10.1038/sdata.2014.33DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4410982PMC
December 2015

Characterization of innate responses to influenza virus infection in a novel lung type I epithelial cell model.

J Gen Virol 2014 Feb 16;95(Pt 2):350-362. Epub 2013 Nov 16.

Seattle Biomedical Research Institute, 307 Westlake Avenue North, Suite 500, Seattle, WA 98109, USA.

Type I alveolar epithelial cells are a replicative niche for influenza in vivo, yet their response to infection is not fully understood. To better characterize their cellular responses, we have created an immortalized murine lung epithelial type I cell line (LET1). These cells support spreading influenza virus infection in the absence of exogenous protease and thus permit simultaneous analysis of viral replication dynamics and host cell responses. LET1 cells can be productively infected with human, swine and mouse-adapted strains of influenza virus and exhibit expression of an antiviral transcriptional programme and robust cytokine secretion. We characterized influenza virus replication dynamics and host responses of lung type I epithelial cells and identified the capacity of epithelial cell-derived type I IFN to regulate specific modules of antiviral effectors to establish an effective antiviral state. Together, our results indicate that the type I epithelial cell can play a major role in restricting influenza virus infection without contribution from the haematopoietic compartment.
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http://dx.doi.org/10.1099/vir.0.058438-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3917066PMC
February 2014

Differential host response, rather than early viral replication efficiency, correlates with pathogenicity caused by influenza viruses.

PLoS One 2013 20;8(9):e74863. Epub 2013 Sep 20.

Seattle Biomedical Research Institute, Seattle, Washington, United States of America.

Influenza viruses exhibit large, strain-dependent differences in pathogenicity in mammalian hosts. Although the characteristics of severe disease, including uncontrolled viral replication, infection of the lower airway, and highly inflammatory cytokine responses have been extensively documented, the specific virulence mechanisms that distinguish highly pathogenic strains remain elusive. In this study, we focused on the early events in influenza infection, measuring the growth rate of three strains of varying pathogenicity in the mouse airway epithelium and simultaneously examining the global host transcriptional response over the first 24 hours. Although all strains replicated equally rapidly over the first viral life-cycle, their growth rates in both lung and tracheal tissue strongly diverged at later times, resulting in nearly 10-fold differences in viral load by 24 hours following infection. We identified separate networks of genes in both the lung and tracheal tissues whose rapid up-regulation at early time points by specific strains correlated with a reduced viral replication rate of those strains. The set of early-induced genes in the lung that led to viral growth restriction is enriched for both NF-κB binding site motifs and members of the TREM1 and IL-17 signaling pathways, suggesting that rapid, NF-κB -mediated activation of these pathways may contribute to control of viral replication. Because influenza infection extending into the lung generally results in severe disease, early activation of these pathways may be one factor distinguishing high- and low-pathogenicity strains.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0074863PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3779241PMC
May 2014

T cell receptor αβ diversity inversely correlates with pathogen-specific antibody levels in human cytomegalovirus infection.

Sci Transl Med 2012 Apr;4(128):128ra42

Division of Geriatric Medicine and Gerontology, Biology of Healthy Aging Program, Johns Hopkins University School of Medicine, 5505 Hopkins Bayview Circle, Baltimore, MD 21224, USA.

A diverse T cell receptor (TCR) repertoire capable of recognizing a broad range of antigenic peptides is thought to be central to effective pathogen-specific immunity by counteracting escape mutations, selecting high-avidity T cells, and providing T cell specificities with comprehensive functional characteristics. However, evidence that TCR diversity is important for the successful control of human infections is limited. A single-cell strategy for the clonotypic analysis of human CD8⁺ TCRαβ repertoires was used to probe the diversity and magnitude of individual human cytomegalovirus (CMV)-specific CD8⁺ T cells recovered directly ex vivo. We found that CD8⁺ TCRαβ repertoire diversity, but not the size of the CD8⁺ T cell response, was inversely related to circulating CMV-specific antibody levels, a measure that has been correlated epidemiologically with differential mortality risks and found here to be higher in persons with detectable (versus undetectable) CMV viral loads. Overall, our findings indicate that CD8⁺ T cell diversity may be more important than T cell abundance in limiting the negative consequences of CMV persistence, demonstrate high prevalence of both TCRα and TCRβ public motif usage, and suggest that a highly diverse TCRαβ repertoire may be an important benchmark and target in the success of immunotherapeutic strategies.
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http://dx.doi.org/10.1126/scitranslmed.3003647DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3593633PMC
April 2012

Enhanced susceptibility of Ago1/3 double-null mice to influenza A virus infection.

J Virol 2012 Apr 8;86(8):4151-7. Epub 2012 Feb 8.

Department of Immunology, St. Jude Children’s Research Hospital, Memphis, Tennessee, USA.

RNA interference (RNAi) is a critical component of many cellular antiviral responses in plants, invertebrates, and mammals. However, its in vivo role in host protection from the negative-sense RNA virus influenza virus type A (flu) is unclear. Here we have examined the role of RNAi in host defense to flu by analyzing Argonaute 1 and 3 double-knockout mice deficient in components of the RNA-induced silencing complex. Compared to littermate controls, flu-infected double-knockout mice exhibited increased mortality, consistent with more severe alveolitis and pneumonitis. These data indicate that optimal resistance to flu requires Argonaute 1 and/or 3. Enhanced mortality of double-knockout mice was not associated either with increased viral replication or with differential pulmonary recruitment or function of innate and adaptive immune cells. Given the absence of detectable immune defects, our results support the notion that the enhanced flu susceptibility of double-knockout mice arises from an intrinsic impairment in the ability of lung cells to tolerate flu-elicited inflammation.
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http://dx.doi.org/10.1128/JVI.05303-11DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3318639PMC
April 2012

Neonatal CD8 T-cell hierarchy is distinct from adults and is influenced by intrinsic T cell properties in respiratory syncytial virus infected mice.

PLoS Pathog 2011 Dec 1;7(12):e1002377. Epub 2011 Dec 1.

Vaccine Research Center, National Institute of Allergy and Infectious Disease, National Institutes of Health, Bethesda, Maryland, United States of America.

Following respiratory syncytial virus infection of adult CB6F1 hybrid mice, a predictable CD8+ T cell epitope hierarchy is established with a strongly dominant response to a K(d)-restricted peptide (SYIGSINNI) from the M2 protein. The response to K(d)M2(82-90) is ∼5-fold higher than the response to a subdominant epitope from the M protein (NAITNAKII, D(b)M(187-195)). After infection of neonatal mice, a distinctly different epitope hierarchy emerges with codominant responses to K(d)M2(82-90) and D(b)M(187-195). Adoptive transfer of naïve CD8+ T cells from adults into congenic neonates prior to infection indicates that intrinsic CD8+ T cell factors contribute to age-related differences in hierarchy. Epitope-specific precursor frequency differs between adults and neonates and influences, but does not predict the hierarchy following infection. Additionally, dominance of K(d)M2(82-90)-specific cells does not correlate with TdT activity. Epitope-specific Vβ repertoire usage is more restricted and functional avidity is lower in neonatal mice. The neonatal pattern of codominance changes after infection at 10 days of age, and rapidly shifts to the adult pattern of extreme K(d)M2(82-90)-dominance. Thus, the functional properties of T cells are selectively modified by developmental factors in an epitope-specific and age-dependent manner.
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http://dx.doi.org/10.1371/journal.ppat.1002377DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3228797PMC
December 2011

Quantitative impact of thymic selection on Foxp3+ and Foxp3- subsets of self-peptide/MHC class II-specific CD4+ T cells.

Proc Natl Acad Sci U S A 2011 Aug 22;108(35):14602-7. Epub 2011 Aug 22.

Department of Microbiology and Center for Immunology, University of Minnesota Medical School, Minneapolis, MN 55455, USA.

It is currently thought that T cells with specificity for self-peptide/MHC (pMHC) ligands are deleted during thymic development, thereby preventing autoimmunity. In the case of CD4(+) T cells, what is unclear is the extent to which self-peptide/MHC class II (pMHCII)-specific T cells are deleted or become Foxp3(+) regulatory T cells. We addressed this issue by characterizing a natural polyclonal pMHCII-specific CD4(+) T-cell population in mice that either lacked or expressed the relevant antigen in a ubiquitous pattern. Mice expressing the antigen contained one-third the number of pMHCII-specific T cells as mice lacking the antigen, and the remaining cells exhibited low TCR avidity. In mice lacking the antigen, the pMHCII-specific T-cell population was dominated by phenotypically naive Foxp3(-) cells, but also contained a subset of Foxp3(+) regulatory cells. Both Foxp3(-) and Foxp3(+) pMHCII-specific T-cell numbers were reduced in mice expressing the antigen, but the Foxp3(+) subset was more resistant to changes in number and TCR repertoire. Therefore, thymic selection of self-pMHCII-specific CD4(+) T cells results in incomplete deletion within the normal polyclonal repertoire, especially among regulatory T cells.
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http://dx.doi.org/10.1073/pnas.1109806108DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3167500PMC
August 2011

Clonally related CD8+ T cells responsible for rapid population of both diffuse nasal-associated lymphoid tissue and lung after respiratory virus infection.

J Immunol 2011 Jul 20;187(2):835-41. Epub 2011 Jun 20.

Department of Infectious Diseases, St. Jude Children's Research Hospital, Memphis, TN 38105, USA.

The immune system has evolved to use sophisticated mechanisms to recruit lymphocytes to sites of pathogen exposure. Trafficking pathways are precise. For example, lymphocytes that are primed by gut pathogens can, in some cases, be imprinted with CCR9 membrane receptors, which can influence migration to the small intestine. Currently, little is known about T cell trafficking to the upper respiratory tract or the relationship between effectors that migrate to the diffuse nasal-associated lymphoid tissue (d-NALT), the lower airways, and the lung. To determine whether a T cell primed by Ag from a respiratory pathogen is imprinted for exclusive trafficking to the upper or lower respiratory tract or whether descendents from that cell have the capacity to migrate to both sites, we inoculated mice by the intranasal route with Sendai virus and conducted single-cell-sequencing analyses of CD8(+) T lymphocytes responsive to a K(b)-restricted immunodominant peptide, FAPGNYPAL (Tet(+)). Cells from the d-NALT, lung airways (bronchoalveolar lavage), lung, and mediastinal lymph node were examined 10 d postinfection to determine TCR usage and clonal relationships. We discovered that 1) Tet(+) cells were heterogeneous but preferentially used TCR elements TRAV6, TRAV16, and TRBD1; 2) both N and C termini of Vα and Vβ TCR junctions frequently encompassed charged residues, perhaps facilitating TCR αβ pairing and interactions with a neutral target peptide; and 3) T cells in the d-NALT were often clonally related to cells in the lower respiratory tract.
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http://dx.doi.org/10.4049/jimmunol.1100125DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3131409PMC
July 2011

Paired analysis of TCRα and TCRβ chains at the single-cell level in mice.

J Clin Invest 2011 Jan 6;121(1):288-95. Epub 2010 Dec 6.

St Jude Children’s Research Hospital, Memphis, Tennessee 38105-3678, USA.

Characterizing the TCRα and TCRβ chains expressed by T cells responding to a given pathogen or underlying autoimmunity helps in the development of vaccines and immunotherapies, respectively. However, our understanding of complementary TCRα and TCRβ chain utilization is very limited for pathogen- and autoantigen-induced immunity. To address this problem, we have developed a multiplex nested RT-PCR method for the simultaneous amplification of transcripts encoding the TCRα and TCRβ chains from single cells. This multiplex method circumvented the lack of antibodies specific for variable regions of mouse TCRα chains and the need for prior knowledge of variable region usage in the TCRβ chain, resulting in a comprehensive, unbiased TCR repertoire analysis with paired coexpression of TCRα and TCRβ chains with single-cell resolution. Using CD8+ CTLs specific for an influenza epitope recovered directly from the pneumonic lungs of mice, this technique determined that 25% of such effectors expressed a dominant, nonproductively rearranged Tcra transcript. T cells with these out-of-frame Tcra mRNAs also expressed an alternate, in-frame Tcra, whereas approximately 10% of T cells had 2 productive Tcra transcripts. The proportion of cells with biallelic transcription increased over the course of a response, a finding that has implications for immune memory and autoimmunity. This technique may have broad applications in mouse models of human disease.
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http://dx.doi.org/10.1172/JCI44752DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3007160PMC
January 2011

T Cell receptor clonotype influences epitope hierarchy in the CD8+ T cell response to respiratory syncytial virus infection.

J Biol Chem 2011 Feb 30;286(6):4829-41. Epub 2010 Nov 30.

Vaccine Research Center, NIAID, National Institutes of Health, Bethesda, Maryland 20892-3017, USA.

CD8+ T cell responses are important for recognizing and resolving viral infections. To better understand the selection and hierarchy of virus-specific T cell responses, we compared the T cell receptor (TCR) clonotype in parent and hybrid strains of respiratory syncytial virus-infected mice. K(d)M2(82-90) (SYIGSINNI) in BALB/c and D(b)M(187-195) (NAITNAKII) in C57Bl/6 are both dominant epitopes in parent strains but assume a distinct hierarchy, with K(d)M2(82-90) dominant to D(b)M(187-195) in hybrid CB6F1/J mice. The dominant K(d)M2(82-90) response is relatively public and is restricted primarily to the highly prevalent Vβ13.2 in BALB/c and hybrid mice, whereas D(b)M(187-195) responses in C57BL/6 mice are relatively private and involve multiple Vβ subtypes, some of which are lost in hybrids. A significant frequency of TCR CDR3 sequences in the D(b)M(187-195) response have a distinct "(D/E)WG" motif formed by a limited number of recombination strategies. Modeling of the dominant epitope suggested a flat, featureless structure, but D(b)M(187-195) showed a distinctive structure formed by Lys(7). The data suggest that common recombination events in prevalent Vβ genes may provide a numerical advantage in the T cell response and that distinct epitope structures may impose more limited options for successful TCR selection. Defining how epitope structure is interpreted to inform T cell function will improve the design of future gene-based vaccines.
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http://dx.doi.org/10.1074/jbc.M110.191437DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3039322PMC
February 2011

Foot-and-mouth disease virus replicates only transiently in well-differentiated porcine nasal epithelial cells.

J Virol 2010 Sep 30;84(18):9149-60. Epub 2010 Jun 30.

Institute for Animal Health, Pirbright Laboratory, Pirbright, Woking, Surrey GU24 0NF, United Kingdom.

Three-dimensional (3D) porcine nasal mucosal and tracheal mucosal epithelial cell cultures were developed to analyze foot-and-mouth disease virus (FMDV) interactions with mucosal epithelial cells. The cells in these cultures differentiated and polarized until they closely resemble the epithelial layers seen in vivo. FMDV infected these cultures predominantly from the apical side, primarily by binding to integrin alphav beta6, in an Arg-Gly-Asp (RGD)-dependent manner. However, FMDV replicated only transiently without any visible cytopathic effect (CPE), and infectious progeny virus could be recovered only from the apical side. The infection induced the production of beta interferon (IFN-beta) and the IFN-inducible gene Mx1 mRNA, which coincided with the disappearance of viral RNA and progeny virus. The induction of IFN-beta mRNA correlated with the antiviral activity of the supernatants from both the apical and basolateral compartments. IFN-alpha mRNA was constitutively expressed in nasal mucosal epithelial cells in vitro and in vivo. In addition, FMDV infection induced interleukin 8 (IL-8) protein, granulocyte-macrophage colony-stimulating factor (GM-CSF), and RANTES mRNA in the infected epithelial cells, suggesting that it plays an important role in modulating the immune response.
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http://dx.doi.org/10.1128/JVI.00642-10DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2937594PMC
September 2010

The intracellular sensor NLRP3 mediates key innate and healing responses to influenza A virus via the regulation of caspase-1.

Immunity 2009 Apr 9;30(4):566-75. Epub 2009 Apr 9.

Department of Immunology, St. Jude Children's Research Hospital, Memphis, TN 38105, USA.

Virus-induced interlukin-1beta (IL-1beta) and IL-18 production in macrophages are mediated via caspase-1 pathway. Multiple microbial components, including viral RNA, are thought to trigger assembly of the cryopyrin inflammasome resulting in caspase-1 activation. Here, we demonstrated that Nlrp3(-/-) and Casp1(-/-) mice were more susceptible than wild-type mice after infection with a pathogenic influenza A virus. This enhanced morbidity correlated with decreased neutrophil and monocyte recruitment and reduced cytokine and chemokine production. Despite the effect on innate immunity, cryopyrin-deficiency was not associated with any obvious defect in virus control or on the later emergence of the adaptive response. Early epithelial necrosis was, however, more severe in the infected mutants, with extensive collagen deposition leading to later respiratory compromise. These findings reveal a function of the cryopyrin inflammasome in healing responses. Thus, cryopyrin and caspase-1 are central to both innate immunity and to moderating lung pathology in influenza pneumonia.
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http://dx.doi.org/10.1016/j.immuni.2009.02.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2765464PMC
April 2009

Full genome sequences of two virulent strains of peste-des-petits ruminants virus, the Côte d'Ivoire 1989 and Nigeria 1976 strains.

Virus Res 2008 Sep 9;136(1-2):192-7. Epub 2008 Jun 9.

Pirbright Laboratory, Institute for Animal Health, Ash Road, Woking, Surrey GU24 0NF, United Kingdom.

Peste-des-petits ruminants virus (PPRV) causes acute febrile illness in both farmed and wild small ruminants, with associated mortality rates of 50-80%. PPRV is a member of the Morbillivirus genus within the Paramyxovirus family and although there are many full length genome sequences available for members of this family, their availability for PPRV in particular is limited. We have determined the full length sequences representing two virulent strains of PPRV, the Côte d'Ivoire 1989 (CI/89) and Nigeria 1976 (Ng76/1) strains. We present an alignment of the promoter regions of these viruses with other available PPRV promoter sequences and have identified domains in PPRV proteins believed to be critical for paramyxovirus promoter attenuation. We have also analysed the proteins of these viruses, comparing them to other available PPRV protein sequences and identified motifs that were previously recognised as being required for the function of other paramyxovirus proteins.
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http://dx.doi.org/10.1016/j.virusres.2008.04.018DOI Listing
September 2008

Reverse genetics for peste-des-petits-ruminants virus (PPRV): promoter and protein specificities.

Virus Res 2007 Jun 9;126(1-2):250-5. Epub 2007 Mar 9.

Institute for Animal Health, Pirbright Laboratory, Ash Road, Woking, Surrey GU24 ONF, United Kingdom.

Peste-des-petits-ruminants virus (PPRV) (family Paramyxoviridae, genus Morbillivirus) causes an acute febrile illness in sheep and goats resulting in significant morbidity and mortality in infected herds. The paramyxoviruses all have negative sense, non-segmented RNA genomes and their host range and pathogenic determinants have been extensively studied using reverse genetics. This technology also enables a more rational approach to be taken with respect to vaccine design. In order to initiate this type of work for PPRV we constructed a PPRV minigenome and studied its expression in transfected cells. As for other morbilliviruses, the minimum requirements for minigenome rescue were shown to be the cis-acting elements of the genome (GP) and antigenome (AGP) promoters as well as the three trans-acting helper proteins N (nucleocapsid), P (phosphoprotein) and L (large polymerase). Homologous PPRV helper proteins were compared to their heterologous analogues from the closely related rinderpest virus (RPV) and heterologous minigenome rescue was found to be a much less efficient process. By engineering two GP/AGP chimeric minigenomes we also identified differences between the two viruses in the specific interactions between the promoters and the transcriptase/replicase complexes. The PPRV minigenome was also shown not to strictly comply with the "rule of six"in vitro.
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http://dx.doi.org/10.1016/j.virusres.2007.01.015DOI Listing
June 2007

Full genome sequence of peste des petits ruminants virus, a member of the Morbillivirus genus.

Virus Res 2005 Jun;110(1-2):119-24

Pirbright Laboratory, Institute for Animal Health, Ash Road, Woking, Surrey GU24 ONF, UK.

Peste des petits ruminants virus (PPRV) causes an acute febrile illness in small ruminant species, mostly sheep and goats. PPRV is a member of the Morbillivirus genus which includes measles, rinderpest (cattle plague), canine distemper, phocine distemper and the morbilliviruses found in whales, porpoises and dolphins. Full length genome sequences for these morbilliviruses are available and reverse genetic rescue systems have been developed for the viruses of terrestrial mammals, with the exception of PPRV. This paper presents the first published full length genome sequence for PPRV. The genome was found to be consistent with the rule-of-six and open reading frames (ORFs) were identified that encoded the eight proteins characteristic of morbilliviruses. At the nucleotide (nt) level, the full length genome of PPRV was most similar to that of rinderpest, the other ruminant morbillivirus. However, at the protein level five of the six structural proteins and the V protein showed a greater similarity to the dolphin morbillivirus (DMV) while only the C and L proteins showed a high relationship to rinderpest.
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http://dx.doi.org/10.1016/j.virusres.2005.01.013DOI Listing
June 2005
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