Emergence of immune escape at dominant SARS-CoV-2 killer T cell epitope

  1. Garry Dolton
  2. Cristina Rius
  3. Md Samiul Hasan
  4. Aaron Wall
  5. Barbara Szomolay
  6. Enas Behiry
  7. Thomas Whalley
  8. Joel Southgate
  9. Anna Fuller
  10. Théo Morin
  11. Katie Topley
  12. Li Rong Tan
  13. Philip J.R. Goulder
  14. Owen B. Spiller
  15. Pierre J. Rizkallah
  16. Lucy C. Jones
  17. Thomas R. Connor
  18. Andrew K. Sewell

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Abstract

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  1. Version published to 10.1016/j.cell.2022.07.002
  2. Version published to 10.1101/2021.06.21.21259010v2 on medRxiv
  3. ScreenIT

    SciScore for 10.1101/2021.06.21.21259010: (What is this?)

    Please note, not all rigor criteria are appropriate for all manuscripts.

    Table 1: Rigor

    EthicsIRB: Our CP study was given a favourable opinion by the Health Research Authority (HRA) Research Ethics Committee London – (Brighton &
    Sex as a biological variable‘M’ and ‘F’ of the patient code denotes male or female, with 3 males and 7 females used for this study.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Cell Line AuthenticationContamination: Cells were regularly tested for mycoplasma contamination (MycoAlert™, Lonza).

    Table 2: Resources

    Antibodies
    SentencesResources
    T-cells were stained with LIVE/DEAD™ fixable violet dead stain (ThermoFisher Scientific) and CD3 (BW264/56) and CD8α (BW135/80) antibodies (all from Miltenyi Biotec).
    CD3
    suggested: None
    CD8α
    suggested: None
    Spike transduced cells were firstly checked for with conjugated anti-rCD2 antibody (OX-34, BioLegend), then in follow-up experiments with 1 μg/test of unconjugated anti-SARS Spike glycoprotein antibody (1A9, Abcam, Cambridge, MA, US).
    anti-rCD2
    suggested: None
    anti-SARS Spike glycoprotein
    suggested: (Abcam Cat# ab273433, RRID:AB_2891068)
    A goat anti-mouse secondary antibody (multiple adsorbed PE conjugated Ig polyclonal; BD Biosciences, Oxford, UK) was used to detect Spike antibody staining.
    anti-mouse
    suggested: None
    , antibody directed against TNF (clone cA2, Miltenyi Biotec) and CD107a antibody (clone H4A3, BD Biosciences) to detect activation-induced degranulation of cytotoxic T-cells.
    TNF
    suggested: None
    CD107a
    suggested: None
    An optimised tetramer staining protocol involving pre-incubation with 50 nM protein kinase inhibitor, Dasatinib (Axon Medchem, Reston, VA, USA) and unconjugated anti-PE antibody (PE001, BioLegend) was used as described previously55–57.
    anti-PE
    suggested: (Santa Cruz Biotechnology Cat# sc-53749, RRID:AB_630095)
    Following tetramer staining, T-cells were stained with LIVE/DEAD™ fixable violet dead stain (ThermoFisher Scientific), anti-CD3 and anti-CD8 antibody and analysed using flow cytometry as above.
    anti-CD8
    suggested: None
    The following day, cells were stained with anti-HLA A2 antibody (clone BB7.2, BioLegend) and incubated at 37°C for 1 h.
    anti-HLA A2
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    Cells were cultured in RPMI 1640 (A549 and T2) or DMEM (HEK293T) medium supplemented with 10% FBS, 2 mM L-glutamine and 100 U/mL penicillin and 100 μg/mL streptomycin (R10 or D10 respectively; all from Merck Group).
    A549
    suggested: None
    Plasmid/PEI mixtures were incubated at room temperature for 15 min, added dropwise to the HEK293T cells (80% confluence in one well of a 6-well plate) and incubated at 37°C in a 5% CO2 humidified atmosphere.
    HEK293T
    suggested: DSMZ Cat# ACC-873, RRID:CVCL_A5EF)
    Experimental Models: Organisms/Strains
    SentencesResources
    Spike genes were designed with a rCD2 co-marker: Xba1-Kozak-Spike-Xho1-P2A-rCD2-Sal1-Stop, and cloned in to pSF (Oxgene, Oxford Genetics Ltd, Littlemore, Oxford, UK).
    rCD2 co-marker: Xba1-Kozak-Spike-Xho1-P2A-rCD2-Sal1-Stop
    suggested: None
    Recombinant DNA
    SentencesResources
    The pSF plasmid was modified by removal of a Xho1 site present in the plasmid backbone and insertion of stop codons after the Sal1 site.
    pSF
    suggested: None
    For transfection, pSF or pELNS (1.52 μg), envelope plasmid (pMD2.G; 0.72 μg) and packaging plasmids (pMDLg/pRRE; 1.83μg and pRSV-REV; 1.83μg) were mixed in 300 μL of Opti-MEM, reduced serum medium (ThermoFisher Scientific) followed by mixing with 1 μg/μl Polyethylenimine (PEI; Merck Group) at a 3:1 PEI: plasmid ratio.
    pMD2
    suggested: None
    pRSV-REV
    suggested: RRID:Addgene_12253)
    Software and Algorithms
    SentencesResources
    Samples were acquired on a FACSCantoII machine (BD Biosciences, Franklin Lakes, NJ, USA) and analyzed in the FlowJo software (Tree Star Inc.
    FlowJo
    suggested: (FlowJo, RRID:SCR_008520)
    Purified products were sequenced on an Illumina MiSeq instrument using the MiSeq v2 reagent kit (Illumina, Cambridge, UK)
    MiSeq
    suggested: (A5-miseq, RRID:SCR_012148)
    Data display: Unless stated otherwise, all data were displayed using GraphPad Prism software.
    GraphPad Prism
    suggested: (GraphPad Prism, RRID:SCR_002798)

    Results from OddPub: Thank you for sharing your code.

    Results from LimitationRecognizer: An explicit section about the limitations of the techniques employed in this study was not found. We encourage authors to address study limitations.

    Results from TrialIdentifier: No clinical trial numbers were referenced.

    Results from Barzooka: We found bar graphs of continuous data. We recommend replacing bar graphs with more informative graphics, as many different datasets can lead to the same bar graph. The actual data may suggest different conclusions from the summary statistics. For more information, please see Weissgerber et al (2015).

    Results from JetFighter: We did not find any issues relating to colormaps.

    Results from rtransparent:
    • Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
    • Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
    • No protocol registration statement was detected.

    Results from scite Reference Check: We found no unreliable references.

    About SciScore

    SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.

  4. Version published to 10.1101/2021.06.21.21259010v1 on medRxiv