Protracted yet coordinated differentiation of long-lived SARS-CoV-2-specific CD8+ T cells during COVID-19 convalescence

  1. Tongcui Ma
  2. Heeju Ryu
  3. Matthew McGregor
  4. Benjamin Babcock
  5. Jason Neidleman
  6. Guorui Xie
  7. Ashley F. George
  8. Julie Frouard
  9. Victoria Murray
  10. Gurjot Gill
  11. Eliver Ghosn
  12. Evan Newell
  13. Sulggi Lee
  14. Nadia R. Roan

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Abstract

CD8+ T cells are important antiviral effectors that can potentiate long-lived immunity against COVID-19, but a detailed characterization of these cells has been hampered by technical challenges. We screened 21 well-characterized, longitudinally-sampled convalescent donors that recovered from mild COVID-19 against a collection of SARS-CoV-2 tetramers, and identified one participant with an immunodominant response against Nuc 322-331 , a peptide that is conserved in all the SARS-CoV-2 variants-of-concern reported to date. We conducted 38- parameter CyTOF phenotyping on tetramer-identified Nuc 322-331 -specific CD8+ T cells, and on CD4+ and CD8+ T cells recognizing the entire nucleocapsid and spike proteins from SARS- CoV-2, and took 32 serological measurements on longitudinal specimens from this participant. We discovered a coordination of the Nuc 322-331 -specific CD8+ T response with both the CD4+ T cell and antibody pillars of adaptive immunity. Nuc 322-331 -specific CD8+ T cells were predominantly central memory T cells, but continually evolved over a ∼6-month period of convalescence. We observed a slow and progressive decrease in the activation state and polyfunctionality of the Nuc 322-331 -specific CD8+ T cells, accompanied by an increase in their lymph-node homing and homeostatic proliferation potential. These results suggest that following a typical case of mild COVID-19, SARS-CoV-2-specific CD8+ T cells not only persist but continuously differentiate in a coordinated fashion well into convalescence, into a state characteristic of long-lived, self-renewing memory.

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    SciScore for 10.1101/2021.04.28.441880: (What is this?)

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

    Table 1: Rigor

    EthicsConsent: Informed consent was obtained from all subjects.
    Sex as a biological variablenot detected.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    Antibodies that required in-house conjugation were conjugated to their corresponding metal isotopes using X8 antibody labeling kits according to manufacturer’s instructions (Fluidigm).
    X8
    suggested: (BMA Biomedicals Cat# T-1046, RRID:AB_1227261)
    Incubation of antigen-bead complexes with patient sera, and subsequent staining by fluorescently-conjugated, isotype-specific antibodies, produces a flow cytometric readout of bead fluorescence which reveals the levels of antigen-specific antibodies and their isotypes.
    antigen-specific
    suggested: None
    The assay was calibrated using mouse monoclonal (IgG2B) antibodies raised against the RBD/S1/S2/NP antigens.
    IgG2B
    suggested: None
    The calibration revealed high specificity and no cross-reactivity between antigens, with the exception of cross- reactivity of anti-RBD antibodies against S1, which was expected as RBD is contained within S1.
    anti-RBD
    suggested: None
    S1
    suggested: None
    Non-specific antibody binding was assessed by incubation of plasma with uncoated (antigen-free) beads.
    antigen-free
    suggested: None
    Software and Algorithms
    SentencesResources
    The antibody cocktail consisted of APC/Cy7-CD3 (SK7, Biolegend), BD Horizonä BV650-CD8 (RPA-T8, BD Biosciences), BD Horizonä BUV737-CD4 (SK3, BD Biosciences), and the LIVE/DEAD Zombie Aqua™
    BD Biosciences
    suggested: (BD Biosciences, RRID:SCR_013311)
    CyTOF data analysis: The CyTOF datasets were normalized to EQTM calibration beads using Fluidigm’s CyTOF software, exported as FCS files, and imported into FlowJo (BD) and Cytobank for gating and downstream analysis.
    FlowJo
    suggested: (FlowJo, RRID:SCR_008520)
    Cytobank was used to calculate the median signal intensity (MSI) of cell populations based on standard two-dimensional dot plots.
    Cytobank
    suggested: (Cytobank, RRID:SCR_014043)
    All of the phenotyping markers were used in tSNE and FlowSOM analysis, except for CD19, which was a parameter used in the upstream gating strategy.
    FlowSOM
    suggested: (FlowSOM, RRID:SCR_016899)
    Line graphs were generated using ggplot2 in R.
    ggplot2
    suggested: (ggplot2, RRID:SCR_014601)

    Results from OddPub: Thank you for sharing your data.

    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

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  2. Version published to 10.1101/2021.04.28.441880 on bioRxiv