Longitudinal Assessment of SARS-CoV-2 Specific T Cell Cytokine-Producing Responses for 1 Year Reveals Persistence of Multi-Cytokine Proliferative Responses, with Greater Immunity Associated with Disease Severity

  1. Jonah Lin
  2. Ryan Law
  3. Chapin S. Korosec
  4. Christine Zhou
  5. Wan Hon Koh
  6. Mohammad Sajjad Ghaemi
  7. Philip Samaan
  8. Hsu Kiang Ooi
  9. FengYun Yue
  10. Anne-Claude Gingras
  11. Antonio Estacio
  12. Megan Buchholz
  13. Patti Lou Cheatley
  14. Katerina Pavinski
  15. Samira Mubareka
  16. Allison J. McGeer
  17. Jerome A. Leis
  18. Jane M. Heffernan
  19. Mario Ostrowski

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Abstract

Cellular-mediated immunity is critical for long-term protection against most viral infections, including coronaviruses. We studied 23 SARS-CoV-2-infected survivors over a one year post symptom onset (PSO) interval by ex vivo cytokine ELISpot assay. All subjects demonstrated SARS-CoV-2-specific IFN-γ, IL-2, and Granzyme B (GzmB) T cell responses at presentation, with greater frequencies in severe disease. Cytokines, mainly produced by CD4+ T cells, targeted all structural proteins (Nucleocapsid, Membrane, Spike) except Envelope, with GzmB > IL-2 > IFN-γ. Mathematical modeling predicted that: 1) cytokine responses peaked at 6 days for IFN-γ, 36 days for IL-2, and 7 days for GzmB, 2) severe illness was associated with reduced IFN-γ and GzmB, but increased IL-2 production rates, 3) males displayed greater production of IFN-γ, whereas females produced more GzmB. Ex vivo responses declined over time with persistence of IL-2 in 86% and of IFN-γ and GzmB in 70% of subjects at a median of 336 days PSO. The average half-life of SARS-CoV-2-specific cytokine-producing cells was modelled to be 139 days (∼4.6 months). Potent T cell proliferative responses persisted throughout observation, were CD4 dominant, and were capable of producing all 3 cytokines. Several immunodominant CD4 and CD8 epitopes identified in this study were shared by seasonal coronaviruses or SARS-CoV-1 in the Nucleocapsid and Membrane regions. Both SARS-CoV-2-specific CD4+ and CD8+ T cell clones were able to kill target cells, though CD8 tended to be more potent.

Importance

Our findings highlight the relative importance of SARS-CoV-2-specific GzmB-producing T cell responses in SARS-CoV-2 control, shared CD4 and CD8 immunodominant epitopes in seasonal coronaviruses or SARS-CoV-1, and indicate robust persistence of T cell memory at least one year after infection. Our findings should inform future strategies to induce T cell vaccines against SARS-CoV-2 and other coronaviruses.

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  1. ScreenIT

    SciScore for 10.1101/2022.01.18.476864: (What is this?)

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

    Table 1: Rigor

    EthicsConsent: Human Subjects and Study Approval: Healthy subjects and individuals with COVID-19 infection as diagnosed by positive nasopharyngeal SARS-CoV-2 PCR were recruited and informed consent was obtained from for blood draws and/or leukapheresis through an REB approved protocol (St.
    Sex as a biological variablenot detected.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    Ex Vivo ELISpot Assay: Ex vivo ELISpot assays were performed using human IFN-γ, IL-2, and granzyme B (GzmB) antibodies (Mabtech).
    IL-2
    suggested: None
    GzmB
    suggested: None
    Briefly, MSIPS4W plates (Millipore) were activated with 35% ethanol and coated with primary antibody: 10 μg/mL for IFN-γ (1-D1K), 10 μg/mL for IL-2 (MT2A91/2C95), and 15 μg/mL for GzmB (MT28).
    MT2A91/2C95
    suggested: None
    GzmB (MT28
    suggested: None
    After incubation, plates were washed with PBS containing 0.05% Tween-20 (BioShop) before the addition of human IFN-γ (7-B6-1), IL-2 (MT8G10), and/or GzmB (MT8610) biotinylated secondary antibodies.
    MT8610
    suggested: None
    For dual colour assays, alkaline phosphatase (ALP) conjugated to IFN-γ secondary antibodies were used with other biotinylated secondary antibodies targeting another cytokine.
    ALP
    suggested: None
    IFN-γ
    suggested: None
    T cells were then co-cultured with irradiated feeder cells containing allogeneic PBMC and BCL, 50 U/mL IL-2 (R&D Systems), 10 ng/mL IL-15 (R&D Systems), and 25 ng/mL mouse anti-human CD3 antibody (UCHT1; BD Bioscience).
    anti-human CD3
    suggested: None
    To determine HLA II-restricted epitopes from CD4+ T cell clones or lines, autologous BCL were blocked with 10 µg/mL of either anti-HLA-A/B/C (BD Biosciences), anti-HLA-DP (Abcam), anti-HLA-DQ (BioLegend), anti-HLA-DR (BioLegend), or anti-HLA-DP/DQ/DR (BioLegend) antibodies for 30 minutes, then pulsed with the specific peptide for 1 hour at 10 µg/mL.
    anti-HLA-A/B/C
    suggested: (Santa Cruz Biotechnology Cat# sc-52422, RRID:AB_629948)
    anti-HLA-DP
    suggested: None
    anti-HLA-DQ
    suggested: None
    anti-HLA-DR
    suggested: None
    anti-HLA-DP/DQ/DR
    suggested: None
    Pulsed BCL (1 x 103 cells/well) were then washed 3 times with R10, mixed with CD4+ T cells (1-2 x 104 cells/well), and tested in IFN-γ ELISpot assay in the presence of 10 µg/mL of anti-HLA antibodies.
    anti-HLA
    suggested: None
    On day 7, PBMCs were first stained with LIVE/DEAD™ fixable blue dead cell stain (Thermo Fisher Scientific) and incubated with Fc receptor blocking solution (Human TruStain FcXTM; BioLegend) before surface staining with fluorochrome-conjugated antibodies to CD3 (APC-Cy7: Clone SK7), CD4 (PerCP-Cy5.5: Clone SK3), and CD8 (PE: Clone HIT8a).
    CD3
    suggested: None
    APC-Cy7
    suggested: None
    CD8
    suggested: None
    Cells were then stained with LIVE/DEAD™ fixable near IR cell stain (Thermo Fisher Scientific) and incubated with Fc receptor blocking solution (Human TruStain FcXTM; BioLegend) before surface staining with fluorochrome-conjugated antibodies to CD4 (BV711: Clone SK3) and CD8 (PE: Clone HIT8a).
    CD4
    suggested: None
    BV711
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    Frozen PBMCs were thawed and incubated with 2.5 mL of supernatant from B95-8 cell lines for 2 hours in a 37°C water bath.
    B95-8
    suggested: None
    Software and Algorithms
    SentencesResources
    After incubation, plates were washed with PBS containing 0.05% Tween-20 (BioShop) before the addition of human IFN-γ (7-B6-1), IL-2 (MT8G10), and/or GzmB (MT8610) biotinylated secondary antibodies.
    BioShop
    suggested: None
    Cells were then fixed using BD Cytofix/Cytoperm and permeabilized using BD Perm/Wash according to the manufacturer’s protocol before staining with anti-IFN-γ (APC: Clone 4S.B3), anti-GzmB (BV421: Clone GB11), and anti-IL-2 (BV711: Clone MQ1-17H1).
    BD Cytofix/Cytoperm
    suggested: None
    Experimental Software and Statistical Analysis: All flow cytometry data were analyzed using FlowJo 10.8.0 software (Treestar, Ashland, OR, USA).
    FlowJo
    suggested: (FlowJo, RRID:SCR_008520)
    Prism 9.0 (GraphPad, San Diego, CA, USA) was used to perform statistical and graphical analyses.
    Prism
    suggested: (PRISM, RRID:SCR_005375)
    GraphPad
    suggested: (GraphPad Prism, RRID:SCR_002798)

    Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).

    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 did not find any issues relating to the usage of bar graphs.

    Results from JetFighter: Please consider improving the rainbow (“jet”) colormap(s) used on page 39. At least one figure is not accessible to readers with colorblindness and/or is not true to the data, i.e. not perceptually uniform.

    Results from rtransparent:
    • No conflict of interest statement was detected. If there are no conflicts, we encourage authors to explicit state so.
    • 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 10.1101/2022.01.18.476864v1 on bioRxiv