Systematic Examination of Antigen-Specific Recall T Cell Responses to SARS-CoV-2 versus Influenza Virus Reveals a Distinct Inflammatory Profile

  1. Jaclyn C Law
  2. Wan Hon Koh
  3. Patrick Budylowski
  4. Jonah Lin
  5. FengYun Yue
  6. Kento T Abe
  7. Bhavisha Rathod
  8. Melanie Girard
  9. Zhijie Li
  10. James M Rini
  11. Samira Mubareka
  12. Allison McGeer
  13. Adrienne K Chan
  14. Anne-Claude Gingras
  15. Tania H Watts
  16. Mario A. Ostrowski

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Abstract

There is a pressing need for an in-depth understanding of immunity to SARS-CoV-2. In this study, we investigated human T cell recall responses to fully glycosylated spike trimer, recombinant N protein, as well as to S, N, M, and E peptide pools in the early convalescent phase and compared them with influenza-specific memory responses from the same donors. All subjects showed SARS-CoV-2–specific T cell responses to at least one Ag. Both SARS-CoV-2–specific and influenza-specific CD4+ T cell responses were predominantly of the central memory phenotype; however SARS-CoV-2–specific CD4+ T cells exhibited a lower IFN-γ to TNF ratio compared with influenza-specific memory responses from the same donors, independent of disease severity. SARS-CoV-2–specific T cells were less multifunctional than influenza-specific T cells, particularly in severe cases, potentially suggesting exhaustion. Most SARS-CoV-2–convalescent subjects also produced IFN-γ in response to seasonal OC43 S protein. We observed granzyme B+/IFN-γ+, CD4+, and CD8+ proliferative responses to peptide pools in most individuals, with CD4+ T cell responses predominating over CD8+ T cell responses. Peripheral T follicular helper (pTfh) responses to S or N strongly correlated with serum neutralization assays as well as receptor binding domain–specific IgA; however, the frequency of pTfh responses to SARS-CoV-2 was lower than the frequency of pTfh responses to influenza virus. Overall, T cell responses to SARS-CoV-2 are robust; however, CD4+ Th1 responses predominate over CD8+ T cell responses, have a more inflammatory profile, and have a weaker pTfh response than the response to influenza virus within the same donors, potentially contributing to COVID-19 disease.

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  1. Version published to 10.4049/jimmunol.2001067
  2. ScreenIT

    SciScore for 10.1101/2020.08.27.20183319: (What is this?)

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

    Table 1: Rigor

    Institutional Review Board StatementConsent: Human subjects and study approval: Written informed consent was obtained from COVID-19 convalescent and healthy blood donors before leukapheresis or peripheral blood samples were obtained.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Sex as a biological variablenot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    To determine whether the addition of agonistic co-stimulatory antibodies increased the sensitivity of detection of ICC by flow cytometry, PBMCs were stimulated with 1ug/ml S or 1 µg/ml BSA, either with or without 2 µg/ml anti-CD28 and 2 µg/ml anti-CD49d (BD Biosciences) for 18h.
    anti-CD28
    suggested: None
    anti-CD49d
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    Inducible stable cell lines were generated in Freestyle 293-F cells (Thermofisher) as previously described (36, 57).
    293-F
    suggested: RRID:CVCL_6642)
    Influenza virus strain A/Peurto Rico/8/1934 (PR8) was grown in embryonated chicken eggs and tissue culture infectious dose determined by infection of MDCK cells (58).
    MDCK
    suggested: CLS Cat# 602280/p823_MDCK_(NBL-2, RRID:CVCL_0422)
    WT SARS-CoV-2 Neutralization Assay: 100 μl of Vero E6 cells were seeded into a 96 well plate at 0.3×106 cells/mL and were incubated overnight for attachment.
    Vero E6
    suggested: None
    Software and Algorithms
    SentencesResources
    Samples were washed twice, then resuspended in FACS buffer and acquired on the BD LSRFortessa X-20 flow cytometer using FACSDiva
    FACSDiva
    suggested: (BD FACSDiva Software, RRID:SCR_001456)
    Data and Statistical analysis: Flow cytometry data were analyzed using FlowJo v10
    FlowJo
    suggested: (FlowJo, RRID:SCR_008520)
    All statistical and graphical analyses were performed using Graphpad Prism v6.
    Graphpad Prism
    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: We detected the following sentences addressing limitations in the study:
    A potential caveat to our findings is that we included only 2 of the SARS-CoV-2 proteins in our cytokine analysis and not the full spectrum of SARS-CoV-2 antigens. However, the cytokine profile we observed in the supernatants of S and N stimulated PBMC is quite similar to that reported by Weiskopf et al for SARS-CoV-2 ARDS patient PBMC collected 3 weeks after ICU admission and stimulated with peptide megapeptide pools covering most of the SARS-CoV-2 proteins (23). There were, however, some differences noted, such as their detection of IL-17A, which we did not detect in our assays. The cytokine profile we detect in the supernatants of SARS-CoV-2 convalescent PBMC after antigen stimulation is similar to the overall cytokine profile reported at the acute phase of infection, including high levels of IL-6, IL-10 and TNF-α (46, 47). This is consistent with the evidence that memory T cells are imprinted by the acute inflammatory milieu (44). Schultheiss et al.(48) recently analyzed total PBMC from SARS-CoV-2 active and early convalescent patients and also noted that total CD4+ T cells showed an altered non-classical Th1 profile, similar to what we observe here with antigen-specific T cell responses. They also noted Th17 responses, which were not consistently observed in the antigen-specific T cells in our cohort. Of note, we observed a disconnect between ICC responses and analysis of T cell responses to S and N based on activation markers. This was not unique to SARS-CoV-2, however,...

    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 pages 49 and 44. 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:
    • 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.

    About SciScore

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  3. Version published to 10.1101/2020.08.27.20183319v1 on medRxiv