Antigenic Determinants of SARS-CoV-2-Specific CD4+ T Cell Lines Reveals M Protein-Driven Dysregulation of Interferon Signaling

  1. Pedro H. Gazzinelli-Guimaraes
  2. Gayatri Sanku
  3. Alessandro Sette
  4. Daniela Weiskopf
  5. Paul Schaughency
  6. Justin Lack
  7. Thomas B. Nutman

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Abstract

We generated CD4 + T cell lines (TCLs) reactive to either SARS-CoV-2 spike (S) or membrane (M) proteins from unexposed naïve T cells from six healthy donor volunteers to understand in fine detail whether the S and M structural proteins have intrinsic differences in driving antigen-specific CD4 + T cell responses. Having shown that each of the TCLs were antigen-specific and antigen-reactive, single cell mRNA analyses demonstrated that SARS-CoV-2 S and M proteins drive strikingly distinct molecular signatures. Whereas the S-specific CD4 + T cell transcriptional signature showed a marked upregulation of CCL1, CD44, IL17RB, TNFRSF18 (GITR) and IGLC3 genes, in general their overall transcriptome signature was more similar to CD4 + T cell responses induced by other viral antigens (e.g. CMV). However, the M protein-specific CD4 + TCLs have a transcriptomic signature that indicate a marked suppression of interferon signaling, characterized by a downregulation of the genes encoding ISG15, IFITM1, IFI6, MX1, STAT1, OAS1, IFI35, IFIT3 and IRF7 (a molecular signature which is not dissimilar to that found in severe COVID-19). Our study suggests a potential link between the antigen specificity of the SARS-CoV-2-reactive CD4 + T cells and the development of specific sets of adaptive immune responses. Moreover, the balance between T cells of significantly different specificities may be the key to understand how CD4 + T cell dysregulation can determine the clinical outcomes of COVID-19.

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  1. Version published to 10.3389/fimmu.2022.883159
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    SciScore for 10.1101/2022.01.20.22269491: (What is this?)

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

    Table 1: Rigor

    EthicsIRB: Study approval: This study (NCT00001230) was approved by the National Institute of Allergy and Infectious Diseases (NIAID) Institutional Review Board.
    Consent: Written informed consent was obtained from all participants.
    Sex as a biological variablenot detected.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    The cells were washed twice with Perm buffer (BioLegend) and resuspended with the intracellular antibody pool containing anti-CD69 (FITC), anti-CD154 (APC), anti-TNF-a (Alexa Fluor 700) and anti-IFN-y (BUV737) (Supplemental Table I) for 30 min at 4°C.
    anti-CD69
    suggested: (BD Biosciences Cat# 560739, RRID:AB_1727505)
    anti-CD154
    suggested: (Thermo Fisher Scientific Cat# 56-1548-42, RRID:AB_2848470)
    anti-TNF-a
    suggested: None
    anti-IFN-y ( BUV737 )
    suggested: None
    Software and Algorithms
    SentencesResources
    Finally, the cells were washed twice with Perm buffer and then acquired using the BD LSRFortessa flow cytometer (BD Biosciences) and FACSDiva software (BD Biosciences) for acquisition.
    FACSDiva
    suggested: (BD FACSDiva Software, RRID:SCR_001456)
    All analyses were performed using FlowJo v10.5.3
    FlowJo
    suggested: (FlowJo, RRID:SCR_008520)
    The remainder of the single cell RNA-Seq (scRNA) analysis was performed with Seurat v3.2.2 [37].
    Seurat
    suggested: (SEURAT, RRID:SCR_007322)
    Differential expression analysis was performed to identify cluster-specific markers and to compare the S and M cell populations using MAST [40], or “Model-based Analysis of Single-cell Transcriptomics”, whereas the cluster-specific canonical pathway enrichment profiles were generated using Ingenuity Pathway Analysis (IPA, Qiagen, Redwood City, CA, USA).
    MAST
    suggested: (MAST, RRID:SCR_016340)
    Ingenuity Pathway Analysis
    suggested: (Ingenuity Pathway Analysis, RRID:SCR_008653)

    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: We found the following clinical trial numbers in your paper:

    IdentifierStatusTitle
    NCT00001230RecruitingHost Response to Infection and Treatment in Filarial Disease…

    Results from Barzooka: We did not find any issues relating to the usage of bar graphs.

    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.
    • Thank you for including a protocol registration statement.

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

    About SciScore

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  3. Version published to 10.1101/2022.01.20.22269491v1 on medRxiv