Identification of Cross-Reactive CD8 + T Cell Receptors with High Functional Avidity to a SARS-CoV-2 Immunodominant Epitope and Its Natural Mutant Variants

  1. Chao Hu
  2. Meiying Shen
  3. Xiaojian Han
  4. Qian Chen
  5. Luo Li
  6. Siyin Chen
  7. Jing Zhang
  8. Fengxia Gao
  9. Wang Wang
  10. Yingming Wang
  11. Tingting Li
  12. Shenglong Li
  13. Jingjing Huang
  14. Jianwei Wang
  15. Ju Zhu
  16. Dan Chen
  17. Qingchen Wu
  18. Kun Tao
  19. Da Pang
  20. Aishun Jin

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Abstract

Despite the growing knowledge of T cell responses and their epitopes in COVID-19 patients, there is a lack of detailed characterizations for T cell-antigen interactions and T cell functions. Using a peptide library predicted with HLA class I-restriction, specific CD8 + T cell responses were identified in over 75% of COVID-19 convalescent patients. Among the 15 SARS-CoV-2 epitopes identified from the S and N proteins, N 361-369 (KTFPPTEPK) was the most dominant epitope. Importantly, we discovered 2 N 361-369 -specific T cell receptors (TCRs) with high functional avidity, and they exhibited complementary cross-reactivity to reported N 361-369 mutant variants. In dendritic cells (DCs) and the lung organoid model, we found that the N 361-369 epitope could be processed and endogenously presented to elicit the activation and cytotoxicity of CD8 + T cells ex vivo . Our study evidenced potential mechanisms of cellular immunity to SARS-CoV-2, illuminating natural ways of viral clearance with high relevancy in the vaccine development.

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  1. ScreenIT

    SciScore for 10.1101/2020.11.02.364729: (What is this?)

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

    Table 1: Rigor

    Institutional Review Board StatementIRB: Ethics Statement: This project “Screening of SARS-CoV-2-specific T cell epitopes” was approved by the ethics committee of Chongqing Medical University.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Sex as a biological variablenot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    Briefly, ELISPOT plates (Millipore, USA) were coated with human IFN-γ antibody (Clone 1-D1K, 2 μg/ml, Mabtech, Sweden) overnight at 4°C.
    IFN-γ
    suggested: None
    Subsequently, the plates were developed with human biotinylated IFN-γ detection antibody (Clone 7-B6-1, 1 μg/ml, Mabtech, Sweden), followed by incubation with Streptavidin-AP (1:1000, Mabtech, Sweden) and BCIP/NBT-plus substrate (Mabtech, Sweden)
    BCIP/NBT-plus substrate ( Mabtech , Sweden)
    suggested: None
    Flow Cytometry Analysis: For staining, 5×105 cells were re-suspended in FACS buffer, and stained with the anti-human antibody cocktail for 30 min at room temperature in the dark.
    anti-human
    suggested: None
    Next, cells were washed with Perm/Wash buffer (Biolegend, USA) twice and stained for 25 min at room temperature with anti-human antibody against IFN-γ (BV421, Clone 4S.B3).
    anti-human antibody against IFN-γ ( BV421
    suggested: None
    CD3+ T cells were stimulated in T cell media with 1 μg/ml anti-CD3 antibody (Clone OKT3) and 1 μg/ml anti-CD28 antibody (Clone 15E8) for 2 days.
    anti-CD3
    suggested: None
    anti-CD28
    suggested: (Millipore Cat# CBL517, RRID:AB_11213666)
    Then, the transduction efficiency was assessed by flow cytometry using anti-mouse TCR-β chain constant region antibody (Clone H57-597).
    anti-mouse TCR-β
    suggested: None
    IFN-γ detection antibody (Clone 4S.B3, 1 μg/ml), IL-2 capture antibody (Clone MQ1-17H12, 4 μg/ml),
    IL-2
    suggested: None
    TNF-α capture antibody (Clone MAb1, 1 μg/ml) and TNF-α detection antibody (Clone MAb11, 1μg/ml).
    TNF-α
    suggested: None
    The ELISA plate was coated with individual anti-cytokine capture antibodies for 12 h, then washed with phosphate-buffered saline with Tween (PBST) buffer for 3 times, followed by blocking with 3% BSA for at least 1 h at room temperature.
    anti-cytokine capture
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    Cell Culture: Lenti-X 293T cells was purchased from Takara Biomedical Technology, COS-7 and K-562 cells were purchased from American Type Culture Collection (
    293T
    suggested: None
    K-562
    suggested: NCI-DTP Cat# K-562, RRID:CVCL_0004)
    Lenti-X 293T and COS-7 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Thermo Fisher Scientific, USA) supplemented with 10% Fetal Bovine Serum (FBS, Thermo Fisher Scientific, USA), 100 IU/ml penicillin and 100 μg/ml streptomycin (Gibco, USA)
    COS-7
    suggested: None
    Software and Algorithms
    SentencesResources
    The sequencing results were analyzed in the IMGT/HLA (https://www.ebi.ac.uk/ipd/imgt/hla/) website.
    IMGT/HLA
    suggested: (IMGT/HLA, RRID:SCR_002971)
    The data were analyzed using FlowJo software (TreeStar Inc, USA).
    FlowJo
    suggested: (FlowJo, RRID:SCR_008520)
    The TCR repertoire was analyzed with the IMGT/V-Quest tool (http://www.imgt.org/) (Brochet et al., 2008).
    IMGT/V-Quest
    suggested: (IMGT/V-QUEST, RRID:SCR_010749)
    http://www.imgt.org/
    suggested: (IMGT - the international ImMunoGeneTics information system, RRID:SCR_012780)
    Statistical Analysis: Statistical analyses of the data were performed using GraphPad Prism version 8.0 (GraphPad Software, Inc. La Jolla, CA, USA) software.
    GraphPad Prism
    suggested: (GraphPad Prism, RRID:SCR_002798)
    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: 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.

    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.

  2. Version published to 10.1101/2020.11.02.364729 on bioRxiv