T cell response to intact SARS-CoV-2 includes coronavirus cross-reactive and variant-specific components

  1. Lichen Jing
  2. Xia Wu
  3. Maxwell P. Krist
  4. Tien-Ying Hsiang
  5. Victoria L. Campbell
  6. Christopher L. McClurkan
  7. Sydney M. Favors
  8. Lawrence A. Hemingway
  9. Charmie Godornes
  10. Denise Q. Tong
  11. Stacy Selke
  12. Angela C. LeClair
  13. Chu-Woo Pyo
  14. Daniel E. Geraghty
  15. Kerry J. Laing
  16. Anna Wald
  17. Michael Gale
  18. David M. Koelle

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Abstract

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  1. Version published to 10.1172/jci.insight.158126
  2. ScreenIT

    SciScore for 10.1101/2022.01.23.22269497: (What is this?)

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

    Table 1: Rigor

    EthicsConsent: Subjects provided informed written consent and studies were Institutional Review Board-approved.
    IRB: Subjects provided informed written consent and studies were Institutional Review Board-approved.
    Sex as a biological variablenot detected.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Experimental Models: Cell Lines
    SentencesResources
    Diamond, Washington University, St. Louis, USA), 229E was cultured on Huh7 cells (90) and NL63 cultured on MK2-LLC cells (CCL-7, ATCC).
    Huh7
    suggested: None
    HeLa cells (CCL-2, ATCC) were transfected with SARS-CoV-2 genes cloned into pDEST103, or pDEST103 control, using FuGene 6 (Promega) and collected at 48 hours.
    HeLa
    suggested: None
    For viral proteins, SARS-CoV-2 individual ORFs in pDEST103 were transfected into and expressed by COS-7 cells (#CRL-1651, ATCC, Manassas, VA), which were lysed with three cycles of freeze- thaw treatment.
    COS-7
    suggested: ATCC Cat# CRL-1651, RRID:CVCL_0224)
    Experimental Models: Organisms/Strains
    SentencesResources
    To measure CD8 T cell recognition of SARS-CoV-2-infected cells, human bronchial epithelial cell 3 immortalized with cyclin dependent kinase 4 and human telomerase reverse transcriptase (HBEC3-KT) (97) transduced with ACE2 (44) (HBE3-KT-A) were infected at MOI 2 in 6-well plates using strain WA1-GFP (gift of Dr. Ralph Baric), or mock-infected, for 24 hours, harvested with Accutase (Thermo-Fisher) and plated at 20,000 cells/well in 96 well U- bottom plates.
    WA1-GFP
    suggested: None
    Recombinant DNA
    SentencesResources
    Full-length SARS-CoV-2 codon-optimized molecular clones non-structural protein (NSP)1-NSP10, NSP12-NSP16, S, ORF3A, ORF3B, E, M, N, ORF6, ORF7A, ORF7B, ORF8, N, ORF9B, ORF9C (also known as ORF9Bwu and ORF14) from Wuhan-Hu-1 (Wu-1, Genbank NC_045512.2), and ORF10 from strain HKU-SZ-005b (Genbank MN975262.1) cloned into pDONR207 or pDONR223 (29) (ThermoFisher, Waltham, MA), were obtained from Addgene (Waterton, MA).
    pDONR207
    suggested: RRID:Addgene_100600)
    pDONR223
    suggested: RRID:Addgene_60532)
    pDEST103 expresses proteins intracellularly as fusions after N-terminal eGFP driven by a CMV promoter.
    pDEST103
    suggested: None
    These were designed with the same 21 aa C-terminal deletion as Wu-1 D614G and D614G/K417N and synthesized and cloned by Twist (South San Francisco, CA) into pHDM to create HDM_Spikedelta21_B.1.351 and HDM_Spikedelta21_B.1.1.7 for transient transfection of eukaryotic cells.
    pHDM
    suggested: None
    HLA A and B cDNA alleles were amplified by RT-PCR (94) or synthesized (Genscript), cloned into pcDNA3.1(-) (ThermoFisher) (96) and sequence-verified.
    pcDNA3.1
    suggested: RRID:Addgene_79663)
    COS-7 in 96-well flat plates were co-transfected with 100 ng/well each HLA cDNA and SARS-CoV-2-p103 plasmids, using FuGene6 (96).
    SARS-CoV-2-p103
    suggested: None
    The ratio of the percent of IFN-γ and/or IL-2 positive CD4 T cells with SARS-CoV-2 antigen compared with pDEST203 empty vector- derived IVTT product, was > 2.
    pDEST203
    suggested: None
    Software and Algorithms
    SentencesResources
    Data were acquired with FACSCANTO and cytokine expression quantified for live, CTV-positive CD3+CD4+CD8- single cells (FlowJo 10.7.1, Becton Dickinson).
    FlowJo
    suggested: (FlowJo, RRID:SCR_008520)
    Statistics and variant abundance: Proportions of ex vivo AIM (+) cells were compared within- person between mock and stimulated conditions using Wilcoxon matched-pairs signed-ranks test (Instat 3.10, GraphPad, San Diego, CA).
    GraphPad
    suggested: (GraphPad Prism, RRID:SCR_002798)
    Correlation between IFN-γ and proliferation results used linear regression and default parameters (Prism 9.1.0, GraphPad).
    Prism
    suggested: (PRISM, RRID:SCR_005375)

    Results from OddPub: Thank you for sharing your data.

    Results from LimitationRecognizer: We detected the following sentences addressing limitations in the study:
    The current study has limitations. We observed no AIM response to whole SARS-CoV-2 in HD, yet, several groups have documented the presence of SARS-CoV-2-reactive T cells in uninfected individuals from either the naïve or sCoV-primed memory repertoires (20, 80). Our approaches may be better suited to memory response to SARS-CoV-2 infection itself but could be adapted to capture these responses, for example by using higher input cell numbers. We noted only moderate preservation of TCR sequences comparing ex vivo sorted AIM (+) T cells and expanded cultures. Biological factors related to TCR repertoire differences could include rare clonotypes that assorted into either the ex vivo TCRseq or expansion fractions of the AIM (+) cells, rather than being represented in both. Indeed, sequencing of AIM (+) cells ex vivo shows broad and diverse responses at the TCR sequence level even to single epitopes, with many clonotypes detected at low levels (81). T cell programming could render some T cells refractory to expansion, with an exhausted T cell phenotype noted for SARS-CoV-2-specific memory T cells in some settings (82). Technical factors could also contribute, ranging from DNA extraction or PCR inefficiencies during TCR sequencing protocols to lack of provision of T cell expansion culture conditions required for some of the AIM (+) cells. Analytically, we discounted T cell clonotypes detected at less than two copies, potentially reducing concurrency between ex vivo and expanded reper...

    Results from TrialIdentifier: We found the following clinical trial numbers in your paper:

    IdentifierStatusTitle
    NCT04776317RecruitingChimpanzee Adenovirus and Self-Amplifying mRNA Prime-Boost P…

    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.

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

    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.

  3. Version published to 10.1101/2022.01.23.22269497v1 on medRxiv