SARS-CoV-2 antigen exposure history shapes phenotypes and specificity of memory CD8+ T cells

  1. Anastasia A. Minervina
  2. Mikhail V. Pogorelyy
  3. Allison M. Kirk
  4. Jeremy Chase Crawford
  5. E. Kaitlynn Allen
  6. Ching-Heng Chou
  7. Robert C. Mettelman
  8. Kim J. Allison
  9. Chun-Yang Lin
  10. David C. Brice
  11. Xun Zhu
  12. Kasi Vegesana
  13. Gang Wu
  14. Sanchit Trivedi
  15. Pratibha Kottapalli
  16. Daniel Darnell
  17. Suzanne McNeely
  18. Scott R. Olsen
  19. Stacey Schultz-Cherry
  20. Jeremie H. Estepp
  21. the SJTRC Study Team
  22. Aditya Gaur
  23. James Hoffman
  24. Motomi Mori
  25. Li Tang
  26. Elaine Tuomanen
  27. Richard Webby
  28. Hana Hakim
  29. Randall T. Hayden
  30. Diego R. Hijano
  31. Resha Bajracharya
  32. Walid Awad
  33. Lee-Ann Van de Velde
  34. Brandi L. Clark
  35. Taylor L. Wilson
  36. Aisha Souquette
  37. Ashley Castellaw
  38. Ronald H. Dallas
  39. Jason Hodges
  40. Ashleigh Gowen
  41. Jamie Russell-Bell
  42. James Sparks
  43. David E. Wittman
  44. Thomas P. Fabrizio
  45. Sean Cherry
  46. Ericka Kirkpatrick Roubidoux
  47. Valerie Cortez
  48. Pamela Freiden
  49. Nicholas Wohlgemuth
  50. Kendall Whitt
  51. Maureen A. McGargill
  52. Joshua Wolf
  53. Paul G. Thomas

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Abstract

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  1. Version published to 10.1038/s41590-022-01184-4
  2. Version published to 10.1101/2021.07.12.21260227v3 on medRxiv
  3. Version published to 10.1101/2021.07.12.21260227v2 on medRxiv
  4. ScreenIT

    SciScore for 10.1101/2021.07.12.21260227: (What is this?)

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

    Table 1: Rigor

    EthicsIRB: Jude Institutional Review Board approved the study.
    Consent: Participants provided written informed consent prior to enrollment and then completed regular questionnaires about demographics, medical history, treatment, and symptoms if positively diagnosed by PCR with SARS-CoV-2.
    Sex as a biological variablenot detected.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    anti-human CD8 BV711-conjugated (Biolegend, 344734, clone SK1)) and TotalSeq-C antibodies (1 µL anti-human CCR7 (Biolegend 353251), 1 µL anti-human CD45RA (Biolegend 304163)) and 2 µL of TotalSeq-C anti-human Hashtag antibodies 1-10 (Biolegend 394661, 394663, 394665, 394667, 394669, 394671, 394673, 394675, 394677, 394679) were added.
    anti-human CD8 BV711-conjugated ( Biolegend , 344734
    suggested: None
    TotalSeq-C
    suggested: None
    anti-human CCR7
    suggested: (BioLegend Cat# 353251, RRID:AB_2800943)
    anti-human CD45RA
    suggested: (BioLegend Cat# 304163, RRID:AB_2800764)
    anti-human Hashtag antibodies 1-10 ( Biolegend 394661 , 394663 , 394665 , 394667 , 394669 , 394671 , 394673 , 394675 , 394677 , 394679
    suggested: None
    In each batch, each PBMC sample was uniquely labeled with a DNA-barcoded hashing antibody (TotalSeq-C anti-human Hashtag antibodies 1-10, Biolegend).
    anti-human Hashtag antibodies 1-10 , Biolegend) .
    suggested: None
    Surface expression of HLA was confirmed via flow cytometry using antibodies against HLA-A, B, C (PE-conjugated, Biolegend 311406, clone W6/32)
    antibodies against HLA-A
    suggested: None
    Transduction was confirmed by expression of mCherry, and surface TCR expression was confirmed via flow cytometry using antibodies against mouse TCRβ constant region (PE-conjugated, Biolegend 109208, clone H57-597) and human CD3 (Brilliant Violet 785-conjugated, Biolegend 344842, clone SK7).
    human CD3
    suggested: (BioLegend Cat# 344842, RRID:AB_2616891)
    Cells were then stained with 1 µL Ghost Dye Violet 510 Viability Dye (Tonbo Biosciences 13-0870-T100) and a cocktail of surface antibodies: 1 µL each of anti-human CD8 (Brilliant Violet 785-conjugated, Biolegend 344740, clone SK1)
    anti-human CD8
    suggested: (BioLegend Cat# 344740, RRID:AB_2566202)
    Following fixation and permeabilization, cells were washed twice with 1X Perm/Wash buffer and stained with a cocktail of intracellular antibodies: 1.25 µL of anti-human IFNγ (Alexa Fluor 647-conjugated, Biolegend 502516, clone 4S.B3) and 1 µL anti-human CD69 (PerCP-eFluor710-conjugated, eBioscience 46-0699-42, clone FN50).
    anti-human IFNγ
    suggested: None
    anti-human CD69
    suggested: None
    For hCoV and SARS-CoV-2 antibody detection, 384-well microtiter plates were coated overnight at 4 °C, with recombinant proteins diluted in PBS.
    SARS-CoV-2
    suggested: None
    ELISA plates were washed and incubated for 30 minutes at room temperature with anti-human secondary antibodies diluted in 1% milk in PBS-T: anti-IgG (1:10,000; Invitrogen, A18805).
    anti-human secondary
    suggested: (Thermo Fisher Scientific Cat# A18805, RRID:AB_2535582)
    anti-IgG
    suggested: (Thermo Fisher Scientific Cat# A18805, RRID:AB_2535582)
    Experimental Models: Cell Lines
    SentencesResources
    K562 cells (ATCC CCL-243) were transduced, then antibiotic selected for one week using 2 µg/mL puromycin in Iscove’s Modified Dulbecco’s Medium (IMDM; Gibco) containing 10% FBS and 1% penicillin/streptomycin.
    K562
    suggested: ATCC Cat# CCL-243, RRID:CVCL_0004)
    Jurkat 76.7 cells (a gift from Wouter Scheper; variant of TCR-null Jurkat 76.7 cells that expresses human CD8 and an NFAT-GFP reporter) were transduced, then antibiotic selected for 1 week using 1 µg/mL puromycin in RPMI (Gibco) containing 10% FBS and 1% penicillin/streptomycin.
    Jurkat 76.7
    suggested: None
    Tetramer generation and Jurkat Cell line staining: Biotinylated HLA-B*15-monomers loaded with NQKLIANQF (SARS-CoV-2) and NQKLIANAF (CCCoV) versions of the peptide were tetramerised using TotalSeq-C-0951-PE-Streptavidin (Biolegend 405261) and TotalSeq-C-0956-APC-Streptavidin (Biolegend 405283).
    Jurkat
    suggested: None
    Recombinant DNA
    SentencesResources
    Lentivirus was generated by transfecting 293T packaging cell line (American Type Culture Collection (ATCC) CRL-3216) with the pLVX lentiviral vector containing the HLA-B*15:01 insert, psPAX2 packaging plasmid (Addgene plasmid #12260), and pMD2.
    pLVX
    suggested: RRID:Addgene_101121)
    This sequence was cloned into the pLVX-EF1α-IRES-Puro lentiviral expression vector (Clontech).
    pLVX-EF1α-IRES-Puro
    suggested: None
    Lentivirus was generated by transfecting 293T packaging cell line (ATCC CRL-3216) with the pLVX lentiviral vector containing the TCR-mCherry insert, psPAX2 packaging plasmid (Addgene plasmid #12260), and pMD2.
    psPAX2
    suggested: RRID:Addgene_12260)
    pMD2
    suggested: None
    Software and Algorithms
    SentencesResources
    Study data are collected and managed using REDCap electronic data capture tools hosted at St. Jude (Harris et al. 2009; 2019).
    REDCap
    suggested: (REDCap, RRID:SCR_003445)
    Resulting GEX matrices were analysed with the Seurat R package version 3.2.3 (Stuart et al. 2019).
    Seurat
    suggested: (SEURAT, RRID:SCR_007322)
    Cells were then washed twice with 1X Perm/Wash buffer and analyzed by flow cytometry on a custom-configured BD Fortessa using FACSDiva software (Becton Dickinson)
    FACSDiva
    suggested: (BD FACSDiva Software, RRID:SCR_001456)
    Flow cytometry data were analyzed using FlowJo software (TreeStar).
    FlowJo
    suggested: (FlowJo, RRID:SCR_008520)

    Results from OddPub: Thank you for sharing your code and data.

    Results from LimitationRecognizer: We detected the following sentences addressing limitations in the study:
    An important limitation of our study is that we could not compare the effect of one vs two doses of mRNA vaccine in individuals with pre-existing immunity. It has been suggested in multiple studies that a second vaccine dose in individuals with pre-existing immunity does not further increase antibody levels from the first dose (Wang et al. 2021; Mazzoni et al. 2021; Krammer et al. 2021; Goel et al. 2021; Ebinger et al. 2021; Camara et al. 2021), but the effect on T cells remains to be studied. We found an increase in the fraction of EMRA T cells in fully vaccinated subjects with pre-existing immunity. Whether or not this increase is associated with more (or less) durable and efficient protection is not clear. Longer term follow-up studies of the durability of memory in vaccine-only, infection-only, and vaccinated after infection groups should closely monitor the phenotype of antigen-specific T cell responses. Precise measurement of epitope-specific T cell and B cell responses is crucial for defining the correlates of SARS-CoV-2 protection, which will inform vaccination strategies to prevent pandemic recurrence as additional SARS-CoV-2 variants emerge. The striking similarity between the phenotypes and constituent repertoires of epitope-specific CD8 T cell responses following infection, vaccination, or infection followed by vaccination, indicate that mRNA vaccines are capable of inducing equivalent memory as an infection episode and further expanding these responses if previou...

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

    IdentifierStatusTitle
    NCT04362995RecruitingSt. Jude Tracking of Viral and Host Factors Associated With …

    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 25, 26 and 22. 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.

    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.

  5. Version published to 10.1101/2021.07.12.21260227v1 on medRxiv