Simultaneous analysis of antigen‐specific B and T cells after SARS‐CoV‐2 infection and vaccination

  1. Krista L. Newell
  2. Mitchell J. Waldran
  3. Stephen J. Thomas
  4. Timothy P. Endy
  5. Adam T. Waickman

Reviewed by

Read the full article See related articles

Discuss this preprint

Start a discussion What are Sciety discussions?

Listed in

Log in to save this article

Abstract

Conventional methods for quantifying and phenotyping antigen‐specific lymphocytes can rapidly deplete irreplaceable specimens. This is due to the fact that antigen‐specific T and B cells have historically been analyzed in independent assays each requiring millions of cells. A technique that facilitates the simultaneous detection of antigen‐specific T and B cells would allow for more thorough immune profiling with significantly reduced sample requirements. To this end, we developed the B and T cell tandem lymphocyte evaluation (BATTLE) assay, which allows for the simultaneous identification of SARS‐CoV‐2 Spike reactive T and B cells using an activation induced marker (AIM) T cell assay and dual‐color B cell antigen probes. Using this assay, we demonstrate that antigen‐specific B and T cell subsets can be identified simultaneously using conventional flow cytometry platforms and provide insight into the differential effects of mRNA vaccination on B and T cell populations following natural SARS‐CoV‐2 infection.

Article activity feed

  1. Version published to 10.1002/cyto.a.24563
  2. ScreenIT

    SciScore for 10.1101/2021.12.08.471684: (What is this?)

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

    Table 1: Rigor

    EthicsField Sample Permit: Sample size was determined based on subject availability and conformity of sample collection to the described timeline.
    Consent: Study approval: All participants provided written informed consent prior to participation in the study, which was performed according to protocols approved by the Institutional Review Board (IRB) of the SUNY Upstate Medical University under IRB number 1587400 for experimental samples and 1839296-1 for pre-pandemic control samples.
    IRB: Study approval: All participants provided written informed consent prior to participation in the study, which was performed according to protocols approved by the Institutional Review Board (IRB) of the SUNY Upstate Medical University under IRB number 1587400 for experimental samples and 1839296-1 for pre-pandemic control samples.
    Sex as a biological variablenot detected.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    Staining and Flow Cytometry: Stimulated PBMC were washed twice in warm complete culture media and transferred to polypropylene 96-well plates for staining in the dark with 50μL of spike tetramer probe cocktail containing 100ng Spike per probe (total 200ng) at 4°C for 1 hour prior to surface staining in FACS wash buffer with the antibodies listed in table S1 at 4°C for 30min.
    30min
    suggested: None
    Plates were washed and antigen-specific antibody binding was detected using anti-human IgG HRP (MilliporeSigma, St. Louis, MO, USA, Cat. SAB3701362), or anti-human IgM HRP (SeraCare, Milford, MA, USA, Cat. 5220-0328).
    antigen-specific
    suggested: None
    anti-human IgG
    suggested: None
    anti-human IgM
    suggested: None
    Software and Algorithms
    SentencesResources
    Stained cells and compensation controls were acquired on a BD Fortessa or Aria II and analyzed using FlowJo (BD), version 10.8.0.
    FlowJo
    suggested: (FlowJo, RRID:SCR_008520)
    Antibody binding data were analyzed by nonlinear regression (One site specific binding with Hill slope) to determine EC50 titers, reported as Kd values, in GraphPad Prism 9.1.0 (GraphPad Software, La Jolla, CA).
    GraphPad Prism
    suggested: (GraphPad Prism, RRID:SCR_002798)
    GraphPad
    suggested: (GraphPad Prism, RRID:SCR_002798)
    Statistical analyses: Statistical analyses were performed using Prism version 9.1.0 (GraphPad), except for correlations.
    Prism
    suggested: (PRISM, RRID:SCR_005375)
    Graphics: Study design and assay workflow schematics were created with BioRender.com.
    BioRender
    suggested: (Biorender, RRID:SCR_018361)

    Results from OddPub: Thank you for sharing your data.

    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: Please consider improving the rainbow (“jet”) colormap(s) used on pages 27 and 29. 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.
    • No funding statement was detected.
    • 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 10.1101/2021.12.08.471684 on bioRxiv