Rapid, simplified whole blood-based multiparameter assay to quantify and phenotype SARS-CoV-2-specific T-cells

  1. Catherine Riou
  2. Georgia Schäfer
  3. Elsa du Bruyn
  4. Rene T. Goliath
  5. Cari Stek
  6. Huihui Mou
  7. Deli Hung
  8. Katalin A. Wilkinson
  9. Robert J. Wilkinson

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Abstract

Rapid tests to evaluate severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-specific T-cell responses are urgently needed to decipher protective immunity and aid monitoring vaccine-induced immunity.

Methods

Using a rapid whole blood assay requiring a minimal amount of blood, we measured qualitatively and quantitatively SARS-CoV-2-specific CD4 T-cell responses in 31 healthcare workers using flow cytometry.

Results

100% of COVID-19 convalescent participants displayed a detectable SARS-CoV-2-specific CD4 T-cell response. SARS-CoV-2-responding cells were also detected in 40.9% of participants with no COVID-19-associated symptoms or who tested PCR-negative. Phenotypic assessment indicated that, in COVID-19 convalescent participants, SARS-CoV-2 CD4 responses displayed an early differentiated memory phenotype with limited capacity to produce interferon (IFN)-γ. Conversely, in participants with no reported symptoms, SARS-CoV-2 CD4 responses were enriched in late differentiated cells, coexpressing IFN-γ and tumour necrosis factor-α and also Granzyme B.

Conclusions

This proof-of-concept study presents a scalable alternative to peripheral blood mononuclear cell-based assays to enumerate and phenotype SARS-CoV-2-responding T-cells, thus representing a practical tool to monitor adaptive immunity due to natural infection or vaccine trials.

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  1. Version published to 10.1183/13993003.00285-2021
  2. ScreenIT

    SciScore for 10.1101/2020.10.30.20223099: (What is this?)

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

    Table 1: Rigor

    Institutional Review Board StatementIRB: The University of Cape Town’s Faculty of Health Sciences Human Research Ethics Committee approved the study (HREC: 207/2020) and written informed consent was obtained from all participants.
    Consent: The University of Cape Town’s Faculty of Health Sciences Human Research Ethics Committee approved the study (HREC: 207/2020) and written informed consent was obtained from all participants.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Sex as a biological variableStudy population: The population studied consisted of healthcare workers (HCW, n=31, 29% male) recruited between July and September 2020, from Groot Schuur Hospital in Cape Town, the hardest hit region of the initial COVID-19 epidemic in South Africa (Mendelson et al., 2020).
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    Briefly, 400 µl whole blood was stimulated with the SARS-CoV-2 S, N and M protein peptide pool at 37°C for 5 hrs in the presence of the co-stimulatory antibodies against CD28 and CD49d (1µg/ml each; BD Biosciences, San Jose, CA, USA) and Brefeldin-A (10µg/ml, Sigma-Aldrich, St Louis, MO, USA).
    antibodies against CD28
    suggested: (Novus Cat# NB100-93558, RRID:AB_1236789)
    CD49d
    suggested: None
    Brefeldin-A
    suggested: None
    Cells were then stained at room temperature for 30 min with antibodies for CD3 BV650, CD4 BV785, CD8 BV510, CD45RA Alexa 488, CD27 PE-Cy5, CD38 APC, HLA-DR BV605, Ki67 PerCP-cy5.5, PD-1 PE, Granzyme B (GrB) BV421, IFNγ BV711, TNFα PE-Cy7 and IL-2 PE/Dazzle 594, as detailed in Supplementary Table 1.
    CD3
    suggested: (RayBiotech Cat# CS-11-0110, RRID:AB_1228004)
    CD4
    suggested: (RayBiotech Cat# CS-11-0132, RRID:AB_1228050)
    CD8
    suggested: (Abcam Cat# ab34397, RRID:AB_2291359)
    CD45RA
    suggested: (Bio-Rad Cat# MCA340A488, RRID:AB_321265)
    HLA-DR
    suggested: (BD Biosciences Cat# 562844, RRID:AB_2744478)
    Ki67
    suggested: None
    TNFα
    suggested: None
    PE-Cy7
    suggested: None
    IL-2 PE/Dazzle 594
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    HEK-293TT cell culture supernatants containing the virions were harvested 3 days post transfection and incubated with heat-inactivated patient plasma at 5-fold serial dilutions for 60 min at 37°C.
    HEK-293TT
    suggested: None
    Plasma/pseudovirus mixtures were then used for transfection of HEK-293T cells stably expressing the ACE2 receptor (Mou et al., 2020).
    HEK-293T
    suggested: None
    Software and Algorithms
    SentencesResources
    Samples were acquired on a BD LSR-II and analysed using FlowJo (v9.9.6, FlowJo LCC, Ashland, OR, USA).
    FlowJo
    suggested: (FlowJo, RRID:SCR_008520)
    Statistical analyses: Graphical representations were performed in Prism (v8.4.3; GraphPad Software Inc, San Diego, CA, USA) and JMP (v14.0.0; SAS Institute, Cary, NC, USA).
    GraphPad
    suggested: (GraphPad Prism, RRID:SCR_002798)
    SAS Institute
    suggested: (Statistical Analysis System, RRID:SCR_008567)
    Statistical tests were performed in Prism.
    Prism
    suggested: (PRISM, RRID:SCR_005375)

    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.

  3. Version published to 10.1101/2020.10.30.20223099v1 on medRxiv