SARS‐CoV‐2‐reactive interferon‐γ‐producing CD8+ T cells in patients hospitalized with coronavirus disease 2019

  1. Estela Giménez
  2. Eliseo Albert
  3. Ignacio Torres
  4. María José Remigia
  5. María Jesús Alcaraz
  6. María José Galindo
  7. María Luisa Blasco
  8. Carlos Solano
  9. María José Forner
  10. Josep Redón
  11. Jaime Signes‐Costa
  12. David Navarro

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Abstract

There is limited information on severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) T‐cell immune responses in patients with coronavirus disease 2019 (COVID‐19). Both CD4+ and CD8+ T cells may be instrumental in resolution of and protection from SARS‐CoV‐2 infection. Here, we tested 25 hospitalized patients either with microbiologically documented COVID‐19 (n = 19) or highly suspected of having the disease (n = 6) for presence of SARS‐CoV‐2‐reactive CD69+ expressing interferon‐γ (IFN‐γ) producing CD8+ T cells using flow‐cytometry for intracellular cytokine staining assay. Two sets of overlapping peptides encompassing the SARS‐CoV‐2 Spike glycoprotein N‐terminal 1 to 643 amino acid sequence and the entire sequence of SARS‐CoV‐2 M protein were used simultaneously as antigenic stimulus. Ten patients (40%) had detectable responses, displaying frequencies ranging from 0.15 to 2.7% (median of 0.57 cells/µL; range, 0.43‐9.98 cells/µL). The detection rate of SARS‐CoV‐2‐reactive IFN‐γ CD8+ T cells in patients admitted to intensive care was comparable ( P  = .28) to the rate in patients hospitalized in other medical wards. No correlation was found between SARS‐CoV‐2‐reactive IFN‐γ CD8+ T‐cell counts and SARS‐CoV‐2 S‐specific antibody levels. Likewise, no correlation was observed between either SARS‐CoV‐2‐reactive IFN‐γ CD8+ T cells or S‐specific immunoglobulin G‐antibody titers and blood cell count or levels of inflammatory biomarkers. In summary, in this descriptive, preliminary study we showed that SARS‐CoV‐2‐reactive IFN‐γ CD8+ T cells can be detected in a non‐negligible percentage of patients with moderate to severe forms of COVID‐19. Further studies are warranted to determine whether quantitation of these T‐cell subsets may provide prognostic information on the clinical course of COVID‐19.

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  1. Version published to 10.1002/jmv.26213
  2. ScreenIT

    SciScore for 10.1101/2020.05.18.20106245: (What is this?)

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

    Table 1: Rigor

    Institutional Review Board StatementIRB: The current study was approved by the Ethics Comittee of Hospital Clínico Universitario
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Sex as a biological variablePatients: Twenty-five non-consecutive patients (14 males and 11 females; median age, 69 years; range, 62 to 87 years) admitted to our center from March 17th to April 24th with clinically suspected Covid-19 were included in the current study.

    Table 2: Resources

    Antibodies
    SentencesResources
    Antibody detection methods: Initial screening for SARS-CoV-2-specific antibodies was carried out by using the 2019-nCoV IgG/IgM Rapid Test (Hangzhou AllTest Biotech Co., Ltd. China), a rapid lateral flow chromatographic immunoassay (LFIC) designed for the qualitative detection of IgG and IgM antibodies in human whole blood or serum.
    IgM
    suggested: None
    Sera obtained at the time of blood collection for SARS-CoV-2 CD8+ T cell immunity analyses were analyzed by the LIAISON® SARS-CoV-2 IgG (DiaSorin, Saluggia, Italy), a fully automated quantitative chemiluminscent assay (CLIA) detecting IgG antibodies against SARS-CoV-2 S protein (S1 and S2 subunits).
    DiaSorin , Saluggia , Italy
    suggested: None
    SARS-CoV-2 S protein ( S1
    suggested: None
    Software and Algorithms
    SentencesResources
    , CA, USA) on the Light Cycler 2.0 instrument, the SARS-COV-2 REALTIME PCR KIT from Vircell Diagnostics (Granada, Spain), the REALQUALITY RQ-2019-nCoV from AB ANALITICA (Padua, Italy), both on the Applied Biosystems 7500 instrument, the SARS-CoV-2 (S gene) – BD MAX™ System (VIASURE Real Time PCR Detection Kits; CerTest, Zaragoza, Spain) and the Abbott RealTime SARS-CoV-2 Assay (Abbott Molecular Diagnostics, Chicago, USA).
    Abbott
    suggested: (Abbott, RRID:SCR_010477)
    Cells were then washed, resuspended in 200 μl of 1% paraformaldehyde in PBS, and analyzed within 2 h on an FACScanto flow cytometer using DIVA v8 software (BD Biosciences Immunocytometry Systems, San Jose, CA, USA).
    BD Biosciences Immunocytometry Systems
    suggested: None
    The analyses were performed using SPSS version 20.0 (SPSS, Chicago, IL, USA).
    SPSS
    suggested: (SPSS, RRID:SCR_002865)

    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: We detected the following sentences addressing limitations in the study:
    In addition to the scarce number of patients in our series, the current study has a major limitation: the specificity of SARS-CoV-2-reactive IFNγ-CD8+ T cells was not proven. Based on sequence analyses, SARS-CoV-2 and seasonal alpha and beta coronaviruses may share HLA-class I-restricted immunogenic epitopes mapping within S1 and M potentially eliciting cross-reactive T cells.19 In support of this assumption, S-reactive CD4+ T cells could be detected in 34% of healthy control individuals who had seemingly not been infected by SARS-CoV-2, albeit at lower frequencies compared to patients with Covid-19, and displaying a differential pattern of cell-surface activation markers. We also tested 4 healthy asymptomatic individuals with no evidence of active or past Covid-19 and found one of them to be reactive, albeit displaying a low frequency of SARS-CoV-2 reactive IFNγ-CD8+ T cells (0.12%) (data not shown). In an epidemiological framework of heavy SARS CoV-2 community transmission, such as the one being currently faced in Spain, recruiting asymptomatic individuals, even if testing negative by RT-PCR or having no evidence of seroconversion, as negative controls, may not be entirely appropriate, as some of these could have been exposed and developed measurable T-cell responses. Unfortunately, we had not cryopreserved blood specimens from healthy individuals with or without documented infection caused by seasonal coronaviruses collected prior to the penetration of SARS-CoV-2 into our ...

    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 10.1101/2020.05.18.20106245 on medRxiv