Impaired ICOS signaling between Tfh and B cells distinguishes hospitalized from ambulatory CoViD-19 patients

  1. Amanda Hanson
  2. Heather Cohen
  3. Hao Wang
  4. Nandini Shekhar
  5. Chinmayee Shah
  6. Abha Dhaneshwar
  7. Bethany W. Harvey
  8. Richard Murray
  9. Christopher J. Harvey

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Abstract

Emerging evidence suggests that SARS-CoV-2 infections are characterized by systemic immune responses that appear to be dysregulated with more severe CoViD-19 disease. Lymphopenia and delayed antibody responses are commonly identified in CoViD-19 subjects, and recent reports have demonstrated abrogation of germinal centers in severe CoViD-19. This work assessed a potential mechanistic basis for impaired humoral responses, focusing on the T follicular helper (Tfh) and B cell interface that is critical for germinal center reactions. Here we demonstrated that Tfh activity is impaired in hospitalized relative to ambulatory CoViD-19 subjects, potentially due to decreased expression of the costimulatory molecule ICOS-L on B cells. Functional impairment manifested as a diminished ability to stimulated Tfh derived IFNγ and IL-21, the latter of which is critical for B cell proliferation and differentiation. Activation of Tfh cells by agonism of the ICOS receptor ex vivo by an agonistic antibody stimulated the generation of IFNγ/IL-21 double positive cells from hospitalized CoViD-19 subjects. This report establishes an immunological defect that differentiates ambulatory from hospitalized CoViD and suggests that agents that could restore impaired mechanisms at the Tfh–B cell interface may be of therapeutic value.

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  1. ScreenIT

    SciScore for 10.1101/2020.12.16.20248343: (What is this?)

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

    Table 1: Rigor

    Institutional Review Board StatementConsent: Participants provided three peripheral blood and serum samples over a 3 to 6-week period and information regarding severity and duration of symptoms was obtained at time of informed consent.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Sex as a biological variablenot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    In flow panel one, samples were re-suspended in flow buffer (PBS+2% FBS, 1 mM EDTA, and 0.1% Sodium azide) containing the following antibodies: CD3-PercpCy5.5 (clone OKT3, Biolegend), CD4-BUV805 (clone SK3, BD Biosciences), CXCR5-APC (clone J252D4, Biolegend), CXCR3-BUV395 (clone IC6/CXCR3, BD Biosciences), PD-1-BV421 (clone EH12.2H7, Biolegend) CD45RA-BUV737 (clone HI100, BD Biosciences), CD62L-PE-Cy7(clone DREG-56, Biolegend), CD19-BV785 (clone SJ25C1, Biolegend), CD38-BV605 (clone HB-7, Biolegend), ICOS-L-BUV661 (clone 2D3/B7H2, BD Biosciences) and eFluor780 fixable viability dye (eBioscience) for 30min at 4°C.
    CXCR3-BUV395
    suggested: None
    PD-1-BV421
    suggested: None
    CD45RA-BUV737
    suggested: None
    CD62L-PE-Cy7
    suggested: None
    CD38-BV605
    suggested: None
    Permeabilized cells were stained with biotinylated anti-human ICOS antibody (clone M13, Jounce Therapeutics) and BCL2-AF488 (clone 100, Biolegend) for 30 min at RT.
    anti-human ICOS
    suggested: (Millipore Cat# ST1691-100UG, RRID:AB_10696600)
    In flow panel two, samples were re-suspended in flow buffer (PBS+2% FBS, 1 mM EDTA, and 0.1% Sodium azide) containing the following antibodies: CD3-PercpCy5.5 (clone OKT3, Biolegend), CD4-BUV805 (clone SK3, BD Biosciences), CXCR5-APC (clone J252D4, Biolegend), CD19-BV785 (clone SJ25C1, Biolegend), CD38-BUV395 (HB-7, BD Biosciences), HLA-DR-BV510 (clone G46-6, BD Biosciences), CD154-PeCy7 (clone 24-31, Biolegend), CD27-FITC (clone: M-T271, BioLegend) and eFluor780 fixable viability dye (eBioscience) for 30min at 4°C.
    CD27-FITC
    suggested: (Miltenyi Biotec Cat# 130-097-924, RRID:AB_2660827)
    CD3/CD28 and CD3/anti-ICOS 36E10 Plate Bound Stimulation + Normal Donor and COVID Cohort CD3/CD28 Bead Stimulation: Plate bound antibody conditions were prepared as follows: In 1X PBS in a 96 well round bottom TC plate, plate bound anti-human CD3 (clone: OKT3, BioXCell) was coated at a final sub-optimal stimulation concentration of 1ug/ml with either plate bound anti-human CD28 (clone:CD28.2, Biolegend) at a final concentration of 10ug/ml, or 10ug/ml final concentration of anti-human ICOS (clone: 36E10, Jounce Therapeutics) for 2 hours at 37C.
    CD3/anti-ICOS
    suggested: None
    clone:CD28.2
    suggested: None
    Cells were then washed with flow buffer and resuspended in a master extracellular staining mix containing the following antibodies: CD3-PerCpCy5.5 (clone: OKT3, Biolegend), CD4-BUV805 (clone SK3, BD Biosciences), CXCR5-APC (clone:J252D4, Biolegend) CD154-PECy7 (clone: 24-31 Biosciences), CD38-BUV395 (clone: HB7, BD Biosciences) and eFluor780 fixable viability dye (eBioscience) for 30 min at 4°C.
    CD4-BUV805
    suggested: None
    CXCR5-APC
    suggested: None
    clone:J252D4
    suggested: None
    CD154-PECy7
    suggested: None
    Permeabilized cells were stained with biotinylated anti-human ICOS antibody (clone: M13, Jounce Therapeutics), IL-21-PE (clone: 3A3-N2, Biolegend), and IFNg-BV605 (clone:B27, Biolegend) for 30 min, RT.
    clone:B27
    suggested: None
    IFNg-BV605
    suggested: None
    Cells were then washed with flow buffer and resuspended in a master extracellular staining mix containing the following antibodies: CD3-PerCpCy5.5 (clone: OKT3, Biolegend), CD4-BUV805 (clone SK3, BD Biosciences), CXCR5-APC (clone:J252D4, Biolegend) CD154-PECy7 (clone: 24-31 Biosciences), CD38-BUV395 (clone: HB7, BD Biosciences) and eFluor780 fixable viability dye (eBioscience) for 30 min at 4°C.
    CD4-BUV805
    suggested: None
    CXCR5-APC
    suggested: None
    clone:J252D4
    suggested: None
    CD154-PECy7
    suggested: None
    Permeabilized cells were stained with biotinylated anti-human ICOS antibody (clone: M13, Jounce Therapeutics), IL-21-PE (clone: 3A3-N2, Biolegend), and IFNg-BV605 (clone:B27, Biolegend) for 30 min, RT.
    clone:B27
    suggested: None
    IFNg-BV605
    suggested: None
    Software and Algorithms
    SentencesResources
    Frosty™ (Thermo Fisher Scientific) or CoolCell® (BioCision, LLC) and stored overnight in a − 80°C freezer and then shipped directly on packed in dry ice.
    CoolCell®
    suggested: None
    In flow panel two, samples were re-suspended in flow buffer (PBS+2% FBS, 1 mM EDTA, and 0.1% Sodium azide) containing the following antibodies: CD3-PercpCy5.5 (clone OKT3, Biolegend), CD4-BUV805 (clone SK3, BD Biosciences), CXCR5-APC (clone J252D4, Biolegend), CD19-BV785 (clone SJ25C1, Biolegend), CD38-BUV395 (HB-7, BD Biosciences), HLA-DR-BV510 (clone G46-6, BD Biosciences), CD154-PeCy7 (clone 24-31, Biolegend), CD27-FITC (clone: M-T271, BioLegend) and eFluor780 fixable viability dye (eBioscience) for 30min at 4°C.
    BD Biosciences
    suggested: (BD Biosciences, RRID:SCR_013311)
    Statistical Analyses: All statistical analyses were performed in Prism (GraphPad Software).
    Prism
    suggested: (PRISM, RRID:SCR_005375)
    GraphPad
    suggested: (GraphPad Prism, RRID:SCR_002798)

    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.

  2. Version published to 10.1101/2020.12.16.20248343v1 on medRxiv