Longitudinal Assessment of SARS-CoV-2 Specific T Cell Cytokine-Producing Responses for 1 Year Reveals Persistence of Multi-Cytokine Proliferative Responses, with Greater Immunity Associated with Disease Severity
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Abstract
Cellular-mediated immunity is critical for long-term protection against most viral infections, including coronaviruses. We studied 23 SARS-CoV-2-infected survivors over a one year post symptom onset (PSO) interval by ex vivo cytokine ELISpot assay. All subjects demonstrated SARS-CoV-2-specific IFN-γ, IL-2, and Granzyme B (GzmB) T cell responses at presentation, with greater frequencies in severe disease. Cytokines, mainly produced by CD4+ T cells, targeted all structural proteins (Nucleocapsid, Membrane, Spike) except Envelope, with GzmB > IL-2 > IFN-γ. Mathematical modeling predicted that: 1) cytokine responses peaked at 6 days for IFN-γ, 36 days for IL-2, and 7 days for GzmB, 2) severe illness was associated with reduced IFN-γ and GzmB, but increased IL-2 production rates, 3) males displayed greater production of IFN-γ, whereas females produced more GzmB. Ex vivo responses declined over time with persistence of IL-2 in 86% and of IFN-γ and GzmB in 70% of subjects at a median of 336 days PSO. The average half-life of SARS-CoV-2-specific cytokine-producing cells was modelled to be 139 days (∼4.6 months). Potent T cell proliferative responses persisted throughout observation, were CD4 dominant, and were capable of producing all 3 cytokines. Several immunodominant CD4 and CD8 epitopes identified in this study were shared by seasonal coronaviruses or SARS-CoV-1 in the Nucleocapsid and Membrane regions. Both SARS-CoV-2-specific CD4+ and CD8+ T cell clones were able to kill target cells, though CD8 tended to be more potent.
Importance
Our findings highlight the relative importance of SARS-CoV-2-specific GzmB-producing T cell responses in SARS-CoV-2 control, shared CD4 and CD8 immunodominant epitopes in seasonal coronaviruses or SARS-CoV-1, and indicate robust persistence of T cell memory at least one year after infection. Our findings should inform future strategies to induce T cell vaccines against SARS-CoV-2 and other coronaviruses.
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SciScore for 10.1101/2022.01.18.476864: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Ethics Consent: Human Subjects and Study Approval: Healthy subjects and individuals with COVID-19 infection as diagnosed by positive nasopharyngeal SARS-CoV-2 PCR were recruited and informed consent was obtained from for blood draws and/or leukapheresis through an REB approved protocol (St. Sex as a biological variable not detected. Randomization not detected. Blinding not detected. Power Analysis not detected. Cell Line Authentication not detected. Table 2: Resources
Antibodies Sentences Resources Ex Vivo ELISpot Assay: Ex vivo ELISpot assays were performed using human IFN-γ, IL-2, and granzyme B (GzmB) antibodies (Mabtech). IL-2suggested: NoneGzmBsuggested: NoneBriefly, MSIPS4W plates (Millipore) were activated … SciScore for 10.1101/2022.01.18.476864: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Ethics Consent: Human Subjects and Study Approval: Healthy subjects and individuals with COVID-19 infection as diagnosed by positive nasopharyngeal SARS-CoV-2 PCR were recruited and informed consent was obtained from for blood draws and/or leukapheresis through an REB approved protocol (St. Sex as a biological variable not detected. Randomization not detected. Blinding not detected. Power Analysis not detected. Cell Line Authentication not detected. Table 2: Resources
Antibodies Sentences Resources Ex Vivo ELISpot Assay: Ex vivo ELISpot assays were performed using human IFN-γ, IL-2, and granzyme B (GzmB) antibodies (Mabtech). IL-2suggested: NoneGzmBsuggested: NoneBriefly, MSIPS4W plates (Millipore) were activated with 35% ethanol and coated with primary antibody: 10 μg/mL for IFN-γ (1-D1K), 10 μg/mL for IL-2 (MT2A91/2C95), and 15 μg/mL for GzmB (MT28). MT2A91/2C95suggested: NoneGzmB (MT28suggested: NoneAfter incubation, plates were washed with PBS containing 0.05% Tween-20 (BioShop) before the addition of human IFN-γ (7-B6-1), IL-2 (MT8G10), and/or GzmB (MT8610) biotinylated secondary antibodies. MT8610suggested: NoneFor dual colour assays, alkaline phosphatase (ALP) conjugated to IFN-γ secondary antibodies were used with other biotinylated secondary antibodies targeting another cytokine. ALPsuggested: NoneIFN-γsuggested: NoneT cells were then co-cultured with irradiated feeder cells containing allogeneic PBMC and BCL, 50 U/mL IL-2 (R&D Systems), 10 ng/mL IL-15 (R&D Systems), and 25 ng/mL mouse anti-human CD3 antibody (UCHT1; BD Bioscience). anti-human CD3suggested: NoneTo determine HLA II-restricted epitopes from CD4+ T cell clones or lines, autologous BCL were blocked with 10 µg/mL of either anti-HLA-A/B/C (BD Biosciences), anti-HLA-DP (Abcam), anti-HLA-DQ (BioLegend), anti-HLA-DR (BioLegend), or anti-HLA-DP/DQ/DR (BioLegend) antibodies for 30 minutes, then pulsed with the specific peptide for 1 hour at 10 µg/mL. anti-HLA-A/B/Csuggested: (Santa Cruz Biotechnology Cat# sc-52422, RRID:AB_629948)anti-HLA-DPsuggested: Noneanti-HLA-DQsuggested: Noneanti-HLA-DRsuggested: Noneanti-HLA-DP/DQ/DRsuggested: NonePulsed BCL (1 x 103 cells/well) were then washed 3 times with R10, mixed with CD4+ T cells (1-2 x 104 cells/well), and tested in IFN-γ ELISpot assay in the presence of 10 µg/mL of anti-HLA antibodies. anti-HLAsuggested: NoneOn day 7, PBMCs were first stained with LIVE/DEAD™ fixable blue dead cell stain (Thermo Fisher Scientific) and incubated with Fc receptor blocking solution (Human TruStain FcXTM; BioLegend) before surface staining with fluorochrome-conjugated antibodies to CD3 (APC-Cy7: Clone SK7), CD4 (PerCP-Cy5.5: Clone SK3), and CD8 (PE: Clone HIT8a). CD3suggested: NoneAPC-Cy7suggested: NoneCD8suggested: NoneCells were then stained with LIVE/DEAD™ fixable near IR cell stain (Thermo Fisher Scientific) and incubated with Fc receptor blocking solution (Human TruStain FcXTM; BioLegend) before surface staining with fluorochrome-conjugated antibodies to CD4 (BV711: Clone SK3) and CD8 (PE: Clone HIT8a). CD4suggested: NoneBV711suggested: NoneExperimental Models: Cell Lines Sentences Resources Frozen PBMCs were thawed and incubated with 2.5 mL of supernatant from B95-8 cell lines for 2 hours in a 37°C water bath. B95-8suggested: NoneSoftware and Algorithms Sentences Resources After incubation, plates were washed with PBS containing 0.05% Tween-20 (BioShop) before the addition of human IFN-γ (7-B6-1), IL-2 (MT8G10), and/or GzmB (MT8610) biotinylated secondary antibodies. BioShopsuggested: NoneCells were then fixed using BD Cytofix/Cytoperm and permeabilized using BD Perm/Wash according to the manufacturer’s protocol before staining with anti-IFN-γ (APC: Clone 4S.B3), anti-GzmB (BV421: Clone GB11), and anti-IL-2 (BV711: Clone MQ1-17H1). BD Cytofix/Cytopermsuggested: NoneExperimental Software and Statistical Analysis: All flow cytometry data were analyzed using FlowJo 10.8.0 software (Treestar, Ashland, OR, USA). FlowJosuggested: (FlowJo, RRID:SCR_008520)Prism 9.0 (GraphPad, San Diego, CA, USA) was used to perform statistical and graphical analyses. Prismsuggested: (PRISM, RRID:SCR_005375)GraphPadsuggested: (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: Please consider improving the rainbow (“jet”) colormap(s) used on page 39. 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:- No conflict of interest statement was detected. If there are no conflicts, we encourage authors to explicit state so.
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Results from scite Reference Check: We found no unreliable references.
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