Research ArticleCORONAVIRUS

Phenotype and kinetics of SARS-CoV-2-specific T cells in COVID-19 patients with acute respiratory distress syndrome

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Science Immunology  26 Jun 2020:
Vol. 5, Issue 48, eabd2071
DOI: 10.1126/sciimmunol.abd2071
  • Fig. 1 Clinical overview of moderate to severe COVID-19 ARDS patients.

    (A) Onset of symptoms, hospitalization status, treatment and follow-up of n=10 COVID-19 ARDS patients included in this study. PBMC samples were obtained weekly after admission to the study. Symbols shown next to the cases match throughout all figures. (B) Percentages of CD3+ T cells within the total LIVE gate measured by flow cytometry performed on PBMC collected 14 days post inclusion. (C) CD4:CD8 ratios measured by flow cytometry performed on PBMC collected 14 days post inclusion. Panels b and c show individual values for n=10 patients versus n=10 HC, as well as the mean ± SD. Asterisk denotes a significant difference. HC = healthy control.

  • Fig. 2 SARS-CoV-2-specific CD4+ T-cell responses in COVID-19 ARDS patients.

    (A, B) Antigen-specific activation of CD4+ T cells after stimulation for 20 hours with MP_S (A) and MP_CD4_R (B), measured via cell surface expression of CD69 and CD137 (gating in Fig. S1). Two left panels show activation percentages (within CD3+CD4+) obtained with the vehicle control (DMSO) and specific stimulation (MP) for HC and COVID-19 patients. The third panel shows the specific activation percentages corrected by subtracting the background present in the DMSO stimulation to allow comparison of both groups. The fourth panel shows the memory phenotype of the CD69+CD137+ responder cells in a donut diagram. Panels show individual values for n=10 patients versus n=10 HC, as well as the mean ± SD. Asterisk denotes a significant difference. HC = healthy control. Symbol shapes of COVID-19 patients are identical between panels, and refer back to Fig. 1.

  • Fig. 3 SARS-CoV-2-specific CD8+ T-cell responses in COVID-19 ARDS patients.

    (A, B, C, D) Antigen-specific activation of CD8+ T cells after stimulation for 20 hours with MP_S (A), MP_CD8_A (C), and MP_CD8_B, measured via cell surface expression of CD69 and CD137 (gating in Fig. S1). MP_CD8 panels (B) show the concatenated responses from panels C and D. Two left panels show activation percentages (within CD3+CD8+ gate) obtained with the vehicle control (DMSO) and specific stimulation (MP) for HC and COVID-19 patients. The third panel shows the specific activation percentages corrected by subtracting the background present in the DMSO stimulation to allow comparison of both groups. The fourth panel shows the memory phenotype of the CD69+CD137+ responder cells in a donut diagram. Panels show individual values for n=10 patients versus n=10 HC, as well as the mean ± SD. Asterisk denotes a significant difference. HC = healthy control. Symbol shapes of COVID-19 patients are identical between panels, and refer back to Fig. 1.

  • Fig. 4 Stimulation index identifies specific responders.

    Antigen-specific activation of CD4+ (A) and CD8+ T cells (B,C) in COVID-19 patients after stimulation for 20 hours with peptide MegaPools (MP) shown as stimulation index (SI). Stimulation index is derived by dividing the percentage obtained with specific stimulation (MP) by the percentage obtained with the vehicle control (DMSO). Values for respective stimulations are shown in Fig. 2 (CD4+, color coded in blue) and Fig. 3 (CD8+, color coded in red). Donors with a SI > 3 (dotted line) are regarded as responders to MP stimulation. Panels show individual values for n=10 patients vs. n=10 HC, as well as the mean ± SD. Asterisk denotes a significant difference. HC = healthy control. Symbol shapes of COVID-19 patients are identical between panels, and refer back to Fig. 1.

  • Fig. 5 SARS-CoV-2-specific cytokine production in COVID-19 ARDS patients.

    (A-J) Antigen-specific production of cytokines measured in cell culture supernatants from PBMC obtained 14 days post ICU admission stimulated (20 hours) with MP_S. Two left panels show quantities obtained with the vehicle control (DMSO) and specific stimulation (MP) for HC and COVID-19 patients. The third panel shows the quantity corrected by subtracting the background present in the DMSO stimulation to allow comparison of both groups. Panels show individual values for n=10 patients versus n=10 HC, as well as the geometric mean. Asterisk denotes a significant difference. HC = healthy control. Additional cytokines (IL-4, IL-17F and IL-21) are shown in Fig. S2. Symbol shapes of COVID-19 patients are identical between panels, and refer back to Fig. 1.

  • Fig. 6 SARS-CoV-2 replication and humoral and cellular immune response kinetics in COVID-19 ARDS patients.

    (A, B, C, D) Sequential measurements of SARS-CoV-2 genomes detected in upper respiratory tract samples by real-time RT-PCR (40-ct, A), SARS-CoV-2-specific serum RBD IgG antibody levels detected by ELISA (OD450, B) and percentage SARS-CoV-2-specific CD4+ and CD8+ T cells after MP_S stimulation of PBMC (C, D), plotted against days post onset of symptoms. Genome levels showed a significant decrease over time, antibody levels and specific CD4+ T-cell frequencies significantly increased (p<0.001, ANOVA repeated measures). A specific increase or decrease of specific CD8+ T cells over time was not detected (p=0.1001, ANOVA repeated measures). Symbol shapes of COVID-19 patients are identical between panels, and refer back to Fig. 1.

  • Fig. 7 Expression of activation markers in representative samples.

    (A, B, C) Representative activation plots showing CD69 and CD137 up-regulation from a non-responder HC (A), cross-reactive HC (B) and reactive COVID-19 sample (C). Each panel shows activation after stimulation with DMSO (negative control) or MP_S. Top row shows CD4+ T cell responses, bottom row shows CD8+ T cell responses. Percentages indicate activated CD69+CD137+ cells as a fraction of either CD4+ or CD8+ T cells.

Supplementary Materials

  • immunology.sciencemag.org/cgi/content/full/5/48/eabd2071/DC1

    Figure S1. Flow cytometry gating strategy.

    Figure S2. SARS-CoV-2-specific cytokine production in COVID-19 ARDS patients.

    Figure S3. Correlations between kinetics of viral loads, virus-specific antibodies and virus-specific T cell responses.

    Table S1. Raw data (in Excel spreadsheet).

  • Supplementary Materials

    The PDF file includes:

    • Fig. S1. Flow cytometry gating strategy.
    • Fig. S2. SARS-CoV-2-specific cytokine production in COVID-19 ARDS patients.
    • Fig. S3. Correlations between kinetics of viral loads, virus-specific antibodies and virus-specific T-cell responses.

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    Other Supplementary Material for this manuscript includes the following:

    • Table S1. Raw data (in Excel spreadsheet).

    Files in this Data Supplement:

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