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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 after inclusion. (C) CD4:CD8 ratios measured by flow cytometry performed on PBMC collected 14 days after inclusion. (B) and (C) show individual values for n = 10 patients versus n = 10 HC, as well as the mean ± SD. Asterisks denote significant differences.

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

    (A and 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 patients with COVID-19. 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. Asterisks denote significant differences. Symbol shapes of patients with COVID-19 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 to 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 (C) and (D). Two left panels show activation percentages (within CD3+CD8+ gate) obtained with the vehicle control (DMSO) and specific stimulation (MP) for HCs and patients with COVID-19. 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. Asterisks denote significant differences. Symbol shapes of patients with COVID-19 are identical between panels and refer back to Fig. 1.

  • Fig. 4 SI identifies specific responders.

    Antigen-specific activation of CD4+ (A) and CD8+ T cells (B and C) in patients with COVID-19 after stimulation for 20 hours with peptide MPs is shown as SI. SI 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 lines) are regarded as responders to MP stimulation. Panels show individual values for n = 10 patients versus n = 10 HC, as well as the mean ± SD. Asterisks denote significant differences. Symbol shapes of patients with COVID-19 are identical between panels and refer back to Fig. 1.

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

    (A to J) Antigen-specific production of cytokines measured in cell culture supernatants from PBMC obtained 14 days after 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 patients with COVID-19. 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. Asterisks denote significant differences. Additional cytokines (IL-4, IL-17F, and IL-21) are shown in fig. S2. Symbol shapes of patients with COVID-19 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 to 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 (Endpoint titer, B), and percentage SARS-CoV-2–specific CD4+ and CD8+ T cells after MP_S stimulation of PBMC (C and D) plotted against days after the onset of symptoms. Genome levels showed a significant decrease over time, and 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 patients with COVID-19 are identical between panels and refer back to Fig. 1.

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

    (A to C) Representative activation plots showing CD69 and CD137 up-regulation from a nonresponder 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, and bottom row shows CD8+ T cell responses. Percentages indicate activated CD69+CD137+ cells as a fraction of either CD4+ or CD8+ T cells. These percentages were used in the generation of Figs. 2 and 3.

Supplementary Materials

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

    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.

    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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