Research ArticleCORONAVIRUS

Longitudinal immune profiling reveals key myeloid signatures associated with COVID-19

See allHide authors and affiliations

Science Immunology  17 Sep 2020:
Vol. 5, Issue 51, eabd6197
DOI: 10.1126/sciimmunol.abd6197
  • Fig. 1 Patient recruitment and categorization.

    (A) Patients were recruited to the study as close to admission as possible and within 7 days. Peripheral blood samples were collected on recruitment and at intervals thereafter. Samples were analyzed immediately, and results were stratified based on their ultimate disease severity. (B) Criteria for patient stratification. NIV, noninvasive ventilation; CPAP, continuous positive airway pressure; COPD, chronic obstructive pulmonary disease.

  • Fig. 2 Whole blood immune profile of patients with COVID-19.

    (A) UMAP of flow cytometry panel broadly visualizing white cells in whole blood. Representative images for healthy individuals and mild, moderate, and severe patients are shown. Key indicates cells identified on the image. (B) Graphs show neutrophil (CD16+CD11bhi), CD14+ monocyte, CD3+ T cell, and CD19+ B cell frequencies in whole blood samples of healthy individuals (n = 28) and recruitment samples from COVID-19 patients with mild (n = 12), moderate (n = 13), and severe (n = 6) disease. (C) Longitudinal time course of (top row) neutrophils (CD16+CD11bhi), (second row) CD14+ monocytes, (third row) CD3+ T cells, and (bottom row) B cells segregated by disease severity. Individual patients are shown as different colors and shapes, with lines connecting data from the same patient. Crossed squares for severe patients are time points in ICU. X-axis values represent the number of days since reported onset of symptoms. (D) Graphs showing frequencies of neutrophils (CD16+CD11bhi), monocytes, T cells, and B cells at the first and last time points in (left) mild/moderate patients (green and blue circles) and (right) severe patients (black circles). Red triangles represent severe patients that had poor outcome (deceased or long-term ICU) and are not included in the statistical test. Graphs show individual patient data, with the bar representing median values. In all graphs, open triangles represent SARS-CoV-2 PCR-negative patients. (B) Kruskal-Wallis with Dunn’s post hoc test: Neutrophils, T cells, and B cells; one-way ANOVA with Holm-Sidak post hoc test: CD14+ monocytes. (D) Paired t test: All except monocyte graph detailing mild and moderate patients, which was tested using Wilcoxon matched-pairs signed-rank test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

  • Fig. 3 Altered phenotype of T and B cells in patients with COVID-19.

    (A and B) Graphs show frequencies of (A) CD8+ and (B) CD4+ T cells in freshly isolated PBMCs of healthy individuals (n = 36) and recruitment samples from COVID-19 patients with mild (n = 17), moderate (n = 18), and severe (n = 9 to 10) disease. (C and D) Representative flow cytometry plots and graph showing frequency of CD8+ T cells that are positive for perforin in healthy individuals (n = 21) and COVID-19 patients with mild (n = 16), moderate (n = 12), and severe (n = 7) disease. (E) Graph showing correlation of perforin+CD8+ T cell frequency with CRP in patients with COVID-19. (F) Graphs show frequencies of CD19+ B cells in freshly isolated PBMCs of healthy individuals (n = 43) and recruitment samples from COVID-19 patients with mild (n = 14), moderate (n = 19), and severe (n = 9) disease. (G) Representative flow cytometry plots and cumulative data show Ki-67 expression by B cells in healthy individuals (n = 39) and patients with COVID-19 (n = 45). Correlation graph shows correlation of Ki-67+ B cells with CRP. (H) Representative flow cytometry plots and cumulative data show frequency of CD27hiCD38hi plasmablasts in healthy individuals (n = 42) and in patients with COVID-19 (n = 66). (I) Correlation graph shows correlation of plasmablasts and IgG+ B cell frequencies. (J) Graph shows frequencies of DN (CD27IgD) B cells in freshly prepared PBMCs of healthy individuals (n = 42) and recruitment samples from COVID-19 patients with mild (n = 14), moderate (n = 19), and severe (n = 9) disease. Graphs show individual patient data, with the bar representing median values. In all graphs, open triangles represent SARS-CoV-2 PCR-negative patients. Mann-Whitney U test (G and H), Kruskal-Wallis with Dunn’s post hoc test (A, D, F, and J), one-way ANOVA with Holm-Sidak post hoc test (B), and Spearman ranked coefficient correlation test (E, G, and I). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

  • Fig. 4 Dysregulation of circulating monocytes in COVID-19.

    (A) Graphs show levels of CD64 expression as assessed by mean fluorescence intensity (MFI) on CD14+ classical monocytes in freshly prepared PBMCs of healthy individuals (n = 25) and recruitment samples from all patients with COVID-19 (n = 58). Patients with COVID-19 were also stratified into mild (n = 12), moderate (n = 10), and severe (n = 8) disease. a.u., arbitrary units. (B) Graphs show frequencies of TNF-ɑ+ CD14+ monocytes after LPS stimulation of freshly prepared PBMCs from healthy individuals (n = 41) and patients with COVID-19 (n = 59). Patients with COVID-19 were also stratified into mild (n = 14), moderate (n = 15), and severe (n = 7) disease. (C) Representative fluorescence-activated cell sorting (FACS) plots demonstrating intracellular COX-2 expression by CD14+ monocytes from healthy individuals and patients with COVID-19. (D and E) Graphs showing (D) frequencies of COX-2+ CD14+ monocytes and (E) COX-2 expression level as determined by MFI in CD14+ monocytes after LPS stimulation of freshly prepared PBMCs from healthy individuals (n = 33) and total COVID-19 patients (n = 51). Patients with COVID-19 were also stratified into mild (n = 12), moderate (n = 11), and severe (n = 6) disease. (F) Representative FACS plots demonstrating intracellular Ki-67 staining by CD14+ monocytes. (G) Graphs show frequencies of Ki-67+ CD14+ monocytes after LPS stimulation of freshly prepared PBMCs from healthy individuals (n = 37) and total COVID-19 patients (n = 60). Patients with COVID-19 were also stratified into mild (n = 14), moderate (n = 14), and severe (n = 8) disease. (H) Correlation of Ki-67 (percentage of monocytes expressing Ki-67) with CRP in patients with COVID-19. (I to K) Longitudinal time course of frequencies of CD14+ monocytes that are positive for (I) TNF-α, (J) COX-2, and (K) Ki-67 after LPS stimulation in mild (green shapes, n = 6 to 7) and severe (black shapes, n = 4 to 6) COVID-19 patients, with lines connecting data from the same patient. On all graphs, x-axis values represent the number of days since onset of symptoms and the dotted line represents the median value from healthy individuals. (L) Graphs showing frequencies of monocytes that are TNF-α+, COX-2, and Ki-67+ after LPS stimulation at the first and last time points in (left) mild patients (green circles) and (right) severe patients (black circles). Graphs show individual patient data, with the bar representing median values. In all graphs, open triangles represent SARS-CoV-2 PCR-negative patients. Mann-Whitney U test (A, B, D, E, and G), Kruskal-Wallis with Dunn’s post hoc test (B, D, E, and G), one-way ANOVA with Holm-Sidak post hoc test (A), Spearman ranked coefficient correlation test (H), and paired t test (L). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

  • Table 1 Clinical characteristics.

    Data are listed as median (IQR)m, where m is the number of missing data points, n (%), or n/N (%), where N is the total number with available data. Representative participants from each severity cohort were used in cross-sectional or longitudinal analysis.

    All patients (49)Mild (18)Moderate (21)Severe (10)
    Age61 (51–71)61.5 (45–72.5)59 (51–68)66 (52–72.5)
    Sex
      Male31 (63.3%)11 (61.1%)13 (62%)7 (70%)
      Female18 (36.7%)7 (38.9%)8 (38%)3 (30%)
    Body mass index27.5 (24.9–30)427.1 (23.6–30)128.3 (25.7–30)226.5 (24.9–30.4)1
    Comorbidity
      Diabetes8/49 (16.3%)3/18 (16.7%)2/21 (9.5%)3/10 (30%)
      Ischemic heart disease5/49 (10.2%)2/18 (11.1%)1/21 (4.8%)2/10 (20%)
      Hypertension14/49 (28.6%)5/18 (27.8%)7/21 (33.3%)2/10 (20%)
      Chronic obstructive
    pulmonary disease
    9/49 (18.4%)4/18 (22.2%)4/21 (19.1%)1/10 (10%)
      Asthma5/49 (10.2%)2/18 (11.1%)3/21 (14.3%)0/10 (0%)
      Malignancy3/49 (6.1%)0/18 (0%)1/21 (4.8%)2/10 (20%)
    Presentation
      Illness onset to admission
    (days)
    7 (4–10)47 (4–8)17.5 (2.8–10.8)35.5 (4–9.5)
      Dyspnea29/41 (70.7%)8/16 (50%)14/16 (87.5%)7/9 (77.8%)
      Cough30/41 (73.2%)11/16 (68.8%)12/16 (75%)7/9 (77.8%)
      Fever28/41 (68.3%)10/16 (62.5%)9/16 (56.3%)9/9 (100%)
      Diarrhea/vomiting14/41 (34.2%)3/16 (18.8%)7/16 (43.8%)4/9 (44.4%)
      Myalgia10/40 (25%)4/15 (26.7%)3/16 (18.8%)3/9 (33.3%)
      Fatigue10/38 (26.3%)3/14 (21.4%)5/15 (33.3%)2/9 (22.2%)
      Day recruited2 (2–3)3 (2–4.5)2 (2–3)2 (2–3)
      Number of time points2 (1–4)2 (1–2)3 (2–3)4.5 (2–5)
      Respiratory rate*20 (18–25)1020 (17–24)521 (18–26)321.5 (17.8–24)2
      Temperature*37.5 (36.9–38.4)1037.1 (36.5–37.4)537.7 (37.1–38.8)338 (37.7–39.2)2
      Systolic blood pressure*125 (117–136)10122 (117–126)5126 (118–136)3137.5 (114.5–156.3)2
    Chest radiograph findings
      Bilateral opacification41/47 (87.2%)11/16 (68.8%)20/21 (95.2%)10/10 (100%)
      Unilateral opacification3/47 (6.4%)2/16 (12.5%)1/21 (4.8%)0/10 (0%)
      No abnormality3/47 (6.4%)3/16 (18.8%)0/21 (0%)0/10 (0%)
    COVID nasopharyngeal test
      Positive42 (86%)15 (83.3%)18 (85.7%)9 (90%)
      Negative7 (14%)3 (16.7%)3 (14.3%)1 (10%)
    Differential full blood count at admission
      White blood cell count (×109/
    liter)
    6.9 (5.7–9.8)6.7 (4.7–7.5)7.1 (6.3–10)7.3 (5.7–10)
      Lymphocytes (×109/liter)1.1 (0.8–1.4)1.2 (0.8–1.3)1.3 (0.8–1.5)0.9 (0.8–1.1)
      Neutrophils (×109/liter)5.1 (3.8–7.4)4.7 (3.2–5.7)5.3 (4.5–7.7)6.3 (4.2–8.6)
      Monocytes (×109/liter)0.4 (0.2–0.7)0.4 (0.2–0.6)0.5 (0.3–0.7)0.3 (0.2–0.5)
      Platelets (×109/liter)244 (188–367)2241 (188–316)2269 (195–366)204 (155–412)
    Highest acute-phase response/liver function tests
      CRP (mg/liter)127 (75–226)88 (38–166)120 (75–201)269 (244–296)
      Alanine aminotransferase (U/
    liter)
    48 (27–87)1657 (28–75)935 (25–79)549 (37–105)2
      Alkaline phosphatase (U/liter)78 (63–96)1679 (63–82)972 (63–90)5110 (71–172)2
      Bilirubin (μM)11 (7–15)1610 (8–14)99 (7–13)513 (10–21)2
    Complications
      Pulmonary embolism5/49 (10.2%)1/18 (5.6%)4/21 (19.1%)0/10 (0%)
      Acute kidney injury3/49 (6.1%)0/18 (0%)2/21 (9.5%)1/10 (10%)
      Mortality6/49 (12.2%)1/18 (5.6%)0/21 (0%)5/10 (50%)

    *Admission observations.

    Supplementary Materials

    • immunology.sciencemag.org/cgi/content/full/5/51/eabd6197/DC1

      Fig. S1. Immune cell types in patients with COVID-19.

      Fig. S2. Serum cytokines and chemokines in patients with COVID-19.

      Fig. S3. T cell activation in patients with COVID-19.

      Fig. S4. B cell subsets in patients with COVID-19.

      Fig. S5. Monocytes in patients with COVID-19.

      Table S1. Raw data file (Excel spreadsheet).

    • The PDF file includes:

      • Fig. S1. Immune cell types in COVID-19 patients.
      • Fig. S2. Serum cytokines and chemokines in COVID-19 patients.
      • Fig. S3. T cell activation in COVID-19 patients.
      • Fig. S4. B cell subsets in COVID-19 patients.
      • Fig. S5. Monocytes in COVID-19 patients.

      [Download PDF]

      Other Supplementary Material for this manuscript includes the following:

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

    Stay Connected to Science Immunology

    Navigate This Article