Table 1.
Demographic, laboratory and severity characteristics of the study cohort.
Fig 1.
Flow cytometric analysis of peripheral blood NK cell subsets and receptors in COVID-19 patients.
Panel A: Flow cytometric gating strategy. Following physical selection (FCS, SSC), CD3-CD19-CD14- cells are gated and further analyzed as NK cells expressing CD16 and CD56. Analysis of the acquisition of 10000 events. Log scale. Panel B: Analysis of peripheral blood NK cell subsets defined by CD16 and CD56 expression in COVID-19 patients (#28, grey columns) and HD (#18, white columns). CD56brightCD16+/- cells are indicated as CD56bright, CD56dimCD16+ NK cells are indicated as CD56dim; CD56-CD16+ NK cells are indicated as CD56-. Histograms show mean±SD. Cells are gated on CD3-CD19-CD14- cells. Significance by Mann-Whitney U-test analysis is indicated. The ratio between CD56brightCD16+/- and CD56dimCD16+ cells is indicated as CD56bright/CD56dim. Open histograms: HD. Greyed histogram: COVID-19. Panel C: Analysis of PB NK cell receptor expression. Histograms indicate the proportion of NK cells expressing each surface molecule. Cells are gated on CD3-CD19-CD14- cells. Significance by Mann-Whitney U-test analysis is indicated. Open histograms: HD. Greyed histogram: COVID-19. Panel D: Molecule density expression of NK cell molecules on PB NK cells. Molecule density is expressed as the MFI ratio. Histograms show mean±SD. Mann-Whitney U-test analysis is indicated.
Fig 2.
Flow cytometric analysis of peripheral blood NK cell development and activation in COVID-19 patients.
Analysis is performed on CD3-CD14-CD19- cells by flow cytometry in COVID-19 patients (#28) and HD (#18). Histograms show mean±SD. Significance by Mann-Whitney U-test analysis is indicated. Open histograms: HD. Greyed histogram: COVID-19. Panel A: Analysis of circulating NK cell development according to NKG2A and KIR expression of PB cells. KIR and NKG2A were analyzed on CD3-CD14-CD19-CD56+CD16+ cells. Maturing = KIR-NKG2A+. Intermediate = KIR+NKG2A+. Mature = KIR+NKG2A-. Memory = CD85j+KIR+ CD57+NKG2A-. Panel B: MFI ratios of KIR expression in NK cells. MFIr = mean fluorescence intensity ratio calculated as (MFI sample–MFI neg control)/MFI neg control. MFIr expresses NK cell molecule density. Panel C: Proportions of NK cells expressing CD69 and HLA-DR. Panel D: MFI ratios of CD69 and HLA-DR on PB NK cells. MFIr = mean fluorescence intensity ratio calculated as (MFI sample–MFI neg control)/MFI neg control. MFIr expresses NK cell molecule density. MFIr expresses NK cell molecule density. Panel E: analysis of the expression of HLA-DR in NKp46+, NKp30+, or NKG2D+ NK cells.
Fig 3.
Flow cytometric and correlation analysis of the expression of activation and tissue-residency molecules on circulating NK cells in COVID-19 patients.
Panel A: Correlation analysis of the expression of CD69 and of HLA-DR on PB CD56+CD16+ NK cells according to least squares linear regression. Slope and 95% confidence limits are plotted. p = 0.01. Panel B: Flow cytometric dot plot analysis of the expression of CD69 and of HLA-DR on circulating NK cells in different patients. CD69 and HLADR are mostly not coexpressed in COVID-19 patients. A representative HD profile is also shown. Panel C: Analysis of CD103 and of CD49d expression on PB NK cells in COVID-19 patients and HD. Histograms show mean±SD. Significant differences are indicated according to the Mann-Whitney U-test. Panel D: Increased coexpression of CD49d and CD69 in circulating CD103+ NK cells in COVID-19 patients. Histograms show mean±SD. Mann-Whitney U-test analysis is shown. Panel E: A tSNE plot showing distinct expression of CD69, CD49d, and CD103 in COVID-19 and HD. Panel E: A t-SNE representation of the coexpression of CD69, CD103, CD49d in PB NK cells. Panel F: Analysis of CXCR6 expression on PB NK cells in a representative sample of COVID-19 patients and HD. Dot plots show CD103 expression on CD3-CD14-CD19- NK cells and their coexpression of CD69 and/or CXCR6. The proportion of positive cells is indicated. COVID-19 patients have relevant circulation of CXCR6+CD69+CD103+NK cells, while HD have no such circulation (here 32.09% vs. 0.63%).
Fig 4.
Functional study of circulating NK cells and of their peripheral turnover through analysis of “inflammatory” CD34+DNAMbrightCXCR4+ precursors.
Panel A: In vitro NK cell cytotoxicity assay by flow cytometric analysis of CD107a expression in a representative COVID-19 patient and HD. A redirected killing (reverse ADCC) is shown, using NKp46 and NKp30 NKG2D, NKG2C, CD16 mAb-mediated triggering in the presence of Fcγ+ P815 cells. Left: no mAb. Center: NCRs (α-NKp46+αNKp30, α- NKG2D, α-NKG2C, α-CD16). Right: the total lytic potential (as tested by maximal stimulation with PMA+ionomycin) of circulating NK cells is decreased in COVID-19 patients. Dots indicate CD107a degranulation 4 hours after NK cell triggering with the indicated stimulus. Panel B: In vitro cytotoxicity of PB NK cells as determined by CD107a expression in COVID-19 patients (n = 8) vs. HD (n = 8) is not inducible. Histograms show mean±SD. Mann-Whitney U-test analysis is shown. Panel C: Perforin expression by NK cells in COVID-19 patients is conserved. Representative flow cytometric analysis of CD3-CD19-CD14-CD56+ NK cells expressing perforin. 10000 events are analyzed. Panel D: The proportion of perforin+ NK cells is conserved in COVID-19 patients. Histograms indicate the proportion of Perf+ NK cells over total CD56+ NK cells, mean±SD. Panel E: Flow cytometric gating strategy and analysis of DNAM-1 and CD34 expression on Lin- CD16- cells in a representative COVID-19 and a HIV-1 patient and a control HD. Lineage = CD3, CD14, CD19, CD56. A representative COVID-19 patient (upper plot), HIV patient (middle plot), and HD (bottom plot) are shown. Box and arrow indicate the area of DNAM-1bright appearance of CD34+ circulating precursors. Panel F: Cumulative circulation of CD34+DNAM-1brightCXCR4+ common lymphocyte precursors in COVID-19 patients (#28) is increased compared to HD (#18) and also to HIV-1 patients (#15). Histograms show mean±SD. Mann-Whitney U-test analysis is shown.
Fig 5.
Ordinal logistic fit of variables with subsequent clinical trajectory according to modified WHO classification.
Panel A: Logistic profiler for the disease course for age and lymphocyte n° at admission according to subsequent disease trajectory. Logistic fit of the model is indicated. The vertical dotted line for given X variables shows a representative current value (red number). Horizontal dotted lines show the current predicted value for the displayed values of the X variables. Panel B: Logistic profiler for disease course according to the proportion of CD34+DNAMbrightCXCR4+ cells, the proportion of NKG2D+ and of NKG2C+ NK cells. Logistic fit of the model is indicated. The vertical dotted line for given X variables shows a representative current value (red number). Horizontal dotted lines show the current predicted value for the displayed values of the X variables. Panel C: Logistic profiler for disease course according to the proportion of CD103+ NK cells and of CD49+CD103+ NK cells. Logistic fit of the model is indicated. The vertical dotted line for given X variables shows a representative current value (red number). Horizontal dotted lines show the current predicted value for the displayed values of the X variables.