Extensive activation, tissue trafficking, turnover and functional impairment of NK cells in COVID-19 patients at disease onset associates with subsequent disease severity

Abstract

The SARS-CoV-2 infection causes severe respiratory involvement (COVID-19) in 5-20% of patients through initial immune derangement, followed by intense cytokine production and vascular leakage. Evidence of immune involvement point to the participation of T, B, and NK cells in the lack of control of virus replication leading to COVID-19. NK cells contribute to early phases of virus control and to the regulation of adaptive responses. The precise mechanism of NK cell dysregulation is poorly understood, with little information on tissue margination or turnover. We investigated these aspects by multiparameter flow cytometry in a cohort of 28 patients hospitalized with early COVID-19. Relevant decreases in CD56brightCD16+/- NK subsets were detected, with a shift of circulating NK cells toward more mature CD56dimCD16+KIR+NKG2A+ and "memory" KIR+CD57+CD85j+ cells with increased inhibitory NKG2A and KIR molecules. Impaired cytotoxicity and IFN-γ production were associated with conserved expression of natural cytotoxicity receptors and perforin. Moreover, intense NK cell activation with increased HLA-DR and CD69 expression was associated with the circulation of CD69+CD103+ CXCR6+ tissue-resident NK cells and of CD34+DNAM-1brightCXCR4+ inflammatory precursors to mature functional NK cells. Severe disease trajectories were directly associated with the proportion of CD34+DNAM-1brightCXCR4+ precursors and inversely associated with the proportion of NKG2D+ and of CD103+ NK cells. Intense NK cell activation and trafficking to and from tissues occurs early in COVID-19, and is associated with subsequent disease progression, providing an insight into the mechanism of clinical deterioration. Strategies to positively manipulate tissue-resident NK cell responses may provide advantages to future therapeutic and vaccine approaches.

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). CD56^brightCD16+/- cells are indicated as CD56^bright, CD56^dimCD16+ NK cells are indicated as CD56^dim; 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 CD56^brightCD16+/- and CD56^dimCD16+ cells is indicated as CD56^bright/CD56^dim. 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+DNAM^brightCXCR4+ 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-1^bright appearance of CD34+ circulating precursors. Panel F: Cumulative circulation of CD34+DNAM-1^brightCXCR4+ 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+DNAM^brightCXCR4+ 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.