key: cord-260852-u0gpklj7
authors: Osman, Mohammed; Faridi, Rehan M.; Sligl, Wendy; Shabani-Rad, Meer-Taher; Dharmani-Khan, Poonam; Parker, Arabesque; Kalra, Amit; Tripathi, Minal Borkar; Storek, Jan; Cohen Tervaert, Jan Willem; Khan, Faisal M.
title: Impaired natural killer cell counts and cytolytic activity in patients with severe COVID-19
date: 2020-10-19
journal: Blood Adv
DOI: 10.1182/bloodadvances.2020002650
sha: 
doc_id: 260852
cord_uid: u0gpklj7

The global pandemic of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)–driven coronavirus disease 2019 (COVID-19) has caused unprecedented human death and has seriously threatened the global economy. Early data suggest a surge in proinflammatory cytokines in patients with severe COVID-19, which has been associated with poor outcomes. We recently postulated that the inflammatory response in patients with severe COVID-19 disease is not inhibited by natural killer (NK) cells, resulting in a “cytokine storm.” Here, we assessed the NK-cell functional activity and the associated cytokines and soluble mediators in hospitalized COVID-19 patients. Significantly impaired NK-cell counts and cytolytic activity were observed in COVID-19 patients when compared with healthy controls. Also, cytokines like interleukin 12 (IL12), IL15, and IL21 that are important for NK-cell activity were not detected systematically. Serum concentrations of soluble CD25 (sCD25)/soluble IL2 receptor α (sIL2-Rα) were significantly elevated and were inversely correlated with the percentage of NK cells. Impaired NK-cell cytolytic activity together with other laboratory trends including elevated sCD25 were consistent with a hyperinflammatory state in keeping with macrophage-activation syndrome. Our findings suggest that impaired counts and cytolytic activity of NK cells are important characteristics of severe COVID-19 and can potentially facilitate strategies for immunomodulatory therapies.

First reported in late 2019, 1,2 the coronavirus disease 2019 , caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), 3 continues to spread across the globe. As of 16 August 2020, 21 756 357 cases have been confirmed with at least 771 635 deaths in over 180 countries worldwide. 4 In addition to fever, nonproductive cough, dyspnea, fatigue, and lymphopenia, severe COVID-19 cases present with pneumonia and acute respiratory distress syndrome (ARDS), multiorgan failure, and even death. 5 To date, there are no preventative vaccines, nor proven therapeutics that have been approved for this disease, though efforts worldwide have shown some promising results. 6, 7 Rapid activation of the innate immune system accompanied by an aggressive inflammatory response, associated with a surge in many proinflammatory cytokines, has been reported, especially in patients developing severe manifestations of COVID-19. 5, 8 This "hypercytokinemia" or "cytokine storm" is thought to be driving the COVID-19-related ARDS, 5, 8 and associated hyperferritinemia is reminiscent of macrophage-activation syndrome (MAS) or secondary hemophagocytic lymphohistiocytosis (HLH; sHLH).

Timely and effective management of severe COVID-19 persists as one of the major unmet needs and can be facilitated by an understanding of the associated hyperinflammation. 9, 10 Although HLH-related laboratory trends may have limited relevance in severe COVID-19, natural killer (NK)-cell functionality could be critical in the evolving COVID-19-MAS/sHLH milieu. 11 NK cells constitute the first line of defense against viral infections and are functionally impaired in MAS/sHLH as an outcome of an intrinsic functional deficiency, lymphopenia, or both. 12 Moreover, NK cells regulate the macrophage turnover and provide a negative feedback mechanism to macrophage overabundance and a more tissueaggressive inflammatory response to infection. 13 The absence of NK-cell-mediated frontline defense is associated with an aggressive secondary hyperinflammatory response driving ARDS in severe COVID-19 patients and forms the hypothetical framework of this study. Here, we evaluated NK-cell counts and activity as well as serum concentrations of associated cytokines and soluble receptors as characteristics of hyperinflammation associated with severe COVID-19.

Seventy-eight healthy controls (42 men, 36 women; median age, 32 years; range, 18-76 years) and 10 hospitalized COVID-19 patients (7 men, 3 women; median age, 67 years; range, 39-87 years), positive for SARS-CoV-2 were included in the study. The patients were SARS-CoV-2 1 by quantitative reverse transcription-polymerase chain reaction assay specific for envelope transcripts. Peripheral blood was collected within 24 to 48 hours of confirmatory polymerase chain reaction testing (5-8 days from the onset of symptoms). All patients were febrile, hypoxic, and were free from any coinfection. Of these, 4 patients required mechanical ventilation. The samples were obtained in the absence of any antiviral or immunomodulation therapies. The University of Alberta Health Research Ethics Board approved this study (Pro00099910).

Flow cytometry-based absolute counts of NK cells (CD45 1 CD3 2 CD56 1 CD16 1/2 ) and their subset distribution (CD56 dim and CD56 bright ) were determined as a routine laboratory investigation and were calculated using clinical hematology laboratory-determined absolute lymphocyte counts. NK-cell functional activity was assessed using a flow cytometry-based CD107a (degranulation) and interferon-g (IFN-g) production assay described earlier. 14 Briefly, peripheral blood mononuclear cells were isolated using the Ficollgradient method. Mononuclear cells were cocultured with erytholeukemic K562 cells in a 2:1 effector-to-target ratio in complete RPMI 1640 medium and mouse anti-human CD107a-fluorescein isothiocyanate (FITC, clone eBioH4A3) for 4 hours at 37°C, 5% CO 2 . Golgi inhibitors (monensin and brefeldin) were added after 1 hour of culture to enable intracellular staining. Cells harvested after culture were stained with monoclonal mouse anti-human antibodies: CD56-allophycocyanin (APC, clone TULY56), CD3-eflour450 (clone 17A2), and IFN-g-phycoerythrin (PE, clone 45-15) before flow cytometry-based enumeration of CD56 1 CD3 2 NK cells that were either degranulating (CD107a 1 ) or producing cytokine (IFN-g 1 ). CD107a 1 and IFN-g 1 NK cells were expressed as a percentage of parent (NK) cells.

Luminex-based multiplex immunoassays were used to determine the serum concentrations of interleukin 12 (IL12), IL15, and IL21 (ThermoFisher Scientific Inc), and soluble CD25 (sCD25)/ soluble IL2 receptor a (sIL2-Ra) (R&D Systems Inc) as per the manufacturer's instructions.

NK-cell counts, functional activities, and serum analytes obtained for COVID-19 patients were compared with those obtained for healthy controls using Mann-Whitney U statistics. Where applicable, Two-tailed P , .05 was considered statistically significant. *P , .05; **P , .0001. ns, not significant. correlation analyses were performed using Spearman rank correlation. Test performance characteristics of all of the assays were determined in the clinically accredited Hematology Translational Laboratory, University of Calgary.

Absolute counts of total NK cells were significantly lower in COVID-19 patients (median, 1.1 3 10 8 /L; range, 0.2 3 10 8 /L to 2.9 3 10 8 /L) than in controls (median, 2 3 10 8 /L; range, 1.1 3 10 8 /L to 8.3 3 10 8 /L; Figure 1 ). Significantly higher percentages of CD56 dim and lower percentages of CD56 bright NK-cell fractions were observed in patients than in controls (Figure 1) . Similarly, higher percent CD16 1 and lower percent CD16 2 populations were observed when comparing the COVID-19 patients to controls (data not shown).

Upon stimulating with K562, the cytolytic responses expressed as the percentage of CD107a 1 NK cells were remarkably impaired with significantly low cytolytic activity per cell in COVID-19 patients as compared with controls ( Figure 1 ). Nine of 10 patients had ,10% CD107a 1 NK cells in response to K652 cells, which was below the healthy control reference range (10% to 38%) established as the 5th to 95th percentile using this assay. Intracellular IFN-g production was not significantly different with 7 of 10 patients having .1% IFN-g-producing NK cells (reference range, 1% to 7%). The observed low NK-cell counts and cytolytic activity is consistent with what is known in MAS/sHLH. 15, 16 Similar observations were recently reported in COVID-19, however, an NKG2A-mediated functional suppression was attributed. 17 Serum concentrations of NK-cell-activating cytokines (IL12, IL15, and IL21) were significantly lower in patients than in controls ( Figure 2 ). These cytokines are typically elevated in patients with MAS/sHLH, suggesting that an alternative mechanism of NK-cell suppression in COVID-19 is in play. Reduced expression of CD107a, Ksp37, granzyme B, and granulysin in addition to impaired production of chemokines, IFN-g, and tumor necrosis factor a (TNFa) was recently shown in ex vivo cells from peripheral blood of COVID-19 patients. 17, 18 Increased immune checkpoints through the upregulation of inhibitory receptors on NK cells contributing to viral escape have been suggested. [18] [19] [20] Furthermore, other RNA viruses causing pulmonary infections such as influenza A have also been reported to reduce NK-cell activity and levels. 21, 22 In vitro stimulation by IL6 (and IL6R) has been shown to result in impaired NK-cell cytotoxicity, which was restored following IL6R blockade using tocilizumab. 23 In the early studies, COVID-19 patients were shown to have higher plasma concentrations of IL6, 5 which significantly correlated with lower NK-cell numbers. 24,25 TNF-a, known to contribute to NK-cell differentiation, 26 is also shown to be upregulated in the plasma of COVID-19 patients. 5 These data suggest that NK-cell recognition and killing of SARS-CoV-2-infected cells may be impaired by the cross talk with monocytes, and antibodies targeting IL6 and TNF signaling may benefit enhanced NK-cell functions in COVID-19 patients. Given the accumulating evidence of an overactive immune system driving severe COVID-19-related ARDS, widespread interest has developed both for anticytokine therapies 27, 28 and early identification of hyperinflammation. 10 Figure 2 ). However, consistent with earlier observations, 30,31 these parameters, and especially those that weigh in heavy in the H-score (eg, hyperferritinemia, sCD25), do not reach the "HLH-high" thresholds. Furthermore, high fever (.39°C) is not exclusive to severe COVID-19 32 and other H-score criteria such as hypertriglyceridemia, organomegaly, and bone marrow hemophagocytosis do not appear to apply to COVID-19. 5, 32 Here, we describe impaired NK-cell cytolytic activity associated with a loss of cytokines important in NK-cell stimulation in severe COVID-19 patients. This impairment and the resulting absence of a negative feedback mechanism for the secondary tissue-aggressive inflammatory reaction may explain the proinflammatory cytokine surge observed in severe COVID-19 patients, as postulated earlier. 11 In its attempt to understand the pathophysiology of the COVID-19 pandemic during these unprecedented times, this study faces several challenges and limitations, a small sample size being one of them. The clinical presentations and laboratory data of only severe COVID-19 patients, representing only a subset of infected individuals, were studied. Finally, given the skewed demographics of disease severity, the healthy controls are not matched for age. Nevertheless, these findings are important as a substantial addition to the rapidly evolving understanding of the pathophysiology in COVID-19 and can be used as relevant biomarkers in patients receiving immunomodulating therapies. 33 In conclusion, this study demonstrates that reduced NK-cell counts and impaired cytolytic activity are important characteristics associated with severe COVID-19-related hyperinflammation. Further prospective studies could address whether NK-cell characteristics, including a genetic predisposition for impaired NK-cell function, could facilitate early identification of severe COVID-19 and guide interventions such as targeted immunomodulation to reduce cytokine storm and hopefully reduce morbidity and mortality.

Coronavirus infections-more than just the common cold

Genomic characterisation and epidemiology of 2019 novel coronavirus: implications for virus origins and receptor binding

Coronaviridae Study Group of the International Committee on Taxonomy of Viruses. The species severe acute respiratory syndrome-related coronavirus: classifying 2019-nCoV and naming it SARS-CoV-2

Coronavirus Disease (COVID-2019): Situation Report-209

Clinical features of patients infected with 2019 novel coronavirus in Wuhan

SARS-CoV-2 mRNA vaccine design enabled by prototype pathogen preparedness

SARS-CoV-2 vaccines: status report

Epidemiological and clinical characteristics of 99 cases of 2019 novel coronavirus pneumonia in Wuhan, China: a descriptive study

Adult haemophagocytic syndrome

HLH Across Speciality Collaboration, UK. COVID-19: consider cytokine storm syndromes and immunosuppression

Fatal COVID-19 infections: is NK cell dysfunction a link with autoimmune HLH?

Defective phosphorylation of interleukin-18 receptor beta causes impaired natural killer cell function in systemic-onset juvenile idiopathic arthritis

Hemophagocytic lymphohistiocytosis: a diagnostic conundrum

NK cell education after allogeneic transplantation: dissociation between recovery of cytokine-producing and cytotoxic functions

Natural killer cell dysfunction is a distinguishing feature of systemic onset juvenile rheumatoid arthritis and macrophage activation syndrome

The inhibitory receptors on NK cells and CTLs are upregulated in adult and adolescent patients with secondary hemophagocytic lymphohistiocytosis

Functional exhaustion of antiviral lymphocytes in COVID-19 patients

A single-cell atlas of the peripheral immune response in patients with severe COVID-19

Sinai Immunology Review Project. Immunology of COVID-19: current state of the science

Impaired type I interferon activity and inflammatory responses in severe Covid-19 patients

Inhibition of human natural killer cell activity by influenza virions and hemagglutinin

Plasma inflammatory cytokines and chemokines in severe acute respiratory syndrome

Inhibition of natural killer cell cytotoxicity by interleukin-6: implications for the pathogenesis of macrophage activation syndrome

Characteristics of peripheral lymphocyte subset alteration in COVID-19 pneumonia

The definition and risks of cytokine release syndrome-like in 11 COVID-19-infected pneumonia critically ill patients: disease characteristics and retrospective analysis

Tumor necrosis factor-a enhances IL-15-induced natural killer cell differentiation

The role of cytokines including interleukin-6 in COVID-19 induced pneumonia and macrophage activation syndrome-like disease

COVID-19 infection and rheumatoid arthritis: Faraway, so close!

Development and validation of the HScore, a score for the diagnosis of reactive hemophagocytic syndrome

Is the HScore useful in COVID-19?

Clinical course and risk factors for mortality of adult inpatients with COVID-19 in Wuhan, China: a retrospective cohort study

Clinical and immunological features of severe and moderate coronavirus disease 2019

High levels of natural killer cells are associated with response to tocilizumab in patients with severe rheumatoid arthritis

The authors thank the Executive Leadership Team of Alberta Precision Laboratories for supporting this study. The authors also thank Stephanie Dookie for help in cytokine analyses.