ABSTRACT
COVID-19 is characterised by a broad spectrum of clinical and pathological features. Natural killer (NK) cells play an important role in innate immune responses to viral infections. Here, we analysed the phenotype and activity of NK cells in the blood of COVID-19 patients using flow cytometry, single-cell RNA-sequencing (scRNA-seq), and a cytotoxic killing assay. In the plasma of patients, we quantified the main cytokines and chemokines. Our cohort comprises COVID-19 patients hospitalised in a low-care ward unit (WARD), patients with severe COVID-19 disease symptoms hospitalised in intensive care units (ICU), and post-COVID-19 patients, who were discharged from hospital six weeks earlier. NK cells from hospitalised COVID-19 patients displayed an activated phenotype with substantial differences between WARD and ICU patients and the timing when samples were taken post-onset of symptoms. While NK cells from COVID-19 patients at an early stage of infection showed increased expression of the cytotoxic molecules perforin and granzyme A and B, NK cells from patients at later stages of COVID-19 presented enhanced levels of IFN-γ and TNF-α which were measured ex vivo in the absence of usual in vitro stimulation. These activated NK cells were phenotyped as CD49a+CD69a+CD107a+ cells, and their emergence in patients correlated to the number of neutrophils, and plasma IL-15, a key cytokine in NK cell activation. Despite lower amounts of cytotoxic molecules in NK cells of patients with severe symptoms, majority of COVID-19 patients displayed a normal cytotoxic killing of Raji tumour target cells. In vitro stimulation of patients blood cells by IL-12+IL-18 revealed a defective IFN-γ production in NK cells of ICU patients only, indicative of an exhausted phenotype. ScRNA-seq revealed, predominantly in patients with severe COVID-19 disease symptoms, the emergence of an NK cell subset with a platelet gene signature that we identified by flow and imaging cytometry as aggregates of NK cells with CD42a+CD62P+ activated platelets. Post-COVID-19 patients show slow recovery of NK cell frequencies and phenotype. Our study points to substantial changes in NK cell phenotype during COVID-19 disease and forms a basis to explore the contribution of platelet-NK cell aggregates to antiviral immunity against SARS-CoV-2 and disease pathology.
Subject(s)
COVID-19 , Humans , Granzymes/metabolism , Perforin/metabolism , Interleukin-15/metabolism , Interleukin-18/metabolism , SARS-CoV-2 , Tumor Necrosis Factor-alpha/metabolism , Blood Platelets/metabolism , Integrin alpha1/metabolism , Killer Cells, Natural , Cytokines/metabolism , Chemokines/metabolism , Interleukin-12/metabolism , Antiviral Agents/metabolism , RNA/metabolismABSTRACT
In rare instances, pediatric SARS-CoV-2 infection results in a novel immunodysregulation syndrome termed multisystem inflammatory syndrome in children (MIS-C). We compared MIS-C immunopathology with severe COVID-19 in adults. MIS-C does not result in pneumocyte damage but is associated with vascular endotheliitis and gastrointestinal epithelial injury. In MIS-C, the cytokine release syndrome is characterized by IFNγ and not type I interferon. Persistence of patrolling monocytes differentiates MIS-C from severe COVID-19, which is dominated by HLA-DRlo classical monocytes. IFNγ levels correlate with granzyme B production in CD16+ NK cells and TIM3 expression on CD38+/HLA-DR+ T cells. Single-cell TCR profiling reveals a skewed TCRß repertoire enriched for TRBV11-2 and a superantigenic signature in TIM3+/CD38+/HLA-DR+ T cells. Using NicheNet, we confirm IFNγ as a central cytokine in the communication between TIM3+/CD38+/HLA-DR+ T cells, CD16+ NK cells, and patrolling monocytes. Normalization of IFNγ, loss of TIM3, quiescence of CD16+ NK cells, and contraction of patrolling monocytes upon clinical resolution highlight their potential role in MIS-C immunopathogenesis.
Subject(s)
COVID-19/complications , Hepatitis A Virus Cellular Receptor 2/metabolism , Interferon-gamma/metabolism , Killer Cells, Natural/immunology , Monocytes/metabolism , Receptors, IgG/metabolism , Systemic Inflammatory Response Syndrome/immunology , T-Lymphocytes/immunology , Adolescent , Alveolar Epithelial Cells/pathology , B-Lymphocytes/immunology , Blood Vessels/pathology , COVID-19/immunology , COVID-19/pathology , Cell Proliferation , Child , Cohort Studies , Complement Activation , Cytokines/metabolism , Enterocytes/pathology , Female , Humans , Immunity, Humoral , Inflammation/pathology , Interferon Type I/metabolism , Interleukin-15/metabolism , Lymphocyte Activation/immunology , Male , Receptors, Antigen, T-Cell/metabolism , SARS-CoV-2/immunology , Superantigens/metabolism , Systemic Inflammatory Response Syndrome/pathologyABSTRACT
Engineered natural killer (NK) cells represent a promising option for immune therapy option due to their immediate availability in allogeneic settings. Severe acute diseases, such as COVID-19, require targeted and immediate intervention. Here we show engineering of NK cells to express (1) soluble interleukin-15 (sIL15) for enhancing their survival and (2) a chimeric antigen receptor (CAR) consisting of an extracellular domain of ACE2, targeting the spike protein of SARS-CoV-2. These CAR NK cells (mACE2-CAR_sIL15 NK cells) bind to VSV-SARS-CoV-2 chimeric viral particles as well as the recombinant SARS-CoV-2 spike protein subunit S1 leading to enhanced NK cell production of TNF-α and IFN-γ and increased in vitro and in vivo cytotoxicity against cells expressing the spike protein. Administration of mACE2-CAR_sIL15 NK cells maintains body weight, reduces viral load, and prolongs survival of transgenic mice expressing human ACE2 upon infection with live SARS-CoV-2. These experiments, and the capacity of mACE2-CAR_sIL15 NK cells to retain their activity following cryopreservation, demonstrate their potential as an allogeneic off-the-shelf therapy for COVID-19 patients who are faced with limited treatment options.