Defining the role of natural killer cells in COVID-19.

Natural killer (NK) cells are critical effectors of antiviral immunity. Researchers have therefore sought to characterize the NK cell response to coronavirus disease 2019 (COVID-19) and the virus that causes it, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The NK cells of patients with severe COVID-19 undergo extensive phenotypic and functional changes. For example, the NK cells from critically ill patients with COVID-19 are highly activated and exhausted, with poor cytotoxic function and cytokine production upon stimulation. The NK cell response to SARS-CoV-2 is also modulated by changes induced in virally infected cells, including the ability of a viral peptide to bind HLA-E, preventing NK cells from receiving inhibitory signals, and the downregulation of major histocompatibility complex class I and ligands for the activating receptor NKG2D. These changes have important implications for the ability of infected cells to escape NK cell killing. The implications of these findings for antibody-dependent NK cell activity in COVID-19 are also reviewed. Despite these advances in the understanding of the NK cell response to SARS-CoV-2, there remain critical gaps in our current understanding and a wealth of avenues for future research on this topic.

NK Cell-Monocyte Cross-talk Underlies NK Cell Activation in Severe COVID-19.

NK cells in the peripheral blood of severe COVID-19 patients exhibit a unique profile characterized by activation and dysfunction. Previous studies have identified soluble factors, including type I IFN and TGF-ß, that underlie this dysregulation. However, the role of cell-cell interactions in modulating NK cell function during COVID-19 remains unclear. To address this question, we combined cell-cell communication analysis on existing single-cell RNA sequencing data with in vitro primary cell coculture experiments to dissect the mechanisms underlying NK cell dysfunction in COVID-19. We found that NK cells are predicted to interact most strongly with monocytes and that this occurs via both soluble factors and direct interactions. To validate these findings, we performed in vitro cocultures in which NK cells from healthy human donors were incubated with monocytes from COVID-19+ or healthy donors. Coculture of healthy NK cells with monocytes from COVID-19 patients recapitulated aspects of the NK cell phenotype observed in severe COVID-19, including decreased expression of NKG2D, increased expression of activation markers, and increased proliferation. When these experiments were performed in a Transwell setting, we found that only CD56bright CD16- NK cells were activated in the presence of severe COVID-19 patient monocytes. O-link analysis of supernatants from Transwell cocultures revealed that cultures containing severe COVID-19 patient monocytes had significantly elevated levels of proinflammatory cytokines and chemokines, as well as TGF-ß. Collectively, these results demonstrate that interactions between NK cells and monocytes in the peripheral blood of COVID-19 patients contribute to NK cell activation and dysfunction in severe COVID-19.

Single-cell RNA-seq methods to interrogate virus-host interactions.

The twenty-first century has seen the emergence of many epidemic and pandemic viruses, with the most recent being the SARS-CoV-2-driven COVID-19 pandemic. As obligate intracellular parasites, viruses rely on host cells to replicate and produce progeny, resulting in complex virus and host dynamics during an infection. Single-cell RNA sequencing (scRNA-seq), by enabling broad and simultaneous profiling of both host and virus transcripts, represents a powerful technology to unravel the delicate balance between host and virus. In this review, we summarize technological and methodological advances in scRNA-seq and their applications to antiviral immunity. We highlight key scRNA-seq applications that have enabled the understanding of viral genomic and host response heterogeneity, differential responses of infected versus bystander cells, and intercellular communication networks. We expect further development of scRNA-seq technologies and analytical methods, combined with measurements of additional multi-omic modalities and increased availability of publicly accessible scRNA-seq datasets, to enable a better understanding of viral pathogenesis and enhance the development of antiviral therapeutics strategies.

SARS-CoV-2 escapes direct NK cell killing through Nsp1-mediated downregulation of ligands for NKG2D.

Natural killer (NK) cells are cytotoxic effector cells that target and lyse virally infected cells; many viruses therefore encode mechanisms to escape such NK cell killing. Here, we interrogate the ability of SARS-CoV-2 to modulate NK cell recognition and lysis of infected cells. We find that NK cells exhibit poor cytotoxic responses against SARS-CoV-2-infected targets, preferentially killing uninfected bystander cells. We demonstrate that this escape is driven by downregulation of ligands for the activating receptor NKG2D (NKG2D-L). Indeed, early in viral infection, prior to NKG2D-L downregulation, NK cells are able to target and kill infected cells; however, this ability is lost as viral proteins are expressed. Finally, we find that SARS-CoV-2 non-structural protein 1 (Nsp1) mediates downregulation of NKG2D-L and that Nsp1 alone is sufficient to confer resistance to NK cell killing. Collectively, our work demonstrates that SARS-CoV-2 evades direct NK cell cytotoxicity and describes a mechanism by which this occurs.

Deep Phenotypic Analysis of Blood and Lymphoid T and NK Cells From HIV+ Controllers and ART-Suppressed Individuals.

T and natural killer (NK) cells are effector cells with key roles in anti-HIV immunity, including in lymphoid tissues, the major site of HIV persistence. However, little is known about the features of these effector cells from people living with HIV (PLWH), particularly from those who initiated antiretroviral therapy (ART) during acute infection. Our study design was to use 42-parameter CyTOF to conduct deep phenotyping of paired blood- and lymph node (LN)-derived T and NK cells from three groups of HIV+ aviremic individuals: elite controllers (N = 5), and ART-suppressed individuals who had started therapy during chronic (N = 6) vs. acute infection (N = 8), the latter of which is associated with better outcomes. We found that acute-treated individuals are enriched for specific subsets of T and NK cells, including blood-derived CD56-CD16+ NK cells previously associated with HIV control, and LN-derived CD4+ T follicular helper cells with heightened expansion potential. An in-depth comparison of the features of the cells from blood vs. LNs of individuals from our cohort revealed that T cells from blood were more activated than those from LNs. By contrast, LNs were enriched for follicle-homing CXCR5+ CD8+ T cells, which expressed increased levels of inhibitory receptors and markers of survival and proliferation as compared to their CXCR5- counterparts. In addition, a subset of memory-like CD56brightTCF1+ NK cells was enriched in LNs relative to blood. These results together suggest unique T and NK cell features in acute-treated individuals, and highlight the importance of examining effector cells not only in blood but also the lymphoid tissue compartment, where the reservoir mostly persists, and where these cells take on distinct phenotypic features.

Multi-omic profiling reveals widespread dysregulation of innate immunity and hematopoiesis in COVID-19.

Our understanding of protective versus pathological immune responses to SARS-CoV-2, the virus that causes coronavirus disease 2019 (COVID-19), is limited by inadequate profiling of patients at the extremes of the disease severity spectrum. Here, we performed multi-omic single-cell immune profiling of 64 COVID-19 patients across the full range of disease severity, from outpatients with mild disease to fatal cases. Our transcriptomic, epigenomic, and proteomic analyses revealed widespread dysfunction of peripheral innate immunity in severe and fatal COVID-19, including prominent hyperactivation signatures in neutrophils and NK cells. We also identified chromatin accessibility changes at NF-κB binding sites within cytokine gene loci as a potential mechanism for the striking lack of pro-inflammatory cytokine production observed in monocytes in severe and fatal COVID-19. We further demonstrated that emergency myelopoiesis is a prominent feature of fatal COVID-19. Collectively, our results reveal disease severity-associated immune phenotypes in COVID-19 and identify pathogenesis-associated pathways that are potential targets for therapeutic intervention.