profiling-of-the-human-natural-killer-cell-receptor-ligand-repertoire

Profiling of the Human Natural Killer Cell Receptor-Ligand Repertoire

Natural killer (NK) cells are among the first responders to viral infections. The ability of NK cells to rapidly recognize and kill virally infected cells ...

Stanford University School of Medicine

In human and murine studies, IFN-γ is a critical mediator immunity to influenza. IFN-γ production is critical for viral clearance and the development of adaptive immune responses, yet excessive production of IFN-γ and other cytokines as part of a cytokine storm is associated with poor outcomes of influenza infection in humans. As NK cells are the main population of lung innate immune cells capable of producing IFN-γ early in infection, we set out to identify the drivers of the human NK cell IFN-γ response to influenza A viruses. We found that influenza triggers NK cells to secrete IFN-γ in the absence of T cells and in a manner dependent upon signaling from both cytokines and receptor-ligand interactions. Further, we discovered that the pandemic A/California/07/2009 (H1N1) strain elicits a seven-fold greater IFN-γ response than other strains tested, including a seasonal A/Victoria/361/2011 (H3N2) strain. These differential responses were independent of memory NK cells. Instead, we discovered that the A/Victoria/361/2011 influenza strain suppresses the NK cell IFN-γ response by downregulating NK-activating ligands CD112 and CD54 and by repressing the type I IFN response in a viral replication-dependent manner. In contrast, the A/California/07/2009 strain fails to repress the type I IFN response or to downregulate CD54 and CD112 to the same extent, which leads to the enhanced NK cell IFN-γ response. Our results indicate that influenza implements a strain-specific mechanism governing NK cell production of IFN-γ and identifies a previously unrecognized influenza innate immune evasion strategy.

Differential Induction of IFN-α and Modulation of CD112 and CD54 Expression Govern the Magnitude of NK Cell IFN-γ Response to Influenza A Viruses.

Dengue virus (DENV) is the most prevalent mosquito-borne virus in the world and a major cause of morbidity in the tropics and subtropics. Upregulation of HLA class I molecules has long been considered a feature of DENV infection, yet this has not been evaluated in the setting of natural infection. Natural killer (NK) cells, an innate immune cell subset critical for mounting an early response to viral infection, are inhibited by self HLA class I, suggesting that upregulation of HLA class I during DENV infection could dampen the NK cell response. Here we addressed whether upregulation of HLA class I molecules occurs during  DENV infection and, if so, whether this suppresses the NK cell response. We found that HLA class I expression was indeed upregulated during acute DENV infection across multiple cell lineages . To better understand the role of HLA class I upregulation, we infected primary human monocytes, a major target of DENV infection, . Upregulation of total HLA class I is dependent on active viral replication and is mediated in part by cytokines and other soluble factors induced by infection, while upregulation of HLA-E occurs in the presence of replication-incompetent virus. Importantly, blocking DENV-infected monocytes with a pan-HLA class I Fab nearly doubles the frequency of degranulating NK cells, while blocking HLA-E does not significantly improve the NK cell response. These findings demonstrate that upregulation of HLA class I during DENV infection suppresses the NK cell response, potentially contributing to disease pathogenesis.

HLA Upregulation During Dengue Virus Infection Suppresses the Natural Killer Cell Response.

Specific causes of preterm birth remain unclear. Several recent studies have suggested that immune changes during pregnancy are associated with the timing of delivery, yet few studies have been performed in low-income country settings where the rates of preterm birth are the highest. We conducted a retrospective nested case-control evaluation within a longitudinal study among HIV-uninfected pregnant Kenyan women. To characterize immune function in these women, we evaluated unstimulated and stimulated peripheral blood mononuclear cells  with the A/California/2009 strain of influenza to understand the influenza-induced immune response. We then evaluated transcript expression profiles using the Affymetrix Human GeneChip Transcriptome Array 2.0. Transcriptional profiles of sufficient quality for analysis were obtained from 54 women; 19 of these women delivered <34 weeks and were defined as preterm cases and 35 controls delivered >37 weeks. The median time to birth from sample collection was 13 weeks. No transcripts were significantly associated with preterm birth in a case-control study of matched term and preterm birth (n = 42 women). In the influenza-stimulated samples, expression of IFNL1 was associated with longer time to delivery-the amount of time between sample collection and delivery (n = 54 women). A qPCR analysis confirmed that influenza-induced IFNL expression was associated with longer time to delivery. These data indicate that during pregnancy,  influenza stimulation results in altered transcriptional response and is associated with time to delivery in cohort of women residing in an area with high preterm birth prevalence.

Influenza-Induced Interferon Lambda Response Is Associated With Longer Time to Delivery Among Pregnant Kenyan Women.

There is an urgent need to better understand the pathophysiology of Coronavirus disease 2019 (COVID-19), the global pandemic caused by SARS-CoV-2, which has infected more than three million people worldwide. Approximately 20% of patients with COVID-19 develop severe disease and 5% of patients require intensive care. Severe disease has been associated with changes in peripheral immune activity, including increased levels of pro-inflammatory cytokines that may be produced by a subset of inflammatory monocytes, lymphopenia and T cell exhaustion. To elucidate pathways in peripheral immune cells that might lead to immunopathology or protective immunity in severe COVID-19, we applied single-cell RNA sequencing (scRNA-seq) to profile peripheral blood mononuclear cells (PBMCs) from seven patients hospitalized for COVID-19, four of whom had acute respiratory distress syndrome, and six healthy controls. We identify reconfiguration of peripheral immune cell phenotype in COVID-19, including a heterogeneous interferon-stimulated gene signature, HLA class II downregulation and a developing neutrophil population that appears closely related to plasmablasts appearing in patients with acute respiratory failure requiring mechanical ventilation. Importantly, we found that peripheral monocytes and lymphocytes do not express substantial amounts of pro-inflammatory cytokines. Collectively, we provide a cell atlas of the peripheral immune response to severe COVID-19.

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

Dengue virus (DENV) is a significant cause of morbidity in many regions of the world, with children at the greatest risk of developing severe dengue. NK cells, characterized by their ability to rapidly recognize and kill virally infected cells, are activated during acute DENV infection. However, their role in viral clearance versus pathogenesis has not been fully elucidated. Our goal was to profile the NK cell receptor-ligand repertoire to provide further insight into the function of NK cells during pediatric and adult DENV infection. We used mass cytometry to phenotype isolate NK cells and PBMCs from a cohort of DENV-infected children and adults. Using unsupervised clustering, we found that pediatric DENV infection leads to a decrease in total NK cell frequency with a reduction in the percentage of CD56CD38 NK cells and an increase in the percentage of CD56perforin NK cells. No such changes were observed in adults. Next, we identified markers predictive of DENV infection using a differential state test. In adults, NK cell expression of activation markers, including CD69, perforin, and Fas-L, and myeloid cell expression of activating NK cell ligands, namely Fas, were predictive of infection. In contrast, increased NK cell expression of the maturation marker CD57 and myeloid cell expression of inhibitory ligands, such as HLA class I molecules, were predictive of pediatric DENV infection. These findings suggest that acute pediatric DENV infection may result in diminished NK cell activation, which could contribute to enhanced pathogenesis and disease severity.

Rosemary Vergara

Department of Medicine

Stanford University School of Medicine

Profiling of the Human Natural Killer Cell Receptor-Ligand Repertoire