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.

Natural Variation in HIV-1 Entry Kinetics Map to Specific Residues and Reveal an Interdependence Between Attachment and Fusion.

HIV-1 entry kinetics reflect the fluid motion of the HIV envelope glycoprotein through at least three major structural configurations that drive virus-cell membrane fusion. The lifetime of each state is an important component of potency for inhibitors that target them. We used the time-of-addition inhibitor assay and a novel analytical strategy to define the kinetics of pre-hairpin exposure (using T20) and co-receptor engagement (via. maraviroc), through a characteristic delay metric, across a variety of naturally occurring HIV Env isolates. Among 257 distinct HIV-1 envelope isolates we found a remarkable breadth of T20 and maraviroc delays ranging from as early as 30 seconds to as late as 60 minutes. The most extreme delays were observed among transmission-linked clade C isolates. We identified four single-residue determinants of late T20 and maraviroc delays that are associated with either receptor engagement or gp41 function. Comparison of these delays with T20 sensitivity suggest co-receptor engagement and fusogenic activity in gp41 act cooperatively but sequentially to drive entry. Our findings support current models of entry where co-receptor engagement drives gp41 eclipse and have strong implications for the design of entry inhibitors and antibodies that target transient entry states. Author Summary: The first step of HIV-1 infection is entry, where virus-cell membrane fusion is driven by the HIV-1 envelope glycoprotein through a series of conformational changes. Some of the most broadly active entry inhibitors work by binding conformations that exist only transiently during entry. The lifetimes of these states and the kinetics of entry are important elements of inhibitor activity for which little is known. We demonstrate a remarkable range of kinetics among 257 diverse HIV-1 isolates and find that this phenotype is highly flexible, with multiple single-residue determinants. Examination of the kinetics of two conformational landmarks shed light on novel kinetic features that offer new details about the role of co-receptor engagement and provide a framework to explain entry inhibitor synergy.