SARS-CoV-2 Coronavirus Is Able To Evade Cytotoxic Responses Of Natural Killer Cells And Cause Immune Dysfunction

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A new study by researchers from Stanford University School of Medicine has found that the SARS-CoV-2 coronavirus is able to evade cytotoxic responses of natural killer cells and cause immune dysfunction.

The study findings were published on a preprint server and are currently being peer reviewed. https://www.biorxiv.org/content/10.1101/2022.06.20.496341v1

NK cell dysfunction in patients with COVID-19. NK cells normally possess antiviral and antifibrotic activities, which are impaired in patients with severe COVID-19, possibly by soluble factors such as IFN-α and/or by inhibitory checkpoint receptors such as NKG2A. Treatments targeting these factors/receptors might potentially reinvigorate NK cells for better control of the disease


 

The role of NK cells in mediating clearance of SARS-CoV-2-infected cells in vivo remains unclear. While several studies have demonstrated that NK cells can reduce the levels of SARS-CoV-2 replication in vitro, no prior study has directly evaluated killing of SARS-CoV-2 infected cells.

Here, we address this critical gap in knowledge and demonstrate that SARS-CoV-2-infected cells escape killing by healthy NK cells in a cell-intrinsic manner, resulting in preferential killing of uninfected bystander cells.

The ability of infected cells to evade NK cell recognition requires infection to proceed long enough to allow an infected cell to express SARS-CoV-2 encoded proteins. We demonstrate that this escape mechanism is driven by downregulation of ligands for NKG2D, a critical activating receptor on NK cells.

Consistent with our findings, Fielding et al. recently reported that NKG2D-L are downregulated on SARS-CoV-2 infected cells, and find that antibody-mediated killing could overcome this evasion mechanism (Fielding et al., 2022). We further demonstrate that this ligand downregulation is driven by the SARS-CoV-2 Nsp1 protein, and show that Nsp1 alone is sufficient to mediate direct NK cell evasion.

This has important implications for NK cell-mediated control of SARS-CoV-2, as preferential escape of infected cells with killing of bystander cells could contribute to SARS-CoV-2 pathogenesis.

These results illustrate the importance of examining the temporal dynamics of the NK cell response to SARS-CoV-2-infected cells. Other studies have assessed the ability of NK cells to suppress viral load by co-culturing NK cells with SARS-CoV-2-infected targets immediately after infection; their results suggest that under these conditions, NK cells can at least partially control viral replication (Krämer et al., 2021; Witkowski et al., 2021; Hammer et al., 2022).

However, our observations demonstrate that NK cells are no longer able to effectively kill infected cells when added to the culture at later time points following infection, after the expression of viral proteins that suppress the innate immune response. The preferential killing of NKG2D-L-positive bystander cells may have important implications for lung pathology during COVID-19. NKG2D-L can be expressed by most cell types (Lanier, 2015) and are upregulated during viral infections, including HIV (Ward et al., 2009) and RSV (Zdrenghea et al., 2012), in response to stress (Borchers et al., 2006).

Therefore, NK cells may actually cause damage to the healthy tissue surrounding infected cells rather than clearing the infection. Our data imply that the timing of NK cell trafficking to the site of infection may be critical in determining whether NK cells are protective or pathogenic in COVID-19, as there is a very narrow window for killing of infected cells before bystander killing could ensue.

Our novel finding that the SARS-CoV-2 protein Nsp1 mediates evasion of NK cell killing has significant implications for both the study of the immune response to coronaviruses and the development of therapeutics for COVID-19. Nsp1 is highly conserved across coronaviruses and is an essential virulence factor; it has been shown to inhibit translation of host antiviral factors across multiple beta-coronaviruses (Kamitani et al., 2006; Züst et al., 2007; Narayanan et al., 2008; Min et al., 2020; Schubert et al., 2020; Vazquez et al., 2021; Yuan et al., 2021). One study found that, among nearly 50,000 SARS-CoV-2 sequences analyzed, only 2.4% had any mutations within Nsp1 (Min et al., 2020). SARS-CoV-2 Nsp1 also shares 84.4% of its sequence identity with SARS-CoV Nsp1.

Moreover, critical motifs within Nsp1 involved in the inhibition of innate immune responses are highly conserved across many beta-coronaviruses(Min et al., 2020). On a practical level, the high degree of conservation of Nsp1 and its importance in coronavirus virulence have already made this protein the focus of several therapeutic strategies (Züst et al., 2007; Afsar et al., 2022; Vora et al., 2022). Our work demonstrates that Nsp1 is an even more attractive target than previously thought, as inhibiting the function of this protein has the potential to fully or partially rescue the NK cell response to SARS-CoV-2-infected cells.

Although Nsp1 is a global inhibitor of host translation, our data demonstrate that it has an outsized effect on NKG2D-L and MHC class I surface expression compared to that of other ligands for NK cell receptors. This appears to be due to the varying stabilities of the different ligands on the cell surface, rather than explicit specificity of Nsp1 for NKG2D-L or MHC class I.

It has been established that NKG2D-L are rapidly turned over on the cell surface and are quickly lost upon treatment with a protein transport inhibitor such as Brefeldin A (Fernández-Messina, Reyburn and Valés-Gómez, 2016; Toledano et al., 2018). MHC class I is similarly transient on the cell surface in the presence of translation inhibition, although its stability varies with haplotype and peptide binding (Yarzabek et al., 2018). CD54, which was not affected by Nsp1, is highly stable for at least 48 hours, even after treatment with similar inhibitors (Braun et al., 1997). Thus, the differential effects of Nsp1 on various ligands for NK cell receptors are likely explained by the varying kinetics of surface turnover.

One of our findings that has been demonstrated by multiple groups is the downregulation of MHC class I upon SARS-CoV-2 infection. The mechanism of this downregulation remains unclear; while we have demonstrated that Nsp1 is responsible for this phenomenon, ORF3a (Arshad et al., 2022), ORF7a (Arshad et al., 2022), ORF6 (Yoo et al., 2021), and ORF8 (Zhang et al., 2021) have also been implicated.

According to the well-established “missing self” model of NK cell activation (Ljunggren and Kärre, 1990; Kärre, 2008), the downregulation of self-MHC can induce NK cell activation through subsequent lack of inhibitory signaling through the killer cell immunoglobulin-like receptors (KIRs).

Therefore, it might be expected that the downregulation of MHC by SARS-CoV-2 would enhance the ability of NK cells to lyse infected cells–precisely the opposite of what we observed in our study. We hypothesize that this can be explained by

1) the relative magnitudes of MHC class I and NKG2D-L downregulation on infected cells and

2) the accepted dogma in the field that missing self alone is not sufficient to cause robust NK cell activation (Vivier et al., 2011; Barrow and Colonna, 2019).

As a result, we propose that the loss of NKG2D-L is the dominant factor in the NK cell response (or lack thereof) to SARS-CoV-2.

Our study has several limitations. In order to focus on NK cell responses in the respiratory tract, we used A549-ACE2 cells, which are an immortalized, malignant cell line. This could therefore have enhanced NK cell targeting of bystander cells. Additionally, while we demonstrated that Nsp1 was sufficient to confer NK cell escape, we were unable to test whether the absence of Nsp1 rescues NK cell killing because knockout of Nsp1 is lethal to the virus.

We also did not fully evaluate why Nsp1 blocks NKG2D-L more effectively than other proteins, but we hypothesize that these proteins are downregulated first as part of the global translation block because they are turned over on the cell surface more quickly and cannot be replaced. Finally, we did not interrogate the ability of every individual SARS-CoV-2 protein to mediate escape from NK cell killing.

This work has significant implications for the ongoing study of COVID-19. Our results deeply interrogate a major flaw in the ability of the immune system to mount a comprehensive immune response to COVID-19. We demonstrate that the timing of the NK cell response to SARS-CoV-2-infected target cells is critical, with NK cells being able to control viral replication early in infection, but not after expression of viral proteins has begun.

This should be further interrogated in vivo to explore whether the kinetics of NK cell trafficking during COVID-19 affect disease outcome. Finally, we reveal that SARS-CoV-2 protein Nsp1 is a major factor in mediating evasion of NK cell killing. This finding reinforces the attractiveness of Nsp1 as a therapeutic target.

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