Numerous studies have already validated that the SARS-CoV-2 virus is able to evade and also disarm various components of the human host’s immune system, in the process leading to immune dysfunction and also what is now being termed as COVID-19 induced immunodeficiency, which all have long term health impacts on individuals exposed to the virus.
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, while killing of uninfected bystander cells is uninhibited. 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.
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. While our experimental system using a cell line with high expression of NKG2D-L could enhance the degree of bystander killing, these findings have important implications for NK cell-mediated control of SARS-CoV-2, as preferential escape of infected cells and possible 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 early after infection; their results suggest that under these conditions, NK cells can at least partially control viral replication 16,18,19.
It is worth noting that these other studies also varied from ours in parameters such as target cell type, cytokine treatment of NK cells, E:T ratio, and duration of co-culture. Our own observations demonstrate that NK cells are no longer able to effectively kill infected cells when added to the culture at 48 hours post-infection, after the expression of viral proteins that suppress the innate immune response.
Therefore, it is possible that NK cells may actually cause damage to the healthy tissue surrounding infected cells rather than clearing the infection, although this hypothesis has not yet been directly tested in primary lung tissue. As NK cells appear to home to the lungs during COVID-19 57–59, our findings indicate that the timing of NK cell trafficking to the site of infection may impact the efficacy of the NK cell response to SARS-CoV-2 infection, as there is a very narrow window for killing of infected cells before bystander killing could ensue.
Interestingly, Witkowski et al. observed that frequency of peripheral blood NK cells in severe COVID-19 patients negatively correlated with viral load; however, this is difficult to interpret in the context of our data because it is unknown whether the increased NK cell frequencies observed resulted from decreased trafficking to the lungs, increased peripheral proliferation, or another mechanism 19.
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 42–47,60. One study found that, among nearly 50,000 SARS-CoV-2 sequences analyzed, only 2.4% had any mutations within Nsp1 44.
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 A32,50. 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 51.
CD54, which was not affected by Nsp1, is highly stable for at least 48 hours, even after treatment with similar inhibitors 52. 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 our data suggests that Nsp1 is responsible for this loss, ORF3a 35, ORF7a 35, ORF6 36, and ORF8 37 have also been implicated.
This could be due to differential downregulation of various HLA molecules by different SARS-CoV-2 proteins. In our study, we grouped together HLAs A, B, and C as there are no commercially available antibody clones that can robustly differentiate HLAs A and B; this is an important limitation of our work.
According to the well-established “missing self” model of NK cell activation 63,64, 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 activation65,66. 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.
While our study focuses on direct lysis of target cells, NK cells can also kill through antibody-dependent cellular cytotoxicity (ADCC). A recent study by Fielding et al. found that antibody-dependent NK cell activation can overcome SARS-CoV-2’s inhibition of direct cytotoxicity, allowing healthy NK cells to mount stronger responses to infected targets. Thus, prior vaccination or infection that results in pre-existing antibodies to SARS- CoV-2 could tip the balance in favor of killing SARS-CoV-2 infected cells. This study also identified downregulation of NKG2D-L on SARS-CoV-2-infected cells through an orthogonal method 20.
This work has significant implications for the ongoing study of COVID-19. Our results deeply interrogate a potential 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.
