Despite clinical success with anti-spike vaccines, the effectiveness of neutralizing antibodies and vaccines has been compromised by rapidly spreading SARS-CoV-2 variants. Viruses can hijack the glycosylation machinery of host cells to shield themselves from the host's immune response and attenuate antibody efficiency. However, it remains unclear if targeting glycosylation on viral spike protein can impair infectivity of SARS-CoV-2 and its variants. We adopted flow cytometry, ELISA, and BioLayer interferometry approaches to assess binding of glycosylated or deglycosylated spike with ACE2. Viral entry was determined by luciferase, immunoblotting, and immunofluorescence assays. Genome-wide association study (GWAS) revealed a significant relationship between STT3A and COVID-19 severity. NF-κB/STT3A-regulated N-glycosylation was investigated by gene knockdown, chromatin immunoprecipitation, and promoter assay. We developed an antibody-drug conjugate (ADC) that couples non-neutralization anti-spike antibody with NGI-1 (4G10-ADC) to specifically target SARS-CoV-2-infected cells. The receptor binding domain and three distinct SARS-CoV-2 surface N-glycosylation sites among 57,311 spike proteins retrieved from the NCBI-Virus-database are highly evolutionarily conserved (99.67%) and are involved in ACE2 interaction. STT3A is a key glycosyltransferase catalyzing spike glycosylation and is positively correlated with COVID-19 severity. We found that inhibiting STT3A using N-linked glycosylation inhibitor-1 (NGI-1) impaired SARS-CoV-2 infectivity and that of its variants [Alpha (B.1.1.7) and Beta (B.1.351)]. Most importantly, 4G10-ADC enters SARS-CoV-2-infected cells and NGI-1 is subsequently released to deglycosylate spike protein, thereby reinforcing the neutralizing abilities of antibodies, vaccines, or convalescent sera and reducing SARS-CoV-2 variant infectivity. Our results indicate that targeting evolutionarily-conserved STT3A-mediated glycosylation via an ADC can exert profound impacts on SARS-CoV-2 variant infectivity. Thus, we have identified a novel deglycosylation method suitable for eradicating SARS-CoV-2 variant infection in vitro. A full list of funding bodies that contributed to this study can be found in the Acknowledgements section

To explore how the immune system controls clearance of SARS-CoV-2, we used a single-cell, mass cytometry–based proteomics platform to profile the immune systems of 21 patients who had recovered from SARS-CoV-2 infection without need for admission to an intensive care unit or for mechanical ventilation. We focused on receptors involved in interactions between immune cells and virus-infected cells. We found that the diversity of receptor repertoires on natural killer (NK) cells was negatively correlated with the viral clearance rate. In addition, NK subsets expressing the receptor DNAM1 were increased in patients who more rapidly recovered from infection. Ex vivo functional studies revealed that NK subpopulations with high DNAM1 expression had cytolytic activities in response to target cell stimulation. We also found that SARS-CoV-2 infection induced the expression of CD155 and nectin-4, ligands of DNAM1 and its paired coinhibitory receptor TIGIT, which counterbalanced the cytolytic activities of NK cells. Collectively, our results link the cytolytic immune responses of NK cells to the clearance of SARS-CoV-2 and show that the DNAM1 pathway modulates host-pathogen interactions during SARS-CoV-2 infection.

Activation of effector T cells leads to upregulation of PD-1, which can inhibit T-cell activity following engagement with its ligand PD-L1. Post-translational modifications (PTM), including glycosylation, phosphorylation, ubiquitination, and palmitoylation, play a significant role in regulating PD-1 protein stability, localization, and interprotein interactions. Targeting PTM of PD-1 in T cells has emerged as a potential strategy to overcome PD-1–mediated immunosuppression in cancer and enhances antitumor immunity. The regulatory signaling pathways that induce PTM of PD-1 can be suppressed with small-molecule inhibitors, and mAbs can directly target PD-1 PTMs. Preliminary outcomes from exploratory studies suggest that focusing on the PTM of PD-1 has strong therapeutic potential and can enhance the response to anti-PD-1.

Protein glycosylation plays a versatile role in regulating homeostasis, such as cell migration, protein sorting, and the immune response. Drugs aimed at targeting glycosylation have strong implications for immunity enhancement, diagnosis, and cancer regression. Programmed death-ligand 1 (PD-L1), expressed in cancer or antigen-presenting cells, binds to programmed cell death protein 1 (PD-1) and suppresses T cells. Glycosylation of PD-L1 at N35, N192, N200, and N219 stabilizes PD-L1 on the cancer cell surface, which contributes to immune evasion by inhibiting T cell activity. To date, at least six glycosyltransferases and four associate proteins are known to regulate PD-L1 glycosylation. Terminal modifications such as poly-N-acetyl-lactosamine (poly-LacNAC), sulfation, and sialylation are commonly found on PD-L1, acting as an immune recognition ligand and regulating certain immune responses. Studies have identified many mechanisms and potential therapeutic targets within the glycosylation pathways of PD-L1, revealing their involvement in cancer pathology, immune evasion, and resistance to immunotherapy. In this review, we covered the glycoforms, terminal moiety, binding lectin, glycosyltransferase, as well as sugar analogs focusing on glycosylated PD-L1. We present a mechanism that originates from the endoplasmic reticulum (ER)-Golgi apparatus (Golgi) and its subsequent translocation to the cell membrane. This pathway determines the immune suppression function of PD-L1 and therefore regulates the immune response such as T cells, monocytes, and macrophages. This collection of findings underscores the significance of glycosylation in the role of PD-L1 in cancer and highlights multiple potential targets and strategies for improving therapeutic intervention and diagnostic techniques.

Patients with severe COVID‐19 often suffer from lymphopenia, which is linked to T‐cell sequestration, cytokine storm, and mortality. However, it remains largely unknown how severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) induces lymphopenia. Here, we studied the transcriptomic profile and epigenomic alterations involved in cytokine production by SARS‐CoV‐2‐infected cells. We adopted a reverse time‐order gene coexpression network approach to analyze time‐series RNA‐sequencing data, revealing epigenetic modifications at the late stage of viral egress. Furthermore, we identified SARS‐CoV‐2‐activated nuclear factor‐κB (NF‐κB) and interferon regulatory factor 1 (IRF1) pathways contributing to viral infection and COVID‐19 severity through epigenetic analysis of H3K4me3 chromatin immunoprecipitation sequencing. Cross‐referencing our transcriptomic and epigenomic data sets revealed that coupling NF‐κB and IRF1 pathways mediate programmed death ligand‐1 (PD‐L1) immunosuppressive programs. Interestingly, we observed higher PD‐L1 expression in Omicron‐infected cells than SARS‐CoV‐2 infected cells. Blocking PD‐L1 at an early stage of virally‐infected AAV‐hACE2 mice significantly recovered lymphocyte counts and lowered inflammatory cytokine levels. Our findings indicate that targeting the SARS‐CoV‐2‐mediated NF‐κB and IRF1‐PD‐L1 axis may represent an alternative strategy to reduce COVID‐19 severity.

In eukaryotic cells, reactive oxygen species (ROS) serve as crucial signaling components. ROS are potentially toxic, so constant adjustments are needed to maintain cellular health. Here we describe a single-cell, mass cytometry-based method that we call signaling network under redox stress profiling (SN-ROP) to monitor dynamic changes in redox-related pathways during redox stress. SN-ROP quantifies ROS transporters, enzymes, oxidative stress products and associated signaling pathways to provide information on cellular redox regulation. Applied to diverse cell types and conditions, SN-ROP reveals unique redox patterns and dynamics including coordinated shifts in CD8⁺ T cells upon antigen stimulation as well as variations in CAR-T cell persistence. Furthermore, SN-ROP analysis uncovers environmental factors such as hypoxia and T cell exhaustion for influencing redox balance, and also reveals distinct features in patients on hemodialysis. Our findings thus support the use of SN-ROP to elucidate intricate redox networks and their implications in immune cell function and disease.