Targeting branched N-glycans and fucosylation sensitizes ovarian tumors to immune checkpoint blockade.

Aberrant glycosylation is a crucial strategy employed by cancer cells to evade cellular immunity. However, it's unclear whether homologous recombination (HR) status-dependent glycosylation can be therapeutically explored. Here, we show that the inhibition of branched N-glycans sensitizes HR-proficient, but not HR-deficient, epithelial ovarian cancers (EOCs) to immune checkpoint blockade (ICB). In contrast to fucosylation whose inhibition sensitizes EOCs to anti-PD-L1 immunotherapy regardless of HR-status, we observe an enrichment of branched N-glycans on HR-proficient compared to HR-deficient EOCs. Mechanistically, BRCA1/2 transcriptionally promotes the expression of MGAT5, the enzyme responsible for catalyzing branched N-glycans. The branched N-glycans on HR-proficient tumors augment their resistance to anti-PD-L1 by enhancing its binding with PD-1 on CD8+ T cells. In orthotopic, syngeneic EOC models in female mice, inhibiting branched N-glycans using 2-Deoxy-D-glucose sensitizes HR-proficient, but not HR-deficient EOCs, to anti-PD-L1. These findings indicate branched N-glycans as promising therapeutic targets whose inhibition sensitizes HR-proficient EOCs to ICB by overcoming immune evasion.

Nanotechnology-based theranostic and prophylactic approaches against SARS-CoV-2.

SARS-CoV-2 (COVID-19) pandemic has been an unpredicted burden on global healthcare system by infecting over 700 million individuals, with approximately 6 million deaths worldwide. COVID-19 significantly impacted all sectors, but it very adversely affected the healthcare system. These effects were much more evident in the resource limited part of the world. Individuals with acute conditions were also severely impacted. Although classical COVID-19 diagnostics such as RT-PCR and rapid antibody testing have played a crucial role in reducing the spread of infection, these diagnostic techniques are associated with certain limitations. For instance, drawback of RT-PCR diagnostics is that due to degradation of viral RNA during shipping, it can give false negative results. Also, rapid antibody testing majorly depends on the phase of infection and cannot be performed on immune compromised individuals. These limitations in current diagnostic tools require the development of nanodiagnostic tools for early detection of COVID-19 infection. Therefore, the SARS-CoV-2 outbreak has necessitated the development of specific, responsive, accurate, rapid, low-cost, and simple-to-use diagnostic tools at point of care. In recent years, early detection has been a challenge for several health diseases that require prompt attention and treatment. Disease identification at an early stage, increased imaging of inner health issues, and ease of diagnostic processes have all been established using a new discipline of laboratory medicine called nanodiagnostics, even before symptoms have appeared. Nanodiagnostics refers to the application of nanoparticles (material with size equal to or less than 100 nm) for medical diagnostic purposes. The special property of nanomaterials compared to their macroscopic counterparts is a lesser signal loss and an enhanced electromagnetic field. Nanosize of the detection material also enhances its sensitivity and increases the signal to noise ratio. Microchips, nanorobots, biosensors, nanoidentification of single-celled structures, and microelectromechanical systems are some of the most modern nanodiagnostics technologies now in development. Here, we have highlighted the important roles of nanotechnology in healthcare sector, with a detailed focus on the management of the COVID-19 pandemic. We outline the different types of nanotechnology-based diagnostic devices for SARS-CoV-2 and the possible applications of nanomaterials in COVID-19 treatment. We also discuss the utility of nanomaterials in formulating preventive strategies against SARS-CoV-2 including their use in manufacture of protective equipment, formulation of vaccines, and strategies for directly hindering viral infection. We further discuss the factors hindering the large-scale accessibility of nanotechnology-based healthcare applications and suggestions for overcoming them.

Siglec-9 Restrains Antibody-Dependent Natural Killer Cell Cytotoxicity against SARS-CoV-2.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection alters the immunological profiles of natural killer (NK) cells. However, whether NK antiviral functions are impaired during severe coronavirus disease 2019 (COVID-19) and what host factors modulate these functions remain unclear. We found that NK cells from hospitalized COVID-19 patients degranulate less against SARS-CoV-2 antigen-expressing cells (in direct cytolytic and antibody-dependent cell cytotoxicity [ADCC] assays) than NK cells from mild COVID-19 patients or negative controls. The lower NK degranulation was associated with higher plasma levels of SARS-CoV-2 nucleocapsid antigen. Phenotypic and functional analyses showed that NK cells expressing the glyco-immune checkpoint Siglec-9 elicited higher ADCC than Siglec-9- NK cells. Consistently, Siglec-9+ NK cells exhibit an activated and mature phenotype with higher expression of CD16 (FcγRIII; mediator of ADCC), CD57 (maturation marker), and NKG2C (activating receptor), along with lower expression of the inhibitory receptor NKG2A, than Siglec-9- CD56dim NK cells. These data are consistent with the concept that the NK cell subpopulation expressing Siglec-9 is highly activated and cytotoxic. However, the Siglec-9 molecule itself is an inhibitory receptor that restrains NK cytotoxicity during cancer and other viral infections. Indeed, blocking Siglec-9 significantly enhanced the ADCC-mediated NK degranulation and lysis of SARS-CoV-2-antigen-positive target cells. These data support a model in which the Siglec-9+ CD56dim NK subpopulation is cytotoxic even while it is restrained by the inhibitory effects of Siglec-9. Alleviating the Siglec-9-mediated restriction on NK cytotoxicity may further improve NK immune surveillance and presents an opportunity to develop novel immunotherapeutic tools against SARS-CoV-2 infected cells. IMPORTANCE One mechanism that cancer cells use to evade natural killer cell immune surveillance is by expressing high levels of sialoglycans, which bind to Siglec-9, a glyco-immune checkpoint molecule on NK cells. This binding inhibits NK cell cytotoxicity. Several viruses, such as hepatitis B virus (HBV) and HIV, also use a similar mechanism to evade NK surveillance. We found that NK cells from SARS-CoV-2-hospitalized patients are less able to function against cells expressing SARS-CoV-2 Spike protein than NK cells from SARS-CoV-2 mild patients or uninfected controls. We also found that the cytotoxicity of the Siglec-9+ NK subpopulation is indeed restrained by the inhibitory nature of the Siglec-9 molecule and that blocking Siglec-9 can enhance the ability of NK cells to target cells expressing SARS-CoV-2 antigens. Our results suggest that a targetable glyco-immune checkpoint mechanism, Siglec-9/sialoglycan interaction, may contribute to the ability of SARS-CoV-2 to evade NK immune surveillance.

Inhibitory Siglec-sialic acid interactions in balancing immunological activation and tolerance during viral infections.

Siglecs are a family of emerging glyco-immune checkpoints. Inhibiting them can enhance the functions of several types of immune cells, whereas engaging them can reduce hyper-inflammation and hyper-activation of immune functions. Siglec-sialoglycan interactions play an important role in modulating immunological functions during cancer, however, their roles in regulating immunological equilibrium during viral infections is less clear. In this review, we discuss the documented and potential roles of inhibitory Siglecs in balancing immune activation and tolerance during viral infections and consider how this balance could affect both the desired anti-viral immunological functions and the unwanted hyper- or chronic inflammation. Finally, we discuss the opportunities to target the Siglec immunological switches to reach an immunological balance during viral infections: inhibiting specific Siglec-sialoglycan interactions when maximum anti-viral immune responses are needed, or inducing other interactions when preventing excessive inflammation or reducing chronic immune activation are the goals.

Differential inhibition of mucin-type O-glycosylation (MTOG) induced by peracetyl N-thioglycolyl-d-galactosamine (Ac5GalNTGc) in myeloid cells.

Investigations on the structure and functional roles of glycosylation - an intricate, complex, and dynamic post translational modification on proteins - in biological processes has been a challenging task. Glycan modifications vary depending on the specific cell type, its developmental stage, and resting or activated state. In the present study, we aim to understand the differences between the mucin-type O-glycosylation (MTOG) of two functionally divergent human cell lines, K562 (chronic myeloid leukemia) and U937 (histiocytic lymphoma), having myeloid origins. MTOG is initiated by the addition of N-acetyl-α-d-galactosamine (GalNAc) to Ser/Thr of glycoproteins. We exploited the metabolic glycan engineering (MGE) strategy using the peracetyl N-thioglycolyl-d-galactosamine (Ac5GalNTGc), a synthetic GalNAc analogue, to engineer the glycoconjugates. Ac5GalNTGc was metabolized and incorporated as N-thioglycolyl-d-galactosamine (GalNTGc) in cell surface glycoproteins in both the cell lines with varying degrees of efficiency. Notably, metabolic incorporation of GalNTGc resulted in differential inhibition of MTOG. It was observed that endogenous glycosylation machinery of K562 is relatively more stringent for selecting GalNTGc whereas U937 is flexible towards this selection. Additionally, we studied how the glycan modifications vary on a given CD antigen in these cell lines. Particularly, MTOG on CD43 was differentially inhibited in K562 and U937 as revealed by glycan-dependent and glycan-independent antibodies. It was observed that the effect of MGE on CD43 was similar to global effects on both cell lines. Consequences of MGE using GalNAc analogues depend on the expression and activity of various glycosyl transferases which determine global glycosylation on cell surface as well as on specific glycoproteins.