An essential mechanism for severe acute respiratory syndrome coronavirus 1 (SARS-CoV-1) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection begins with the viral spike protein ...binding to the human receptor protein angiotensin-converting enzyme II (ACE2). Here, we describe a stepwise engineering approach to generate a set of affinity optimized, enzymatically inactivated ACE2 variants that potently block SARS-CoV-2 infection of cells. These optimized receptor traps tightly bind the receptor binding domain (RBD) of the viral spike protein and prevent entry into host cells. We first computationally designed the ACE2–RBD interface using a two-stage flexible protein backbone design process that improved affinity for the RBD by up to 12-fold. These designed receptor variants were affinity matured an additional 14-fold by random mutagenesis and selection using yeast surface display. The highest-affinity variant contained seven amino acid changes and bound to the RBD 170-fold more tightly than wild-type ACE2. With the addition of the natural ACE2 collectrin domain and fusion to a human immunoglobulin crystallizable fragment (Fc) domain for increased stabilization and avidity, the most optimal ACE2 receptor traps neutralized SARS-CoV-2–pseudotyped lentivirus and authentic SARS-CoV-2 virus with half-maximal inhibitory concentrations (IC50s) in the 10- to 100-ng/mL range. Engineered ACE2 receptor traps offer a promising route to fighting infections by SARS-CoV-2 and other ACE2-using coronaviruses, with the key advantage that viral resistance would also likely impair viral entry. Moreover, such traps can be predesigned for viruses with known entry receptors for faster therapeutic response without the need for neutralizing antibodies isolated from convalescent patients.
Bacteria, such as Fusobacterium nucleatum, are present in the tumor microenvironment. However, the immunological consequences of intra-tumoral bacteria remain unclear. Here, we have shown that ...natural killer (NK) cell killing of various tumors is inhibited in the presence of various F. nucleatum strains. Our data support that this F. nucleatum-mediated inhibition is mediated by human, but not by mouse TIGIT, an inhibitory receptor present on all human NK cells and on various T cells. Using a library of F. nucleatum mutants, we found that the Fap2 protein of F. nucleatum directly interacted with TIGIT, leading to the inhibition of NK cell cytotoxicity. We have further demonstrated that tumor-infiltrating lymphocytes expressed TIGIT and that T cell activities were also inhibited by F. nucleatum via Fap2. Our results identify a bacterium-dependent, tumor-immune evasion mechanism in which tumors exploit the Fap2 protein of F. nucleatum to inhibit immune cell activity via TIGIT.
Immunoglobulin G (IgG) antibodies contain a complex N-glycan embedded in the hydrophobic pocket between its heavy chain protomers. This glycan contributes to the structural organization of the Fc ...domain and determines its specificity for Fcγ receptors, thereby dictating distinct cellular responses. The variable construction of this glycan structure leads to highly-related, but non-equivalent glycoproteins known as glycoforms. We previously reported synthetic nanobodies that distinguish IgG glycoforms. Here, we present the structure of one such nanobody, X0, in complex with the Fc fragment of afucosylated IgG1. Upon binding, the elongated CDR3 loop of X0 undergoes a conformational shift to access the buried N-glycan and acts as a 'glycan sensor', forming hydrogen bonds with the afucosylated IgG N-glycan that would otherwise be sterically hindered by the presence of a core fucose residue. Based on this structure, we designed X0 fusion constructs that disrupt pathogenic afucosylated IgG1-FcγRIIIa interactions and rescue mice in a model of dengue virus infection.
Natural killer cells (NK) are a component of innate immunity well known for their potent ability to kill virus-infected or neoplastically transformed cells following stimulation of the NK cell ...receptor NKG2D. One of the various ligands of NKG2D is MICB, a stress-induced ligand that has been found to be upregulated on the surface of tumor cells. However, there is little knowledge about how this upregulation may occur or how it may be selected against in tumors as a mechanism of immune escape. Here, we report that the metastasis-associated microRNA (metastamir) miR-10b directly binds to the 3' untranslated region of MICB and downregulates its expression. Notably, antagonizing miR-10b action enhanced NKG2D-mediated killing of tumor cells in vitro and enhanced clearance of tumors in vivo. Conversely, overexpression of miR-10b downregulated MICB and impaired elimination of tumor cells. Together, our results define MICB as a novel immune target of miR-10b, implying a direct link between metastasis capability and immune escape from NK cells.
PML-RARA and AML1-ETO are important oncogenic fusion proteins that play a central role in transformation to acute myeloid leukemia (AML). Whether these fusion proteins render the tumor cells with ...immune evasion properties is unknown. Here we show that both oncogenic proteins specifically downregulate the expression of CD48, a ligand of the natural killer (NK) cell activating receptor 2B4, thereby leading to decreased killing by NK cells. We demonstrate that this process is histone deacetylase (HDAC)-dependent, that it is mediated through the downregulation of CD48 messenger RNA, and that treatment with HDAC inhibitors (HDACi) restores the expression of CD48. Furthermore, by using chromatin immuoprecepitation (ChIP) experiments, we show that AML1-ETO directly interacts with CD48. Finally, we show that AML patients who are carrying these specific translocations have low expression of CD48.
•PML-RARA and AML1-ETO evade NK cell recognition by specifically downregulating the expression of CD48.•The findings are relevant to AML patients bearing these specific translocations.
Human cytomegalovirus (HCMV) is a major human pathogen, causing serious diseases in immunocompromised populations and congenially infected neonates. One of the main immune cells acting against the ...virus are Natural Killer (NK) cells. Killing by NK cells is mediated by a small family of activating receptors such as NKp30 that interact with the cellular ligand B7-H6. The outcome of B7-H6-NKp30 interaction was, so far, mainly studied with regard to NK recognition and killing of tumors. Here, we demonstrated that the expression of B7-H6 is upregulated following HCMV infection and that HCMV uses two of its genes: US18 and US20, to interfere with B7-H6 surface expression, in a mechanism involving endosomal degradation, in order to evade NK cell recognition.
Herpes simplex virus 1 (HSV1) is a ubiquitous human pathogen that utilizes variable mechanisms to evade immune surveillance. The glycosylphosphatidylinositol (GPI) anchoring pathway is a multistep ...process in which a myriad of different proteins are covalently attached to a GPI moiety to be presented on the cell surface. Among the different GPI-anchored proteins there are many with immunological importance. We present evidence that the HSV1-encoded miR H8 directly targets PIGT, a member of the protein complex that covalently attaches proteins to GPI in the final step of GPI anchoring. This results in a membrane down-modulation of several different immune-related, GPI-anchored proteins, including ligands for natural killer-activating receptors and the prominent viral restriction factor tetherin. Thus, we suggest that by utilizing just one of dozens of miRNAs encoded by HSV1, the virus can counteract the host immune response at several key points.
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•HSV1 miR H8 targets PIGT of the GPI anchoring pathway•Expression of the anti-viral protein tetherin is reduced and viral spread enhanced•Expression of GPI-anchored activating NK cell ligands is reduced•Recognition and elimination by NK cells decrease
Herpes simplex virus 1 is a ubiquitous human pathogen and the cause of several ailments. Enk et al. found that the HSV1-encoded miR H8 targets the GPI anchoring pathway, reducing expression of several immune-modulating proteins, thus enhancing viral spread and enabling evasion of natural killer cell elimination.
Expression of the stress-induced ligands MICA, MICB and ULBP 1-6 are up-regulated as a cellular response to DNA damage, excessive proliferation or viral infection; thereby, they enable recognition ...and annihilation by immune cells that express the powerful activating receptor NKG2D. This receptor is present not exclusively, but primarily on NK cells. Knowledge about the regulatory mechanisms controlling ULBP expression is still vague. In this study, we report a direct interaction of the oncogenic RNA binding protein (RBP) IMP3 with ULBP2 mRNA, leading to ULBP2 transcript destabilization and reduced ULBP2 surface expression in several human cell lines. We also discovered that IMP3 indirectly targets MICB with a mechanism functionally distinct from that of ULBP2. Importantly, IMP3-mediated regulation of stress-ligands leads to impaired NK cell recognition of transformed cells. Our findings shed new light on the regulation of NKG2D ligands and on the mechanism of action of a powerful oncogenic RBP, IMP3.
MicroRNAs are key players in most biological processes. Some microRNAs are involved in the genesis of tumors and are therefore termed oncomiRs, while others, termed metastamiRs, play a significant ...role in the formation of cancer metastases. Previously, we identified ten different cellular microRNAs that downregulate the expression of MICB, a ligand of the activating NK receptor NKG2D. Interestingly, several of the ten MICB-targeting microRNAs, such as miR-10b, are involved in tumor formation and metastasis. In this work, we identify a complex interplay between these different microRNAs. Specifically, we demonstrate that three of the MICB-targeting microRNAs: miR-20a, miR-17-5p and miR-93, also target the same site in the 3'UTR of TWIST1, a transcription factor implicated in cancer metastasis. Additionally, we show that miR-520d-5p targets a different site in the 3'UTR of TWIST1. We next show that the miR-520d-5p-mediated decrease of TWIST1 expression results in reduced expression of one of its targets, miR-10b, and in the restoration of E-Cadherin expression, which in turn results in reduced cellular motility and invasiveness. Finally, we show that miR-520d-5p leads to reduced proliferation of tumor cells, and that high levels of miR-520d-5p correlate with higher survival rates of cancer patients.
HCMV is a highly sophisticated virus that has developed various mechanisms for immune evasion and viral dissemination throughout the body (partially mediated by neutrophils). NK cells play an ...important role in elimination of HCMV-infected cells. Both neutrophils and NK cells utilize similar sets of chemokine receptors to traffic, to and from, various organs. However, the mechanisms by which HCMV attracts neutrophils and not NK cells are largely unknown. Here, we show a unique viral protein, vCXCL1, which targets three chemokine receptors: CXCR1 and CXCR2 expressed on neutrophils and CXCR1 and CX3CR1 expressed on NK cells. Although vCXCL1 attracted both cell types, neutrophils migrated faster and more efficiently than NK cells through the binding of CXCR2. Therefore, we propose that HCMV has developed vCXCL1 to orchestrate its rapid systemic dissemination through preferential attraction of neutrophils and uses alternative mechanisms to counteract the later attraction of NK cells.
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•vCXCL1 binds the chemokine receptor CX3CR1 expressed on CD56Dim CD16Pos NK cells•vCXCL1 binds CXCR2 on neutrophils and CXCR1 on both NK cells and neutrophils•Neutrophils migrate faster and more efficiently through the binding of vCXCL1 to CXCR2•HCMV uses vCXCL1 to attract neutrophils and orchestrate its dissemination in the body
Viral CXCL1 (vCXCL1) is a chemokine produced following infection with human cytomegalovirus. Yamin et al. show here that vCXCL1 binds to three chemokine receptors: CXCR1, CXCR2, and CX3CR1 and that neutrophils migrate faster and more efficiently than NK cells toward vCXCL1 through binding to CXCR2 expressed by neutrophils only.
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