Genome-wide CRISPR screens reveal a specific ligand for the glycan-binding immune checkpoint receptor Siglec-7.

Glyco-immune checkpoint receptors, molecules that inhibit immune cell activity following binding to glycosylated cell-surface antigens, are emerging as attractive targets for cancer immunotherapy. Defining biologically relevant ligands that bind and activate such receptors, however, has historically been a significant challenge. Here, we present a CRISPRi genomic screening strategy that allowed unbiased identification of the key genes required for cell-surface presentation of glycan ligands on leukemia cells that bind the glyco-immune checkpoint receptors Siglec-7 and Siglec-9. This approach revealed a selective interaction between Siglec-7 and the mucin-type glycoprotein CD43. Further work identified a specific N-terminal glycopeptide region of CD43 containing clusters of disialylated O-glycan tetrasaccharides that form specific Siglec-7 binding motifs. Knockout or blockade of CD43 in leukemia cells relieves Siglec-7-mediated inhibition of immune killing activity. This work identifies a potential target for immune checkpoint blockade therapy and represents a generalizable approach to dissection of glycan-receptor interactions in living cells.

The conserved arginine residue in all siglecs is essential for Siglec-7 binding to sialic acid.

Siglecs are sialic acid (Sia)-binding immunoglobulin-like lectins; the majority of Siglecs functions as transmembrane receptors on the immune cells via Sia residues. Recently, a new Sia binding site in Siglec-7, termed site 2, where arginine (R) 67 was critical, was identified by computational modeling and biochemical analyses, relative to the primary Sia binding site, termed site 1, containing critical R124. Here, the presence of a new essential R94 residue, which is completely conserved among all identified Siglecs, was demonstrated. A mutation of R94 residue in Siglec-7 led to the disappearance of the Sia binding property, similar to a site 1 mutation (R124A). R94 is close to R67 in site 2, and site 2 mutations at either of them abolished the ligand-binding properties to both gangliosides and glycoproteins. These data suggest that, in addition to site 1, the conserved R residue among Siglecs in site 2 is another functional site.

CD68+ macrophages as crucial components of the foreign body reaction demonstrate an unconventional pattern of functional markers quantified by analysis with double fluorescence staining.

Implants like meshes for the reinforcement of tissues implement the formation of a persistent inflammation with an ambient fibrotic reaction. In the inflammatory infiltrate several distinct cell types have been identified, but CD68+ macrophages are supposed to be most important. To investigate the collaboration among the various cell types within the infiltrate we performed at explanted meshes from humans double fluorescence staining with CD68 as a constant marker and a variety of other antibodies as the second marker. The list of second markers includes lymphocytes (CD3, CD4, CD8, CD16, CD56, FoxP3, and CD11b) stem cells (CD34), leucocytes (CD45, CD15), macrophages (CD86, CD105, CD163, and CD206); deposition of EC matrix (collagen-I, collagen-III, MMP2, and MMP8); Ki67 as a marker for proliferation; and the tyrosine-protein kinase receptor AXL. The present study demonstrates within the inflammatory infiltrate the abundant capability of CD68+ cells to co-express a huge variety of other markers, including those of lymphocytes, varying between 5 and 83% of investigated cells. The observation of co-staining was not restricted to a specific polymer but was seen with polypropylene fibers as well as with fibers made of polyvinylidene fluoride, although with differences in co-expression rates. The persisting variability of these cells without the functional reduction toward differentiated mature cell types may favor the lack of healing at the interface of meshes.

The Roles of Siglec7 and Siglec9 on Natural Killer Cells in Virus Infection and Tumour Progression.

The function of natural killer (NK) cells, defending against virus infection and tumour progression, is regulated by multiple activating and inhibiting receptors expressed on NK cells, among which sialic acid-bind immunoglobulin-like lectins (Siglecs) act as a vital inhibitory group. Previous studies have shown that Siglec7 and Siglec9 are expressed on NK cells, which negatively regulate the function of NK cells and modulate the immune response through the interaction of sialic acid-containing ligands. Siglec7 and Siglec9 are very similar in distribution, gene encoding, protein sequences, ligand affinity, and functions in regulating the immune system against virus and cancers, but differences still exist between them. In this review, we aim to discuss the similarities and differences between Siglec7 and Siglec9 and analyze their functions in virus infection and tumour progression in order to develop better anti-viral and anti-tumor immunotherapy in the future.

The mucin-selective protease StcE enables molecular and functional analysis of human cancer-associated mucins.

Mucin domains are densely O-glycosylated modular protein domains that are found in a wide variety of cell surface and secreted proteins. Mucin-domain glycoproteins are known to be key players in a host of human diseases, especially cancer, wherein mucin expression and glycosylation patterns are altered. Mucin biology has been difficult to study at the molecular level, in part, because methods to manipulate and structurally characterize mucin domains are lacking. Here, we demonstrate that secreted protease of C1 esterase inhibitor (StcE), a bacterial protease from Escherichia coli, cleaves mucin domains by recognizing a discrete peptide- and glycan-based motif. We exploited StcE's unique properties to improve sequence coverage, glycosite mapping, and glycoform analysis of recombinant human mucins by mass spectrometry. We also found that StcE digests cancer-associated mucins from cultured cells and from ascites fluid derived from patients with ovarian cancer. Finally, using StcE, we discovered that sialic acid-binding Ig-type lectin-7 (Siglec-7), a glycoimmune checkpoint receptor, selectively binds sialomucins as biological ligands, whereas the related receptor Siglec-9 does not. Mucin-selective proteolysis, as exemplified by StcE, is therefore a powerful tool for the study of mucin domain structure and function.

The localization of CD3, CD79a, CD68 and S100 protein immunoreactive cells in hemal nodes of Saanen goat (Capra hircus).

We investigated the structure of hemal nodes in Saanen goats using immunohistochemical staining. We examined the distribution of CD3 positive T lymphocytes, CD79a positive B lymphocytes, CD68 positive macrophages and S100 protein positive follicular dendritic cells. Hemal nodes of six healty adult female goats were used. Hemal nodes were removed from the thoracic and abdominal cavities. The oval to round hemal nodes were observed especially between the abdominal aorta and vena cava, and near the kidneys and adrenal glands. Tissue sections were stained with Crossmon's modified triple stain to demonstrate general histological structure. The avidin-biotin-peroxidase technique using anti-CD3, anti-CD79a, anti-CD68 and anti-S100 primary antibodies was used for immunohistochemistry. Many CD3 positive T lymphocytes were found in the germinal center of the lymph follicles and in the lymphatic cords of hemal nodes; CD3 positive cells also were observed in the sinuses. CD79a and CD68 positive cells were found at the germinal center of the lymph follicles. In the lymph follicles near the subcapsular sinuses, CD79a and CD68 positive cells were found especially in e areas bordering the mantle zone. S100 positive cells were found in the lymph follicles, lymphatic cords and sinuses.

The CD163 long-range scavenger receptor cysteine-rich repeat: expression, purification and X-ray crystallographic characterization.

Scavenger receptors (SRs) play critical roles in various physiological and pathological pathways. One of them, CD163, is a multifunctional endocytic receptor and is characterized by a long-range scavenger receptor cysteine-rich (SRCR) repeat. However, the structural and functional details of this long-range SRCR repeat have not yet been elucidated. In this study, the CD163 long-range SRCR repeat was expressed in Drosophila Schneider 2 cells. The recombinant protein was homogeneous after purification by metal-affinity, cation-exchange and size-exclusion chromatography. Single crystals were obtained using 20% PEG 4000, 0.15 M potassium sodium tartrate tetrahydrate pH 8.5 and diffracted to 3.30 Šresolution. As the first view of a long-range SRCR repeat, this work lays the structural basis for a deep understanding of SRs and their multiple functions.

CD163 knockout pigs are fully resistant to highly pathogenic porcine reproductive and respiratory syndrome virus.

Porcine reproductive and respiratory syndrome virus (PRRSV) causes severe economic losses to current swine production worldwide. Highly pathogenic PRRSV (HP-PRRSV), originated from a genotype 2 PRRSV, is more virulent than classical PRRSV and further exacerbates the economic impact. HP-PRRSV has become the predominant circulating field strain in China since 2006. CD163 is a cellular receptor for PRRSV. The depletion of CD163 whole protein or SRCR5 region (interaction site for the virus) confers resistance to infection of several PRRSV isolates in pigs or cultured host cells. In this study, we described the generation of a CD163 knockout (KO) pig in which the CD163 protein was ablated by using CRISPR/Cas9 gene targeting and somatic cell nuclear transfer (SCNT) technologies. Challenge with HP-PRRSV TP strain showed that CD163 KO pigs are completely resistant to viral infection manifested by the absence of viremia, antibody response, high fever or any other PRRS-associated clinical signs. By comparison, wild-type (WT) controls displayed typical signs of PRRSV infection and died within 2 weeks after infection. Deletion of CD163 showed no adverse effects to the macrophages on immunophenotyping and biological activity as hemoglobin-haptoglobin scavenger. The results demonstrated that CD163 knockout confers full resistance to HP-PRRSV infection to pigs without impairing the biological function associated with the gene.

Chemical Synthesis and Evaluation of a Disialic Acid-Containing Dextran Polymer as an Inhibitor for the Interaction between Siglec 7 and Its Ligand.

A new sialic acid (Sia)-containing glycopolymer-a fluorescent probe with high-density disialic acid (diSia) on the surface of polysaccharide dextran (diSia-Dex)-was synthesized as a key molecule to regulate the Sia recognition lectins, Siglecs, that are involved in the immune system. According to our original methods, diSia was synthesized by α-selective sialylation, and a dextran template possessing terminal acetylenes and amino groups was prepared. A diSia and a fluorescent molecule were subsequently introduced to surface-modified dextran by Hüisgen reaction and amidation, respectively. The modulatory activity of Siglec7 was evaluated by using synthetic probes. DiSia-Dex showed high binding avidity toward Siglec7, with a KD value of 5.87×10-10 m, and a high inhibitory activity for the interaction between Siglec7 and a ligand (GD3), with a IC50 value of 1.0 nm. Notably, diSia-Dex was able to release Siglec7 from the pre-existing Siglec7-GD3 complex, possibly due to its unique properties of a slow dissociation rate and a high association rate. Together, these data show that diSia-Dex can be widely applicable as a modulator of Siglec7 functions.

Clinical significance of sCD163 and its possible role in asthma (Review).

Macrophages exert important functions in the balance and efficiency of immune responses, and participate in innate and adaptive immunity. The proinflammatory actions of macrophages are implicated in autoimmune diseases. Unlike classically activated M1 macrophages, the alternatively activated cluster of differentiation (CD)163+ and CD206+ M2 macrophages are involved in tissue repair and wound healing, and use oxidative metabolism to support their long­term functions. CD163 is a member of the scavenger receptor superfamily, categorized into class B, and its soluble(s) form, sCD163, is a marker of activated M2 macrophages. CD163 is selectively expressed in cells of the monocyte and macrophage lineages; however, its biological role has yet to be elucidated. The expression of sCD163 is markedly induced by anti­inflammatory mediators, such as glucocorticoids and interleukin­10, whereas it is inhibited by proinflammatory mediators, such as interferon­Î³. These findings suggest that CD163 may serve as a potential target for the therapeutic modulation of inflammatory responses. The concentration of sCD163 in blood is associated with acute and chronic inflammatory processes in autoimmune disorders of connective tissue, fat metabolism and cardiovascular diseases, and it can be used for the assessment of cancer prognosis. A role for sCD163 in the pathogenesis of asthma has also been proposed. The present review serves to present the available knowledge concerning the implication of sCD163 in the pathophysiological mechanisms of asthma, and evaluate its potential as a biomarker and possible therapeutic target for asthma.