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Nature Chemical Biology Aug 2023Mammalian cell surface and secreted glycoproteins exhibit remarkable glycan structural diversity that contributes to numerous physiological and pathogenic interactions....
Mammalian cell surface and secreted glycoproteins exhibit remarkable glycan structural diversity that contributes to numerous physiological and pathogenic interactions. Terminal glycan structures include Lewis antigens synthesized by a collection of α1,3/4-fucosyltransferases (CAZy GT10 family). At present, the only available crystallographic structure of a GT10 member is that of the Helicobacter pylori α1,3-fucosyltransferase, but mammalian GT10 fucosyltransferases are distinct in sequence and substrate specificity compared with the bacterial enzyme. Here, we determined crystal structures of human FUT9, an α1,3-fucosyltransferase that generates Lewis and Lewis antigens, in complex with GDP, acceptor glycans, and as a FUT9-donor analog-acceptor Michaelis complex. The structures reveal substrate specificity determinants and allow prediction of a catalytic model supported by kinetic analyses of numerous active site mutants. Comparisons with other GT10 fucosyltransferases and GT-B fold glycosyltransferases provide evidence for modular evolution of donor- and acceptor-binding sites and specificity for Lewis antigen synthesis among mammalian GT10 fucosyltransferases.
Topics: Animals; Humans; Fucosyltransferases; Glycosyltransferases; Lewis Blood Group Antigens; Polysaccharides; Mammals
PubMed: 37202521
DOI: 10.1038/s41589-023-01345-y -
International Journal of Molecular... Dec 2023Cancer management still requires more potent and safer treatments, of which immunomodulatory receptors on the lymphocyte surface have started to show promise in new... (Review)
Review
Cancer management still requires more potent and safer treatments, of which immunomodulatory receptors on the lymphocyte surface have started to show promise in new cancer immunotherapies (e.g., CTLA-4 and PD-1). CD6 is a signal-transducing transmembrane receptor, mainly expressed by all T cells and some B and NK cell subsets, whose endogenous ligands (CD166/ALCAM, CD318/CDCP-1, Galectins 1 and 3) are overexpressed by malignant cells of different lineages. This places CD6 as a potential target for novel therapies against haematological and non-haematological malignancies. Recent experimental evidence for the role of CD6 in cancer immunotherapies is summarised in this review, dealing with diverse and innovative strategies from the classical use of monoclonal antibodies to soluble recombinant decoys or the adoptive transfer of immune cells engineered with chimeric antigen receptors.
Topics: Activated-Leukocyte Cell Adhesion Molecule; Antigens, CD; Antigens, Differentiation, T-Lymphocyte; Immunotherapy; Neoplasms; T-Lymphocytes
PubMed: 38139340
DOI: 10.3390/ijms242417510 -
Molecular Therapy : the Journal of the... Nov 2023Chimeric antigen receptor (CAR)-T cells represent a promising frontier in cancer immunotherapy. However, the current process for developing new CAR constructs is time...
Chimeric antigen receptor (CAR)-T cells represent a promising frontier in cancer immunotherapy. However, the current process for developing new CAR constructs is time consuming and inefficient. To address this challenge and expedite the evaluation and comparison of full-length CAR designs, we have devised a novel cloning strategy. This strategy involves the sequential assembly of individual CAR domains using blunt ligation, with each domain being assigned a unique DNA barcode. Applying this method, we successfully generated 360 CAR constructs that specifically target clinically validated tumor antigens CD19 and GD2. By quantifying changes in barcode frequencies through next-generation sequencing, we characterize CARs that best mediate proliferation and expansion of transduced T cells. The screening revealed a crucial role for the hinge domain in CAR functionality, with CD8a and IgG4 hinges having opposite effects in the surface expression, cytokine production, and antitumor activity in CD19- versus GD2-based CARs. Importantly, we discovered two novel CD19-CAR architectures containing the IgG4 hinge domain that mediate superior in vivo antitumor activity compared with the construct used in Kymriah, a U.S. Food and Drug Administration (FDA)-approved therapy. This novel screening approach represents a major advance in CAR engineering, enabling accelerated development of cell-based cancer immunotherapies.
Topics: Humans; Receptors, Chimeric Antigen; Protein Domains; Receptors, Antigen, T-Cell; T-Lymphocytes; Neoplasms; Immunoglobulin G; Immunotherapy, Adoptive; Antigens, CD19
PubMed: 37705245
DOI: 10.1016/j.ymthe.2023.09.008 -
Biomolecules Jul 2023Cell surface HLA-I molecules (Face-1) consist of a polypeptide heavy chain (HC) with two groove domains (G domain) and one constant domain (C-domain) as well as a light... (Review)
Review
Cell surface HLA-I molecules (Face-1) consist of a polypeptide heavy chain (HC) with two groove domains (G domain) and one constant domain (C-domain) as well as a light chain, B2-microglobulin (B2m). However, HCs can also independently emerge unfolded on the cell surface without peptides as B2m-free HC monomers (Face-2), B2m-free HC homodimers (Face 3), and B2m-free HC heterodimers (Face-4). The transport of these HLA variants from ER to the cell surface was confirmed by antiviral antibiotics that arrest the release of newly synthesized proteins from the ER. Face-2 occurs at low levels on the normal cell surface of the lung, bronchi, epidermis, esophagus, breast, stomach, ilium, colorectum, gall bladder, urinary bladder, seminal vesicles ovarian epithelia, endometrium, thymus, spleen, and lymphocytes. They are upregulated on immune cells upon activation by proinflammatory cytokines, anti-CD3 antibodies, antibiotics (e.g., ionomycin), phytohemagglutinin, retinoic acid, and phorbol myristate acetate. Their density on the cell surface remains high as long as the cells remain in an activated state. After activation-induced upregulation, the Face-2 molecules undergo homo- and hetero-dimerization (Face-3 and Face-4). Alterations in the redox environment promote dimerization. Heterodimerization can occur among and between the alleles of different haplotypes. The glycosylation of these variants differ from that of Face-1, and they may occur with bound exogenous peptides. Spontaneous arthritis occurs in HLA-B27+ mice lacking B2m (HLA-B27+ B2m-/-) but not in HLA-B27+ B2m+/- mice. The mice with HLA-B27 in Face-2 spontaneous configuration develop symptoms such as changes in nails and joints, hair loss, and swelling in paws, leading to ankyloses. Anti-HC-specific mAbs delay disease development. Some HLA-I polyreactive mAbs (MEM series) used for immunostaining confirm the existence of B2m-free variants in several cancer cells. The upregulation of Face-2 in human cancers occurs concomitantly with the downregulation of intact HLAs (Face-1). The HLA monomeric and dimeric variants interact with inhibitory and activating ligands (e.g., KIR), growth factors, cytokines, and neurotransmitters. Similarities in the amino acid sequences of the HLA-I variants and HLA-II β-chain suggest that Face-2 could be the progenitor of both HLA classes. These findings may support the recognition of these variants as a neo-HLA class and proto-HLA.
Topics: Female; Male; Humans; Animals; Mice; HLA-B27 Antigen; HLA Antigens; Cell Membrane; Cytokines; Anti-Bacterial Agents; Antibodies, Monoclonal
PubMed: 37627243
DOI: 10.3390/biom13081178 -
Anesthesiology Jan 2024The Prevalence of Hepatitis B Markers in Anesthesia Personnel. By Berry AJ, Isaacson IJ, Hunt D, Kane MA. Anesthesiology 1984; 60:6-9 The prevalence of hepatitis B viral...
The Prevalence of Hepatitis B Markers in Anesthesia Personnel. By Berry AJ, Isaacson IJ, Hunt D, Kane MA. Anesthesiology 1984; 60:6-9 The prevalence of hepatitis B viral markers has increased in some groups of medical workers who are exposed to blood from patients carrying the virus, but this has not been studied critically in physicians and others who administer anesthesia. Physician anesthesiologists (M.D.) and nurse anesthetists and anesthesia assistants (non-M.D.) at four university-affiliated hospitals were evaluated for hepatitis B markers as determined by seropositivity for hepatitis B surface antigen, antibody to the hepatitis B surface antigen, or antibody to the hepatitis B core antigen. In the 86 subjects (38 M.D., 48 non-M.D.) who represented 80.4% of possible participants, the overall prevalence of serologic markers of hepatitis B was 23.3%. The frequency did not differ between M.D. (23.7%) and non-M.D. (22.9%) groups or between men (20.3%) and women (26.9%). Of 81 subjects who had no clinical history of hepatitis, 16 (19.8%) had positive serologic markers. The frequency of seropositivity increased with time since graduation from medical school (M.D.) or nursing school or college (non-M.D.). The prevalence of serologic markers of hepatitis B virus in this study of anesthesia personnel is five to eight times that of the general population but is similar to that of other medical workers who frequently are exposed to blood.
Topics: Male; Humans; Female; Hepatitis B Surface Antigens; Hepatitis B; Anesthesiology; Physicians; Anesthesiologists
PubMed: 38085155
DOI: 10.1097/ALN.0000000000004779 -
JCI Insight Dec 2023Regulatory T cells (Tregs) have potential for the treatment of autoimmune diseases and graft rejection. Antigen specificity and functional stability are considered...
Regulatory T cells (Tregs) have potential for the treatment of autoimmune diseases and graft rejection. Antigen specificity and functional stability are considered critical for their therapeutic efficacy. In this study, expansion of human Tregs in the presence of porcine PBMCs (xenoantigen-expanded Tregs, Xn-Treg) allowed the selection of a distinct Treg subset, coexpressing the activation/memory surface markers HLA-DR and CD27 with enhanced proportion of FOXP3+Helios+ Tregs. Compared with their unsorted and HLA-DR+CD27+ double-positive (DP) cell-depleted Xn-Treg counterparts, HLA-DR+CD27+ DP-enriched Xn-Tregs expressed upregulated Treg function markers CD95 and ICOS with enhanced suppression of xenogeneic but not polyclonal mixed lymphocyte reaction. They also had less Treg-specific demethylation in the region of FOXP3 and were more resistant to conversion to effector cells under inflammatory conditions. Adoptive transfer of porcine islet recipient NOD/SCID IL2 receptor γ-/- mice with HLA-DR+CD27+ DP-enriched Xn-Tregs in a humanized mouse model inhibited porcine islet graft rejection mediated by 25-fold more human effector cells. The prolonged graft survival was associated with enhanced accumulation of FOXP3+ Tregs and upregulated expression of Treg functional genes, IL10 and cytotoxic T lymphocyte antigen 4, but downregulated expression of effector Th1, Th2, and Th17 cytokine genes, within surviving grafts. Collectively, human HLA-DR+CD27+ DP-enriched Xn-Tregs expressed a specific regulatory signature that enabled identification and isolation of antigen-specific and functionally stable Tregs with potential as a Treg-based therapy.
Topics: Mice; Humans; Animals; Swine; T-Lymphocytes, Regulatory; Mice, SCID; Mice, Inbred NOD; HLA-DR Antigens; Forkhead Transcription Factors
PubMed: 37874660
DOI: 10.1172/jci.insight.162978 -
Nature Biotechnology Jul 2023Identification of CD8 T cell epitopes is critical for the development of immunotherapeutics. Existing methods for major histocompatibility complex class I (MHC class I)...
Identification of CD8 T cell epitopes is critical for the development of immunotherapeutics. Existing methods for major histocompatibility complex class I (MHC class I) ligand discovery are time intensive, specialized and unable to interrogate specific proteins on a large scale. Here, we present EpiScan, which uses surface MHC class I levels as a readout for whether a genetically encoded peptide is an MHC class I ligand. Predetermined starting pools composed of >100,000 peptides can be designed using oligonucleotide synthesis, permitting large-scale MHC class I screening. We exploit this programmability of EpiScan to uncover an unappreciated role for cysteine that increases the number of predicted ligands by 9-21%, reveal affinity hierarchies by analysis of biased anchor peptide libraries and screen viral proteomes for MHC class I ligands. Using these data, we generate and iteratively refine peptide binding predictions to create EpiScan Predictor. EpiScan Predictor performs comparably to other state-of-the-art MHC class I peptide binding prediction algorithms without suffering from underrepresentation of cysteine-containing peptides. Thus, targeted immunopeptidomics using EpiScan will accelerate CD8 T cell epitope discovery toward the goal of individual-specific immunotherapeutics.
Topics: Ligands; Cysteine; Histocompatibility Antigens Class I; Peptides; Epitopes, T-Lymphocyte; Protein Binding
PubMed: 36593401
DOI: 10.1038/s41587-022-01566-x -
The Journal of Biological Chemistry Aug 2023The expression of trophoblast cell surface antigen-2 (Trop-2) is enhanced in many tumor tissues and is correlated with increased malignancy and poor survival of patients...
The expression of trophoblast cell surface antigen-2 (Trop-2) is enhanced in many tumor tissues and is correlated with increased malignancy and poor survival of patients with cancer. Previously, we demonstrated that the Ser-322 residue of Trop-2 is phosphorylated by protein kinase Cα (PKCα) and PKCδ. Here, we demonstrate that phosphomimetic Trop-2 expressing cells have markedly decreased E-cadherin mRNA and protein levels. Consistently, mRNA and protein of the E-cadherin-repressing transcription factors zinc finger E-Box binding homeobox 1 (ZEB1) were elevated, suggesting transcriptional regulation of E-cadherin expression. The binding of galectin-3 to Trop-2 enhanced the phosphorylation and subsequent cleavage of Trop-2, followed by intracellular signaling by the resultant C-terminal fragment. Binding of β-catenin/transcription factor 4 (TCF4) along with the C-terminal fragment of Trop-2 to the ZEB1 promoter upregulated ZEB1 expression. Of note, siRNA-mediated knockdown of β-catenin and TCF4 increased the expression of E-cadherin through ZEB1 downregulation. Knockdown of Trop-2 in MCF-7 cells and DU145 cells resulted in downregulation of ZEB1 and subsequent upregulation of E-cadherin. Furthermore, wild-type and phosphomimetic Trop-2 but not phosphorylation-blocked Trop-2 were detected in the liver and/or lung of some nude mice bearing primary tumors inoculated intraperitoneally or subcutaneously with wild-type or mutated Trop-2 expressing cells, suggesting that Trop-2 phosphorylation, plays an important role in tumor cell mobility in vivo, too. Together with our previous finding of Trop-2 dependent regulation of claudin-7, we suggest that the Trop-2-mediated cascade involves concurrent derangement of both tight and adherence junctions, which may drive metastasis of epithelial tumor cells.
Topics: Animals; Humans; Mice; beta Catenin; Cadherins; Cell Line, Tumor; Down-Regulation; Epithelial-Mesenchymal Transition; Galectin 3; Gene Expression Regulation, Neoplastic; MCF-7 Cells; Mice, Nude; RNA, Messenger; Trophoblasts; Zinc Finger E-box-Binding Homeobox 1
PubMed: 37380081
DOI: 10.1016/j.jbc.2023.104971 -
Nature Cancer Dec 2023Despite recent advances in the treatment of acute myeloid leukemia (AML), there has been limited success in targeting surface antigens in AML, in part due to shared...
Despite recent advances in the treatment of acute myeloid leukemia (AML), there has been limited success in targeting surface antigens in AML, in part due to shared expression across malignant and normal cells. Here, high-density immunophenotyping of AML coupled with proteogenomics identified unique expression of a variety of antigens, including the RNA helicase U5 snRNP200, on the surface of AML cells but not on normal hematopoietic precursors and skewed Fc receptor distribution in the AML immune microenvironment. Cell membrane localization of U5 snRNP200 was linked to surface expression of the Fcγ receptor IIIA (FcγIIIA, also known as CD32A) and correlated with expression of interferon-regulated immune response genes. Anti-U5 snRNP200 antibodies engaging activating Fcγ receptors were efficacious across immunocompetent AML models and were augmented by combination with azacitidine. These data provide a roadmap of AML-associated antigens with Fc receptor distribution in AML and highlight the potential for targeting the AML cell surface using Fc-optimized therapeutics.
Topics: Humans; Antibodies, Monoclonal; Antigens, Surface; Leukemia, Myeloid, Acute; Receptors, Fc; Receptors, IgG; Ribonucleoproteins, Small Nuclear; Tumor Microenvironment
PubMed: 37872381
DOI: 10.1038/s43018-023-00656-2 -
Journal of Thrombosis and Haemostasis :... Sep 2023Endoglin, alias CD105, is a human membrane glycoprotein highly expressed in vascular endothelial cells. It is involved in angiogenesis and angiogenesis-related diseases,... (Review)
Review
Endoglin, alias CD105, is a human membrane glycoprotein highly expressed in vascular endothelial cells. It is involved in angiogenesis and angiogenesis-related diseases, including the rare vascular pathology known as hereditary hemorrhagic telangiectasia type 1. Although endoglin acts as an accessory receptor for members of the transforming growth factor-β family, in recent years, emerging evidence has shown a novel functional role for this protein beyond the transforming growth factor-β system. In fact, endoglin has been found to be an integrin counterreceptor involved in endothelial cell adhesion processes during pathological inflammatory conditions and primary hemostasis. Furthermore, a circulating form of endoglin, also named as soluble endoglin, whose levels are abnormally increased in different pathological conditions, such as preeclampsia, seems to act as an antagonist of membrane-bound endoglin and as a competitor of the fibrinogen-integrin interaction in platelet-dependent thrombus formation. These studies suggest that membrane-bound endoglin and circulating endoglin are important components involved in vascular homeostasis and hemostasis.
Topics: Female; Humans; Pregnancy; Antigens, CD; Endoglin; Endothelial Cells; Integrins; Receptors, Cell Surface; Telangiectasia, Hereditary Hemorrhagic; Transforming Growth Factor beta; Transforming Growth Factors; Vascular Cell Adhesion Molecule-1
PubMed: 37315795
DOI: 10.1016/j.jtha.2023.06.007