Enrichment and nLC-MS/MS Analysis of Head and Neck Cancer Mucinome Glycoproteins.

Mucin-domain glycoproteins expressed on cancer cell surfaces play central roles in cell adhesion, cancer progression, stem cell renewal, and immune evasion. Despite abundant evidence that mucin-domain glycoproteins are critical to the pathobiology of head and neck squamous cell carcinoma (HNSCC), our knowledge of the composition of that mucinome is grossly incomplete. Here, we utilized a catalytically inactive point mutant of the enzyme StcE (StcEE447D) to capture mucin-domain glycoproteins in head and neck cancer cell line lysates followed by their characterization using sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE), in-gel digestion, nano-liquid chromatography-tandem mass spectrometry (nLC-MS/MS), and enrichment analyses. We demonstrate the feasibility of this workflow for the study of mucin-domain glycoproteins in HNSCC, identify a set of mucin-domain glycoproteins common to multiple HNSCC cell lines, and report a subset of mucin-domain glycoproteins that are uniquely expressed in HSC-3 cells, a cell line derived from a highly aggressive metastatic tongue squamous cell carcinoma. This effort represents the first attempt to identify mucin-domain glycoproteins in HNSCC in an untargeted, unbiased analysis, paving the way for a more comprehensive characterization of the mucinome components that mediate aggressive tumor cell phenotypes. Data associated with this study have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the data set identifier PXD029420.

Multidimensional separation and analysis of alpha-1-acid glycoprotein N-glycopeptides using high-field asymmetric waveform ion mobility spectrometry (FAIMS) and nano-liquid chromatography tandem mass spectrometry.

Bottom-up nLC-MS/MS-based glycoprotein mass spectrometry workflows rely on the generation of a mixture of non-glycosylated and glycosylated peptides via proteolysis of glycoproteins. Such methods are challenged by suppression of hydrophilic glycopeptide ions by more abundant, hydrophobic, and readily ionizable non-glycosylated peptides. Commercially available high-field asymmetric waveform ion mobility spectrometry (FAIMS) devices have recently been introduced and present a potential benefit for glycoproteomic workflows by enabling orthogonal separation of non-glycosylated peptides and glycopeptides following chromatographic separation, and prior to MS/MS analysis. However, knowledge is lacking regarding optimal FAIMS conditions for glycopeptide analyses. Here, we document optimal FAIMS compensation voltages for the transmission and analysis of human alpha-1-acid glycoprotein (AGP) tryptic N-glycopeptide ions. Further, we evaluate the effect of FAIMS on AGP glycopeptide assignment confidence by comparing the number of assigned glycopeptides at different confidence levels using a standard nLC-MS/MS method or an otherwise identical method employing FAIMS. Optimized methods will potentiate glycoproteomic analyses by increasing the number of unique glycopeptide identifications and the confidence of glycopeptide assignments. Data are available via ProteomeXchange with identifier PXD036667. Analysis of alpha-1-acid glycoprotein (AGP) tryptic digests via nLC-FAIMS-MS/MS (top) led to the establishment of ideal FAIMS voltages for the analysis of AGP N-glycopeptides (bottom), suggesting that FAIMS can improve the depth of glycoproteome characterization. Pairs of CV magnitudes are shown along the x-axis.

Identification of α1,2-fucosylated signaling and adhesion molecules in head and neck squamous cell carcinoma.

Head and neck cancer is the seventh most common cancer in the world, and most cases manifest as head and neck squamous cell carcinoma. Despite the prominent role of fucosylated carbohydrate antigens in tumor cell adhesion and metastasis, little is known about the functional role of fucose-modified glycoproteins in head and neck cancer pathobiology. Inactivating polymorphisms of the fut2 gene, encoding for the α1,2-fucosyltransferase FUT2, are associated with an increased incidence of head and neck cancer among tobacco users. Moreover, the presence of the α1,2-fucosylated Lewis Y epitope, with both α1,2- and α1,3-linked fucose, has been observed in head and neck cancer tumors while invasive regions lose expression, suggesting a potential role for α1,2-fucosylation in the regulation of aggressive tumor cell characteristics. Here, we report an association between fut2 expression and head and neck cancer survival, document differential surface expression of α1,2-fucosylated epitopes in a panel of normal, dysplastic, and head and neck cancer cell lines, identify a set of potentially α1,2-fucosylated signaling and adhesion molecules including the epidermal growth factor receptor (EGFR), CD44 and integrins via tandem mass spectrometry, and finally, present evidence that EGFR is among the α1,2-fucosylated and LeY-displaying proteins in head and neck cancer. This knowledge will serve as the foundation for future studies to interrogate the role of LeY-modified and α1,2-fucosylated glycoproteins in head and neck cancer pathogenesis. Data are available via ProteomeXchange with identifier PXD029420.

sLeX Expression Delineates Distinct Functional Subsets of Human Blood Central and Effector Memory T Cells.

Sialyl Lewis X (sLeX) regulates T cell trafficking from the vasculature into skin and sites of inflammation, thereby playing a critical role in immunity. In healthy persons, only a small proportion of human blood T cells express sLeX, and their function is not fully defined. Using a combination of biochemical and functional studies, we find that human blood sLeX+CD4+T cells comprise a subpopulation expressing high levels of Th2 and Th17 cytokines, chemokine receptors CCR4 and CCR6, and the transcription factors GATA-3 and RORγT. Additionally, sLeX+CD4+T cells exclusively contain the regulatory T cell population (CD127lowCD25high and FOXP3+) and characteristically display immune-suppressive molecules, including the coinhibitor receptors PD-1 and CTLA-4. Among CD8+T cells, sLeX expression distinguishes a subset displaying low expression of cytotoxic effector molecules, perforin and granzyme ß, with reduced degranulation and CD57 expression and, consistently, marginal cytolytic capacity after TCR engagement. Furthermore, sLeX+CD8+T cells present a pattern of features consistent with Th cell-like phenotype, including release of pertinent Tc2 cytokines and elevated expression of CD40L. Together, these findings reveal that sLeX display is associated with unique functional specialization of both CD4+ and CD8+T cells and indicate that circulating T cells that are primed to migrate to lesional sites at onset of inflammation are not poised for cytotoxic function.

Defibrotide inhibits donor leucocyte-endothelial interactions and protects against acute graft-versus-host disease.

Allogeneic hematopoietic stem cell transplantation (allo-HCT) is an effective therapy for the treatment of high-risk haematological malignant disorders and other life-threatening haematological and genetic diseases. Acute graft-versus-host disease (aGvHD) remains the most frequent cause of non-relapse mortality following allo-HCT and limits its extensive clinical application. Current pharmacologic agents used for prophylaxis and treatment of aGvHD are not uniformly successful and have serious secondary side effects. Therefore, more effective and safe prophylaxis and therapy for aGvHD are an unmet clinical need. Defibrotide is a multi-target drug successfully employed for prophylaxis and treatment of veno-occlusive disease/sinusoidal obstruction syndrome. Recent preliminary clinical data have suggested some efficacy of defibrotide in the prevention of aGvHD after allo-HCT. Using a fully MHC-mismatched murine model of allo-HCT, we report here that defibrotide, either in prophylaxis or treatment, is effective in preventing T cell and neutrophil infiltration and aGvHD-associated tissue injury, thus reducing aGvHD incidence and severity, with significantly improved survival after allo-HCT. Moreover, we performed in vitro mechanistic studies using human cells revealing that defibrotide inhibits leucocyte-endothelial interactions by down-regulating expression of key endothelial adhesion molecules involved in leucocyte trafficking. Together, these findings provide evidence that defibrotide may represent an effective and safe clinical alternative for both prophylaxis and treatment of aGvHD after allo-HCT, paving the way for new therapeutic approaches.

Glycoengineering of chimeric antigen receptor (CAR) T-cells to enforce E-selectin binding.

Tissue colonization (homing) by blood-borne cells critically hinges on the ability of the cells to adhere to vascular endothelium with sufficient strength to overcome prevailing hemodynamic shear stress. These adhesive interactions are most effectively engendered via binding of the endothelial lectin E-selectin (CD62E) to its cognate ligand, sialyl Lewis-X (sLe X ), displayed on circulating cells. Although chimeric antigen receptor (CAR) T-cell immunotherapy holds promise for treatment of various hematologic and non-hematologic malignancies, there is essentially no information regarding the efficiency of CAR T-cell homing. Accordingly, we performed integrated biochemical studies and adhesion assays to examine the capacity of human CAR T-cells to engage E-selectin. Our data indicate that CAR T-cells do not express sLe X and do not bind E-selectin. However, enforced sLe X display can be achieved on human CAR T-cells by surface fucosylation, with resultant robust E-selectin binding under hemodynamic shear. Importantly, following intravascular administration into mice, fucosylated human CAR-T cells infiltrate marrow with 10-fold higher efficiency than do unfucosylated cells. Collectively, these findings indicate that custom installation of sLe X programs tissue colonization of vascularly administered human CAR T-cells, offering a readily translatable strategy to augment tissue delivery, thereby lowering the pertinent cell dosing and attendant cell production burden, for CAR T-cell immunotherapy applications.

Ligation of the CD44 Glycoform HCELL on Culture-Expanded Human Monocyte-Derived Dendritic Cells Programs Transendothelial Migration.

The success of dendritic cell (DC)-based immunotherapeutics critically hinges on the capacity of the vascularly administered cells to enter tissues. Transendothelial migration (TEM) is dictated by an ordered cascade of receptor/ligand interactions. In this study, we examined the key molecular effectors of TEM of human monocyte-derived DCs (mo-DCs) generated by clinically relevant methods: CD14 selection (CD14-S) and plastic adherence selection (PA-S). Without chemokine input, CD14-S cells undergo greater TEM than PA-S cells over TNF-α-stimulated HUVECs. TEM of CD14-S mo-DCs is E-selectin/very late Ag-4 (VLA-4) dependent, and engagement of E-selectin ligands activates VLA-4 on CD14-S mo-DCs but not on PA-S mo-DCs. E-selectin binding glycoforms of P-selectin glycoprotein ligand-1 (PSGL-1) (i.e., cutaneous lymphocyte Ag [CLA]) and CD44 (i.e., hematopoietic cell E-selectin/L-selectin ligand [HCELL]) are both expressed on CD14-S mo-DCs, but only CLA is expressed on PA-S mo-DCs. To elucidate the effect of CD44 or PSGL-1 engagement, mo-DCs were pretreated with their ligands. Ligation of CD44 on CD14-S mo-DCs triggers VLA-4 activation and TEM, whereas PSGL-1 ligation does not. HCELL expression on CD14-S mo-DC can be enforced by cell surface exofucosylation, yielding increased TEM in vitro and enhanced extravasation into bone marrow in vivo. These findings highlight structural and functional pleiotropism of CD44 in priming TEM of mo-DCs and suggest that strategies to enforce HCELL expression may boost TEM of systemically administered CD14-S mo-DCs.

Distinct human α(1,3)-fucosyltransferases drive Lewis-X/sialyl Lewis-X assembly in human cells.

In humans, six α(1,3)-fucosyltransferases (α(1,3)-FTs: FT3/FT4/FT5/FT6/FT7/FT9) reportedly fucosylate terminal lactosaminyl glycans yielding Lewis-X (LeX; CD15) and/or sialyl Lewis-X (sLeX; CD15s), structures that play key functions in cell migration, development, and immunity. Prior studies analyzing α(1,3)-FT specificities utilized either purified and/or recombinant enzymes to modify synthetic substrates under nonphysiological reaction conditions or molecular biology approaches wherein α(1,3)-FTs were expressed in mammalian cell lines, notably excluding investigations using primary human cells. Accordingly, although significant insights into α(1,3)-FT catalytic properties have been obtained, uncertainty persists regarding their human LeX/sLeX biosynthetic range across various glycoconjugates. Here, we undertook a comprehensive evaluation of the lactosaminyl product specificities of intracellularly expressed α(1,3)-FTs using a clinically relevant primary human cell type, mesenchymal stem cells. Cells were transfected with modified mRNA encoding each human α(1,3)-FT, and the resultant α(1,3)-fucosylated lactosaminyl glycoconjugates were analyzed using a combination of flow cytometry and MS. The data show that biosynthesis of sLeX is driven by FTs-3, -5, -6, and -7, with FT6 and FT7 having highest potency. FT4 and FT9 dominantly biosynthesize LeX, and, among all FTs, FT6 holds a unique capacity in creating sLeX and LeX determinants across protein and lipid glycoconjugates. Surprisingly, FT4 does not generate sLeX on glycolipids, and neither FT4, FT6, nor FT9 synthesizes the internally fucosylated sialyllactosamine VIM-2 (CD65s). These results unveil the relevant human lactosaminyl glycans created by human α(1,3)-FTs, providing novel insights on how these isoenzymes stereoselectively shape biosynthesis of vital glycoconjugates, thereby biochemically programming human cell migration and tuning human immunologic and developmental processes.

Cell-Specific Variation in E-Selectin Ligand Expression among Human Peripheral Blood Mononuclear Cells: Implications for Immunosurveillance and Pathobiology.

Both host defense and immunopathology are shaped by the ordered recruitment of circulating leukocytes to affected sites, a process initiated by binding of blood-borne cells to E-selectin displayed at target endothelial beds. Accordingly, knowledge of the expression and function of leukocyte E-selectin ligands is key to understanding the tempo and specificity of immunoreactivity. In this study, we performed E-selectin adherence assays under hemodynamic flow conditions coupled with flow cytometry and Western blot analysis to elucidate the function and structural biology of glycoprotein E-selectin ligands expressed on human PBMCs. Circulating monocytes uniformly express high levels of the canonical E-selectin binding determinant sialyl Lewis X (sLeX) and display markedly greater adhesive interactions with E-selectin than do circulating lymphocytes, which exhibit variable E-selectin binding among CD4+ and CD8+ T cells but no binding by B cells. Monocytes prominently present sLeX decorations on an array of protein scaffolds, including P-selectin glycoprotein ligand-1, CD43, and CD44 (rendering the E-selectin ligands cutaneous lymphocyte Ag, CD43E, and hematopoietic cell E-selectin/L-selectin ligand, respectively), and B cells altogether lack E-selectin ligands. Quantitative PCR gene expression studies of glycosyltransferases that regulate display of sLeX reveal high transcript levels among circulating monocytes and low levels among circulating B cells, and, commensurately, cell surface α(1,3)-fucosylation reveals that acceptor sialyllactosaminyl glycans convertible into sLeX are abundantly expressed on human monocytes yet are relatively deficient on B cells. Collectively, these findings unveil distinct cell-specific patterns of E-selectin ligand expression among human PBMCs, indicating that circulating monocytes are specialized to engage E-selectin and providing key insights into the molecular effectors mediating recruitment of these cells at inflammatory sites.

Imaging specific cellular glycan structures using glycosyltransferases via click chemistry.

Heparan sulfate (HS) is a polysaccharide fundamentally important for biologically activities. T/Tn antigens are universal carbohydrate cancer markers. Here, we report the specific imaging of these carbohydrates using a mesenchymal stem cell line and human umbilical vein endothelial cells (HUVEC). The staining specificities were demonstrated by comparing imaging of different glycans and validated by either removal of target glycans, which results in loss of signal, or installation of target glycans, which results in gain of signal. As controls, representative key glycans including O-GlcNAc, lactosaminyl glycans and hyaluronan were also imaged. HS staining revealed novel architectural features of the extracellular matrix (ECM) of HUVEC cells. Results from T/Tn antigen staining suggest that O-GalNAcylation is a rate-limiting step for O-glycan synthesis. Overall, these highly specific approaches for HS and T/Tn antigen imaging should greatly facilitate the detection and functional characterization of these biologically important glycans.

A Glycovariant of Human CD44 is Characteristically Expressed on Human Mesenchymal Stem Cells.

The clinical effectiveness of systemically administered human mesenchymal stem cells (hMSCs) depends on their capacity to engage vascular endothelium. hMSCs derived from bone marrow (BM-hMSCs) natively lack endothelial binding capacity, but express a CD44 glycovariant containing N-linked sialyllactosamines that can be α(1,3)-fucosylated using fucosyltransferase-VI (FTVI) to enforce sLeX decorations, thereby creating hematopoietic cell E-/L-selectin ligand (HCELL). HCELL expression programs potent shear-resistant adhesion of circulating cells to endothelial beds expressing E-selectin. An alternative source of hMSCs is adipose tissue (A-hMSCs), and we assessed whether A-hMSCs bind E-selectin and/or possess sialyllactosamine-decorated CD44 accessible to α(1,3)-fucosylation. Similar to BM-hMSCs, we found that A-hMSCs natively lack E-selectin ligands, but FTVI-mediated cell surface α(1,3)-fucosylation induces sLeX expression and robust E-selectin binding secondary to conversion of CD44 into HCELL. Moreover, treatment with the α(1,3)-fucosyltransferase-FTVII also generated expression of HCELL on both BM-hMSCs and A-hMSCs, with sLeX decorations created on N-linked glycans of the "standard" CD44 (CD44s) isoform. The finding that hMSCs from both source tissues each lack native E-selectin ligand expression prompted examination of the expression of glycosyltransferases that direct lactosaminyl glycan synthesis. These studies reveal that both types of hMSCs conspicuously lack transcripts encoding α(1,3)-fucosyltransferases, but equally express glycosyltransferases critical to creation of sialyllactosamines. Collectively, these data indicate that assembly of a sialyllactosaminyl-decorated CD44s glycovariant is a conserved feature of hMSCs derived from adipose tissue and marrow, thus identifying a CD44 glycosignature of these cells and supporting the applicability of cell surface α(1,3)-fucosylation in programming migration of systemically administered A-hMSCs to sites of tissue injury/inflammation. Stem Cells 2017;35:1080-1092.

Production via good manufacturing practice of exofucosylated human mesenchymal stromal cells for clinical applications.

BACKGROUND: The regenerative and immunomodulatory properties of human mesenchymal stromal cells (hMSCs) have raised great hope for their use in cell therapy. However, when intravenously infused, hMSCs fail to reach sites of tissue injury. Fucose addition in α(1,3)-linkage to terminal sialyllactosamines on CD44 creates the molecule known as hematopoietic cell E-/L-selectin ligand (HCELL), programming hMSC binding to E-selectin that is expressed on microvascular endothelial cells of bone marrow (BM), skin and at all sites of inflammation. Here we describe how this modification on BM-derived hMSCs (BM-hMSCs) can be adapted to good manufacturing practice (GMP) standards. METHODS: BM-hMSCs were expanded using xenogenic-free media and exofucosylated using α(1,3)-fucosyltransferases VI (FTVI) or VII (FTVII). Enforced fucosylation converted CD44 into HCELL, and HCELL formation was assessed using Western blot, flow cytometry and cell-binding assays. Untreated (unfucosylated), buffer-treated and exofucosylated BM-hMSCs were each analyzed for cell viability, immunophenotype and differentiation potential, and E-selectin binding stability was assessed at room temperature, at 4°C, and after cryopreservation. Cell product safety was evaluated using microbiological testing, karyotype analysis, and c-Myc messenger RNA (mRNA) expression, and potential effects on genetic reprogramming and in cell signaling were analyzed using gene expression microarrays and receptor tyrosine kinase (RTK) phosphorylation arrays. RESULTS: Our protocol efficiently generates HCELL on clinical-scale batches of BM-hMSCs. Exofucosylation yields stable HCELL expression for 48 h at 4°C, with retained expression after cell cryopreservation. Cell viability and identity are unaffected by exofucosylation, without changes in gene expression or RTK phosphorylation. DISCUSSION: The described exofucosylation protocol using xenogenic-free reagents enforces HCELL expression on hMSCs endowing potent E-selectin binding without affecting cell viability or native phenotype. This described protocol is readily scalable for GMP-compliant clinical production.

Staining of E-selectin ligands on paraffin-embedded sections of tumor tissue.

BACKGROUND: The E-selectin ligands expressed by cancer cells mediate adhesion of circulating cancer cells to endothelial cells, as well as within tissue microenvironments important for tumor progression and metastasis. The identification of E-selectin ligands within cancer tissue could yield new biomarkers for patient stratification and aid in identifying novel therapeutic targets. The determinants of selectin ligands consist of sialylated tetrasaccharides, the sialyl Lewis X and A (sLeX and sLeA), displayed on protein or lipid scaffolds. Standardized procedures for immunohistochemistry make use of the antibodies against sLeX and/or sLeA. However, antibody binding does not define E-selectin binding activity. METHODS: In this study, we developed an immunohistochemical staining technique, using E-selectin-human Ig Fc chimera (E-Ig) to characterize the expression and localization of E-selectin binding sites on paraffin-embedded sections of different cancer tissue. RESULTS: E-Ig successfully stained cancer cells with high specificity. The E-Ig staining show high reactivity scores in colon and lung adenocarcinoma and moderate reactivity in triple negative breast cancer. Compared with reactivity of antibody against sLeX/A, the E-Ig staining presented higher specificity to cancer tissue with better defined borders and less background. CONCLUSIONS: The E-Ig staining technique allows the qualitative and semi-quantitative analysis of E-selectin binding activity on cancer cells. The development of accurate techniques for detection of selectin ligands may contribute to better diagnostic and better understanding of the molecular basis of tumor progression and metastasis.

T-lymphocyte homing: an underappreciated yet critical hurdle for successful cancer immunotherapy.

Advances in cancer immunotherapy have offered new hope for patients with metastatic disease. This unfolding success story has been exemplified by a growing arsenal of novel immunotherapeutics, including blocking antibodies targeting immune checkpoint pathways, cancer vaccines, and adoptive cell therapy (ACT). Nonetheless, clinical benefit remains highly variable and patient-specific, in part, because all immunotherapeutic regimens vitally hinge on the capacity of endogenous and/or adoptively transferred T-effector (Teff) cells, including chimeric antigen receptor (CAR) T cells, to home efficiently into tumor target tissue. Thus, defects intrinsic to the multi-step T-cell homing cascade have become an obvious, though significantly underappreciated contributor to immunotherapy resistance. Conspicuous have been low intralesional frequencies of tumor-infiltrating T-lymphocytes (TILs) below clinically beneficial threshold levels, and peripheral rather than deep lesional TIL infiltration. Therefore, a Teff cell 'homing deficit' may arguably represent a dominant factor responsible for ineffective immunotherapeutic outcomes, as tumors resistant to immune-targeted killing thrive in such permissive, immune-vacuous microenvironments. Fortunately, emerging data is shedding light into the diverse mechanisms of immune escape by which tumors restrict Teff cell trafficking and lesional penetrance. In this review, we scrutinize evolving knowledge on the molecular determinants of Teff cell navigation into tumors. By integrating recently described, though sporadic information of pivotal adhesive and chemokine homing signatures within the tumor microenvironment with better established paradigms of T-cell trafficking under homeostatic or infectious disease scenarios, we seek to refine currently incomplete models of Teff cell entry into tumor tissue. We further summarize how cancers thwart homing to escape immune-mediated destruction and raise awareness of the potential impact of immune checkpoint blockers on Teff cell homing. Finally, we speculate on innovative therapeutic opportunities for augmenting Teff cell homing capabilities to improve immunotherapy-based tumor eradication in cancer patients, with special focus on malignant melanoma.

Glycoengineering of E-Selectin Ligands by Intracellular versus Extracellular Fucosylation Differentially Affects Osteotropism of Human Mesenchymal Stem Cells.

Human mesenchymal stem cells (MSCs) hold great promise in cellular therapeutics for skeletal diseases but lack expression of E-selectin ligands that direct homing of blood-borne cells to bone marrow. Previously, we described a method to engineer E-selectin ligands on the MSC surface by exofucosylating cells with fucosyltransferase VI (FTVI) and its donor sugar, GDP-Fucose, enforcing transient surface expression of the potent E-selectin ligand HCELL with resultant enhanced osteotropism of intravenously administered cells. Here, we sought to determine whether E-selectin ligands created via FTVI-exofucosylation are distinct in identity and function to those created by FTVI expressed intracellularly. To this end, we introduced synthetic modified mRNA encoding FTVI (FUT6-modRNA) into human MSCs. FTVI-exofucosylation (i.e., extracellular fucosylation) and FUT6-modRNA transfection (i.e., intracellular fucosylation) produced similar peak increases in cell surface E-selectin ligand levels, and shear-based functional assays showed comparable increases in tethering/rolling on human endothelial cells expressing E-selectin. However, biochemical analyses revealed that intracellular fucosylation induced expression of both intracellular and cell surface E-selectin ligands and also induced a more sustained expression of E-selectin ligands compared to extracellular fucosylation. Notably, live imaging studies to assess homing of human MSC to mouse calvarium revealed more osteotropism following intravenous administration of intracellularly-fucosylated cells compared to extracellularly-fucosylated cells. This study represents the first direct analysis of E-selectin ligand expression programmed on human MSCs by FTVI-mediated intracellular versus extracellular fucosylation. The observed differential biologic effects of FTVI activity in these two contexts may yield new strategies for improving the efficacy of human MSCs in clinical applications. Stem Cells 2016;34:2501-2511.

G-CSF induces membrane expression of a myeloperoxidase glycovariant that operates as an E-selectin ligand on human myeloid cells.

The host defense response critically depends on the production of leukocytes by the marrow and the controlled delivery of these cells to relevant sites of inflammation/infection. The cytokine granulocyte-colony stimulating factor (G-CSF) is commonly used therapeutically to augment neutrophil recovery following chemo/radiation therapy for malignancy, thereby decreasing infection risk. Although best known as a potent inducer of myelopoiesis, we previously reported that G-CSF also promotes the delivery of leukocytes to sites of inflammation by stimulating expression of potent E-selectin ligands, including an uncharacterized ∼65-kDa glycoprotein. To identify this ligand, we performed integrated biochemical analysis and mass spectrometry studies of G-CSF-treated primary human myeloid cells. Our studies show that this novel E-selectin ligand is a glycoform of the heavy chain component of the enzyme myeloperoxidase (MPO), a well-known lysosomal peroxidase. This specialized MPO glycovariant, referred to as "MPO-E-selectin ligand" (MPO-EL), is expressed on circulating G-CSF-mobilized leukocytes and is naturally expressed on blood myeloid cells in patients with febrile leukocytosis. In vitro biochemical studies show that G-CSF programs MPO-EL expression on human blood leukocytes and marrow myeloid cells via induction of N-linked sialofucosylations on MPO, with concomitant cell surface display of the molecule. MPO-EL is catalytically active and mediates angiotoxicity on human endothelial cells that express E-selectin. These findings thus define a G-CSF effect on MPO chemical biology that endows unsuspected functional versatility upon this enzyme, unveiling new perspectives on the biology of G-CSF and MPO, and on the role of E-selectin receptor/ligand interactions in leukocyte migration and vascular pathology.

Fulfilling Koch's postulates in glycoscience: HCELL, GPS and translational glycobiology.

Glycoscience-based research that is performed expressly to address medical necessity and improve patient outcomes is called "translational glycobiology". In the 19th century, Robert Koch proposed a set of postulates to rigorously establish causality in microbial pathogenesis, and these postulates can be reshaped to guide knowledge into how naturally-expressed glycoconjugates direct molecular processes critical to human well-being. Studies in the 1990s indicated that E-selectin, an endothelial lectin that binds sialofucosylated carbohydrate determinants, is constitutively expressed on marrow microvessels, and investigations in my laboratory indicated that human hematopoietic stem cells (HSCs) uniquely express high levels of a specialized glycoform of CD44 called "hematopoietic cell E-/L-selectin ligand" (HCELL) that functions as a highly potent E-selectin ligand. To assess the role of HCELL in directing HSC migration to marrow, a method called "glycosyltransferase-programmed stereosubstitution" (GPS) was developed to custom-modify CD44 glycans to enforce HCELL expression on viable cell surfaces. Human mesenchymal stem cells (MSCs) are devoid of E-selectin ligands, but GPS-based glycoengineering of CD44 on MSCs licenses homing of these cells to marrow in vivo, providing direct evidence that HCELL serves as a "bone marrow homing receptor". This review will discuss the molecular basis of cell migration in historical context, will describe the discovery of HCELL and its function as the bone marrow homing receptor, and will inform on how glycoengineering of CD44 serves as a model for adapting Koch's postulates to elucidate the key roles that glycoconjugates play in human biology and for realizing the immense impact of translational glycobiology in clinical medicine.

Congenital disorder of fucosylation type 2c (LADII) presenting with short stature and developmental delay with minimal adhesion defect.

Leukocyte adhesion deficiency type II is a hereditary disorder of neutrophil migration caused by mutations in the guanosine diphosphate-fucose transporter gene (SLC35C1). In these patients, inability to generate key fucosylated molecules including sialyl Lewis X leads to leukocytosis and recurrent infections, in addition to short stature and developmental delay. We report two brothers with short stature and developmental delay who are compound heterozygotes for novel mutations in SLC35C1 resulting in partial in vivo defects in fucosylation. Specifically, plasma glycoproteins including immunoglobulin G demonstrated marked changes in glycoform distribution. While neutrophil rolling on endothelial selectins was partially impeded, residual adhesion proved sufficient to avoid leukocytosis or recurrent infection. These findings demonstrate a surprising degree of immune redundancy in the face of substantial alterations in adhesion molecule expression, and show that short stature and developmental delay may be the sole presenting signs in this disorder.

HCELL Expression on Murine MSC Licenses Pancreatotropism and Confers Durable Reversal of Autoimmune Diabetes in NOD Mice.

Type 1 diabetes (T1D) is an immune-mediated disease resulting in destruction of insulin-producing pancreatic beta cells. Mesenchymal stem cells (MSCs) possess potent immunomodulatory properties, garnering increasing attention as cellular therapy for T1D and other immunologic diseases. However, MSCs generally lack homing molecules, hindering their colonization at inflammatory sites following intravenous (IV) administration. Here, we analyzed whether enforced E-selectin ligand expression on murine MSCs could impact their effect in reversing hyperglycemia in nonobese diabetic (NOD) mice. Although murine MSCs natively do not express the E-selectin-binding determinant sialyl Lewis(x) (sLe(x) ), we found that fucosyltransferase-mediated α(1,3)-exofucosylation of murine MSCs resulted in sLe(x) display uniquely on cell surface CD44 thereby creating hematopoietic cell E-/L-selectin ligand (HCELL), the E-selectin-binding glycoform of CD44. Following IV infusion into diabetic NOD mice, allogeneic HCELL(+) MSCs showed threefold greater peri-islet infiltrates compared to buffer-treated (i.e., HCELL(-) ) MSCs, with distribution in proximity to E-selectin-expressing microvessels. Exofucosylation had no effect on MSC immunosuppressive capacity in in vitro assays; however, although engraftment was temporary for both HCELL(+) and HCELL(-) MSCs, administration of HCELL(+) MSCs resulted in durable reversal of hyperglycemia, whereas only transient reversal was observed following administration of HCELL(-) MSCs. Notably, exofucosylation of MSCs generated from CD44(-/-) mice induced prominent membrane expression of sLe(x) , but IV administration of these MSCs into hyperglycemic NOD mice showed no enhanced pancreatotropism or reversal of hyperglycemia. These findings provide evidence that glycan engineering to enforce HCELL expression boosts trafficking of infused MSCs to pancreatic islets of NOD mice and substantially improves their efficacy in reversing autoimmune diabetes. Stem Cells 2013;33:1523-1531.

Progress and obstacles towards generating hematopoietic stem cells from pluripotent stem cells.

PURPOSE OF REVIEW: Human pluripotent stem cells (PSCs) have the potential to provide an inexhaustible source of hematopoietic stem cells (HSCs) that could be used in disease modeling and in clinical applications such as transplantation. Although the goal of deriving definitive HSCs from PSCs has not been achieved, recent studies indicate that progress is being made. This review will provide information on the current status of deriving HSCs from PSCs, and will highlight existing challenges and obstacles. RECENT FINDINGS: Recent strides in HSC generation from PSCs has included derivation of developmental intermediates, identification of transcription factors and small molecules that support hematopoietic derivation, and the development of strategies to recapitulate niche-like conditions. SUMMARY: Despite considerable progress in defining the molecular events driving derivation of hematopoietic progenitor cells from PSCs, the generation of robust transplantable HSCs from PSCs remains elusive. We propose that this goal can be facilitated by better understanding of the regulatory pathways governing HSC identity, development of HSC supportive conditions, and examining the marrow homing properties of PSC-derived HSCs.