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.

Major differences in glycosylation and fucosyltransferase expression in low-grade versus high-grade bladder cancer cell lines.

Bladder cancer is the ninth most frequently diagnosed cancer worldwide, and there is a need to develop new biomarkers for staging and prognosis of this disease. Here we report that cell lines derived from low-grade and high-grade bladder cancers exhibit major differences in expression of glycans in surface glycoproteins. We analyzed protein glycosylation in three low-grade bladder cancer cell lines RT4 (grade-1-2), 5637 (grade-2), and SW780 (grade-1), and three high-grade bladder cancer cell lines J82COT (grade-3), T24 (grade-3) and TCCSUP (grade-4), with primary bladder epithelial cells, A/T/N, serving as a normal bladder cell control. Using a variety of approaches including flow cytometry, immunofluorescence, glycomics and gene expression analysis, we observed that the low-grade bladder cancer cell lines RT4, 5637 and SW780 express high levels of the fucosylated Lewis-X antigen (Lex, CD15) (Galß1-4(Fucα1-3)GlcNAcß1-R), while normal bladder epithelial A/T/N cells lack Lex expression. T24 and TCCSUP cells also lack Lex, whereas J82COT cells express low levels of Lex. Glycomics analyses revealed other major differences in fucosylation and sialylation of N-glycans between these cell types. O-glycans are highly differentiated, as RT4 cells synthesize core 2-based O-glycans that are lacking in the T24 cells. These differences in glycan expression correlated with differences in RNA expression levels of their cognate glycosyltransferases, including α1-3/4-fucosyltransferase genes. These major differences in glycan structures and gene expression profiles between low- and high-grade bladder cancer cells suggest that glycans and glycosyltransferases are candidate biomarkers for grading bladder cancers.

Antibodies from Lampreys as Smart Anti-Glycan Reagents (SAGRs): Perspectives on Their Specificity, Structure, and Glyco-genomics.

The repertoire of glycans expressed by individual cells and tissues is enormous, and various estimates indicate that thousands of different glycans and "glycan determinants" are critical for functional recognition by glycan-binding proteins. Defining the steady-state expression and functional impacts of the human glycome will require a concerted worldwide effort, along with the development of new immunological, genetic, chemical, and biochemical technologies. Here, we describe the generation of smart anti-glycan reagents (SAGRs), recombinant antibodies that recognize novel glycan determinants. The antibodies are generated by the sea lamprey (Petromyzon marinus), through immunization with glycoconjugates, cells, and even tissues. SAGRs represent a versatile immunological tool for defining the expression of glycans in cells and tissues. We also present a comparison of lamprey-derived anti-carbohydrate antibodies that have been characterized to date. Finally, we explore the unique glyco-genome of the lamprey itself as it compares to those of humans and mice and how it may relate to the lamprey's inherent capacity to produce antibodies to mammalian glycans.

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.

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.

ST3Gal-4 is the primary sialyltransferase regulating the synthesis of E-, P-, and L-selectin ligands on human myeloid leukocytes.

The precise glycosyltransferase enzymes that mediate selectin-ligand biosynthesis in human leukocytes are unknown. This knowledge is important because selectin-mediated cell tethering and rolling is a critical component of both normal immune response and various vascular disorders. We evaluated the role of 3 α(2,3)sialyltransferases, ST3Gal-3, -4, and -6, which act on the type II N-Acetyllactosamine structure (Galß1,4GlcNAc) to create sialyl Lewis-X (sLe(X)) and related sialofucosylated glycans on human leukocytes of myeloid lineage. These genes were either silenced using lentiviral short hairpin RNA (shRNA) or functionally ablated using the clustered regularly interspaced short palindromic repeat/Cas9 technology. The results show that ST3Gal-4, but not ST3Gal-3 or -6, is the major sialyltransferase regulating the biosynthesis of E-, P-, and L-selectin ligands in humans. Reduction in ST3Gal-4 activity lowered cell-surface HECA-452 epitope expression by 75% to 95%. Glycomics profiling of knockouts demonstrate an almost complete loss of the sLe(X) epitope on both leukocyte N- and O-glycans. In cell-adhesion studies, ST3Gal-4 knockdown/knockout cells displayed 90% to 100% reduction in tethering and rolling density on all selectins. ST3Gal-4 silencing in neutrophils derived from human CD34(+) hematopoietic stem cells also resulted in 80% to 90% reduction in cell adhesion to all selectins. Overall, a single sialyltransferase regulates selectin-ligand biosynthesis in human leukocytes, unlike mice where multiple enzymes contribute to this function.

Glycosphingolipids on Human Myeloid Cells Stabilize E-Selectin-Dependent Rolling in the Multistep Leukocyte Adhesion Cascade.

OBJECTIVE: Recent studies suggest that the E-selectin ligands expressed on human leukocytes may differ from those in other species, particularly mice. To elaborate on this, we evaluated the impact of glycosphingolipids expressed on human myeloid cells in regulating E-selectin-mediated cell adhesion. APPROACH AND RESULTS: A series of modified human cell lines and primary neutrophils were created by targeting UDP-Glucose Ceramide Glucosyltransferase using either lentivirus-delivered shRNA or CRISPR-Cas9-based genome editing. Enzymology and mass spectrometry confirm that the modified cells had reduced or abolished glucosylceramide biosynthesis. Glycomics profiling showed that UDP-Glucose Ceramide Glucosyltransferase disruption also increased prevalence of bisecting N-glycans and reduced overall sialoglycan expression on leukocyte N- and O-glycans. Microfluidics-based flow chamber studies demonstrated that both the UDP-Glucose Ceramide Glucosyltransferase knockouts and knockdowns display ≈60% reduction in leukocyte rolling and firm adhesion on E-selectin bearing stimulated endothelial cells, without altering cell adhesion to P-selectin. Consistent with the concept that the glycosphingolipids support slow rolling and the transition to firm arrest, inhibiting UDP-Glucose Ceramide Glucosyltransferase activity resulted in frequent leukocyte detachment events, skipping motion, and reduced diapedesis across the endothelium. Cells bearing truncated O- and N-glycans also sustained cell rolling on E-selectin, although their ability to be recruited from free fluid flow was diminished. CONCLUSIONS: Glycosphingolipids likely contribute to human myeloid cell adhesion to E-selectin under fluid shear, particularly the transition of rolling cells to firm arrest.

Silencing α1,3-fucosyltransferases in human leukocytes reveals a role for FUT9 enzyme during E-selectin-mediated cell adhesion.

Leukocyte adhesion during inflammation is initiated by the binding of sialofucosylated carbohydrates expressed on leukocytes to endothelial E/P-selectin. Although the glycosyltransferases (glycoTs) constructing selectin-ligands have largely been identified using knock-out mice, important differences may exist between humans and mice. To address this, we developed a systematic lentivirus-based shRNA delivery workflow to create human leukocytic HL-60 cell lines that lack up to three glycoTs. Using this, the contributions of all three myeloid α1,3-fucosyltransferases (FUT4, FUT7, and FUT9) to selectin-ligand biosynthesis were evaluated. The cell adhesion properties of these modified cells to L-, E-, and P-selectin under hydrodynamic shear were compared with bone marrow-derived neutrophils from Fut4(-/-)Fut7(-/-) dual knock-out mice. Results demonstrate that predominantly FUT7, and to a lesser extent FUT4, forms the selectin-ligand at the N terminus of leukocyte P-selectin glycoprotein ligand-1 (PSGL-1) in humans and mice. Here, 85% reduction in leukocyte interaction was observed in human FUT4(-)7(-) dual knockdowns on P/L-selectin substrates. Unlike Fut4(-/-)Fut7(-/-) mouse neutrophils, however, human knockdowns lacking FUT4 and FUT7 only exhibited partial reduction in rolling interaction on E-selectin. In this case, the third α1,3-fucosyltransferase FUT9 played an important role because leukocyte adhesion was reduced by 50-60% in FUT9-HL-60, 70-80% in dual knockdown FUT7(-)9(-) cells, and ∼85% in FUT4(-)7(-)9(-) triple knockdowns. Gene silencing results are in agreement with gain-of-function experiments where all three fucosyltransferases conferred E-selectin-mediated rolling in HEK293T cells. This study advances new tools to study human glycoT function. It suggests a species-specific role for FUT9 during the biosynthesis of human E-selectin ligands.

Lipoxin A4 inhibits immune cell binding to salivary epithelium and vascular endothelium.

Lipoxins are formed by leukocytes during cell-cell interactions with epithelial or endothelial cells. Native lipoxin A(4) (LXA(4)) binds to the G protein-coupled lipoxin receptors formyl peptide receptor 2 (FPR2)/ALX and CysLT1. Furthermore, LXA(4) inhibits recruitment of neutrophils, by attenuating chemotaxis, adhesion, and transmigration across vascular endothelial cells. LXA(4) thus appears to serve as an endogenous "stop signal" for immune cell-mediated tissue injury (Serhan CN; Annu Rev Immunol 25: 101-137, 2007). The role of LXA(4) has not been addressed in salivary epithelium, and little is known about its effects on vascular endothelium. Here, we determined that interleukin-1ß (IL-1ß) and tumor necrosis factor-α (TNF-α) receptor activation in vascular endothelium and salivary epithelium upregulated the expression of adhesion molecules that facilitates the binding of immune cells. We hypothesize that the activation of the ALX/FPR2 and/or CysLT1 receptors by LXA(4) decreases this cytokine-mediated upregulation of cell adhesion molecules that enhance lymphocyte binding to both the vascular endothelium and salivary epithelium. In agreement with this hypothesis, we observed that nanomolar concentrations of LXA(4) blocked IL-1ß- and TNF-α-mediated upregulation of E-selectin and intercellular cell adhesion molecule-1 (ICAM-1) on human umbilical vein endothelial cells (HUVECs). Binding of Jurkat cells to stimulated HUVECs was abrogated by LXA(4). Furthermore, LXA(4) preincubation with human submandibular gland cell line (HSG) also blocked TNF-α-mediated upregulation of vascular cell adhesion molecule-1 (VCAM-1) in these cells, and it reduced lymphocyte adhesion. These findings suggest that ALX/FPR2 and/or CysLT1 receptor activation in endothelial and epithelial cells blocks cytokine-induced adhesion molecule expression and consequent binding of lymphocytes, a critical event in the pathogenesis of Sjögren's syndrome (SS).

von Willebrand factor self-association on platelet GpIbalpha under hydrodynamic shear: effect on shear-induced platelet activation.

The function of the mechanosensitive, multimeric blood protein von Willebrand factor (VWF) is dependent on its size. We tested the hypothesis that VWF may self-associate on the platelet glycoprotein Ibα (GpIbα) receptor under hydrodynamic shear. Consistent with this proposition, whereas Alexa-488-conjugated VWF (VWF-488) bound platelets at modest levels, addition of unlabeled VWF enhanced the extent of VWF-488 binding. Recombinant VWF lacking the A1-domain was conjugated with Alexa-488 to produce ΔA1-488. Although ΔA1-488 alone did not bind platelets under shear, this protein bound GpIbα on addition of either purified plasma VWF or recombinant full-length VWF. The extent of self-association increased with applied shear stress more than ∼ 60 to 70 dyne/cm(2). ΔA1-488 bound platelets in the milieu of plasma. On application of fluid shear to whole blood, half of the activated platelets had ΔA1-488 bound, suggesting that VWF self-association may be necessary for cell activation. Shearing platelets with 6-µm beads bearing either immobilized VWF or anti-GpIbα mAb resulted in cell activation at shear stress down to 2 to 5 dyne/cm(2). Taken together, the data suggest that fluid shear in circulation can increase the effective size of VWF bound to platelet GpIbα via protein self-association. This can trigger mechanotransduction and cell activation by enhancing the drag force applied on the cell-surface receptor.

Bone vascular niche E-selectin induces mesenchymal-epithelial transition and Wnt activation in cancer cells to promote bone metastasis.

How disseminated tumour cells engage specific stromal components in distant organs for survival and outgrowth is a critical but poorly understood step of the metastatic cascade. Previous studies have demonstrated the importance of the epithelial-mesenchymal transition in promoting the cancer stem cell properties needed for metastasis initiation, whereas the reverse process of mesenchymal-epithelial transition is required for metastatic outgrowth. Here we report that this paradoxical requirement for the simultaneous induction of both mesenchymal-epithelial transition and cancer stem cell traits in disseminated tumour cells is provided by bone vascular niche E-selectin, whose direct binding to cancer cells promotes bone metastasis by inducing mesenchymal-epithelial transition and activating Wnt signalling. E-selectin binding activity mediated by the α1-3 fucosyltransferases Fut3/Fut6 and Glg1 are instrumental to the formation of bone metastasis. These findings provide unique insights into the functional role of E-selectin as a component of the vascular niche critical for metastatic colonization in bone.

Optimizing human Treg immunotherapy by Treg subset selection and E-selectin ligand expression.

While human Tregs hold immense promise for immunotherapy, their biologic variability poses challenges for clinical use. Here, we examined clinically-relevant activities of defined subsets of freshly-isolated and culture-expanded human PBMC-derived Tregs. Unlike highly suppressive but plastic memory Tregs (memTreg), naïve Tregs (nvTreg) exhibited the greatest proliferation, suppressive capacity after stimulation, and Treg lineage fidelity. Yet, unlike memTregs, nvTregs lack Fucosyltransferase VII and display low sLeX expression, with concomitant poor homing capacity. In vitro nvTreg expansion augmented their suppressive function, but did not alter the nvTreg sLeX-l°w glycome. However, exofucosylation of the nvTreg surface yielded high sLeX expression, promoting endothelial adhesion and enhanced inhibition of xenogeneic aGVHD. These data indicate that the immature Treg glycome is under unique regulation and that adult PBMCs can be an ideal source of autologous-derived therapeutic Tregs, provided that subset selection and glycan engineering are engaged to optimize both their immunomodulation and tropism for inflammatory sites.

Using CRISPR-Cas9 to quantify the contributions of O-glycans, N-glycans and Glycosphingolipids to human leukocyte-endothelium adhesion.

There is often interest in dissecting the relative contributions of the N-glycans, O-glycans and glycosphingolipids (GSLs) in regulating complex biological traits like cell signaling, adhesion, development and metastasis. To address this, we developed a CRISPR-Cas9 toolkit to selectively truncate each of these commonly expressed glycan-types. Here, O-glycan biosynthesis was truncated by knocking-out Core 1 ß3Gal-T Specific Molecular Chaperone (COSMC), N-glycans by targeting the ß1,2 GlcNAc-transferase (MGAT1) and GSLs by deleting UDP-glucose ceramide glucosyltransferase (UGCG). These reagents were applied to reveal the glycoconjugates regulating human myeloid cell adhesion to selectins under physiological shear-flow observed during inflammation. These functional studies show that leukocyte rolling on P- and L-selectin is ablated in cells lacking O-glycans, with N-glycan truncation also increasing cell rolling velocity on L-selectin. All three glycan families contributed to E-selectin dependent cell adhesion with N-glycans contributing to all aspects of the leukocyte adhesion cascade, O-glycans only being important during initial recruitment, and GSLs stabilizing slow cell rolling and the transition to firm arrest. Overall, the genome editing tools developed here may be broadly applied in studies of cellular glycosylation.

Distinct glycosyltransferases synthesize E-selectin ligands in human vs. mouse leukocytes.

The binding of selectins to carbohydrate epitopes expressed on leukocytes is the first step in a multi-step cell adhesion cascade that controls the rate of leukocyte recruitment at sites of inflammation. The glycans that function as selectin-ligands are post-translationally synthesized by the serial action of Golgi resident enzymes called glycosyltransferases (glycoTs). Whereas much of our current knowledge regarding the role of glycoTs in constructing selectin-ligands comes from reconstituted biochemical investigations or murine models, tools to assess the impact of these enzymes on the human ligands are relatively underdeveloped. This is significant since the selectin-ligands, particularly those that bind E-selectin, vary between different leukocyte cell populations and they are also different in humans compared with mice. To address this shortcoming, a recent study by Buffone et al. (2013) outlines a systematic strategy to knockdown upto three glycoTs simultaneously in human leukocytes. The results suggest that the fucosyltransferases (FUTs) regulating selectin-ligand synthesis may be species-specific. In particular, they demonstrate that FUT9 plays a significant role during human, but not mouse, leukocyte-endothelial interactions. Overall, this article discusses the relative roles of the FUTs during human L-, E-, and P-selectin-ligand biosynthesis, and the potential that the knockdown strategy outlined here may assess the role of other glycoTs in human leukocytes also.